HA20-lys10在大肠埃希菌中的融合表达纯化和凝胶阻滞试验  

Fusion Expression and Purification of HA20-lys10 in E. coli and Its GeI Retardation Experiments

作　　者：孙兴会[1] 张宇清[1] 王霞[1] 侯敏[1] 谭理[1] 李平[1] 朱运松[1] 

机构地区：[1]复旦大学上海医学院生物化学与分子生物学系,上海200032

出　　处：《复旦学报（医学版）》2004年第1期32-35,共4页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金(30170398)

摘　　要：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。Purpose To express and purify the fused protein containing 20 residues of amino-terminal of influenza hemagglutinin HA2 and ten lysines, and investigate its ability of binding plasmid. Methods The fused gene HA20-lys10 containing gene of 20 residues of amino-terminal of influenza hemagglutinin HA2 and ten lysines was amplified by PCR, and was cloned into prokaryotic expression vector pET32a( + ) . The re-combinant expression plasmid pET-HA-K was transformed into E. coli BL21 (DE3). When A600 reached 0. 6, IPTG was added to induce the expression of recombinant protein. The expressed protein was purified by one-step affinity chromatography, and its ability of binding plasmid was investigated via gel retardation experiments. Results After induction for 3 hours by 1 mmol/L IPTG, there was a high level expression of recombinant protein, and most of them were soluble. After purification by affinity chromatography, the purity of HA20-lys10 was up to 85 % , and 20μg HA20-lys10 could retard plasmid completely. Conclusions These results indicate that HA20-lys10 is hopeful to be used in receptor-mediated gene transfer to improve the efficiency.

关 键 词：HA20-Iys10 大肠埃希菌 凝胶阻滞试验 融合基因 基因表达 基因纯化 

分 类 号：R378.21[医药卫生—病原生物学] Q78[医药卫生—基础医学]