Prokaryotic Expression Of The Main Structural Proteins And Preparation Of Monoclonal Antibodies Against P Protein Of Spring Viremia Of Carp Virus

Spring viremia of carp virus(SVCV),which causes an cute hemorrhagic and highly contagious disease mainly in cyprinids,is the viral pathogen of spring viremia of carp(SVC). SVCV is classfied in the family Rhabdoviridae,genus Vesiculovirus.The SVCV genome is approximately11kb in length and encodes five structural proteins:the nucleoprotein(N),the phosphoprotein(P),the matrix protein(M),the glycoprotein(G),and a RNA-dependent polymerase(L).Outbreaks of SVC disease had historically caused the large losses on the fish farming industry in Europe,Asian and North America.The current study aimed to obtain G,N,and P structural protein genes of SVCV using molecular cloning technology,express in prokaryotic cells.The recombinant proteins were purified,and they laid a foundation of the development of SVCV diagnostic methods.BALB/c mices were immunized three times with the recombinant P protein.Then we are going to prepare monoclonal antibody(McAb) against P protein by hybridoma technique.These McAbs may have important consequences for futher investigation of the structure and function of SVCV P protein.1.Clone and prokaryotic expression of the SVCV G、 N and P genesAccording to the nucleotide sequence of the SVCV publicted in GeneBank,three pairs of primers were designed to amplify the G、 N and P genes specifically which incorporate the restriction endonuclease sites Nco I at the5’ end of the ORF and Xho I at the3’end of the ORF. The G、 N and P genes of SVCV were amplified by RT-PCR, after sequencing, cloned into the pET-28a(+) prokaryotic expression vetor.Then the recombinant plasmid were transformed into E.coli Rosseta.The recombinant proteins were expressed by the induction with IPTG and identified by SDS-PAGE and Western Blotting.The result of SDS-PAGE showed that the G、N and P genes were all satisfactorily expressed in E.coli,and the molecular weight of expressed products were about43K、47KD and37KD,matched with the expected results.Western Blotting indicated that all the fusion protein were identified by the positive antiserum against SVCV.2. Preparation and identification of monoclonal antibodies against SVCV P proteinThe recombinant P protein was purified by affinity chromatography.BALB/c mice were immunized with the purified recombinant SCVC P protein with Freund’s adjuvant.From the fusion of myeloma cells SP2/0with spleen cells of the mice immunized with the recombinant P protein,we screened hybridoma cells producing anti-phosphoprotein McAbs by ELISA and selected two hybridioma clones named as2E1and3A9.The activity of McAbs for SVCV phosphoprotein in hybridoma culture supernatants were assessed by Western Blotting and indirect fluorescent antibody(IFA) testing.As a result,both of the McAbs could recognize the two native phosphoprotein,NSl and NS2,in SVCV-infected FHM cells. Hybridomas were propagated and inoculated into the peritoneal cavity of BALB/c mice after pristan treatment.Approximately10days after inoculation,ascites with high McAbs titers were collected.The antibody of titer of ascites wrer1:16000and1:32000using ELISA method.Thus,these McAbs could be clinically useful for quick diagnosis during the SVCV outbreaks and facilitate more fundamental studies on the structure and function of SVCV P protein.