Expression And Immunogenicity Of Multi-dermer Copy Of M2e Of Avain Influenza In E.coli

Highly pathogenic avian influenza(HPAI) was a devastating disease of poultry leading to high mortality caused by some H5 and H7 subtypes influenza A viruses. HAPI was classified as one of the list A animal disease by OIE and China Ministry of Agriculture.Seasonal antigenic variation of the major influenza surface glycoproteins,hemagglutinin(HA) and neuraminidase(NA),pose a major obstacle to control the viral disease by vaccination,because immunization with currently available influenza vaccines induces antibodies only to the virus strains included in the vaccine.However,as the virus changes by mutations(drift) and gene re-assortment(shift),these antibodies loose step by step their efficacy to prevent the disease.Therefore,influenza infections can occur repeatedly throughout life and protection by vaccination requires annual administration with updated vaccines.We have developed a universal influenza A vaccine based on the extracellular domain of the third integral membrane protein,called M2e.M2-protein is scarcely present on virus particles,but occurs abundantly on virus infected cells.M2e is highly conserved in all influenza A virus strains.Therefore,the M2e based vaccine is expected to protect against any new epidemic or pandemic strain.The H9M2e and H5M2e were synthesized based on avain influenza m2e gene and E coli condon bias.The two gene was inserted into plasmid pSIMPLE and the multidermer copy M2e clone plasmid was constructed based on the fact isocaudamer enzyme existed.After that,the target gene was subcloned into an expression vector pET-32a(+).Then the recombinant expression plasmid was was digested by BglⅡ/XhoⅠand sequenced.The results showed that 6 expression plasmid included 1,2,3,4,5 and 6 copies of M2e tandem of avain influenza respectively were successfully constructed.The positive recombinant plasmid was named as pET-(H9M2e-H5M2e)₁-Fc,pET-(H9M2e-H5M2e)₂-Fc,pET-(H9M2e-H5M2e)₃-Fc,pET-(H9M2e-H5M2e)₄-Fc,pET-(H9M2e-H5M2e)₅-Fc,pET-(H9M2e-H5M2e)₆-Fc. Then the positive recombination plasmid was transformed into E.coli BL21(DE3), and induced with IPTG..The expression for the fusion proteins which named as 2M2e,4M2e,6M2e,8M2e,10M2e,12M2e were analyzed by SDS-PAGE.Results indicated that the relative molecular mass of the fusion proteins were measured to be 40.9,47.0,53.1,59.3,65.3 and 71.4 kDa.Three fusion proteins 4M2e,8M2e 12M2e were purified by Ni-sepharose affinity chromatography,which could be applied to learn its immunogenicity,a specific antigenicity of the three purified protein were demonstrated by Western blotting.21-days-old unimmunized chicken were divided into 7 groups.The three fusion proteins 4M2e,8M2e and 12M2e with Freund' adjuvant were divied as group A,B,C,the protein 12M2e with vash oil and CpG adjuvant were divied as group D and E, the protein 12M2e unpurified with vash oil was devided as group F,and the negative group was devied as group G.The protein was injected intramuscularly at the dose of 150ug. Animals were boostered three weeks post the primary immunization.Blood samples were Collected every weeks after first immunization and M2e specific antibodies in the serum were detected with indirect ELISA established.The expression of CD3, CD4 and CD8 on the periphera blood lymphocytes surface was evaluated by monitoring fluorimetric changes of the corresponding FITC/PE conjugated monoclonal antibodyby flow cytometry on 3 weeks,5 weeks and 7 weeks after the fist immunization.Results showed that the fusion protein 12M2e with Freund's adjuvant could induce the highest level of antibodies.This serum was used in Viral neutralization test accomplished on SPF egg embryos and Madin-Darby canine kidney(MDCK) cell,at the same time,indirected fluorescence assay(IFA) was tested on MDCK infected by H9N2 viruses.M2 protein-specific antibodies were able to inhibit virus adsorption and penetration of virions,and were able to inhibit the replication of influenza virus.Fluorescence microscopy showed that the anti-M2e sera could bind the M2 expressed on the surface of the infected MDCK cells.