Production Of Polyclonal Antibody Of MHV-N Protein And Development Of An Indirect ELISA For Detection Of Anti-MHV Antibodies

Mouse hepatitis virus (Mouse hepatitis virus, MHV) belongs to the family coronaviridae, is a positive strand RNA virus infected mice, and widely exists in various experimental mouse groups. Different types, different strains of virus in different age, lines and immune status of the mice can cause hepatitis, encephalomyelitis and enteritis and so on many different symptoms. Typically, most infected mice were hidden infection, but the mixed infection with certain microorganisms, or under certain conditions to stimulate the outbreaks of disease frequently. It is more difficult to eliminate this virus infection in rodent laboratory animal. Rats of mouse hepatitis virus infection can change the parameters of immune response, interference experiment, so the establishment of rapid diagnostic method is very necessary.In this study, according to the MHV-A59N gene sequence published in GenBank (AY700211), we designed a pair of specific primers, amplified MHV major antigen fragment of N gene (NS) by RT-PCR, then connect the target fragment into pMD18-T carrier. After double enzyme digestion and sequence analysis, the target gene fragment was cloned into prokaryotic expression vector pET-32a, and constructed the recombinant plasmid pET-32a-N, and transformed E. coli Rossetta. SDS-PAGE verified MHV N recombinant fusion protein was successfully expressed induced by IPTG. Its molecular weight is43KD, which is consistent with the expected size. At last, the purified MHV N protein used as antigen to prepare the polyclonal antibody with the high reaction titer of106, Western blot and indirect immunofluorescence showed it had very high specificity and sensitivity against MHV.The purified fusion protein NP (S) as coating antigen, an indirect ELISA for the detection of mouse hepatitis virus antibody was developed and optimized. The results shows that the optimal dilution of antigen was300ng-mL-1. The serum dilution and the optimal reaction time were1:200and120min. The enzyme labelled antibody best diluted to the concentration and the optimal reaction time were1:5000and60min. The optimal of blocking solutions was5%skimmed milk powder. The results of repeatability test and sensitivity test showed that the indirect ELISA method had good reproducibility and good sensitivity.180serum samples were collected in Harbin and detected by this indirect ELISA method, the results showed the positive rate is7.22%.In conclusions, the polyclonal antibody preparation and the development of indirect ELISA with high sensitivity and repeatability, it laid the foundation for the further study of MHV bioproperties and development of MHV antibody diagnostic kit.