Significance of the proposed study

Bacteria are the leading fish pathogens which cause high mortalities (Brock and
Lightner, 1990). Fish farming is a high-risk, capital-intensive industry that is sitespecific and requires technical expertise. It is clearly more difficult to be
financially successful in fish farming than in conventional farming of livestock or
horticulture because of bacterial and fungal pathogens. In India south east to south
west covered by costal areas and people are more dependent in fish farming. The
deliberate synthesis of nanomaterials using the potential bacterial strain is at best a
recent phenomenon to get control over drug resistant bacterial pathogens
(Saravanan and Nanda et.al 2010).
Construction of a cDNA library for the parasite would therefore represent a major
research advance, and is the central goal of this study. The library will establish a
genetic information base for M. anguillicaudatus, containing protein-encoding
sequences from the genome, which can then be used by researchers to analyse
functions of specific genes. The process of constructing the cDNA library
encompasses several topics of research. The library must be screened for positive M.

anguillicaudatus clones, from which some are selected for in vivo excision of the
pBK-CMV phagemid vector (containing the insert) from the ZAP Express vector.
Plasmid DNA containing the insert must be isolated from the vector, and then
analysed to determine the sequence of the cloned cDNA. A full-length cDNA
sequence can then be constructed using Rapid Amplification of cDNA Ends (RACE).
Finally, bioinformatics programs are used to analyse the DNA sequences in order to
predict corresponding protein sequences, which can be compared with other genes
whose functions are known.
Non-specific immune system or innate immune system is the first line of defense
mechanism against pathogenic diseases by various infectious microbial organisms.
According to Soderhall and Cerenius Soderhall 1992crustaceans are lacking in
adaptive immune system, so they completely depend on their innate immune system
that includes both cellular and humoral immune systems. Cellular defense system
depends on cell immune system, including encapsulation, nodule formation, and

phagocytosis whereas humoral immune system possesses prophenoloxidase activating

mechanism, clotting mechanism and production of antimicrobial peptides (AMPs)
Jesu 2013 . AMPs or host defense peptides are an evolutionarily conserved
component of the innate immune mechanism and are present in all the living things.
They are important immune genes with a capability to normalize and/or destroy
invading microbial pathogens [2]. These AMPs are commonly small cationic proteins
that have antimicrobial response and have the ability to enhance immunity by
functioning as immunomodulators. AMPs are very suitable candidate for development
of novel antibiotics due to their antimicrobial nature against Gram-negative, Grampositive, fungal, some viral and protozoan pathogens [3] and can also serve as
templates in the development of therapeutic agents [4].Immune gene plays an
important role in the action of programmed cell death (PCD), which is described by
cell shrinkage, chromatin condensation, DNA fragmentation, and formation of
apoptotic bodies. PCD is a normal
physiological function by which additional or repaired cells are terminated in order to
regulate the homeostasis of various tissues and organs
So far no such a report has been published from of Misgurnus anguillicaudatus
Immune gene. To gain insight into the characterization of Immune gene and its role in
M anguillicaudatus

, a full length cDNA of Immune gene is identified from the

M anguillicaudatus

cDNA library constructe by Genome Sequencing FLX (GS FLX)

technology. The transcriptional differentiation of Immune gene mRNA has been analyze using
A. hydrophila challenge. Furthermore, overexpression and purification of recombinant protein
were conducte using Escherichia coli BL21 (DE3) bacterial expression system.

Bacterial strains and culture conditions

The sh isolates were isolated from diseased sh from the cultured pond in E Zhou,
Hubei province, China. The disease symptoms were deep hemorrhagic ulcers in the
midbody and tail regions. Aeromonas selective agar, i.e., Aeromonas Medium Base
(Oxoid, Ltd.), supplemented with ampicillin SR136, was used to obtain Aeromonas
isolates from sh ulcer scraps. For comparison, a number of water samples were
collected from the same ponds as the sh within a 2-month time period. These
environmental samples were processed by using the same selective medium, except
that no ampicillin was added, due to the low-level ampicillin resistance among

environmental isolates. The presumed Aeromonas isolates were conrmed by oxidase

and catalase test and by determining the sensitivity to the vibriostatic reagent 0/129
(150 g ml1 ; Sigma, St. Louis, Mo.). The isolates were identied to the species level
by traditional biochemical methods, including tests for esculin hydrolysis, lysine
decarboxylase, arginine dihydrolase, and ornithine decarboxylase; tests for acid
production from arabinose, glucose, sucrose, and mannitol; and tests for susceptibility
to ampicillin and cephalothineAll isolates were stored in 30% glycerol in brain heart
infusion (BHI) broth at 70C until further use, subsequently recultured on BHI agar
plates (Becton Dickinson Microbiology Systems), and then incubated overnight at
37C.
For bacterial challenge, the fish were injected intraperitonealy with A. hydrophila
(5 _ 106 CFU/ml) suspended in 1X phosphate buffer saline (100 ml/fish).. Samples
were collected before (0 h), and after injection (3, 6, 12, 24 and 48 h) and were
immediately snap-frozen in liquid nitrogen and stored at _80 _C until total RNAwas
isolated. Using a sterilized syringe, the blood (0.5e1.0 ml per fish) was collected from
the fish caudal fin and immediately centrifuged at 4000 _ g for 10 min at 4 _C to
allow blood cell collection for total RNA extraction. PBS (1X) were prepared and
served as control (100 ml/fish). All samples were analyzed in three duplications and
the best representative data is expressed here. The methodology followed for this, is
described elsewhere [Livak 2001].

RNA Isolation
Homogenize tissue samples in 1 ml of TRIZOL reagent per 50 to
100 mg of tissue using a glass-Teflon or power homogenizer (e.g.
Polytron, Tekmars TISSUEMIZER). The sample volume should not
exceed 10% of the volume of TRIZOL Reagent used for the
homogenization. Rinse cell monolayer with ice cold PBS once. Lyse
cells directly in a culture dish by adding 1 ml of TRIZOL Reagent per
3.5 cm diameter dish and scraping with cell scraper. Pass the cell
lysate several times through a pipette. Vortex thoroughly. The
amount of TRIZOL reagent added is based on the area of the culture
dish (1 ml per 10 cm2) and not on the number of cells present. An
insufficient amount of TRIZOL Reagent may result in DNA
contamination of the isolated RNA. Pellet cells by spinning at 300 X
g for 5 min. Lyse cells with TRIZOL Reagent by repetitive pipetting or
by passing through syringe and needle. Use 1 ml of the reagent per
5-10 X 106 of animal cells. Incubate the homogenized sample for 5
minutes at room temperature to permit the complete dissociation of
nucleoprotein complexes. Centrifuge to remove cell debris. Transfer
the supernatant to new tube. Add 0.2 ml of chloroform per 1 ml of
TRIZOL Reagent. Cap sample tubes securely. Vortex samples
vigorously for 15 seconds and incubate them at room temperature
for 2 to 3 minutes. Centrifuge the samples at no more than 12,000 x

g for 15 minutes at 2 to 80C. Following centrifugation, the mixture

separates into lower red, phenol-chloroform phase, an interphase,
and a colorless upper aqueous phase. RNA remains exclusively in
the aqueous phase. Transfer upper aqueous phase carefully without
disturbing the interphase into fresh tube. Measure the volume of the
aqueous phase (The volume of the aqueous phase is about 60% of
the volume of TRIZOL Reagent used for homogenization). Precipitate
the RNA from the aqueous phase by mixing with isopropyl alcohol.
Use 0.5 ml of isopropyl alcohol per 1 ml of TRIZOL Reagent used for
the initial homogenization. Incubate samples at 15 to 30 oC for 10
minutes and centrifuge at not more than 12,000 x g for 10 minutes
at 2 to 4oC. The RNA precipitate, often invisible before
centrifugation, forms a gel-like pellet on the side and bottom of the
tube. Remove the supernatant completely. Wash the RNA pellet
once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml
of TRIZOL Reagent used for the initial homogenization. Mix the
samples by vortexing and centrifuge at no more than 7,500 x g for 5
minutes at 2 to 8 oC. Repeat above washing procedure once.
Remove all leftover ethanol. Air-dry or vacuum dry RNA pellet for 510 minutes. Do not dry the RNA pellet by centrifuge under vacuum.
It is important not to let the RNA pellet dry completely as this will
greatly decrease its solubility. Partially dissolved RNA samples have
an A260/A280 ratio < 1.6. Dissolve RNA in DEPC-treated water by
passing solution a few times through a pipette tip. Dilute 1 l of RNA
with 39 l of DEPC-treated water (1:40 dilution). Using 10 l
microcuvette, take OD at 260 nm and 280 nm to determine sample
concentration and purity. The A260/A280 ratio should be above 1.6.
Apply the convention that 1 OD at 260 equals 40 g /ml RNA.

cDNA synthesis
During the synthesis of cDNA we also setup 2 negative controls, a
very important step in the process. Our negative controls are prepared
with the same mix as is used with our study group and consist of: 1)
One negative consisting of a duplicate of one sample (selected at
random) but without the RT enzyme added. 2) One negative consisting
of the RT enzyme and synthesis mix but with no RNA and just
nuclease free water.
500ng RNA of each sample prepared in volume of 10ul and placed on
ice. 1 negative control prepared without RNA in 10ul water (labelled
RNA-VE) 1 negative control containing 500ng RNA from any one
sample (labelled RT-VE) If you wish, make extra control sample to
use for the Standard curve and label SC (Alternatively you can use a

pool of your control cDNA for this). 9ul of mix added to each sample
and 2 negative controls All samples heated to 65C for 5 minutes and
returned to ice 1ul RT enzyme added to all samples (NOT added
to RT-VE) All samples heated at 37C for 60 minutes. Samples frozen
or 1:30 dilution made if running 96 well plates in which case SC, 1:20
dilution made. For 384 well plate follow other protocol.
Bioinformatics analysis
The Basic Local Alignment Tool (BLAST) program was used to search similar
nucleotide and protein sequences. The open reading frame (ORF) and amino acid
sequence was obtained by using DNAssit 2.2. Characteristic domains or motifs were
identified using the PROSITE profile database. Identity, similarity and gap
percentages were calculated using FASTA program. The N-terminal transmembrane
sequence was determined by DAS transmembrane prediction program (http://
www.sbc.su.se/wmiklos/DAS). Signal peptide analysis was done using the SignalP
worldwide P server (http://www.cbs.dtu.dk). Pairwise and multiple sequence
alignment were analyzed using the ClustalW version 2 program. The phylogenetic
relationship of the MrAK-1 was determined using the neighbor-joining (NJ) method
and MEGA 4.1 program.
Gene expression analysis by real time PCR
Total RNA was extracted from the tissues of each animal using TRI Reagent
following manufacturers protocol (Guangzhou Dongsheng Biotech, China). Total
RNA was treated with RNase free DNA set (5 Prime GmbH, Hamburg, Germany) to
remove the contaminating DNA. The total RNA concentration was measured
spectrophometrically (NanoVue Plus Spectrophotometer, GE Healthcare UK Ltd,
England). First-strand cDNA was synthesized from total RNA by M-MLV reverse
transcriptase (Promega, USA) following the manufacturers protocol with AOLP
primer (50GGCCACGCGTCG ACTAGTAC(T)16(A/C/G)30). The relative expression
of immune gene in the gill, hepatopancreas, stomach, intestine, brain and muscle were
measured by quantitative real time polymerase chain reaction (qRT-PCR). qRT-PCR
was carried out using a ABI 7500 Real-time Detection System (Applied Biosystems)
in 20 ml reaction volume containing 4 ml of cDNA from each tissue, 10 ml of Fast
SYBR_ Green Master Mix, 0.5 ml of each primer (20 pmol/ml) and 5 ml dH2O.

The qRT-PCR cycle profilewas 1 cycle of 95 _C for 10 s, followed by 35

cycles of 95 _C for 5 s, 58 _C for 10 s and 72 _C for 20 s and finally 1 cycle of
95 _C for 15 s, 60 _C for 30 s and 95 _C for 15 s. The same qRT-PCR cycle

profile was used for the internal control gene, b-actin. All the primers used in
this study were designed by Primer3 and BLASTprogramme(http://www.ncbi.
nlm.nih.gov/tools/primer-blast/). Gene specific primers were designed using
highly conserved regions from M. anguillicaudatus gene. b-actin primers
(forward primer; 50-accaccgaaattgctccatcctct-30 and reverse primer; 50acggtcacttgtt caccatcggcatt-30) were designe based on the EST of a 1357 base
pairs sequence (GenBank Accession No. AY651918) from M.
anguillicaudatus. After the PCR program, data were analyze with ABI 7500
SDS software (Applied Biosystems). To maintain consistency, the baselinewas
set automatically by the software. The comparative CT method (2_ddCT
method)was used to analyze the expression level of Immune gene [25]. All
samples were obtained and analyzed in three replicates, and the results are
expressed as relative fold of one sample as mean _ standard deviation. For
comparison of relative Immune gene mRNA expression, statistical analysis is
performe using one-wayANOVA and meancomparisons were performe by
Tukeys Multiple Range Test using SPSS 11.5 at the 5% significant level.