中国生物工程杂志

The influenza A virus matrix protein2 gene(M2) which deleted transmembrane region was amplified by overlap extending PCR, and the multi-epitope gene of hemagglutinin(HA) was PCR amplified with seven continuous synthesized segments by designing primer. The two gene segments were separately cloned into pMD18-T vector to sequence analysis and prokarytic expression vector pET28a+ to construct the recombinant plasmid pET28a+-M2d-HA. 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 about 45% of the total cellular protein. The inclusion bodies were washed with 1% Triton X-100 solution twice, and dissolved in 8 mol/L urea solution. The solution protein was purified by Ni2+ affinity chromatography, and refolded by dilution renaturation, then purified by Q Sepharose FF cation exchange column. The purity of the protein was over 90% by HPLC analysis. The result of western-blot showed it has good antigenicity and specificity. These results strongly supported for the further study of the broad-spectrum influenza virus subunit vaccine.