谷胱甘肽-S-转移酶-人表皮生长因子融合蛋白基因的表达及其产物的生物活性  

Expression of gene of GST-hEGF fusion protein and bioactivity of its product

作　　者：智庆文[1] 李前[2] 王淑豪[2] 王玉霞[2] 李仕贵[3] 孙曼霁[2] 

机构地区：[1]山东省科学院生物研究所,济南250014 [2]军事医学科学院毒物药物研究所,北京100850 [3]四川农业大学水稻研究所,四川温江611130

出　　处：《中国药理学与毒理学杂志》2005年第2期113-117,共5页Chinese Journal of Pharmacology and Toxicology

摘　　要：目的　通过融合蛋白的表达使人表皮生长因子(hEGF)易于纯化。方法　构建基于tac启动子的pGEX 4T-1-hEGF原核表达载体,转化大肠杆菌BL2 1(DE3 ) ,以异丙基 1 硫代-β-D半乳糖苷诱导,表达谷胱甘肽 S 转移酶(GST) hEGF融合蛋白。结果　表达产物占细菌总蛋白的3 4%。部分为可溶性的,部分形成包涵体。菌体裂解上清液经GlutathioneSepharose 4B纯化后,SDS PAGE电泳出现一条3 2ku蛋白条带,与GST hEGF融合蛋白的计算分子量相符。菌体裂解液中的可溶性GST hEGF的产率为63mg·L- 1 。将其添加到培养的HEK-2 93细胞中,能明显促进该细胞的分裂和生长。结论　成功地构建并表达了GST-hEGF融合蛋白基因,其表达产物GSTAIM To simplify the purification of the recombinant human epidermal growth factor (hEGF) by the expression of fused protein. METHODS pGEX 4T-1-hEGF prokaryotic expression vector bearing a tac promotor was constructed and transformed to Escherichia coli BL21 (DE 3). GST-hEGF fusion protein was expressed under the induction of IPTG. RESULTS The expression product, partially soluble and partially in form of inclusion body, amounted 34% of the total protein of the cells. The supernatant of the cell lysate was subjected to Glutathione Sepharose 4B affinity chromatography. The purified protein showed in SDS-PAGE a single band with a molecular weight of 32 ku, compatible with the estimated value of GST-hEGF molecule. The yield of the soluble GST-hEGF in the supernatant of cell lysate came to 63 mg·L^(-1). The soluble GST-hEGF fusion protein obviously promoted the cell growth and proliferation when added to the cultured human embryo kidney cells (HEK-293). CONCLUSION The GST-hEGF fusion protein gene was successfully constructed and expressed, GST-hEGF well manifested the biological activity in promoting cell proliferation.

关 键 词：融合蛋白质类 谷胱甘肽转移酶 生长物质 基因表达 

分 类 号：Q786[生物学—分子生物学]