Prokaryotic Expression, Purification And Identification Of N Gene Of Cvs Strain Of Rabies Virus

Rabies is a fatal zoonosis caused by rabies virus, once diagnosed positive the mortality is nearly 100%. In recent years, it is reported that about 50,000 human died annually from rabies all over the world. In China, incidence and mortality of rabies shows a rising trend, which has greatly affected public health and social development. Therefore, great effort should be made to control the problem. At present ,there are many methods available to detect Rabies virus or antibody,but they all have drawback such and that. So, it is recommended new diagnostic method be exploited.In this study, the nucleoprotein was obtained by RT-PCR and cloned into pMD-18-T plasmid. The result of sequencing and analyzing with DNAstar showed that the complete length of N gene was 1353bp, encoded 450 amino acid. Compared to rabies strain CVS-11, PV-11,SRV9,SAD-B19,HEP-Flury ,ERA the NP nucleotide homology was 84.4%～99.5%,and amino acid homology was 97.3%～99.3% respectively.The N gene was subcloned into the prokaryotic vector pET28a(+). The positive recombinant pET-N was transformed into E.coli Rossetta(DE3) and induced by IPTG at 37℃with final concentration of 0.1mg/ml, 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. Western blot analysis results proved that the N protein of RV CVS strain was successfully expressed. Then the protein was purified and protein of high purity was obtained. Than the anti-rabies virus N protein antibody was obtained by injecting the NP into mice. .This experiment laid a foundation for use of monoclonal antibody and Indirect ELISA.