Prokaryotic Expression Of Influenza A (H1N1)2009Virus Hemagglutinin Gene, Preparation Of Antibodies And Application

Influenza A H1N1(2009) influenza virus was a novel influenza virus through naturalcombination of several influenza virus genes, whose genome contained gene fragments fromhuman influenza virus, avian influenza virus and swine influenza virus. Its genome was anegative-sense RNA, and comprised of eight separate segments of single-stranded, whichencoded10proteins. Hemagglutinin (HA) was a surface glycoprotein encoded by the fourthsegment, and was the major protective antigen, which could stimulate body to producespecific neutralizing antibody. Therefore, HA was not only the preferred antigen of geneticengineering vaccine, but also the major antigen in the diagnosis of influenza virus. Then, theresearch on HA of influenza virus could contribute to influenza prventation and control, anddiagnostic reagent research and development.In this study, the full-length gene sequence of H1N1(2009) HA was found fromGenebank. Superior gene sequence was determined through analysis of antigenic epitopes andoptimization of codon. Then the aim gene was synthesized. The aim gene was joined toexpression vector pET-30Xa/LIC. Then it was transformed into E. coli BL21(DE3), disgestedby restriction enzyme and sequenced. HA fusion protein induced by IPTG was expressedsuccessfully after SDS-PAGE analysis. The optimal conditions were: temperature37℃, IPTGfinal concentration of0.05mmol/L, inducing time6h. The fusion protein was in the form ofinclusion body, and relative molecular weight was about63kD. Western-blot analysis showedthat it had better reactive ability. The purity of the combinant protein was up to90%throughNi-affinity chromatography and protein refolding.Taking refolding protein as immunogen to inoculate BALB/C mouse of6-8weeks old,the immunized spleed cells of BALB/C mouse and myeloma cells (SP2/0) were performed tofuse together, then fused cell was screened by limitted dilution. After screening, there were3hybridoma cell strains named HA-B8, HA-D1, HA-H2which can secret anti-HA antibodiesstably. Ascites were prepared, purified and identified as IgG1immunoglobulin, which hadbetter specificity, and its titer was105.New Zealand white rabbits were immunized with the refolding protein and rabbitanti-HA polyclonal antibody was prepared. Its titer was1:32through agar gel diffusiondetection. Polyclonal antibody was performed by ammonium sulphate precipitation andaffinity chromatography, and antibody with high purity and better specificity was obtained. Then it was labeled with horseradish peroxidase (HRP) using the sodium periodateoxidization method.Taking purified monoclonal antibody as coating antibody and labeled polyclonalantibody as enzyme-labeled antibody, a primary detection method of double antibodysandwich ELISA for Influenza A H1N1(2009) virus was established. The experiment showedthat the detection method had better specificity without cross-reaction with Influenza virusH5N1, H9N2, EV71virus, and Measles virus.In this study, HA protein was expressed successfully in E.coli; the recombinant proteinwas used to immunize BALB/C mice and New Zealand white rabbits in order to preparepolyclonal antibody and monoclonal antibody. Double antibody sandwich ELISA fordetecting Influenza A H1N1(2009) was initially established on the basis of antibodypreparation.