Cloning And Expression Of G Gene And N Gene Of CVS Strain Of Rabies Virus

Rabies is a major lethal zoonosis of significant public health concern in many parts of the world,especially in China.It causes a fatal encephalomyelitis for which there is no treatment once the clinical symptoms have appeared.Vaccine inoculation is a useful way to control this disease.Although traditional vaccines have shown their efficiency in controlling the incidence of rabies,the security issues to be concerned.So developing novel vaccine that more efficient and cheaper has become a research hotspot.In this study,the glycoprotein(G) gene and nucleoprotein(N) were obtained by RT-PCR and cloned into pMD18-T plasmid,respectively.After sequencing and analyzing with DNAstar,The results showed that the complete length of G gene was 1575bp,which encoded 524 amino acids,and the complete length of N gene was 1353bp,encoded 450 amino acids.Compared to strains CVS-11,PV-11,SRV 9, SAD-B19,HEP-Flury,ERA,3aG,the GP nucleotide homology was 89.0%～98.8%, and amino acid homologies are 87.2%～97.0%.the NP nucleotide homology was 92.6%～99.6%,and amino acid homologies were 97.6%～99.3%.The N gene was subcloned into the prokaryotic vector pET28a(+).The positive recombinant pET-N was transformed into E.coli BL21 and induced by IPTG at 37℃, SDS-PAGE was performed to analyze the N gene production.Results showed that the protein was highly expressed in E.coli,and the molecular weight was 54kDa. The mass spectrum results proved that the N protein of RV CVS strain was successfully expressed.Then the anti-rabies virus N protein antibody was obtained by injecting the NP into rabbits.This may provide tools for the diagnostic reagent development.The GP gene was inserted into the transfer vector pVL1393 to generate plasmid pVL-G.After co-transfection with Bsu36 I linearized BmBacPAK6 genomic DNA into Bm-N cells by lipofection,the recombinant baculovirus with the GP gene was obtained and plaque screening was further conducted.The silkworm chrysalis was infected with the recombinant virus.The sandwich-ELISA results showed that the high titer of the GP antigen expression was 1:4096,it was over more than 10 times to the inactivated antigen.This experiment has laid a foundation for use of silkworm-baculovirus expression system for RV gene engineering vaccine.