人白细胞介素6缺失突变体的克隆、表达与纯化及其初步功能鉴定(英文)

利用人白细胞介素6(hIL-6)cDNA上Pst Ⅰ酶切位点,在不改变相位的情况下,缺失IL-6分子中127—174位氨基酸残基,以pBV220为表达载体构建并筛选出重组子pBV*-DIL-6,编码12kD的IL-6缺失突变体。带有缺失IL-6分子的重组表达载体转化E.coli HB101株,42℃热诱导后,菌体裂解物经SDS-PAGE鉴定,表明携有pBV*-DIL-6的受体菌在约12 kD位置有一特异条带,与理论推算值基本相符;表达的蛋白命名为rDIL-6。薄层扫描结果显示rDIL-6占菌体总蛋白的30％,实现了IL-6突变体在大肠杆菌中的高表达。重组蛋白主要以包涵体形式存在。ELISA方法证明重组蛋白可与rIL-6R-28特异结合;另外利用只与IL-6结合的McAB CLB.IL-6/14证明rDIL-6能竞争性抑制IL-6与rIL-6R-28的结合,说明目的蛋白与IL-6竞争结合IL-6Rα。7TD1细胞学分析表明rDIL-6不具有IL-6支持杂交瘤细胞生长特性,高浓度的重组蛋白可以部分中和IL-6对7TD1细胞生长刺激作用,表明缺失突变体具有一定的拮抗作用。

Cloning, Expression and Purification of a Human IL-6 Deletion Mutant in E . coli and Its Preliminary Functional Identification

Internal deletion of the human IL-6 has been generated in the region encoding residues 127 -174 at the cDNA level. With pBV220 as expressing vector, the recombinant pBV -DIL-6 encoding the deletion mutant (12 kD) of hIL-6 has been constructed. The resulted recombinant plasmids were then used to transform E.coli strain HB101, and the expression in the PLPR promoter system, which is temperature-regu- latable, was achieved. The expressed protein, mainly recovered as inclusion bodies, amounts to 30% of the total bacterial protein. After purification and renaturation, the biological activity of the expressed product was measured by MTT method in an IL-6-dependent cell line 7TD1. The results show that the amino acid residues of IL-6 127 to 174 are crucial for IL-6 activity. Receptor binding assay in vitro indicated that the entire region was not involved in forming the receptor binding surface. Furthermore, we demonstrated the antagonistic effect of the mutant to IL-6 using 7TD1 assay.