人γ干扰素 /β 肿瘤坏死因子融合蛋白 His6融合表达及一步纯化

将人 V干扰素 /U肿瘤坏死因子融合蛋白 (hVTNF-U )重组基因克隆于表达载体 pET28,构建成 T7 lac启动子控制下的 His6融合表达质粒 ,转化溶源菌 E. coli BL21( DE3).经 IPTG(1mM )诱导表达 , 阳性菌在 SDS-PAGE泳图上出现一条粗表达带 ,其分子量 (32kDa) 与目的蛋白 H is6-V TN F-U ) 理论分 子量相符. 薄层扫描与溶解性分析显示 ,表达产物占菌体总蛋白的 45% 以上 ,主要为不溶性的包涵体 ( IBs).离心分离 IBs产物 ,溶于 7M尿素中 ,然后通过 Ni柱进行亲和层析 ,即可获得一步纯化的表达产物 (纯度为 96%、回收率为 91% ).纯化产物再经复性缓冲液稀释复性 ,其细胞毒比活性与抗病毒比活性分 别达到 1. 2× 10 7～ 2. 0× 107u /mgp和 6. 6× 10 5 ～ 7. 2× 105 u/mgp.

Over-expressionof the His6-

The hIFN-V/TNF-Ufusion protein(hVTNF-U ) recombinant gene was cloned to the expression vector pET28, and a T7lac promoter-based fusion expression plasmid w as constructed that directed the synthesis of hVTN F-Uin E. coli as fusion with a stretch of six consecutive histidine residues( H is6-tag) at N terminus. SDS-PAGE analysis revealed that with IPT G(1m M) induction the strain bearing the plasmid highly expresses the target protein ( 32kD) which molecular w eight is same as the theory molecular w eight of His6-VTNF-U , and expression level of the product( as insoluble inclusion bodies, IBs) is above 45% of the total bacterial proteins. After cell lysis, the IBs is pelleted by centrifugation, dissolves in 7M urea, then purifies in one step by Ni column( Ni2 + -sepharose 6B). And the purity ofmore than 96% and the recovery of 91% w ere obtained. The purified product was refolded under low temperature( < 10℃ ). The specific cytotoxic activity and the specific antiviral activity of the renaturation product are 1. 2× 10 7 ～ 2. 0× 107 u/mgp and 6. 6× 105～ 7 . 2× 105 u/mgp respectively