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目的 表达和纯化流感病毒血凝素HA2氨基末端20个氨基酸与10个赖氨酸的融合蛋白HA20-lys10,并观察其与质粒结合的能力。方法 用PCR方法构建流感病毒血凝素HA2氨基末端20个氨基酸与10个赖氨酸的融合基因HA20—lys10,并克隆于pET32a(+)原核表达载体。将重组表达质粒pET-HA-K转化大肠埃希菌BL21(DE3),当A(Abs)_(600nm)达到0.6时,加入终浓度为1mmol/L IPTG诱导目的蛋白的表达。表达产物用亲和层析一步法进行纯化,然后用凝胶阻滞试验观察重组蛋白结合质粒的能力。结果 IPTG诱导后可获得相对分子质量约为27 000的目的蛋白,表达蛋白以可溶形式存在,占菌体蛋白20％以上。表达菌超声后,表达产物经亲和层析一步纯化后可获得纯度达85％以上目的蛋白。凝胶阻滞试验发现,纯化的重组蛋白在一定条件下可以与质粒结合,使质粒的迁移率明显改变。结论 融合蛋白HA20—lys10有望用于受体介导基因转移,以提高基因转移的效率。
Objective To express and purify HA20-lys10, a fusion protein of amino-terminal 20 amino acids and 10 lysine of influenza virus hemagglutinin HA2, and to observe its ability to bind to plasmids. Methods The fusion gene HA20-lys10 between the amino-terminal 20 amino acids and 10 lysine of influenza virus hemagglutinin HA2 was constructed by PCR and cloned into prokaryotic expression vector pET32a (+). The recombinant plasmid pET-HA-K was transformed into Escherichia coli BL21 (DE3). When A (Abs) _ (600nm) reached 0.6, IPTG at a final concentration of 1mmol / L was added to induce the expression of the target protein. The expression product was purified by one-step affinity chromatography, and then the ability of the recombinant protein to bind to the plasmid was observed by gel retardation assay. Results After induced by IPTG, the target protein of about 27 000 in molecular weight was obtained. The expressed protein existed in soluble form, accounting for more than 20% of the bacterial protein. After the expression of bacteria by ultrasound, the expression product was purified by affinity chromatography step to obtain the purity of more than 85% of the target protein. Gel retardation test found that the purified recombinant protein can be combined with the plasmid under certain conditions, the plasmid mobility was significantly changed. Conclusion The fusion protein HA20-lys10 is expected to be used for receptor-mediated gene transfer in order to improve the efficiency of gene transfer.