PSZ 19:16 (Pind.

1/07)

UNIVERSITI TEKNOLOGI MALAYSIA

DECLARATION OF THESIS / UNDERGRADUATE PROJECT PAPER AND COPYRIGHT

Authors full name : Date of birth Title : :

ALI ADEL DAWOOD 1st NOVEMBER 1966 CLONING OF INFLUENZA B NS1 GENE IN Escherichia THE EFFECT OF ETHNO/LINGO DIVERSITY ON coli

Academic Session:

2009/2010

I declare that this thesis is classified as:

CONFIDENTIAL RESTRICTED OPEN ACCESS

(Contains confidential information under the Official Secret Act 1972)* (Contains restricted information as specified by the organization where research was done)* I agree that my thesis to be published as online open access (full text)

I acknowledged that Universiti Teknologi Malaysia reserves the right as follows: 1. The thesis is the property of Universiti Teknologi Malaysia. 2. The Library of Universiti Teknologi Malaysia has the right to make copies for the purpose of research only. 3. The Library has the right to make copies of the thesis for academic exchange. Certified by:

SIGNATURE

SIGNATURE OF SUPERVISOR

S2925947
(NEW IC NO. /PASSPORT NO.) Date :

DR. CHAN GIEK FAR

NAME OF SUPERVISOR Date :

3rd DECEMBER 2010

3rd DECEMBER 2010

NOTES :

If the thesis is CONFIDENTAL or RESTRICTED, please attach with the letter from the organization with period and reasons for confidentiality or restriction.

I hereby declare that I have read this thesis and in my/our opinion this thesis is sufficient in terms of scope and quality for the award of the degree of Master of Science (Biotechnology).

Signature Name of Supervisor Date

: ..................................................................... : Dr. CHAN GIEK FAR : 3rd DECEMBER 2010

CLONING OF INFLUENZA B NS1 GENE IN Escherichia coli

ALI ADEL DAWOOD

A dissertation submitted in partial fulfillment of the requirements for the award of the degree of Master of Science (Biotechnology)

FACULTY OF BIOSCIENCES AND BIOENGINEERING UNIVERSITI TEKNOLOGI MALAYSIA

NOVEMBER 2010

ii

Hereby, I declare that this thesis entitled Cloning of influenza B NS1 gene in Escherichia coli is the result of my own research except as cited in the references. The thesis has not been accepted for any degree and is not concurrently submitted in candidature of any other degree.

Signature Name Date

: ................................ : Ali Adel Dawood : 3rd DECEMBER 2010

iii

To my beloved parents& my wife; Thanks for the dim of light when all I see were darkness Thanks for giving me the best things in my life and finally Thanks for your sacrifices to make me a better person each day.

iv

ACKNOWLEDGEMENT

In the Name of Allah, the Most Benevolent, Most Merciful

Firstly, I thank Allah for giving me the patience, persistency and his blessings throughout my completing this project. I would like to express my utmost gratitude and special appreciation to my supervisor Dr. Chan Giek Far, for encouragement, guidance, support, advices and care helped me in all the time of research and writing of this thesis.

I am deeply indebted and grateful to my family especially my mother, father and my wife for their love, support and praying for my success in every time, their patience and kindness in helping and guiding me in every part of this project. No word can express my appreciation for their love.

Special thanks to all the staffs of the Faculty of Biosciences and Bioengineering and members of the labs for not only helping me with my study but also making my stay in the lab a very pleasurable and memorable one.

Sincere appreciation and thanks are also extended to all staff of Mosul College of Medicine especially members of the Department of Anatomy for their encouragement, guidance, advices even in some words.

Last but not least, I would like to thank to people and everyone who has helped me direct or indirectly towards completing this research project.

ABSTRACT

The non-structural NS1 protein of influenza B virus is a multifunctional virulence protein which is involved in the transport of viral RNA. Inside the host cell, NS1B antagonizes and inhibits the / interferon system which is induced as host antiviral response. Moreover, it prevents the activation of double-stranded-RNA activated protein kinase (PKR) by binding to dsRNA and inhibits the maturation of GAS8 (gene of tumor suppressor). NS1 protein is utilized as a target for diagnostic of influenza viruses in infected animals. pUC57 carrying NS1 synthetic gene of influenza B was attempted to be transformed into Escherichia coli strain BL21(DE3). NS1B was extracted and attempted to be cloned into prokaryotic expression vectors pET-32b, pET-32a, pQE-81L and pQE-80L, respectively using restriction digestion enzymes (SacI, PstI and HindIII). Then, recombinant DNA was attempted to be transformed into Escherichia coli strains BL21(DE3) and DH5.

vi

ABSTRAK

Protein non-struktur NS1 virus influenza B adalah virulensi pelbagai fungsi dan terlibat dalam pengangkutan RNA virus. Di dalam sel perumah, NS1B menghalang sistem interferon / yang menyebabkan tindakbalas antivirus oleh perumah. Selain itu, protein NS1 mencegah pengaktifan doublestranded RNA-aktif protein kinase (PKR) dengan cara mengikat dsRNA dan menghalang pematangan GAS8 (gen tumor supresor). NS1 protein digunakan sebagai target untuk diagnostik terhadap virus influenza pada haiwan yang dijangkiti. pUC57 membawa gen NS1 sintetik influenza B diklonkan ke dalam Escherichia coli strain BL21(DE3). NS1B diekstraksi dan diklon ke vektor ekspresi prokariotik pET-32b, PET-32a, pQE-81L dan pQE-80L masing-

masing dengan menggunakan enzim pembatas (SacI, PstI dan HindIII). Kemudian DNA rekombinan ditransformasikan ke dalam strain Escherichia coli BL21 (DE3) dan DH5.

vii

TABLE OF CONTENTS

CHAPTER

TITLE

PAGE

DECLARATION DEDICATION ACKNOWLEDGEMENTS ABSTRACT ABSTRAK TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF SYMBOLS/ABBREVIATIONS

ii iii iv v vi vii xii xiv xvi xix 1 1 1 2 2 3 3

LIST OF APPENDICES 1
INTRODUCTION 1.1 Influenza 1.2 Influenza virus 1.3 Problem statement of the study 1.4 Objective of the study 1.5 Scope of the study

1.6 Significant of the study

LITERATURE REVIEW 2.1

4 4 4

Overview of influenza
2.1.1 Influenza infection

viii

2.1.2 2.1.3

Treatment

Preventative measures for human

Influenza virus

7 7 8 8 8 10 11 14

2.1.4

Isolation of influenza virus

2.2 Influenza virus 2.2.1 2.2.2 2.2.3 Viral genome

Types of influenza virus

Lifecycle of influenza virus

2.3 Influenza B virus 2.4 Nonstructural (NS1) protein 2.4.1

Nonstructural protein NS1 of

Influenza B virus

15

2.4.2

function and influences of NS1 protein 2.4.2.1 NS1B binds to ISG-15 protein 2.4.2.2 NS1B inhibits and antagoni-sts / interferon system 2.4.2.3

17

18

19

NS1 inhibits nuclear export of mRNA 21

2.4.2.4

NS1 protein interacts with GAS8 22

2.4.2.5

NS1 protein inhibits premRNA 23

2.4.2.6

NS1 protein prevents activation of PKR 24

2.5

Previous studies of cloning and expression of NS1 gene 25

MATERIALS AND METHODS 3.1

29 29

Experimental Design

ix

3.2 Materials 3.2.1 3.2.2 3.2.3 3.2.4 3.2.5

35 35 35 35 37 38 40 40

Bacterial strains
Chemicals pET vectors pQE vectors NS1B Synthetic Gene

3.3 Methods 3.3.1

Liquid and solid media preparation

3.3.1.1 Luria Bertani agar and

broth
3.3.1.2 Preparation of ampicillin

40

stock solution
3.3.2 3.4

40 41

Preparation of competent cells

Transformation pUC57-NS1B into E. coli BL21(DE3) competent cells 41 42 43 44 44 44 45 46 46 47

3.5

Isolation of pUC57-NS1B plasmid Agarose gel electrophoresis Isolation of pET-32b plasmid Single digestion

3.6 3.7
3.8

3.9 DNA Extraction

3.10 Determination of DNA concentration

3.11 Ligation NS1B with pET-32b 3.12 Transformation of recombinant product 3.13 Isolation of pQE-81L plasmid 3.14 Amplification of NS1B gene using

Polymerase Chain Reaction (PCR)

3.14.1 Primer design 3.14.2 Polymerase chain reaction 3.15

47 47 48

Double digestion using PstI and HindIII restriction enzymes in one step 49

3.16

Double digestion using restriction

enzymes in two steps

3.17 3.18

50 51 52 52

Ligation and transformation into DH5 Screening of positive colonies

3.18.1 Chelex 100 extraction method 3.18.2 PCR amplification and gel

electrophoresis to detect NS1B

3.19 3.20

53 53

Isolation of pET-32a plasmid Amplification of NS1B gene using Polymerase Chain Reaction (PCR)
3.20.1 Primer design

54 54

3.21

Double digestion using SacI and HindIII restriction enzymes in one step 55

3.22

Double digestion using SacI and HindIII restriction enzymes in two steps 55

3.23

Ligation, transformation into E. coli BL21 (DE3) and and screening of colonies 57 57

3.24 3.25

Isolation plasmid (pQE-80L) PCR amplification, double digestion, ligation, transformation into DH5 and screening of colonies

58

RESULTS AND DISCUSSION 4.1

59

Transformation of pUC57-NS1B into E. coli BL21(DE3) 59

4.2

Cloning of pET-32b-NS1B into E. coli BL21(DE3) 59

4.3

Cloning of pQE-81L-NS1B into E. coli DH5 61

4.4

Cloning of pET-32a-NS1B into E. coli BL21(DE3) 65

4.5

Cloning of pQE-80L-NS1B into E. coli

xi

DH5
4.6

68 69

Confirmation of ligation

CONCLUSION AND FUTURE WORKS 5.1 Conclusion 5.2 Future works

71 71 72

REFERENCES Appendices A-B

74 79-80

xii

LIST OF TABLES

TABLE NO.

TITLE

PAGE

3.1

Single digestion components in PCR tube using HindIII 44

3.2

Ligation mix components of pET-32b and NS1B 46

3.3

The pUC57 primers for PCR of NS1B gene from pUC57-NS1B 48 48 49

3.4 3.5 3.6

PCR reaction components of pUC57-NS1B PCR cycle Double digestion components in PCR tube using PstI and HindIII

50

3.7

Double digestion components in PCR tube using HindIII(1st step) 50

3.8

Double digestion components in PCR tube using PstI(2nd step) 51 53

3.9 3.10

The pQE primers for PCR Primers containing SacI and HindIII restriction enzyme sites used to amplify NS1B gene

54

3.11

Double digestion components in PCR tube using SacI and HindIII 55

3.12

Double digestion components in PCR tube using HindIII(1st step) 56

xiii

3.13

Double digestion components in PCR tube using SacI(2nd step) 56

3.14

Primers containing SacI and HindIII restriction enzyme sites used to amplify NS1B gene 57

xiv

LIST OF FIGURES

FIGURE NO.

TITLE

PAGE

2.1 2.2 2.3 2.4 2.5

Life cycle of influenza virus Influenza virus structure Electron microscope image of influenza B Influenza virus particles on cells lining Homology modeling of RNA binding domain of NS1B

10 11 13 14

16 30

3.1 3.2

Flow chart of experimental design Cloning of pET-32b-NS1B into E. coli BL21(DE3)

31 32

3.3 3.4

Cloning of pQE-81L-NS1B into E. coli DH5 Cloning of pET-32a-NS1B into E. coli BL21(DE3)

33 34 36 37 38 39 60

3.5 3.6 3.7 3.8 3.9 4.1 4.2

Cloning of pQE-80L-NS1B into E. coli DH5 pET-32 vectors pQE-80L vector pQE-81L vector pUC57-NS1B gene construct Restriction digestion by HindIII. Transformation of pET-32b-NS1B into competent E. coli BL21(DE3)

61

4.3

Plasmid isolation and digestion of pUC57-

xv

NS1B and pQE-81L 4.4 4.5 PCR amplification of NS1B Transformation of pQE-81L-NS1B into competent DH5 4.6 4.7 PCR of recombinant pQE-81L-NS1B Miniprep of pUC57-NS1B and PCR amplification of NS1B 4.8 Transformation of pET-32a-NS1B into competent E. coli BL2(DE3) 4.9 4.10 PCR of recombinant pET-32a-NS1B Transformation of pQE-80L-NS1B into competent DH5 4.11 4.12 PCR of recombinant pQE-80L-NS1B PCR amplification

62 63

64 65

66

67 67

68 69 70

xvi

LIST OF SYMBOLS/ ABBREVIATIONS

AMP ATP bp BSA / C cDNA del NS1 dH2O dNTP DNA dsRNA E. coli EDTA ELISA g GAS GST HA His-Tag IFN IPTG IRF

Adenosine monophosphate Adenosine triphosphate Base pairs Bovine Serum Albumin Alfa / Beta interferon Degree Celsius Clone deoxyribonucleic acid Deletion nonstructural 1 Deionized water Deoxynucleoside triphosphate Deoxyribonucleic acid double-stranded RNA Escherichia coli Ethylene diamenetetraacetate Enzyme linked immunosorbent assay Gram Growth arrest specific gene Glutathione S-transferase Hemagglutinine Histidine tagged Interferon Isopropyl -D-1-thiogalactopyranoside Interferon regulator factor

xvii

ISG kb KDa LB LPAI M mM MCS ml mg min. g l m MgCl2 mRNA NA NEP NES ng NLS NMR NS1 OD PACT

Interferon stimulate gene Kilo base Kilo dalton Luria Bertani Low pathogenicity avian influenza Molar Milmolar Multi cloning site Milliliter Milligram Minutes Microgram Microliter Micromter Magnesium chloride Messenger ribonucleic acid Neuraminidase Nuclear export protein Nuclear export sequence Nanogram Nuclear localization sequence Nuclear Magnetic Resonance Nonstructural 1 Optical density Protein activator of the interferon-induced protein kinase

PCR PKR RNA

Polymerase chain reaction Protein kinase Ribonucleic acid

xviii

RNP rpm RT-PCR SDS-PAGE

Ribonucleoprotein Rotation per minute Reverse transcription polymerase chain reaction Sodium Dodecyl Sulphate- Polyacrilamide Gel Electrophoresis

sec. SIV ssRNA TAE Tris U6 SnRNA UV V

Seconds Simian immunodeficiency virus Single strand RNA Tris-acetate-EDTA 2-hydroxymethyl-2-methyl-1,3-propanediol U6 small nuclear ribonucleoprotein Ultraviolet Volts

xix

LIST OF APPENDICES

APPENDEX

TITLE

PAGE

NS1B gene sequence (870 bp) of Influenza B virus (B/Taiwan/45/2007)

79

NS1B protein sequence (281 amino acids)

80

CHAPTER 1

INTRODUCTION

1.1

Influenza

Influenza is a contagious respiratory viral illness of global importance. The disease was caused by influenza viruses known as flu. The most common symptoms of the flu are chills, fever, sore throat, muscle pains, severe headache, coughing, weakness and general discomfort. Some influenza viruses can cause more severe diseases than the common cold like pneumonia. Influenza viruses spread around the world and can be transmitted through the air by coughs, sneezes, creating aerosols containing the virus. This can also be transmitted by direct contact with infected animals or humans (Metreveli et al., 2006; Spickler et al., 2009).

1.2

Influenza virus

Influenza viruses have unique features of reverse sense single strand RNA. They have been classified into three distinct types: A, B and C. Influenza B viruses are mainly found in humans. These viruses can cause epidemics in human populations, but have not been responsible for pandemics. Influenza B viruses

comprised of single group of hemagglutinin and neuraminidase antigens since their first isolation in 1940 (Nerome et al., 1998). Influenza B viruses are categorized into lineages rather than subtypes and are also classified into strains. Influenza B viruses undergo antigenic drift, though it occurs more slowly than in influenza A viruses (Metreveli el al., 2006; Spickler et al., 2009). Twelve antigenic variants were distinguished by a panel of monoclonal antibodies appeared to circulate in the 19811982 epidemic season in Japan. The evolutionary lineages of influenza B viruses since 1988 have been represented by two epidemic strains B/Victoria/2/87 and B/Yamagata/16/88 (Nerome et al., 1998).

1.3

Problem statements of the study

The main problem of this study is to clone NS1B gene in pET-32b, pET32a, pQE-81L, and pQE-80L vectors for transformation into Escherichia coli BL21(DE3) and DH5.

1.4

Objective of the study

The objective of this research was to clone of NS1B synthetic gene into pET-32b, pET-32a, pQE-81L, and pQE-80L vectors. The recombinant constructs were transformed into E. coli hosts.

1.5

Scope of the study

The scope of this study encompassed the cloning of NS1B gene of influenza B into pET-32b, pET-32a, pQE-81L, and pQE-80L vectors, which are subsequently transformed into competent E. coli BL21(DE3) and DH5.

1.6

Significant of the study

Recombinant NS1 fusion protein of high purity is more significant for detection of antigenicity. Successful cloning and overexpression of NS1 gene are useful for specific diagnostic and further applications. This reverse genetic system will allow studies to explore the functions of NS1B domains during the replication cycle and to assess their contributions to the pathogenesis and virulence of influenza B virus.

CHAPTER 2

LITERATURE REVIEW

2.1

Overview of influenza

2.1.1

Influenza infection

Historically, Influenza A is responsible for occasional pandemics affecting millions of people worldwide. Its often associated with considerable morbidity and mortality. During the years 1918-1920, influenza virus caused one of the most destructive disease outbreaks in world history and later become known as the Spanish flu pandemic. It resulted in the death of an approximately 50-100 million people thus, this influenza pandemic killed more people than the World War 1 (Metreveli et al., 2006).

Since, the diseases occurred in the United States in 1924-1925, and then emerged in 1929 in a much milder form. This virus continued to circulate in humans until 1957. It was then replaced by another human influenza virus called the Asian flu virus. Pandemic influenza A viruses emerged three times during the last century: in 1918 (H1N1 subtype), in 1957 (H2N2), and in 1968 (H3N2).

Currently both H3N2 and H1N1, a late fifties variants that re-appeared in 1977and remained co-circulate in humans (Metreveli et al., 2006).

Influenza B can also cause similar symptoms as Influenza A, but generally in a milder form. Influenza C viruses infect mammals only and generally without cause disease. They are genetically distinct from A and B types (Metreveli et al., 2006).

The human influenza viruses are transmitted from person to person. Infected adults usually begin to shed influenza A viruses the day before the symptoms appear, and are infectious for 3-5 days after initial signs. Young children can shed virus up to six days before, and 10 days or more after they become ill. Severely immunocompromised individuals may remain infectious for weeks or months. Humans can transmit influenza viruses to ferrets, and occasionally to swine and rare cases from person-to-person spread, including a localized outbreak among recruits at a military base which have been reported in humans infected with swine influenza viruses. No cases of sustained transmission have been reported in humans infected with the avian influenza viruses. Fecal shedding of the avian H5N1 virus has been documented in a child with diarrhea. Transmission of this virus across the placenta may also be possible (Spickler et al., 2009).

In poultry, there are two forms of disease. Low pathogenicity avian influenza (LPAI) viruses generally cause asymptomatic infections and mild respiratory. High pathogenicity avian influenza (HPAI) viruses cause severe disease that can kill up to 90-100% of a poultry flock. The severity of zoonotic avian influenza varies with the virus. Generally, avian influenza viruses do not spread efficiently in mammals, and infections remain limited to individual animals or small groups (Spickler et al., 2009).

Other avian influenza viruses can also undergo cross-species transmission. LPAI H9N2 viruses have become endemic in poultry in parts of Asia and the Middle East may be of particular concern. Recently, they were found in pigs with respiratory disease and fatal paralysis in China. In addition, H9N2 viruses have been infected humans. As of January 2009, human H9N2 infections have been significantly less severe than those caused by avian HPAI H5N1 viruses. Epidemics occur every few years, due to small changes in the influenza viruses. Human pandemics, resulting from antigenic shifts, were most recently reported in 1918, 1957 and 1968 (Spickler et al., 2009).

2.1.2

Treatment

Antiviral drugs are available for influenza treatment in the United States. Amantadine and rimantadine (adamantanes) are active against human influenza A viruses, if treatment begun within the first 48 hours. Zanamivir and oseltamivir are used effective for both influenza A and influenza B. Treatment usually results in milder symptoms and recovery one day sooner. Side effects including neuropsychiatric events may appear. Drug resistance develops rapidly in viruses exposed to amantadine or rimantadine and may emerge during treatment. For example, during the 2006-2008 flu seasons, human influenza viruses circulating in the United States and Canada exhibited high resistance to amantadine and rimantadine. Laboratory studies have shown that influenza viruses can also become resistant to zanamivir and oseltamivir. However, this appears to be less common than resistance to adamantanes (Spickler et al., 2009).

2.1.3

Preventative measures for human influenza viruses

An annual vaccine is available for influenza A and B. Both inactivated (injected) and live (intranasal) vaccines may available. The vaccine is given in the fall before the flu season. It contains the viral strains which are most likely to produce epidemics during the following winter, and is updated annually (Spickler et al., 2009).

2.1.4

Isolation of influenza virus

Human influenza A and influenza B infections can be diagnosed by virus isolation, detection of antigens or nucleic acids, or retrospectively by serological test. The viruses can be isolated in cell lines or chicken embryos, and also can be identified by hemagglutination inhibition tests. Antigens can be detected in respiratory secretions by immunofluorescence or enzyme-linked immunosorbent assays (ELISA). Commercial rapid diagnostic test kits such as (Directigen Flu A test) can provide a diagnosis within 30 minutes. Reverse transcription polymerase chain reaction (RT-PCR) techniques are also available; besides the serological tests which include complement fixation, hemagglutination inhibition and immunodiffusion. A rising titer is necessary to diagnose human influenza when using serological tests. RT-PCR can be used for the diagnosis of influenza C and avian influenza viruses (Spickler et al., 2009).

2.2

Influenza virus

2.2.1

Viral genome

The viral genome consists of eight segments of reverse single stranded RNA or called negative polarity. A total of 11 known proteins are encoded by the genome. The three largest segments (1, 2 and 3) encode polymerase proteins, PB1, PB2 and PA that are responsible for RNA synthesis. Segment numbers 4 and 6 encode surface proteins which are hemagglutinin (HA) and neuraminidase (NA) that are involved in attachment to the host cell and fusion between the viral envelope and cellular membrane, and release of virus particles. The 5th segment encodes a nucleoprotein (NP) that protects the viral RNA, but also has other functions. The 7th segment encodes the M1 and M2 proteins. M1 is a matrix protein that covers the inner surface of the viral membrane. The last segment encodes two proteins: non-structural protein 1 (NS1) and nuclear export protein (NEP). NS1 and NEP are involved in various aspects in the process of taking over the host cell (Metreveli et al., 2006).

2.2.2

Types of influenza virus

Influenza viruses have been classified into three distinct types A, B and C. Influenza viruses are found in a number of species including birds, swine, horses, dogs beside humans. Influenza A viruses include the avian, swine, equine and canine influenza viruses, as well as the human influenza A viruses. Influenza A viruses were classified into subtypes based on two surface antigens, the hemagglutinin (H) and neuraminidase (N) proteins. There are 16 hemagglutinin antigens (H1 to H16) and nine neuraminidase antigens (N1 to N9). These two

proteins are involved in cell attachment and release virions from cells, and are also major targets for the immune response. Limited subtypes are found in each species of mammal. Influenza A viruses were also classified into strains. Strains of influenza viruses are described as their types, hosts, place of first isolation, strain number, year of isolation, and antigenic subtype. For example, the prototype strain of the H7N7 subtype of equine influenza virus which first was isolated in Czechoslovakia in 1956 is A/eq/Prague/56 (H7N7) (Spickler et al., 2009).

Limited information is available on the subtypes found in other species of birds. Subtypes that have been found in ratites include H3N2, H4N2, H4N6, H5N1 H5N2, H5N9, H7N1, H7N3, H9N2, H10N4 and H10N7. Isolates from cage birds usually contain H3 or H4. However, infections with high pathogenicity subtypes containing H7 or H5 can also occur (Spickler et al., 2009).

Human influenza A viruses are mainly found in humans, and they can also infect ferrets and sometimes swine. Experimental infections have been reported in raccoons. Human viruses can also replicate in the nasal epithelium of experimentally infected horses. H1N1, H1N2 and H3N2 viruses are currently in general circulation in humans. The H1N2 viruses appeared most recently. These viruses were first seen in human populations in 2001, probably as a result of genetic reassortment between the H3N2 and H1N1 viruses. H2N2 viruses circulated in the human population between 1957 and 1968 (Spickler et al., 2009).

Influenza C viruses are not classified into subtypes, but are classified into strains. Each strain is antigenically stable, and accumulates few changes over time (Spickler et al., 2009).

10

2.2.3

Lifecycle of influenza virus

Life cycle of influenza virus is started through attachment to sialic acid receptor of the host cell. However, strains vary in their affinities for different sialyloligosaccharides. Hemagglutinin is a viral glycoprotein binds to the cell receptor sialic acid. The virus receptor bound is taken into the cell by endocytosis. Viral envelope fuses with the lipid bilayer of the vesicle and releasing the viral ribonucleoprotein (RNP) into the cell cytoplasm. Then RNP is transported into the nucleus. New viral proteins are translated from transcribed mRNA and the viral RNA is replicated from the negative stranded templates. The new viral RNA is encapsidated by the nucleoprotein and transported to the cell surface where envelope hemagglutinin and neuraminidase components are incorporated from the cell membrane. Progeny virion is formed and then released from the cell by budding (Metreveli el al., 2006).

Figure 2.1

Life cycle of influenza virus (Metreveli el al., 2006)

11

2.3

Influenza B virus

Influenza B virus is an envelope virus, belonging to the family Orthomyxoviridae. It contains 8 segments of negative sense single-stranded RNA virion (Palese et al., 2007). Until now, it is still unknown why influenza B does not have many natural hosts like influenza A and can be antigenily drifted (Hai et al., 2008). Influenza B viruses have only one subtype and evolve under three major lineages: B/Lee/40virus like variants, B/Victoria/2/87- like variants and B/Yamagata/16/88- like strains (Nerome et al., 1998; Hai et al., 2008).

Figure 2.2

Influenza virus structure

(http://micro.magnet.fsu.edu/cells/viruses/influenzavirus.html)

Influenza B viruses comprised of a single group of hemagglutinin and neuraminidase antigens since the first isolation in 1940. For this difference, it has been shown that numerous influenza A viruses circulate in wild and domestic

12

animals such as wide variety of bird species, swine, equine, seals, whales and mink. Indeed, since has reported in early 1970s. This is in sharp contrast that influenza B viruses have not been isolated from animals other than humans. These differences in epizootic background may lead to evidence that influenza B viruses have no antigenic shift observed (Nerome et al., 1998).

Despite the lack of antigenic shift, a number of antigenic variants of B viruses have been isolated during periods of widespread influenza virus activity since its first isolation in 1940. Particularly, it is noteworthy that a higher number of monoclonal variants of influenza B viruses have been detected in the same epidemic season than influenza A viruses. For example, twelve antigenic variants distinguished by a panel of monoclonal antibodies appeared to circulate in the 19811982 epidemic seasons in Japan. Thus, evolution of influenza B virus is definitely characterized by coexistence of several antigenic variants in the same epidemic period of influenza A virus in which a single dominant virus predominates in a single epidemic period (Nerome et al., 1998).

The first demonstrated of influenza B viruses have evolved in multiple lineages which can cocirculate for considerable periods of time. The continuing analysis of recent epidemic viruses from evolutionary point of view showed that there have been two distinct lineages of influenza B viruses since 1988, being represented by the two epidemic strains B/Victoria/2/87 and B/Yamagata/16/88, respectively. The evolutionary rate of influenza B viruses was reported to be lower than of human influenza A viruses. It is still uncertain why the former virus is able to survive for a long period of time without antigenic drift in the hemagglutinin glycoprotein (Nerome et al., 1998).

Common cold is caused by influenza B and is milder than type A viruses. However, some cases lead to severe diseases but rarely morbidity (Spickler et al., 2009). Most virion segments encode structural protein from reverse RNA. On the other hand, the segment number 8 encodes nonstructural

13

proteins typically 890 nucleotides which encode for two different proteins: nonstructural protein NS1 (26 KDa) and nuclear export protein NEP (11 KDa) which was previously known as NS2 (Spickler et al., 2009, Palese et al., 2007).

Until now, it is still uncertain why former virus is able to survive for a long period time without antigenic drift. Recently, antigenic analysis along with investigation of genomic structure, systematic deletion and insertion mutations have occurred in the same nucleotide regions of hemagglutinin molecule of influenza B viruses (Nerome et al., 1998).

Figure 2.3

Electron microscope image of influenza B

(http://www.wellcome.ac.uk/Education-resources/Teaching-and-education/BigPicture/All- issues/Epidemics/WTD028128.htm)

14

Figure 2.4

Influenza virus particles on cells lining

(http://www.wellcome.ac.uk/Education-resources/Teaching-and-education/BigPicture/All- issues/Epidemics/WTD028128.htm)

2.4

Nonstructural (NS1) protein

Influenza NS1A protein consists of 230-237 amino acids, which are divided into two major domains: N-terminal RNA-binding domain, which comprises the first 73 amino acids residues forming symmetric homodimer insolution. On the contrary, the influenza NS1B protein comprises of 281 amino acids. The first 93 amino acids is N-terminus. This domain is responsible for

15

binding with nucleolin. Other domain is located in C-terminal of protein body. This domain is known as an effector domain which interacts with many viral and cellular factors. It involves in translation and post-transcriptional processing of RNA (Manasatienkij et al., 2008).

NS1 protein persists only in the infected animal cells and not in vaccinated animals, so that, these vaccinated animals are not producing NS1 protein specific antibodies and this is allows differentiation between vaccinated and natural infected animals (Fang-kun et al ., 2008). Hence, this may path the way of utilizing NS1 protein as a target for diagnostic of influenza viruses.

The crystal structure of the NS1B protein has not yet been solved. Computer modeling based on the crystal structure of the N-terminal domain of the NS1A protein has been allowed alignment of the N-terminal RNA-binding domains of the two proteins, which are thought to possess similar -helical structures (Donelan et al., 2004).

2.4.1

Nonstructural protein NS1 of influenza B virus

The RNA-binding domain (N-terminus domain) of the NS1B protein is 93 amino acids in length and thus 20 amino acids larger than that of the NS1A protein. The RNA binding domain has a molecular weight of approximately 22.7 KDa and the three-dimensional structure has not yet been determined. Nonetheless, sequence alignment of the RNA-binding domains of the NS1A and NS1B proteins suggested that the RNA binding domain of the NS1B protein exhibits a dimeric six-helical chain fold similar to that of the NS1A protein, except that there are large loops between the three - helices in each chain (Yuan et al., 2002).

16

The three-dimensional structure of the NS1B (1-93) RNA-binding domain is based on NMR solution structure of the NS1A (1-73) RNA-binding domain and a sequence alignment of NS1A. The large surface loops between the -helices in the NS1B model contain 21 amino acids residues. Because these loops appear as insertions in the sequence alignment, these amino acids are left unconstrained in the structure calculations, so that homology modeling approach does not predict a specific structure for these loops. The ribbon representations of the predicted structure of NS1B (1-93) were shown in (Fig. 2.5). The loop 1 (residues 2436) of polypeptide chain 1 is located between the N and C-terminus domain respectively, of the chain 2. Loop 2 (residues 6471) of polypeptide chain 1 is located near the N-terminus of the same chain, and by symmetry loop 2 of chain 2 is located near the N-terminus of chain 2 (Yuan et al., 2002).

Figure 2.5: Homology modeling of RNA binding domain of NS1B R53, R50, R53and R50 are the RNA binding residues (Yuan et al., 2002)

17

2.4.2

Functions and influences of NS1 protein

Previous studies suggested two nonexclusive explanations for the strong general impact of the NS1B gene deletion on viral replication. First, the NS1B protein may play an important role in counteracting the activity of antiviral gene products that are expressed in Vero cells in an IFN-independent manner. A second possible explanation for restriction of the NS1B virus in Vero cells might be a critical contribution of the NS1B protein to the principal viral replication process rather than an effect on the host cell (Dauber et al., 2003).

The nonstructural NS1A protein (26 KDa) of influenza virus has been shown to enter and accumulate in the nucleus of virus-infected cells independently of any other influenza viral protein (Greenspan and Palese, 1988). NS1A contains two nuclear localization sequences (NLS1 and NLS2), and a nuclear export sequence (NES). A nucleolar localization sequence (NoLS) has been reported for some strains, and is concomitant with NLS2 (Hale et al., 2008).

NLS mediate the active nuclear import of NS1 via binding to cellular importin-. Translocation of NS1 into the nucleus is extremely rapid. NLS1 is highly conserved, monopartite, and involves three residues also involved in binding dsRNA. NLS2 is absent from the NS1 proteins of a large number of virus strains. It is difficult to describe a function to this sequence with regard to viral replication. Concurrent with NLS2 is a functional nucleolar localization signal (NoLS), which includes additional basic residues. Despite this, the nucleolar function of NS1 is unknown (Hale et al., 2008).

The NLSs of the influenza B virus NS1 proteins are located within the RNA-binding domains at the N-terminal 93 amino acids. NLSs overlapping with an RNA-binding domain have been described for other viral regulatory proteins, including UL69 of human cytomegalovirus and human immunodeficiency virus that promote nuclear export of the respective viral mRNAs. RNA binding and

18

nuclear imports are mutually exclusive in the Rev protein, thereby ensuring that exported viral mRNAs do not return immediately to the nucleus. Although, it has not yet been determined whether, the NS1B protein is involved in the transport of viral RNA. It is possible that this overlap indicates a similar regulatory mechanism. The NLS of the NS1B protein was necessary but not sufficient to mediate speckle association, which is required the whole N-terminal domain (Schneider et al., 2008).

At the early infection, NS1B protein revealed a highly dynamic intracellular localization and accumulates in nuclear speckle which is an important for efficient virus replication. At the late stage of infection, NS1B protein accumulates in the cytoplasm which depends on the interaction with another transported proteins and RNAs which could be regulated by an exposed export signal (Schneider et al., 2008).

2.4.2.1

NS1B binds to ISG-15 protein

The RNA-binding domain at the N-terminal residue and the adjacent 94103 regions are required for the binding of interferon stimulate gene (ISG-15) (Yuan et al., 2001 and Krug et al., 2003). This site is located in one of the surface loops of this domain. The NS1B proteins are required for dsRNA binding (Yuan et al., 2002). However, ISG-15 and dsRNA can bind to the N-terminal 103 amino acid long fragment of the NS1B protein. ISG-15 protein does not have detectable effect on the binding of dsRNA. The N-terminal region of the NS1B protein binds not only dsRNA but also a specific cellular protein (Yuan et al., 2002). Unique biological activities of the NS1B protein are indicated by its deficiency to inhibit pre- mRNA processing, which has ability to bind to the antiviral response gene product ISG-15 and to inhibit its conjugation to cellular proteins (Yuan et al., 2001).

19

NS1B protein has prevented the conjugation of the ubiquitin-like ISG-15 protein to cellular target proteins, which may additionally benefit the activation of such antiviral reactions (Donelan et al., 2004). Moreover, NS1 protein was showed bind non-specifically to all ssRNAs and has greater binding activity (Hatada et al., 1992).

The major goals is to determine the role of the N-terminal, 93 amino acid RNA-binding domain in ISG-15 binding, whether it is required because it maintains the dimerization, and hence proper folding of the NS1B protein, or whether it contains part of the binding site for the ISG-15 protein. However, the binding dsRNA to the N-terminal domains of the NS1A and NS1B proteins are well established in vitro (Hatada et al., 1992, Lu et al., 1994). The specific function of this dsRNA binding during virus infection remains elusive. The NS1 proteins are at a distinct disadvantage in competing with these cellular proteins for dsRNA (Yuan et al., 2002).

2.4.2.2

NS1B inhibits and antagonists / interferon system NS1 protein is involved in virulence and inhibition of the / interferon

system during virus infection (Donelan et al., 2004; Garcia-Sastre et al., 1998). Efficient replication of influenza viruses and most other viruses necessitates are suppressed of antiviral responses mediated by the / interferon system, which is an important part of the immune responses of vertebrates (Dauber et al., 2006).

The NS1B protein is identified as a viral factor that antagonizes interferon induction and boosts viral replication (Dauber et al., 2003). On the contrary, Dauber and his coworkers found that analysis of these viral mutants demonstrated inhibition of / interferon production is largely independent of the dsRNA binding activity of NS1 but critically relies on the presence of the C-

20

terminal part of the protein. The dsRNA shielding does not appear to be sufficient to prevent IFN induction by influenza B virus (Dauber et al., 2006).

The roles of dsRNA binding by NS1B protein are to promote viral replication and to inhibit of antiviral responses. Dauber (2003) has generated a set of isogenic influenza B viruses with mutations that either affected NS1 dsRNA binding or introduced a large C-terminal truncation. By using in vitro binding and reporter gene assays, it was established in previous studies that the larger Cterminal part (amino acid 94 to 281) of NS1B protein contains can suppress activation of the IFN- promoter by Sendai virus infection (Donelan et al., 2004).

The observed lack of activity of the N-terminal domain of NS1 towards IFN inhibition in influenza B virus-infected cells underscores the important role of the C-terminal part of the protein in this process, which presumably involves an RNA-independent mechanism. Further analyses of the intracellular RNA and protein targets of the NS1 domains appear to be promising strategy not only to learn more about details of the IFN suppressive activities of influenza viruses but also about the factors and mechanisms that drive cellular responses to virus infections in general (Dauber et al., 2006).

NS1B protein is not related to its ability to antagonize the interferon (IFN) induced host antiviral response, since NS1/B/Lee virus is significantly impaired in its ability to replicate even in IFN-deficient systems, such as VERO cells and 6-day-old eggs (Dauber et al., 2003). Deletion of the truncated NS1B gene residues from (1-93) protein showed that RNA-binding activity correlated with interferon promoter inhibition. In addition, a recombinant influenza B virus with NS1 deleted induces higher levels of IRF-3 activation. NS1B protein

prevents the nuclear translocation of interferon regulator factor (IRF-3) and especially interferon induction in virus-infected cells. It was shown that, the expression of the NS1B protein complements the growth of influenza A virus with NS1A deleted (Donelan et al., 2004).

21

Inhibition of IFN- synthesis correlates with the inhibition of IRF-3 nuclear translocation. This study found that the C-terminal domain of the NS1B protein possesses IFN antagonist activity, so that deletion of this region would result in a virus that is more attenuated than the wild-type virus. Viruses containing mutations within the NS1B N-terminal domain that adversely affect binding to dsRNA may also prove to be promising live attenuated vaccine candidates (Donelan et al., 2004).

On the other hand, the role of dsRNA binding by the influenza B virus NS1 protein is to promote viral replication and to inhibit of antiviral responses. However, dsRNA binding was necessary for inhibiting protein kinase (PKR) activation and enable efficient viral replication. The blockade of PKR activities by both of the NS1 A and B is a common theme in many virus families, highlighting that this enzyme is a key factor of the cellular antiviral response (Dauber et al., 2006).

2.4.2.3

NS1 inhibits nuclear export of mRNA

The NS1 protein also functions inhibition rather than facilitation of nuclear export of mRNA (Qian et al., 1993). The RNA-binding domain binds to the poly (A) sequence in mRNAs. The effector domain and RNA binding domain of NS1 protein represses transcription. Interactions between proteins that regulate nuclear mRNA export and their nuclear targets will be varied and complex (Qian et al., 1993).

Cytoplasmic as well as nuclear localization of the NSI protein occurred only when the effector domain was present and was inactivated by a point mutation. A possible explanation from the observation even wild-type NSI protein has some affinity for one or more cytoplasmic targets. Specifically, the small

22

amount of the wild-type NS1 protein that is present in the cytoplasm has been shown to be associated with ribosomes and polysomes. It has not been established whether this ribosome associated NS1 protein has any function. This study

showed that interactions between proteins that regulate nuclear mRNA export and their nuclear targets will be varied and complex depending on the interaction of transcription factors with their protein targets (Qian et al., 1993).

2.4.2.4

NS1 protein interacts with GAS8

Growth arrest-specific (GAS) genes are expressed preferentially in cultured cells that have entered a quiescent state following serum deprivation or growth. Eleven GAS genes have been identified from a variety of biological functions, including the control of microfilament organization, nerve cell growth or differentiation, apoptosis, tyrosine kinase receptor activity, and negative and positive control of the cell cycle. GAS8 is located at 16q24.3 and was found to be a common deletion present in breast and prostate carcinomas. It was shown as a potential tumor suppressor gene. The GAS8 gene consists of 11 exons spanning approximately 25 kb. GAS8 protein associates with microtubules in vitro and in vivo (Zhao et al., 2009).

Zhao and coworkers (2009) found that neither RNA binding domain nor the effector domain of NS1 protein interacts with GAS8. Deletion analysis revealed that the N-terminal 260 amino acids of GAS8 were able to interact with NS1. Examination of the localization of GAS8 protein in cells revealed that two types of localization existed: Golgi and cytoplasmic. This phenomenon was even observed in the same types of cells. The Golgi apparatus localization is dependent on intact microtubules and in cell-cycle regulated. Although, the function of GAS8 has not yet been determined, the effect of the association between GAS8 and NS1 on virus infection is unknown. The mammalian GAS8 gene is a possible tumor

23

suppressor that was previously identified as one of several genes that are upregulated upon growth arrest. GAS8 was expressed in HeLa, CV-1, A549, and DU145 cells. NS1 inhibited the maturation of GAS8 mRNA. This is consistent with the fact that transient expression of NS1 in mammalian cells leads to retention of polyadenylation RNA in the nucleus and inhibition of pre-mRNA splicing (Zhao et al., 2009).

2.4.2.5

NS1 protein inhibits pre-mRNA

Lu and coworkers (1994) found the NS1 protein inhibits pre-mRNA splicing both in vitro and in vivo by associating with the spliceosomes that are formed from pre-mRNA. He found that NS1 protein associates with U6 snRNA (one of the basic protein of spliceosome) in infected cells. A fraction of NS1 was also detected in association with ribosomes and polysomes in cytoplasmic fractions of infected cells (Lu et al., 1994).

The nuclear export and splicing of pre-mRNAs are competing processes. Genetics of these processes have been best studied in yeast by the isolation and characterization of pre-mRNA processing. These studies found that some of the protein factors involved in the splicing reaction might act as RNA helicases and suggesting that dsRNA unwinding processes are necessary for spliceosome assembly and release of the mRNA from the splicing complex. NS1 mRNA is poorly spliced to yield NS2 mRNA. However, inactivation of the NS1 gene led to a substantial increase in the splicing efficiency, as shown by the relative accumulations of NS1 and NS2 mRNAs (Fortes et al., 1994).

24

2.4.2.6

NS1 protein prevents activation of PKR

NS1 protein prevents the activation of double-stranded-RNA (dsRNA) activated protein kinase (PKR) by binding to dsRNA. Activation of PKR results in down regulation of cellular translation and is believed to be part of an antiviral defense strategy in mammalian cells. Host proteins interacting with the NS1 protein during infection have been characterized. The identification of such proteins is of great interest. Hence, they may influence the host range and virulence of influenza virus strains. Such a hypothesis is supported by studies of influenza viruses with mutated or heterologous NS1 alleles which showed altered growth characteristics in different cells. By using the yeast interaction trap, it has identified NS1-I by its ability to bind to NS1. It was demonstrated the conservation of the interaction of NS1-I with six different human and avian influenza virus NS1 proteins, suggesting that there is a role for the NS1NS1-I interaction during the virus life cycle. Overexpression of a truncated of NS1-I protein would affect the normal course of infection and detailed mapping of the interacting domains of NS1. NS1-I interaction should reveal whether one or more of the temperature- sensitive mutations and identified NS1 protein can be correlated with reduced ability to bind NS1-I (Wolf et al., 1996).

PKR needs to be activated via a conformational change that is brought about by binding to either of its activators, double-stranded RNA (dsRNA) or the PACT protein. Other studies were carried out a series of in vitro experiments to determine whether the NS1 protein could be utilized a common mechanism to inhibit PKR activation by both PACT and dsRNA, despite their different modes of activation. They demonstrated that the direct binding of the NS1A protein to the N-terminal region of PKR can serve as such a common mechanism and this binding does not require the RNA binding activity of the NS1A protein (Li et al., 2006).

25

2.5

Previous studies of cloning and expression of NS1 gene

NS1 gene from various influenza viruses had been cloned. This gene was successfully expressed in Escherichia coli (Binns et al., 1996, Young et al., 1983, Hatada et al., 1992). Antibody of NS1 was detected in serum sample from ponies experimental infected cell with influenza virus without the animals being vaccinated with whole inactivated virion. While determination of NS1 antigens were appeared to be located in the C-terminal half of the protein. However, NS1 protein appears only in infected cells. Using NS1 protein as a diagnostic marker for influenza virus infections in the presence of high levels of antibodies of influenza hemagglutinin generated by recent vaccination is an attractive alternative. NS1 protein from equine influenza virus its entirety or in part as a fusion protein with glutathione S-transferase (GST) in a diagnostic tests for detection this type of virus (Binns et al., 1996).

Electrophoresis in slab gels (12.5%) in the presence of SDS was used to observed NS1 protein. ELISA was carried out on sera experimental samples to test for the presence of anti-NS1 antibodies. ELISA results on control animals positive showed 60% of detection level which were similar to the 79% of detection for naive animals, while in vaccinated animals was 26% (Binns et al., 1996).

NS1 gene was isolated from equine influenza virus A/equine 2/Suffolk /89(H3N8), cloned into pGEX-3X vector and transformed in E. coli TG1 competent cell. Recombinant protein was observed by SDS-PAGE and immunoblot analysis was carried out to detect the antigens. ELISA diagnostic was usefulness depending upon duration of the antibodies response to NS1 following the initial infection. Detection efficiency of about 70% would be useful for batch testing training yards (Birch-Machin et al., 1997).

26

The antigenic determinants of NS1 protein are situated in C-terminal domain. Highly homologous regions are present at the position of the Miami/63 and Suffolk /89 NS1 proteins and clearly indicate that these proteins should be capable of eliciting a similar immune response. Combination antibody response with a cytotoxic T cell response if elicited would be indicated that NS1 protein could be an important component of future vaccines in equines and other species (Birch-Machin et al., 1997).

pET-28 vector was employed to express the NS1 gene under the control of the T7 promoter and fused with HisTag sequences. NS1 gene from Influenza A (H9N2) was cloned and highly expressed to about 37.4% of the total cellular protein, and nearly 95% after purification. Utilizing IPTG inducer besides lactose inducer had no effect on the growth of host cells (Fang-kun et al., 2008). The antigenicity of the recombinant protein was demonstrated in Western-blotting test by using positive sera from pigs. This way was not only laying the foundation for development of ELISA antibody test for differentiation diagnosis between vaccinated and naturally infected pigs, but also facilitating the monitoring and eradication of SIV. This procedure focused on the antibody titer in pigs infected with influenza virus without interference by vaccinated antibody titers and this allows the differentiation between the vaccinated and the naturally infected pigs (Fang-kun et al .,2008).

pQE-80L, the other type of vector with 6xHis-tag, was used to express NS1 protein of influenza A/Chicken/TH/KU14/04 (H5N1). The NS1 gene was inserted into pQE-80L using T4 DNA ligase. Ligation product was transformed into subcloning efficiency DH5 competent cell by heating shock of the cells at 42C for 20 seconds. The transformed E. coli was plated on LB agar containing 100g/ml ampicillin. The transformants were screened by PCR using specific primers. IPTG was used to induce E. coli. The recombinant NS1 proteins were purified by precipitation and affinity chromatography and characterized by SDSPAGE and western blot analysis. The amount of NS1 protein purified by the

27

precipitation method was less than that purified under denatured conditions and contained a small amount of E. coli protein. To confirm the expression, the fusion protein was reacted with anti-histidine monoclonal antibodies and was indicated specifically with antibodies raised against the NS1 protein (Manasatienkij et al., 2008). The recombinant proteins produced in E. coli were not post-translationaly modified. However, in some cases, it may be more effective antigens than the recombinant proteins produced in insect cell systems (Manasatienkij et al., 2008, Spencer et al., 2007). It was unknown whether the lack of modification in the NS1 protein would enhance its serological activities (Manasatienkij et al., 2008).

The plasmid pGEX- NS1B encodes a protein consisting of GST fused to the NS1 protein of influenza B/Yamagata/1/73 virus. The wild type NS1 cDNA was subcloned into the pGEX-5X-1 vector between the XhoI and EcoRI restriction sites. The pGEX-NS1B (1-93) plasmid was constructed by in frame insertion of the GST ORF in front of the coding region from 1 to 93 amino acids of the NS1B protein. Similarly, pGEX-NS1 (94-281) was constructed by fusing the GST ORF to a cDNA encoding from 94 to 281 amino acids of the NS1B protein. Fusion proteins were expressed in E. coli BL21 and purified using glutathione-sepharose beads. NS1B mutant cDNAs were also cloned into pCAGGS vectors for expression in mammalian cells. On the other hand, NS1A was expressed in trans by transfecting MDCK cells with plasmid pCAGGS as a result, these cells support del NS1A virus replication. In order to determine whether expression of the NS1B protein complements the growth of del NS1A virus, del NS1A transfected MDCK cells with pCAGGS-NS1B plasmid. Expression of NS1B protein was able to complement the growth of the del NS1A virus to levels slightly lower than those seen when the NS1A protein was expressed in trans. Hence, the results indicated that the NS1 protein of influenza B virus can functionally replace the NS1 protein of influenza A virus in this assay (Donelan et al., 2004).

28

Other rapid diagnostic method for detection of antigens in nasal swabs of test animals and immune-PCR were established. It is a highly sensitive way to detect NS1 protein (Ozaki et al., 2001).

29

CHAPTER 3

MATERIALS AND METHODS

3.1

Experimental Design

pUC57 plasmid carrying NS1B synthetic gene was cloned and transformed into competent E. coli strain BL21(DE3). Figure 3.1 shows the general flow chart of experimental design for each vector that started from plasmid isolation, restriction digestion, agarose gel electrophoresis, ligation, transformation and screening of colonies. In the first attempt (Figure 3.2), pET32b was amplified in E. coli strain BL21(DE3). Plasmid miniprep was done for pUC57-NS1B and pET-32b. Then, single digestion was done using HindIII enzyme. Ligation, transformation and screening colonies were done on clones of BL21(DE3). In the second attempt (Figure 3.3), plasmid miniprep was done for pUC57-NS1B and pQE-81L, which was amplified in E. coli strain DH5. Double digestion, was done using PstI and HindIII enzymes. Ligation, transformation and screening colonies were done on clones of DH5. In the third attempt (Figure 3.4), plasmid miniprep was done for pUC57-NS1B and pET-32a. NS1B gene containing SacI and HindIII restriction enzyme sites were PCR amplified from pUC57-NS1B. Double digestion was done using SacI and HindIII enzymes. Ligation, transformation and screening colonies were done on clones of BL21(DE3). In the fourth attempt (Figure 3.5), plasmid miniprep was done for pUC57-NS1B and pQE-80L. Double digestion was done using SacI and HindIII

30

enzymes. Ligation, transformation and screening colonies were done on clones of DH5.

Isolation of pUC57-NS1B and expression vectors from E. coli strains

Restriction digestion using Hind III Restriction digestion using SacI, PstI and HindIII enzymes

DNA agarose gel electrophoresis and extraction

Ligation of NS1B gene with vectors and transformation into E. coli competent strains

Screening of colonies and PCR amplification

Plasmid isolation, digestion, gel electrophoresis

Figure 3.1

Flow chart of experimental design

31

pET-32b
HindIII HindIII

pUC57-NS1B
NS1B gene HindIII

T7 promoter

Single digestion with HindIII

Single digestion with HindIII

pET-32b vector (5899bp)

NS1B gene (870bp)

Ligation

NS1B HindII I T7 promoter HindII I

pET-32b NS1B

Transformation into E. coli BL21(DE3)

Screening of colonies

Figure 3.2

Cloning of pET-32b-NS1B into E. coli BL21(DE3)

32

pQE-81L
PstI HindIII

PCR amplification of NS1B gene using pUCfor and pUCrev primers

NS1B gene PstI

pQE for

HindIII

Double digestion with PstI and HindIII

pQE-81L vector (4753bp)

NS1B gene (870bp)

Ligation
NS1B PstI HindIII

pQE for

pQE-81L NS1B

Transformation into E. coli DH5 Screening of colonies Cloning of pQE-81L-NS1B into E. coli DH5

Figure 3.3

33

pET-32a
SacI HindIII

PCR amplification of NS1B gene using NS1BforSacI and NS1BrevHindIII primers

NS1B gene SacI HindIII

T7 promoter

Double digestion with SacI and HindIII

pET-32a vector (5900bp)

NS1B gene (870bp)

Ligation

NS1B HindIII SacI T7 promoter

pET-32a NS1B

Transformation into E. coli BL21(DE3)

Screening of colonies

Figure 3.4

Cloning of pET-32a-NS1B into E. coli BL21(DE3)

34

pQE-80L

PCR amplification of NS1B gene using NS1BforSacI and NS1BrevHindIII primers

HindIII SacI NS1B gene HindIII

SacI

pQE for

Double digestion with SacI and HindIII

pQE-80L vector (4751bp)

NS1B gene (870bp)

Ligation

NS1B
SacI HindIII

pQE for pQE-80L NS1B Transformation into E. coli DH5 Screening of colonies Figure 3.5 Cloning of pQE-80L-NS1B into E. coli DH5

35

3.2

Materials

3.2.1

Bacterial strains

Two strains of Escherchia coli were used in this study - BL21(DE3) and DH5. These strains are commonly used as a host for cloning and protein expression in molecular biology labs because they have the simplest genomic and foreign DNA can be transformed easily. On the other hand, use of E. coli as a host enables large quantity production of recombinant proteins (Leonhartsberger, 2006).

3.2.2

Chemicals

Most chemicals were of analytical and molecular biology grade obtained from Sigma-Aldrich, Merck, GenScript, Promega, QIAGEN, Yeastern Biotech and GeneAll. All restriction enzymes, DNA polymerase were purchased from Promega. Ligase enzyme was obtained from Yeastern Biotech. Primers used for PCR amplification were synthesized by 1st Base Laboratories Sdn. Bhd. Plasmid miniprep and gel extraction kits were purchased from GeneAll and QIAGEN. The pET and pQE vectors were ordered from Novagen and QIAGEN, respectively.

3.2.3

pET vectors

pET is a bacterial plasmid designed to enable quick high quantity production of desired protein. pET-32a (5900bp), and pET-32b (5899bp) contain

36

several elements like: LacI gene, T7 promoter which is specific to T7 RNA polymerase, Lac operator which is poly linker, f1 origin of replication, ampicillin resistance gene and colE1 origin of replication (Blaber et al.,1998).

pET-32a is designed for cloning and high-level expression of peptide sequences comprise of 109 amino acids Trx Tag thioredoxin protein. Cloning sites are available for cloning and producing fusion proteins. The plasmid also contains cleavable His Tag and S Tag sequences for detection and purification (LaVallie et al., 1993).

Figure 3.6

pET-32 vectors

(http://www.lablife.org/p?a=vdb_view&id=g2.nm7d74YBP4q6Utqf6tNu_Ho-).

37

3.2.4

pQE vectors

The pQE series have been designed to allow tightly regulated 6xHistagged protein expression in any E. coli host strain. The vectors are based on the pQE-30 series and include a lacIq repressor gene. pQE-80L and pQE-81L vectors have a cis-lacIq gene that overexpresses the lac repressor, strongly suppressing protein expression from the lac promoter before induction with IPTG (QIAGEN, 2001).

Figure 3.7

pQE-80L vector

(ttp://www.lablife.org/p?a=vdb_view&id=g2%2eL3YawzFqJIzZU2TasKbNJ)

38

Figure 3.8

pQE-81L vector

(http://www.lablife.org/p?a=vdb_view&id=g2%2eL3YawzFqJIzZU2TasKbNJ)

3.2.5

NS1B Synthetic Gene

The synthetic NS1B gene was installed from synthetic oligonucleotides and PCR products by GenScript (USA). Fragment was cloned into pUC57 into EcoRV site.

39

MCS of pUC57

Figure 3.9

pUC57-NS1B gene construct (GenScript, 2009)

40

3.3

Methods

3.3.1

Liquid and solid media preparation

3.3.1.1

Luria Bertani agar and broth

LB medium was prepared by dissolving tryptone (4g), yeast extract (2g) and sodium chloride (4g) in deionized water and it was made up to 400 ml. For LB agar, 2% of agar was added. The contents were mixed and autoclaved at 121 C for 20 minutes. For LB broth or agar containing 50g/ml ampicillin, 10 ml of ampicillin from stock solution of 2mg/ml was added to the media. For LB broth or agar containing 100g/ml ampicillin, 20 ml of ampicillin from stock solution of 2mg/ml was added to the media. The ampicillin solution was added to the medium after it was cooled to 55C.

3.3.1.2

Preparation of ampicillin stock solution

Ampicilin powder (Sigma-Aldrich) of 0.1g was dissolved in 50 ml of sterile deionized water and filtered by using Sartobind nylon filter of size 0.2m.

41

3.3.2

Preparation of competent cells

DNA is very hydrophilic molecule that does not normally pass through a bacterial cell membrane. Bacteria need to be made "competent" in order to uptake foreign DNA. Two strains of E. coli BL21(DE3) and DH5 hosts were used in transformation. E. coli was grown overnight in 10ml of LB broth at 37C with shaking at 200rpm. The cells were chilled for 10 minutes on ice. Then, the cells were centrifuged at 4C for 10 minutes at 3000 rpm. After that, the supernatant was discarded and the pellet was gently resuspended in 10ml of 0.1M calcium chloride. Then, cells were incubated for 20 minutes on ice. The cells were spun again at 4C for 10 minutes at 3000 rpm. For long term storage, the cells were resuspended in 1ml of 0.1M calcium chloride plus 15% glycerol and aliquoted into 0.2ml suspension and stored at -80C. It is the best to use fresh competent cell for transformation.

3.4 cells

Transformation pUC57-NS1B into E. coli BL21(DE3) competent

pUC57-NS1B (2l) was added to competent cells and kept on ice for approximately 20 minutes and this was heat shocked at 42C for 45seconds. Immediately, the tube was placed back on ice for at least 2 minutes. Then, 1ml of LB broth was added and incubated with agitation at 37C for 1 hour at 200rpm. The cells were very briefly spun and 700l of supernatant was removed. The cells were resuspended and plated out evenly using a sterile glass spreader at 100l on LB agar containing 50g/ml ampicillin. The plates were incubated overnight at 37C. Each colony was streaked on LB agar containing 100g/ml ampicillin and the plates were incubated overnight at 37C.

42

3.5

Isolation of pUC57-NS1B plasmid

E. coli BL21(DE3) containing pUC57-NS1B was cultured overnight in LB broth containing 100g/ml ampicillin at 37C with shaking at 200rpm. The plasmids were isolated using plasmid miniprep kit from GeneAll. The cells were harvested by centrifuging at 4000rpm for 5 minutes. The supernatant was removed and the pellet was resuspended by pipetting thoroughly in 250l of buffer S1. The solution was transformed to a new 1.5ml microcentrifuge tube. Then, 250l of S2 buffer was added. The solution was gently mix by inverting tube for 10 times and incubated for 2 minutes until the cell suspension became clear. 350l of buffer S3 was added to the solution and immediately inverted gently for 10 times before centrifuged for 10 minutes at 14000rpm. PD column was placed in a collection tube. Clear solution was transferred to the PD column by pipetting while white pellet formed remained in the microcentrifuge tube. The PD column was spun at 8000rpm for 30 seconds at room temperature. The flowthrough was discarded and the PD column was placed back in the collection tube. 500l of buffer AW was added to the PD column and spun at 8000rpm for 30 seconds. The flow-through was discarded and the PD column was placed back in the collection tube. Then the PD column was washed by adding 700l of buffer PW. The column was then centrifuged at 8000rpm for 30 seconds. The flowthrough was discarded and the PD column was placed back in the collection tube. Additional centrifugation was done at 14000rpm for 1 minute. Next, PD column was transferred into a new 1.5ml of microcentrifuge tube.

DNA was eluted by adding 50l of elution buffer to the centre of the PD column. After 2-minutes stand at room temperature, the tube was centrifuged at 14000rpm for 1 minute. The product was run on 1% agarose gel to check whether the plasmid DNA is successfully isolated from E. coli before being kept at -20C.

43

3.6

Agarose gel electrophoresis

Agarose gel electrophoresis is widely used in molecular biology labs to separate DNA strands by size, and to estimate the size of the separated strand by comparing with the known fragments (DNA ladder). This is achieved by pulling negatively charged DNA molecules through an agarose matrix with an electric field. The concentration of the agarose depends on the size of the DNA fragments to be separated.

50X of TAE buffer was prepared from EDTA (186g, pH 8), Tris base (242g), and glacial acetic acid (57.1ml), and the solution was made up to 1000ml with deionized water. The final pH was 8.5 and autoclaved at 121C for 20 minutes. 1X TAE buffer was diluted from 50X TAE buffer by mixing 20ml of 50X TAE buffer with 980ml of distilled water. 0.5g of agarose powder was added to 50ml of 1X TAE buffer and was boiled in microwave for 1 minute and then left to cool down. 5l of ethidium bromide was added to the agarose gel. The gel casting tray was assembled in gel tank and well forming comb was inserted into slots on casting tray. The agarose gel was poured into the gel casting tray. After the gel was solidified in 30 minutes, the comb was removed carefully. The tray containing agarose gel was placed into electrophoresis chamber and 1X TAE buffer was added to cover the gel.

To prepare sample, 5l of the DNA was mixed with 1l of 6X loading dye. 6l of 1kb DNA ladder was used as a marker. The sample and the marker were loaded separately into wells and the gel was run at 100V for 50 minutes. After gel running, power supply was turn off. The gel was removed from the casting tray and was viewed on a UV transilluminator and documented by using GeneFlash.

44

3.7

Isolation of pET-32b plasmid

The same protocol as described in section 3.5 for plasmid miniprep was used to isolate pET-32b from E. coli BL21(DE3).

3.8

Single digestion

pUC57-NS1B and pET-32b were digested using HindIII and the following components were mixed in PCR tube:

Table 3.1: Single digestion components in PCR tube using HindIII

pUC57-NS1B Acetylated BSA Buffer E HindIII enzyme Total

16.8l 0.2l 2l 1l 20l

pET-32b Acetylated BSA Buffer E HindIII enzyme

16.8l 0.2l 2l 1l 20l

The reactions were incubated at 37C for 5 hours. The digestion products were then purified by gel extraction to remove the buffer and restriction enzymes.

3.9

DNA Extraction

The digestion product needs to be purified before further use. Following electrophoresis, the NS1B band with the size of 870bp and pET-32b at size nearly 6000bp were cut out with a clean sharp scalpel from the gel. The gel slices were

45

separately put inside 2ml microcentrifuge tube. The tubes were weighed before and after putting in the gel. Three times volume of QG solution buffer was added to one volume of gel as mentioned in the protocol of QIAGEN gel extraction kit. The solution was incubated at 50C for 10 minutes and vortexed at each 2 minutes. One volume of isopropanol was added to the sample and mixed. The sample was applied to the QIAquick column and centrifuged at 13000rpm for 1 minute. The flow-through was discarded and the column was placed back. This step was repeated till all the sample was passed through the column. Then, 0.5ml of QG buffer was added to column and centrifuged at 13000rpm for 1 minute. The flow-through was discarded. Then, the column was placed back, washed by adding 0.7ml of PE buffer and centrifuged at 13000rpm for 1 minute. The flowthrough was discarded and centrifuged again to remove all PE buffer. The column was transferred to a new 1.5ml microcentrifuge tube and 50l of elution buffer was added to the centre of column. It was stood at room temperature for 2-5 minutes and then centrifuged at 13000rpm for 1 minute. The product was kept in 20C.

3.10

Determination of DNA concentration

The measuring of DNA concentration was carried out by using Nanodrop Spectrophotometer. The DNA concentration and purity was measured by using 1 l of the sample. An OD260: OD280 ratio of purified plasmid is to be found in the expected range of 1.8 and 2.0. 1 l of distilled water was used to initialize the instrument and 1l of elution buffer was used as a blank.

46

3.11

Ligation NS1B with pET-32b

T4 DNA Ligase was used in ligating the vector and the insert gene. DNA ligases catalyze the formation of a phosphodiester bond between adjacent nucleotide with the hydrolysis of ATP to AMP and inorganic phosphate. NS1B and digested pET-32b with HindIII were ligated and the following components were mixed in PCR tube:

Table 3.2: Ligation mix components of pET-32b and NS1B 1st mix pET-32b NS1B 10X ligation buffer 10X ligation buffer YEA T4 DNA ligase dH2O Total A B 1l 3l 1l 1l 1l 3l 10l 2nd mix 2l 4l 1l 1l 1l 1l 10l

The reactions were incubated in thermocycler at 22C for 20 minutes followed by inactivation at 65C for 10 minutes. The ligation mix was kept at 4C for transformation. It is better to directly transform the ligation mix into competent cells.

3.12

Transformation of recombinant product

The constructed recombinant plasmid prepared as described in section 3.11 was transformed into E. coli BL21(DE3) competent cells. The ligation mix (10l) was added to 100l of competent cells and incubated on ice for 20 minutes.

47

The cells were heat shocked at 42C for 45 seconds. Tube was immediately placed back on ice for at least 2 minutes. 1ml of LB broth was added to the tube. Then, tube was incubated at 37C for 1 hour with shaking at 200rpm. Cells were collected by centrifugation at 10,000 rpm for 30 seconds. 0.7ml was removed from supernatant and the cells were resuspended. The cells (100l) were spread out on LB agar containing 50g/ml ampicillin. IPTG (5l) and X-gal (10l) were spread on the plates for 1 hour prior to use. Some cells were plated out on LB agar as a control. The plates were incubated at 37C for overnight.

3.13

Isolation of pQE-81L plasmid

The same protocol as described in section 3.5 for plasmid miniprep was used to isolate pQE-81L from E. coli DH5.

3.14 (PCR)

Amplification of NS1B gene using Polymerase Chain Reaction

3.14.1

Primer design

Primers were designed based on the upstream and downstream region of NS1B gene of pUC57 vector. The characteristics of the primers are as described in Table 3.3.

48

Table 3.3: The pUC57 primers for PCR of NS1B gene from pUC57-NS1B

Primers Sequences (5)3-

Length(bp)

% of GC Content

Melting Temp.(C)

pUC for 5GTAAAACGACGG CCAGTGA-3 pUC rev 5CAGGAAACAGCT ATGACC-3

19

52-6

60.2

18

50.0

57.6

3.14.2

Polymerase Chain Reaction (PCR)

Using micropipette, a PCR reaction was prepared by pipetting the following reagents into PCR tube:

Table 3.4: PCR reaction components of pUC57-NS1B

Go Taq DNA polymerase 5X Green buffer dNTP MgCl2 Forward primer (pUCfor) Reverse primer (pUCrev) dH2O pUC57- NS1B Total

0.25l 10l 1l 5l 0.2l 0.2l 32.35l 1l 50l

49

The PCR process was initiated with initial denaturation step at 94C for 5 minutes. The process continued with the denaturation, annealing and extension steps. The reaction conditions for each step in PCR cycle were as follows:

Table 3.5: PCR cycle

Steps Denaturation Annealing Extension

Temperature 94C 50C 72C

Time 30 sec. 30 sec. 1 min.

The above cycle was run for 25 cycles. Then the cycle was continued by further extension at 72C for 7 minutes. Then, the sample was run on gel electrophoresis as described in section 3.6. The PCR products were purified by using the protocols as mentioned in section 3.9 prior to digestion with PstI and HindIII restriction enzymes.

3.15 step

Double digestion using PstI and HindIII restriction enzymes in one

NS1B amplified gene and pQE-81L were digested using PstI and HindIII restriction enzymes and the following components were mixed in a PCR tube:

50

Table 3.6: Double digestion components in PCR tube using PstI and HindIII

pQE-81L or NS1B gene Acetylated BSA 10X Multicore enzyme buffer HindIII enzyme PstI enzyme Total

15.8l 0.2l 2l 1l 1l 20l

The reactions were incubated at 37C for 5 hours. Then, gel electrophoresis was done and extracted using the same protocols mentioned in sections 3.6 and 3.9. The extracted product was used for ligation reaction.

3.16

Double digestion using restriction enzymes in two steps

The following components were mixed in PCR tube for the first digestion for NS1B amplified gene and pQE-81L: Table 3.7: Double digestion components in PCR tube using HindIII(1st step)

pQE-81L or NS1B gene Acetylated BSA Buffer E HindIII enzyme Total

15.8l 0.2l 2l 2l 20l

The reaction was incubated at 37C for 5 hours. Then, the reaction was observed by gel electrophoresis and extracted using the same protocols as

51

mentioned in sections 3.6 and 3.9. The extracted product was used for the second digestion. The following components were mixed in PCR tube:

Table 3.8: Double digestion components in PCR tube using PstI(2nd step)

First digestion product Acetylated BSA Buffer H PstI enzyme Total

15.8l 0.2l 2l 2l 20l

The reaction was incubated at 37C for 5 hours. Then, the reaction was observed by gel electrophoresis and extracted using the same protocols as mentioned in sections 3.6 and 3.9. The extracted product was used for ligation reaction.

3.17

Ligation and transformation into DH5 Ligation and transformation of pQE-81L-NS1B into E. coli DH5

competent cells were done using the same protocol as mentioned in sections 3.11 and 3.12.

52

3.18

Screening of positive colonies

Colonies grown on LB agar containing 30g/ml ampicillin were isolated and further streaked on LB agar containing 100g/ml ampicillin. The plates were incubated at 37C for overnight. Colony PCR was used as the screening method.

3.18.1

Chelex 100 extraction method

Chelex 100 resin is highly purified, and most suitable for DNA applications. It is composed of styrene divinylbenzene copolymers with paired iminodiacetate ions. Chelex 100 is very effective in binding metal contaminants with a high selectivity for divalent ions, without altering the concentration on nonmetal ions. One benefit of using the Chelex extraction is that divalent heavy metals which introduce DNA damage at high temperature can be removed. The extraction was set up under aqueous alkaline conditions, as the Chelex 100 suspension was prepared in TE buffer (http://www.nfstc.org/pdi/Subject03/pdi_s03_m03_01.htm).

10% Chelex 100 was used to extract DNA. 100l of chelex solution was added into 1.5ml microcentrifuge tube and the number of tubes was prepared according to the numbers of colonies to be screened. Each tube was labeled as the number of colonies. A scrap of each colony was removed using toothpick, resuspended and vortexed with the Chelex suspension for 5 to 10 seconds. The samples were incubated at 95C for 5 minutes with agitation 400rpm. Then, the samples were centrifuged at 12000rpm for 5 minutes. The supernatant was used for PCR amplification. The samples were kept in 4C for further studies.

53

3.18.2

PCR amplification and gel electrophoresis to detect NS1B

For screening of colonies, sequencing primers of pQE-81L vector were used the characteristics of the primers used are as in Table 3.9. The PCR

amplification and gel electrophoresis were as mentioned in sections 3.14.2 and 3.6.

Table 3.9: The pQE primers for PCR

Primers Sequences (5)3-

Length(bp)

% of GC Content

Melting Temp.(C)

pQE for 5CGGATAACAATTTC ACACAG-3 pQE rev 5GTTCTGAGGTCATT ACTGG-3

20

40.0

56.3

19

47.4

58.0

3.19

Isolation of pET-32a plasmid

The same protocol as described in section 3.5 for plasmid miniprep was used to isolate pET-32a from E. coli BL21(DE3).

54

3.20 (PCR)

Amplification of NS1B gene using Polymerase Chain Reaction

PCR amplification of NS1B gene from pUC57-NS1B was done using the same protocol mentioned in section 3.14.

3.20.1

Primer design

The forward and reverse primers were designed to contain SacI and HindIII restriction sites for endonucleases digestion. The characteristics of the primers design are shown in Table 3.10.

Table 3.10: Primers containing SacI and HindIII restriction enzyme sites used to amplify NS1B gene

Primers Sequences (5)3-

Length(bp)

% of GC Content

Melting Temp.(C)

NS1B forSacI 5-GAGCTCATGGCGAA CAAT-3 NS1B revHindIII 5-AAGCTTCTAATTGT CTCC-3

18

50.0

57.0

18

38.9

53.1

55

3.21 step

Double digestion using SacI and HindIII restriction enzymes in one

NS1B amplified gene and pET-32a were digested using SacI and HindIII and the following components were mixed in PCR tube:

Table 3.11: Double digestion components in PCR tube using SacI and HindIII

pET-32a or NS1B gene Acetylated BSA 10X Multicore enzyme buffer HindIII enzyme SacI enzyme Total

15.8l 0.2l 2l 1l 1l 20l

The reaction was incubated at 37C for 5 hours. Then, the reaction was observed by gel electrophoresis and extracted using the same protocols as mentioned in sections 3.6 and 3.9. The extracted products were used for ligation reaction.

3.22 steps

Double digestion using SacI and HindIII restriction enzymes in two

The following components were mixed in PCR tube for the first digestion for NS1B amplified gene and pET-32a:

56

Table 3.12: Double digestion components in PCR tube using HindIII(1st step)

pET-32a or NS1B gene Acetylated BSA Buffer E HindIII enzyme Total

15.8l 0.2l 2l 2l 20l

The reaction was incubated at 37C for 5 hours. Then, the reaction was observed by gel electrophoresis and extracted using the same protocols as mentioned in sections 3.6 and 3.9. The extracted product was used for the second digestion. The following components were mixed in PCR tube: Table 3.13: Double digestion components in PCR tube using SacI(2nd step)

First digestion product Acetylated BSA Buffer J SacI enzyme Total

15.8l 0.2l 2l 2l 20l

The reaction was incubated at 37C for 5 hours. Then, the reaction was observed by gel electrophoresis and extracted using the same protocols as described in sections 3.6 and 3.9. The extracted product was used for ligation reaction.

57

3.23 colonies

Ligation, transformation into E. coli BL21 (DE3) and screening of

Ligation, transformation into E. coli BL21(DE3) and colony screening were done using the same protocols as mentioned in sections 3.11, 3.12 and 3.18. The primers used for PCR were T7 promoter and T7 terminator primer. The characteristics of the primers used are shown in Table 3.14.

Table 3.14: Primers containing SacI and HindIII restriction enzyme sites used to amplify NS1B gene

Primers Sequences (5)3-

Length(bp)

% of GC Content

Melting Temp.(C)

T7

promoter

5-

20

40.0

56.3

TAATACGACTCACA CAAGGG-3 T7 terminator 519 52.5 60.2

GCTAGTTATTGCTC AGCGG-3

3.24

Isolation plasmid (pQE-80L)

The same protocol as described in section 3.5 for plasmid miniprep was used to isolate pET-32a from E. coli BL21(DE3).

58

3.25

PCR amplification, double digestion, ligation, transformation into

DH5 and screening of colonies

The same protocols as described in sections 3.20, 3.21, 3.22, 3.17 and 3.18 were used.

59

CHAPTER 4

RESULTS AND DISCUSSION

4.1

Transformation of pUC57-NS1B into E. coli BL21(DE3)

Transformation of pUC57-NS1B synthetic gene construct into competent E. coli BL21(DE3) was successfully done. The colonies were maintained using LB agar containing 100g/ml ampicillin and incubated overnight at 37C. Plasmid isolation of pUC57-NS1B from E. coli BL21(DE3) was done using GeneAll miniprep kit and the presence of pUC57-NS1B was verified by agarose gel electrophoresis.

4.2

Cloning of pET-32b-NS1B into E. coli BL21(DE3)

Single restriction digestion was done for pUC57-NS1B and pET-32b by using HindIII enzyme which recognizes 5AAGCTT 3 sequence.

The restriction digestion of pUC57-NS1B was successful and NS1B gene (870 bp) was separated and this is shown in Figure 4.1. pET-32b was also linearized and its presence was verified by the band of 5.9kb in Figure 4.1.

60

8000 6000 5000 3000 2500 2000 1500 1000 750

pET-32b (5.9kb)

pUC57 (2.7kb)

NS1B (0.87kb)

Figure 4.1

Restriction digestion by HindIII.

M: 1 kb Marker, Lane 1, 2: pUC57 and NS1B gene, Lane 3: pET-32b

Subsequently, the NS1B gene was cloned into pET-32b vector and transformed into competent E. coli BL21(DE3). After overnight incubation, colonies were observed and this is shown in Figure 4.2.

61

Colonies

Figure 4.2 BL21(DE3)

Transformation of pET-32b-NS1B into competent E. coli

Colonies were streaked on LB agar containing 100 g/ml ampicillin and spread with IPTG and X-gal. X-gal is used to indicate whether a cell expresses the -galactosidase enzyme, which is encoded by the lacZ gene, in a technique called blue/white screening or X-gal screening. X-gal dye is cleaved by galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole

(http://www.fermentas.com/en/products/all/reagents/r094-xgal). All colonies were observed to be blue in color. Hence, the insertion of NS1B gene into pET-32b vector gave negative result. This may due to recircularization of the vector. The attempts to increase the ratio of gene: vector insert was done. However, the cloning steps were not successful.

4.3

Cloning of pQE-81L-NS1B into E. coli DH5

We attempted the directional cloning approach of NS1B gene into pQE81L vector. Double digestion was done for pQE-81L by using PstI and HindIII enzymes. As for pUC57-NS1B, it was first digested with HindIII enzyme and the result is shown in Figure 4.3.

62

M
6000 5000 3000 2500 2000 1500 1000 750

Lane 1

pQE-81L (4.8kb)

NS1B (0.87kb)

Figure 4.3

Plasmid isolation and digestion of pUC57-NS1B and pQE-81L

M: 1kb Marker, Lane 1 and 2: pUC57-NS1B digested with HindIII, Lane 3: Miniprep of pUC57-NS1B, Lane 4: Double digestion of pQE-81L, Lane 5: Miniprep of pQE-81L. PstI enzyme recognizes the 5 CTGCAG 3' sequence and HindIII enzyme recognizes 5AAGCTT 3 sequence. Figure 4.6 shows the isolation and digestion of the vector and gene. Lane 1 and 2 shows the successful digestion of pUC57-NS1B with HindIII enzyme and the band of NS1B is of correct size (870bp). Lane 4 shows the double digestion of pQE-81L by using PstI and HindIII and the band is of correct size (4.8kb) but in this way still unknown whether the digestion correct complete or not. Plasmid miniprep for both pUC57NS1B and pQE-81L were verified in lane 3 and 5.

Because the concentration of NS1B gene extracted was low (4.7 ng/l) and this would affect the next step of digestion, so PCR amplification was done to increase the concentration of NS1B by using primers designed for pUC57. A band

63

of the correct size was shown in lane 1 of Figure 4.4, indicating successful amplification. Then, PCR product was extracted, double digested with PstI and HindIII and used for cloning into pQE-81L. The transformed colonies were shown in Figure 4.5.

M Lane 2 5 6

Lane 1 7

NS1B

1000 750

Figure 4.4

PCR amplification of NS1B

64

Colonies

Figure 4.5

Transformation of pQE-81L-NS1B into competent DH5

After transformation, colony PCR was carried out with pQEfor (5CGGATAACAATTTCACACAG-3') and pQErev (5'-

GTTCTGAGGTCATTACTGG-3') primers. However, all the colonies screened gave negative result. As shown in Figure 4.6, the size of amplified band is smaller than 300bp, indicating that only the pQE-81L vector was transformed to cells without the cloning of NS1B gene. The right band size to be expected is nearly 1000bp.

65

M clone 21 22 23 24 25 26 27 28 29 30 31 32

1000

300

Figure 4.6

PCR of recombinant pQE-81L-NS1B

4.4

Cloning of pET-32a-NS1B into E. coli BL21(DE3)

PCR amplification was done on pUC57-NS1B to amplify NS1B gene with a newly designed primers NS1BforSacI (5-GAGCTCATGGCGAACAAT3 )and NS1BrevHindIII (5'-AAGCTTCTAATTGTCTCC-3') which introduced SacI and HindIII restriction sites to NS1B for directional cloning of the gene into pET-32a. The amplified NS1B gene is shown in Figure 4.7.

66

M 3 4

Lane 1

Lane 2

3000 2500 1000 NS1B

Figure 4.7

Miniprep of pUC57-NS1B and PCR amplification of NS1B

M: 1kb marker, Lane1: Miniprep of pUC57-NS1B, Lane 2: PCR amplification of NS1B gene The HindIII enzyme recognizes 5AAGCTT 3 sequence while SacI enzyme recognizes 5 GAGCTC 3 sequence. The gene was extracted, double digested with SacI and HindIII and used for cloning into pET-32a. The transformed colonies were shown in Figure 4.8. The colonies were screened by PCR. pET-32a vectors universal primers, namely TAATACGACTCACTATAGGG-3') and T7 T7 promoter (5'terminator (5'-

GCTAGTTATTGCTCAGCGG-3'). All colonies screened gave negative result. As shown in Figure 4.9, the size of amplified band is smaller than 750 bp, indicating that only the pET-32a vector was transformed into the cells without the cloning of NS1B gene. The right band size to be expected is nearly 1500 bp.

67

Colonies

Figure 4.8 BL2(DE3)

Transformation of pET-32a-NS1B into competent E. coli

M 1 2 3 4 5 6 7 8 9

10 11 12 13 14

1500

750

Figure 4.9

PCR of recombinant pET-32a-NS1B

M: 1 kb Marker, Lane 1-14: Clone from 62-75

68

4.5

Cloning of pQE-80L-NS1B into E. coli DH5

The amplified NS1B gene containing SacI and HindIII sites was extracted, digested and cloned into pQE-80L vector. The transformed colonies were shown in Figure 4.10. After transformation into DH5, colony PCR was done using pQEfor and pQErev primers. All screened colonies yield negative result. The result is shown in Figure 4.11. This result showed that only the pQE80L vector was transformed into cells without the cloning of NS1B. The right band size to be expected is nearly1000bp.

Colonies

Figure 4.10

Transformation of pQE-80L-NS1B into competent DH5

69

Clone 83 84 85 86 87 88 89 90 91 92

300

Figure 4.11

PCR of recombinant pQE-80L-NS1B

M: 1kb Marker, Clone number from 83-92

4.6

Confirmation of ligation

Ligation and transformation of pET-32a-NS1B into E. coli BL21(DE3) and pQE-80L-NS1B into E. coli DH5 yield negative result. Therefore, to confirm whether ligation had happened, PCR amplification was done on the ligation. 1l of ligation mix for pET-32a-NS1B and pQE-80L-NS1B were amplified. The result shown in Figure 4.12 indicated that the fragments without insert were amplified.

70

M
90

Lane
86 87

1
88

2
89

83 84 85 91 92

750

300

Figure 4.12

PCR amplification

M: 1kb Marker, Lane 1: fragment without gene insert from pET-32-NS1B, Lane 2: fragment without gene insert from pQE-80L-NS1B

71

CHAPTER 5

CONCLUSION AND FUTURE WORKS

5.1

Conclusion

Nonstructural protein NS1 is used for identification and differentiation between the vaccinated and naturally infected animals. Vaccinated animals have not produced nonstructural protein (Fang-kun et al., 8002). The function of NS1 protein during viral multiplication remains unclear, although a possible regulatory role in viral replication has been suggested from studies of temperature sensitive mutant of NS1 (Binns et al., 1993). Dauber and coworkers (2003) found that reverse genetic system studies may allow to explore the function of NS1B during the replication cycle and to assess their contributions to the pathogenesis and virulence of influenza B (Dauber et al., 2003).

pUC57 plasmid carrying NS1B gene was successful transformed and isolated from E. coli BL21(DE3). Designed primers used for PCR of NS1B showed successful amplification.

First screening of pET-32b-NS1B colonies using white/blue method gave negative result. Similar negative results were observed when screening colonies of pQE-81L-NS1B, pET-32a-NS1B and pQE-80L-NS1B. Screening of colonies for pQE-81L-NS1B and pQE-80L-NS1B revealed a band under the size

72

300bp, which is not as the expected band size of 1000bp. Screening of colonies for pET-32a-NS1B showed a band of the size nearly 750bp, which is not as the expected band size of 1500bp. Screening of colonies for all vectors used showed only plasmids without any insertion of the NS1B gene.

Cloning NS1B into pET-32b using single restriction digestion with HindIII, pQE-81L using double restriction digestion with (PstI and HindIII), pET32a using double restriction digestion with (SacI and HindIII), pQE-80L using double restriction digestion with (SacI and HindIII) gave unexpected result. This result may relate to re-ligation of digested vector for single digestion and uncompleted digestion for vectors of double restriction digestion.

5.2

Future works

A further construction of NS1 gene into pET32a, pET-32b, pQE-80L and pQE-81L vectors should be performed with best precaution of procedures to avoid any contamination and the use of appropriate restriction enzymes with high efficiency to get recombinant construction. The NS1B gene can also be cloned into other expression vectors according to the open reading frame and use other host.

In the future, it is suggested that the overexpression of the constructed recombinant into expression system are being studied and work on purification can also be performed using immobilized affinity chromatography resin. NS1B protein can be analyzed by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis.

73

More studies to determine immunogenicity of influenza NS1B fusion protein by using Western blotting or Enzyme-linked immunosorbent assay (ELISA) methods should be carried out.

74

References

Binns, Matthew, and Ely. (1996). Expression of the non-structural protein NS1 111of influenza virus and detection of anti-NS1 antibody in serum. European 111Patent Office. Application no: 96300681.2

Birch-machin, I., Rowan, A., Jane Pick, J., Mumford, J., and Binns, M. (1997). 111Expression of the non structural protein NS1 of equine influenza a virus :

111detection of Anti-NS1antibody in post infection equine sera. Journal of 111Virology methods: 255-263.

Blaber, M. (1998). Spring. Web Page for Lecture 25 of Molecular Biology and 111Biotechnology Course: Prokaryotic Expression Vectors.

Dauber, B., Heins, G., and Wolff, T. (2003). The Influenza B Virus 111Nonstructural NS1 Protein Is Essential for Efficient Viral Growth and 111Antagonizes Beta Interferon Induction. Journal of Virology: p. 18651872.

Dauber, B., Schnider, J., and Wolff, T. (2006). Double-Stranded RNA Binding of 111Influenza B Virus Nonstructural NS1 Protein Inhibits Protein Kinase R but 111Is Not Essential To Antagonize Production of Alpha/Beta Interferon. Journal 111of Virology: p. 1166711677. Donelan, N., Dauber, B., Wang, X., Basler, C., Wolff, T., and Garca-Sastre, A. 111(2004). The N-and C- Terminal Domains of the NS1 protein of Influenza B 111Virus Can Independently Inhibit IRF-3 and Beta Interferon Promoter 111activation. Journal of Virology: p.11574-11582.

75

Fang-Kun, W., Xio-Fang, Y., Yi-cheng, W., Cun, Z., Li-huan, X., and Si-dang, 111L. (2008). Cloning, Prokaryotic Expression, and Antigenicity Analysis of 111NS1 Gene of H9N2 Swine Influenza Virus. gricultural Sciences in China : 1117(7): 895-900.

Fortes, P., Beloso, A., and Ortin, J. (1994). Influenza VirusNs1 protein inhibits 111pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport. The 111EMBO Journal: vol.13 no.3 pp. 704-712. Garca-Sastre, A., Egorov, A., Matassov, D., Brandt, S., Levy, D., Durbin, J., 111Palese, P., and Muster, T. (1998). Influenza A virus lacking the NS1 gene 111replicates in interferon-deficient systems. Virology 252:324330.

Greenspan, D., Palese P., and Krystal, M. (1988).

Two Nuclear Location

111Signals in the Influenza Virus NS1 Nonstructural Protein. Journal of 111Virology, p: 3020-3026.

Hai, R., Martinez-Sobrido, L., Fraser, K., Allyon, J., Garcia-Saster, A., and 111Palese, P. (2008). Influenza B Virus NS1-Truncated Mutants: Live111Attenuated Vaccine Approach. Journal of Virology: p. 1058010590.

Hale, B., Randall, R., Ortin, J., and Jackson, D. (2008). The multifunctional NS1 111 protein of influenza A viruses. Journal of General Virology: p. 89, 23591112376.

Hatada, E., Takezawa, T., and Fukuda, R. (1992). Specific binding of influenza 111A virus NS1protein to the virus minus-sense RNA in vitro. Journal of 111General virology:73,17-25.

76

Krug, R., Yuan, W., Noah, D., and Latham, A. (2003). Intracellular warfare 111between human influenza viruses and human cells: the roles of the viral NS1 111protein.Virology, 309:181-189.

LaVallie, R., DiBlasio, A., Kovacic, S., Grant, L., Schendel, F. and McCoy, M. 111(1993) Bio/Technology 11, 187193.

Leonhartsberger, S. (2006). E. coli Expression System Efficiency Secretes 111Recombinant Proteins into Culture Broth. Bioprocess internantional. 4(4), 11110-14.

Li, S., Min, J., Krug, R., and Sen, G. (2006). Binding of the influenza A virus 111NS1 protein to PKR mediates the inhibition of its activation by either PACT 111or double-stranded RNA. Journal of Virology: 349, 13-21.

Lu, Y., Qian, X., and Krug, R. (1994).The influenza virus NS1 protein: a novel 111inhibitor of pre-mRNA splicing. Genes Dev: 1817-1828.

Manasatienkij, W., Lekcharoensuk, P., and Upragarin, N. (2008). Expression 111and Purification of NS1 Protein of Highly Pathogenic Avian Influenza Virus 111H5N1 in Escherichia coli. Kasetsart J. (Nat. Sci.) 42: 485 494.

Metreveli, G. (2006). Expression of influenza virus nonstructural protein 1 111(NS1). Swedish University of Agricultural Sciences.

Nerome, R., Hiromoto, Y., Sugita, S., Tanabe, N., Ishida, M., Mastumoto, M., 111Lindstrom, S., Takahashi, T., and Nerome, K. (1998). Evolutionary 111characteristics of influenza B virus since its first isolation in 1940: dynamic 111circulation of deletion and insertion mechanism.Arch Virol.143:1569-1583.

77

Ozaki, H., Sugiura, T., Sugita, S., Imagawa, H., and Kida, H. (2001).Detection 111of antibodies to the nonstructural protein (NS1)of influenza A virus allows 111distinction between vaccinated and infected horses .Veterinary Microbiology 11182:111-119.

Palese, P., and Shaw, L. (2007). Orthomyxoviridae: the viruses and their 111replication, p. 16471689.

Qian, X., Alonso-caplen, F., and Krug, R. (1993). Two Functional Domains of 111the Influenza Virus NS1 Protein Are Required for Regulation of Nuclear 111Export of mRNA. Journal of Virology: p. 2433-2441.

Schneider, J., Dauber, B., Melen, K., Julkunen, I., and Wolff, T. (2008). 111Analysis of Influenza B Virus NS1 Protein Trafficking Reveals a Novel 111Interaction with Nuclear Speckle Domains. Journal of Virology; p. 701711.

Spickler, A. (2009). FluGrippe, avian influenza, Grippe Aviair, Fowl Plaque, 111Swine influenza, Hog Flu, Pig Flu, Equine influenza, Canine influenza. 111Lowa State University, College of Veterinary Medicine, Ames, Lowa.

Wolff, T., Oneili, R., and Palese, P. (1996). Interaction Cloning of NS1-I, a 111Human Protein That Binds to the Nonstructural NS1 Proteins of Influenza A 111and B Viruses. Journal of virology; p. 53635372.

Young, F., Desselberger, U., Palese, P., Ferguson, B., Shatzman, A. and 111Rosenberg, M. (1983). Efficient expression of influenza virus NS1 111nonstructureal proteins in Escherichia coli. Proc. Natl. Acad. Sci. USA. 80, 1116105- 6109.

78

Yuan, W., and Krug, M. (2001). Influenza B virus NS1 protein inhibits 111conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein. 111EMBO J. 20, 362371.

Yuan, W., Aramini, J., Montelione, G., and Krug, R. (2002). Structural Basis 111for Ubiquitin-like ISG 15 Protein Binding to the NS1 Protein of Influenza B 111Virus: AProteinProtein Interaction Function That Is Not Shared by the 111Corresponding N-terminal Domain of the NS1 Protein of Influenza 111AVirus.Virology 304:291-301.

Zhao, L., Xu, L., Zhou, X., Zhu, Q., Yang, Z., Zhang, C., Zhu, X., Yu, M., 11 Zhang, Y., Zhao, X., Huang, P. (2009). Interaction of influenza virus NS1 11.protein with growth arrest-specific protein 8. Virology Journal: 6:218.

79

APPENDIX A

NS1B gene sequence (870 bp) of Influenza B virus (B/Taiwan/45/2007)

HindIII PstI

aagcttctgcag atg gcg aac aat atg acc aca aca caa att gag gtg ggt ccg gga gca acc aat gcc acc ata aac ttt gaa gca gga att ctg gag tgc tat gaa agg ctt tca tgg caa aga gcc ctt gac tac cct ggt caa gac cgc cta aac aga cta aag aga aaa tta gag tca aga ata aag act cac aac aaa agt gag cct gaa agt aaa agg atg tcc ctt gaa gag aga aaa gca att gga gta aaa atg atg aaa gta ctc cta ttt atg aat ccg tct gct gga att gaa ggg ttt gag cca tac tgt atg aaa agt tcc tca aat agc aac tgt acg aaa tac aat tgg acc gat tac cct tca aca cca ggg agg tac ctt gat gac ata gaa gaa gaa cca gag gat gtt gat ggc cca act gaa ata gta tta agg gac atg aac aac aaa gat gca agg caa aag ata aag gag gaa gta aac act cag aaa gaa ggg aag ttc cgt ttg aca ata aaa agg gat atg cgt aat gta ttg tcc ttg aga gtg ttg gta aac gga aca ttc ctc aaa cac ccc aat gga tac aag tcc tta tca act ctg cat aga ttg aat gca tat gac cag agt gga agg ctt gtt gct aaa ctt gtt gcc act gat gat att aca gtg gag gat gaa gaa gat ggc cat cgg atc ctc aac tca ctc ttc gag cgt ctt aat gaa gga cat tca aag cca att cga gca gct gaa act gcg gtg gga gtc tta tcc caa ttt ggt caa gag cac cga tta tca cca gaa gag gga gac aat tag aagcttctgcag
HindIII PstI

a c g t

: : : :

Adenine Cytosine Guanine Thymine

80

APPENDIX B

NS1B protein sequence (281 amino acids)

MANNMTTTQIEVGPGATNATINFEAGILECYERLSWQRALDYPGQDRLN RLKRKLESRIKTHNKSEPESKRMSLEERKAIGVKMMKVLLFMNPSAGIEG FEPYCMKSSSNSNCTKYNWTDYPSTPGRYLDDIEEEPEDVDGPTEIVLRD MNNKDARQKIKEEVNTQKEGKFRLTIKRDMRNVLSLRVLVNGTFLKHPN GYKSLSTLHRLNAYDQSGRLVAKLVATDDITVEDEEDGHRILNSLFERLN EGHSKPIRAAETAVGVLSQFGQEHRLSPEEGDN

A C D E F G H I K L

: : : : : : : : : :

Alanine Cysteine Aspartic acid Glutamic acid Phenylalanine Glycine Histidine Isoleucine Lysine Leucine

M N P Q R S T V W Y

: : : : : : : : : :

Methionine Asparagine Proline Glutamine Arginine Serine Threonine Valine Tryptophan Tyrosine