Expression And Purification Of A Novel Influenza Virus Subunit Vaccine And Analysis Of Its Preliminary Immunological Effect

Influenza(Flu) is a kind of severe respiratory disease caused by influenza virus(â…£) which is highly infectious. Flu explodes suddenly in short-term time and spreads qickly, which can cause global epidemic and lead to great economic burdon and human health losses. Hemagglutinin(HA) is one of the major surface antigens ofâ…£. The high variation of HA causes that the influenza virus vaccine have to be replaced of a new vaccine every year, so as to bring many difficulties to production of the vaccine. The influenza A virus matrix protein2 gene(M2) is a transmembrane protein. It has highly conserved antigenic epitopes and is a potential candidate antigen for the influenza vaccine with cross protection. In present, the avian influenza viruse spreads gradually to many countries, and threats the human health more greatly. Therefore, it is greatly significant to develop a new broad-spectrum influenza virus vaccine with cross-protection for controlling the flu epidemic.In our studies, M2d gene which deleted M2 transmembrane region was selected from influenza A virus, And a multi-epitope gene of HA was synthesized, which contains two B cell epitopes and one Th cell epitope from influenza virus (A/New Caledonia/20/99 H1N1), and two B cell epitopes and two Th cell epitopes from influenza virus (A/California/7/2004 H3N2). The extracelluar domain gene of M2 and the multi-epitope gene of HA were optimized for better expression of the recombiant M2d-HA gene in E.coli according to E. coli codon bias database. M2d gene was amplified by overlap extending PCR; meanwhile, the multi-epitope gene of HA was also amplified by self-pimering PCR. The M2d gene and the HA multi-epitope gene were ordinally inserted into prokarytic expression vector pET28a+ to construct the recombinant plasmid pET28a+-M2d-HA. The result was confirmed by DNA sequencing.The recombinant plasmid was transformed into E.coli BL21(DE3), and the high expression strain was obtained by screening monoclones. The recombinant protein existed as inclusion bodies, which accounted for 45% of the total cellular protein. The recombinant protein was purified by Ni²⁺ affinity chromatography, and refolded by dilution renaturation, then purified by Q Sepharose FF cation exchange column. The result of HPLC analysis showed the purity of the recombinant protein was over 90%. The result of western blot showed it had antigenicity and specificity. The result of ELISA illuminated the recombinant protein with Freund's adjuvant could induce to produce high antiserum level of specific antibody in immunized Kunming mice. The immunofluorescence test demonstrated the binding of the antiserums with influenza virus(H1N1) infected MDCK cells. These results suggested that the recombinant protein could provide good antigenicity.In this study, the highly purified M2d-HA/pHistag fusion protein was obtained, and the antiserum against recombiant M2d-HA/pHistag fusion protein showed good immunoreactivity. These results were helpful for the further study of the broad-spectrum influenza virus subunit vaccine, and strongly supported for the development and basic study of influenza virus vaccine.