Clonning And Expression Of E M And N Genes Of SARS-CoV And Evaluation Of Their Immunogenicity

Objective To construct eukaryotic and prokaryotic expression plasmids of envelop gene(E) , membrane gene(M) and nucleocapsid(N) gene of SARS-CoV, and to evaluate their immunogenicity.Method E, M and N genes were obtained by PCR , and then cloned into eukaryotic expression vector pVAX1. The mice and rabbits were immunized by intramuscular injection with the recombinant plasmids .The antigen-specific antibodies were determined by ELISA .The weight change of the animal was observed .Their organs were taken , and pathological change were observed .E,M and N genes were also cloned into prokaryotic expression vector pGEX-4T-l.The condition under which the recombinant proteins were expressed was compared . The recombinant N protein as an antigen , the antibodies in the serum from the mice which were immunized with recombinant plasmid pVAX1-N.Result The sequences of recombinant plasmids were identical to the sequences in the Genebank . Of the animals immunized with recombinant plasmid pVAX1-E, pVAX1-M and pVAXl-N , specific antibodies against SARS-CoV increased significantly in sera. The weight change and pathological result of the animal injected with recombinant plasmid pVAX1-E, pVAXl-M and pVAXl-N showed little difference with negative contral. A highly expressed recombinant N protein could be observed by SDS-PAGE . The plasmid pGEX-4T-l/E and pGEX-4T-l/M expressed the specific proteins at a very low degree through western blot analysis using mouse-anti-GST.tag as antibody. Recombinant N protein could react with sera from mice immunized with pVAXl-N by western blot. Conclusions Recombinant plasmids pVAX1-E, pVAXl-M and pVAXl-N had...