Expression Of Influenza A Virus Nuclear Export Protein And Preparation Of Anti-NEP Antibodies

Influenza A virus is a species of influenza virus A genus of the Orthomyxoviridae family of viruses,which can cause zoonotic diseases.Influenza A virus can evade the attack of the immune system through antigenic drift,and causing a epidemic.Sometimes it can form a new subtype through antigenic shift,and cause a pandemic.Influenza vaccine has only limited effect,and with the emergence of drug-resistant strains of influenza A virus,discovering new drug targets and developing new drugs for influenza virus is also one of the research hotspots.With the understanding of influenza virus,more researchers pay attention to nuclear export protein(NEP).NEP is a protein encoded by segment eight of influenza A virus,about 14kD.The main function of NEP is mediating the viral ribonucleoprotein(vRNP)complexes from the cell nucleus to the cytoplasm.And it is expected to be a new target for the prevention and treatment of the flu.The use of antibodies to study the function of proteins is a classic method in molecular biology,including immunoprecipitation,immunoblotting and so on.In particular,monoclonal antibodies are favored by researchers because of their specificity.Purpose:Obtaining the gene of NEP,preparing and purifing NEP recombinant proteins,and to provide basic materials for preparation and identification of hybridomas.Anti-NEP monoclonal antibodies were prepared to establish the basis for further research of influenza virus.Methods:(1)The gene of NEP is cloned from influenza virus by RT-PCR and PCR technology.(2)Construct two recombinant plasmids,pET-28a-NEP and pGEX-4t-1-NEP,express NEP-HIS and GST-NEP recombinant proteins by prokaryotic expression system,and purify the target proteins.(3)GST-NEP is used to immunize mice,after cell fusion,use NEP-HIS recombinant protein to screen hybridomas that secret antibodies against NEP.Finally,the specificity of monoclonal antibodies is detected by Western blotting.Results:(1)The gene of NS of influenza virus was cloned by RT-PCR,and then the gene of NEP was cloned by PCR successfully using NS gene as a template.(2)pET-28a-NEP and pGEX-4t-1-NEP recombinant plasmids were constructed,and NEP recombinant proteins was successfully expressed in E.coli,and purified the recombinant proteins.(3)Mice were immunized with GST-NEP,After 3 rounds of normal immunization,the serum titers were elevated.Spleen cells were successfully fused with myeloma cells,and hybridoma cell lines were established.The positive clones were screened by NEP-HIS protein.Finally,2 hybridoma cell lines secreting anti-NEP antibodies were obtained,named 1E10 and 2A4.Immunoblotting showed that antibodies of 1E10 could specifically bind to the NEP of influenza A virus.Conclusion:pET-28a-NEP and pGEX-4t-1-NEP recombinant plasmids were constructed by genetic engineering,and a high purity recombinant proteins was obtained.Two hybridomas that can secret anti-NEP antibodies stablely were successfully obtained,Western blot showed that antibodies of 1E10 could bind to NEP encoded by influenza virus finally.It will lay the foundation for further research on influenza virus,in vitro diagnosis and a new target anti-influenza drugs.