Research On The Immunogenicity Of The Grass Carp Reovirus Capsid Proteins

Grass carp (Ctenopharyngodon idellus), member of cyprinid family is a popularcommercial fish species in China, and accounts for about23%of the annual aquculturalproduction in China. Grass carp reovirus (GCRV) is the causative pathogen of grasscarp hemorrhagic disease.The GCRV can infect the occurrence grass carp fingerlinghemorrhagic disease periodicly, and the death rate is as high as90%, also can infect theherring, pseudorasbora parva and gobiocypris rarus. So far, there are currently noeffective drugs or measures to control the viral infection. Prevention and control of thedisease is impeded largely due to the lack of research in economic subunit vaccinedevelopment, and therefore it’s very important significance to study antibody detectionagainst grass carp reovirus and genetic engineering subunit vaccine.In this paper, the research mainly including the following aspects:1, The cloning, prokaryotic expression and antibody preparation of VP5andVP7proteins, and their ability of neutralizing the virus analysis experimentThe11dsRNA fragmental genome of Grass carp reovirus (GCRV) is enclosed infive inner core proteins and two outer capsid proteins. The Glutathione S-transferase(GST) fusion protein expression vector pGEX-4T-3was employed to clone andexpression of GCRV outer capsid gene vp5and gene vp7, which were amplified byRT–PCR from infected Grass carp. The recombinant GST-fusion protein rVP5and rVP7were induced by1mM IPTG in DH5α and confirmed by SDS-PAGE and Western blotassays. An expected90-kDa rVP5and52-kDa rVP7were highly expressed respectively,and were mainly exhibited in the formation of the inclusion body. After purification, rVP5and rVP7was intraperitoneally injected to the experimental mice to produceanti-rVP5polyclonal serum and anti-rVP7polyclonal serum respectively. In vitromicroneutralization and TCID50assays indicated that both polyclonal antibody againstrVP5and rVP7could neutralize GCRV, and suggested that both rVP5and rVP7had thepotential to be used as subunit vaccine against GCRV infection. The present study pavedthe way for further characterization of the immunogenicity of viral outer capsid proteinVP7in grass carp Ctenopharyngodon idellus and could be based to develop antibody orantigen detection assays for GCRV pathogen.2, The immunological detection methods based on the immunogenicity the VP7of Grass carp reovirusOutbreaks of grass carp reovious (GCRV) infection in grass carp are still a seriousproblem worldwide. Frequent outbreak of grass carp hemorrhagic disease, caused bygrass carp reovious (GCRV) infection, poses as a serious problem in the production ofgrass carp Ctenopharyngodon idella. Although various nucleic acids-based diagnosticmethods have been shown effective, lack of commercial monoclonal antibody againstgrass carp IgM has impeded the development of any reliable immunoassays in detectionof GCRV infection. The study was carried out to develop and characterizes polyclonalantibodies and monoclonal antibodies against grass carp Ctenopharyngodon idella IgM,and preparation and screening of monoclonal antibodies against the constant region ofgrass carp IgM protein. Heavy chain constant (CH) regions of grass carp IgM, CH1andCH2, were highly expressed by pET-16B vector in E coli.. His6-tagged rCH1and rCH2were purified through chromatography and served as antigens to raise ponoclonalantibodies in mice. The recombinant proteins rCH1and rCH2were confirmed byWestern blot analysis with anti-His-tag monoclonal antibody and the polyclonalantibodies against CH1or CH2proteins.The results suggested that the polyclonal antibodies could recognize the heavy chainof grass carp IgM. Two hybridomas, designated as2E8against rCH1and3A2againstrCH2, were screened through enzyme-linked immunosorbent assay. The2E8and3A2 MAbs could efficiently recognize antibody against grass carp reovirus (GCRV) in serumof infected grass carps in various specific detections including the double indirectenzyme-linked immunosorbent assay (D-ELISA), reverse transcription-polymerasechain reaction(RT-PCR),western blotting, and dot blot analysis. In comparison to aconventional RT-PCR method, validity of the detection methods were furtherdemonstrated by test on clinical fish sera samples collected from grass carp farm inJiangxi and Guangdong Provinces during disease pandemic in2011. The resultssuggested that the monoclonal antibody showed highly specificity and sensitivity toGCRV pathogen and it could be used for the detection of GCRV and the structuralproteins analysis.In conclusion, the detection methods especially Dot-blot establishedhere could be used for specific and sensitive serodiagnosis of GCRV infection. Inconclusion, the the monoclonal antibodies aganist rCH1and rCH2characterized in thisstudy paved the way for the development of immunoassays for pathogens in grass carp.