hKGF2在不同原核载体中的构建、表达和纯化分析  

Construction,expression and purification of HKGF2 in different prokaryotic vectors

作　　者：游力[1] 余德荣[2] 许晓群[2] 王郡甫[2] 高春义[2] 赵跃然[1] 

机构地区：[1]山东大学附属省立医院生殖医学中心,济南250021 [2]山东省医学科学院基础医学研究所,济南250062

出　　处：《山东大学学报（医学版）》2008年第12期1115-1119,共5页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金资助课题(30371304)

摘　　要：目的比较人角质细胞生长因子2(hKGF2)在不同原核表达载体和宿主菌中的表达及相应蛋白纯化及生物学活性。方法RT-PCR法从培养的人胚胎肺成纤维细胞提取成熟hKGF2 cDNA,克隆测序后,分别与pBV220和pET-30a(+)表达载体连接,转化不同宿主菌进行诱导表达,聚丙烯酰胺凝胶电泳(SDS-PAGE)及Western blot进行蛋白鉴定。针对不同原核表达载体分别用阳离子交换层析和镍亲和层析(Ni-NTA)纯化目的蛋白,电泳分析蛋白纯化结果,并分别检测其生物学活性。结果目的基因片段克隆入pBV220载体后在E.coliJM109中表达量最高,约占菌体总蛋白的15%;克隆入pET-30a(+)载体在E.coliBL21(DE3)中表达量约占菌体总蛋白的25%;均为可溶性表达,不同方法纯化后蛋白纯度达95%以上,均能有效促进人胚胎肾上皮细胞增殖。结论hKGF2基因在不同的宿主菌中蛋白表达不同,以融合蛋白表达纯化效果更好。Objective To explore expressions of hKGF2 （human keratinocyte growth factor 2）in different prokaryotic vectors and host bacteria and their corresponding purification and bioactivity. Methods The mature hKGF2 gene was amplified by RT-PCR from cultured human embryonic lung fibroblast. After DNA cloning and sequencing, the hKGF2 gene was ligated with pBV220 and pET-30a（＋）, and then transformed into different host bacteria. The expressed protein was determined by SDS-PAGE and Western blot. The protein in different vectors was purified by cation-exchange chromatography and Ni-affinity chromatograph, and determined by electrophoretic analysis, followed by bioaetivity detection with MTT. Results The expression level of hKGF2 in pBV220 was highest when transformed into E. coli DH5a, which amounted to 15% of total bacterial proteins and the target protein level in pET-30a（ ＋ ） transformed into E. coli BL21 （ DE3）reached 25 %. The purity of the target protein was over 95 % and all purified proteins could promote the proliferation of human embryonic kidney epithelial cells. Conclusions The purified pro- rein yield of hKGF2 in E. coli BL21（DE3）/pEr30a（ ＋ ） is significantly higher than that in E. coli JM109/pBV220.

关 键 词：人角质细胞生长因子2 pBV220载体 pET-30a(+)载体 基因表达 蛋白纯化 

分 类 号：Q789[生物学—分子生物学]