甲型H1N1流感病毒HA基因与沙门菌fljB基因的融合表达与鉴定

目的 研发基于鞭毛蛋白的新型甲型H1N1流感疫苗。方法 分别以鼠伤寒沙门菌SL7202基因组和含有甲型H1N1流感病毒HA基因的重组质粒pET-HA为模板, 通过 PCR扩增Ⅱ相鞭毛蛋白fljB和HA1-2基因，再通过overlap PCR将这两基因片段拼接成HA1-2-fljB，将其插入原核表达质粒pET30a(+)，构建重组质粒pET-HA1-2-fljB，并转化表达宿主菌E.coli BL21(DE3) 中，以 IPTG诱导表达。SDS-PAGE和Western blotting鉴定融合蛋白HA1-2-fljB的表达。结果 正确构建出重组质粒pET-HA1-2-fljB，SDS-PAGE和Western blotting结果显示，融合蛋白分子量约94 KD，并具有良好的免疫反应性，能强烈诱导HEK293-mTLR5细胞分泌IL-8。结论 成功表达具有TLR5生物学活性的融合蛋白HA1-2-fljB, 为研究甲型H1N1流感疫苗奠定了基础。

Fusion expression and identification of hemagglutin gene of H1N1 subtype influenza virus and fljB gene of Salmonella typhimurium

The aim of this study was to develop a novel kind of flagellin-based influenza A virus subtype H1N1vaccine. The genes coding for fljB and HA1-2 were respectively amplified by polymerase chain reaction (PCR) and the two genes were merged into HA1-2-fljB by overlap PCR, and then the gene was inserted to vector pET30a(+) to construct the recombinant plasmid. The recombinant plasmid was transformed into expressive vector E.coli BL21(DE3) and was induced with IPTG. The expression of fusion protein was analyzed by SDS-PAGE and Western blotting. The results indicated that the recombinant plasmid pET-HA1-2-fljB was constructed. The fusion protein, which molecule weight was about 94 KD, had a good immunoreactivity and strongly induced HEK293-mTLR5 cells to secrete IL-8. In conclusion, a flagellin-based fusion protein HA1-2-fljB with TLR5 bioactivity was expressed correctly, and these results would establish the basis for the further research in influenza virus subtype H1N1 vaccine.