Preparation And Analysis Of Antibodies Against The Outer Capsid Proteins Of Taura Syndrome Virus And Grass Carp Reovirus

Aquatic animal viral diseases brought serious harm to the aquaculture industry. Asthe major viral pathogens infecting penaeus shrimp and grass carp, Taura syndromevirus（TSV）and Grass carp reovirus（GCRV）had heavily affected the sustainingdevelopment of aquaculture. Therefore, it is particula rly necessary to investigate thestructural proteins of the two aquatic animal viruses. TSV is a member of the genusAparavirus. Its genome size is about10kb, containing two open reading fragments（ORFs）. ORF1encodes non-structural proteins and ORF2encodes three structuralproteins: VP1, VP2and VP3. GCRV is assigned to the genus Aquaticreovirus. Itsgenomic RNAs encode seven structural proteins and five kinds of non-structuralproteins. Protein VP7of GCRV is a structural protein which is encoded by vp7gene inS10genomic fragment. The VP5-VP7heterodimer forms the outer coat protein layer ofGCRV. In this study, the vp2and vp3genes of TSV and the vp7gene of GCRV weresuccessfully expressed in prokaryotic cells. The corresponding proteins were purifiedand injected to test animals for raising polyclonal and monoclonal antibodies. The maincontents were as follows:1. Cloning, expression and antibodies preparation of TSV capsid protein genes vp2andvp3According to the complete genome sequence of TSV published on GeneBank, twopairs of primers were designed respectively. Total RNAs, which were extracted frompenaeus shrimp infected with TSV, were used as the template for amplifying the mainstructural protein genes vp2and vp3of TSV by RT-PCR. Capsid protein genes vp2andvp3were cloned into pET-16b-1and pGEX-4t-3expression vector respectively, andtransformed into Escherichia coli BL21and DH5α for protein expression. After IPTGinduction and SDS-PAGE analysis, recombinant protein VP2and VP3of about42kDaand48kDa in molecule weight were produced in E. coli. It was showed that the twotarget proteins were all expressed in the form of inclusion bodies. Protein bands in gelwere tapped and purified. The purified proteins were used as immunogens for thepreparation of polyclonal antibodies: P-vp2and P-vp3. Two antigenic peptidesoriginated from TSV capsid protein VP2and VP3, which were synthesized respectively, were used as antigen for production of monoclonal antibodies. After BALB/c miceimmunization, cell fusion and subclone screenings, hybridoma cells were obtained.M–vp2and M-vp3were obtained after the culture supernatant purification.Our immunoassays demonstrated that the four kinds of antibodies all showedspecific immunoreactivities to corresponding recombinant proteins by Western blotassay without cross-reaction with other proteins in E.coli. Immunodot-blot resultsshowed that both monoclonal antibodies and polyclonal antibodies could detect thenatural VP2and VP3proteins from TSV-infected shrimps， in the meantime, thespecificity of monoclonal antibodies against the structural protein of TSV were higherthan the polyclonal antibodies, the titer of the monoclonal antibody M-vp2was slightlyhigher than the monoclonal antibody M-vp3. The results provide the technicalfoundation for the establishment of the TSV immunoassay methods and also provide thepossibility for the realization of the TSV immunology prevention and control.2. Expression and monoclonal antibody preparation of GCRV outer capsid protein VP7The VP7expression plasmid was transformed into Escherichia coli DH5αcompetent cells. After induction by IPTG and analysis by SDS-PAGE, the recombinantprotein VP7was obtained. SDS-PAGE analysis results showed that VP7protein wasexpressed in the form of inclusion bodies. Protein bands in gel were tapped and purified.The purified proteins were used as immunogen for the preparation of polyclonalantibodies (P-vp7). Mouse spleen cells were fused with SP2/0tumor. After twoscreenings and re-clone,12hybridoma cells were obtained. By ELISA and Western blot,the best hybridoma cell was screened. Mouse ascites and cell culture supernatants weredealt with the bitterness-saturated ammonium sulfate method, the monoclonal antibodyagainst VP7protein of GCRV was obtained, and the subtype of the monoclonalantibody was identificated.The microneutralization and TCID50assay indicated that the polyclonal antibodiescould possess the specially ability to bind to a native form of GCRV viruses, and couldit efficiently block viral infection. ELISA and Western blot results showed that theminimum detectable concentration of monoclonal antibody was0.737μg/ml and had nocross-reaction with GST-tagged protein. Antibody subtype identification showed thatthe antibody subtype was IgG2b. The antibody was used to detect GCRV infected grasscarp samples which were collected from different parts by ELISA method, and RT-PCR assay was set as a control. ELISA results showed the positive rate of detected sampleswas consistent with the RT-PCR results. It suggested that the monoclonal antibodyshowed highly specificity and sensitivity to GCRV pathogen and it could be used forthe detection of GCRV and the structural proteins analysis. This study providesinformation for the development of immunoassays technology research of GCRV and itcould also provide the basic data for the immunoreaction between GCRV virus and itshosts in the future.