人肝细胞生长因子受体原核载体的构建表达及纯化  

Prokaryotic expression and purification of human MET

作　　者：邱栾 赵珂[2] 董小明[2] 高瑞[2] 张飞翔[2] 张欣悦[1] 詹轶群[2] 李长燕[2] 李建雄[1] QIU Luan ZHAO Ke DONG Xiaoming GAO Rui ZHANG Feixiang ZHANG Xinyue ZHAN Yiqun LI Changyan LI Jianxiong(Department of Radiation Oncology, Hainan Branch of Chinese PLA General Hospital, Sanya 572013, Hainan Province, China Beijing Institute of Radiation Medicine, Beijing 100850, China)

机构地区：[1]解放军总医院海南分院放射治疗科,海南三亚572013 [2]军事医学科学院放射与辐射研究所,北京100850

出　　处：《解放军医学院学报》2017年第7期657-661,共5页Academic Journal of Chinese PLA Medical School

基　　金：国家重点研发计划(2016YFC0105700);国家自然科学基金(81273543;81001042);北京市科技新星计划(Z141107001814122)~~

摘　　要：目的克隆人肝细胞生长因子受体(MET)基因,构建不同结构域(1-507、1-587、553-970、553-1057、1018-1390)的原核表达载体,获得其原核表达产物,并纯化相应蛋白质,为基于MET不同结构域的小分子抑制剂筛选提供依据。方法采用PCR技术从人肝细胞癌细胞系Hep G2 cDNA文库中扩增出人MET基因(1-507、1-587、553-970、553-1057、1018-1390),将其克隆到p GEX-4T-2载体中,在大肠埃希菌BL21中表达后,对原核表达产物进行纯化,以SDS-PAGE和Western blot鉴定表达与纯化产物。结果从人肝细胞癌细胞系Hep G2 cDNA文库中扩增获得分别约1 524 bp、1 764 bp、1 257 bp、1 518 bp和1 119 bp的DNA片段,并成功构建在p GEX-4T-2载体上,经测序与目的序列完全一致;在BL21中诱导表达出相对分子质量约为82 kU、91 kU、72 kU、82 kU、67 kU的目的蛋白,经纯化后获得了纯度较高的重组蛋白GST-c-MET。结论成功获得了MET不同结构域的重组蛋白GST-c-MET(1-507、1-587、553-970、553-1057、1018-1390),为后续研究MET小分子抑制剂筛选奠定了实验基础。Objective To construct the prokaryotic expression vectors of human MET （1-507, 1-587, 553-970, 553-1057, 1018-1390） and obtain their purified proteins, and provide basis for screening small molecule inhibitors to human MET. Methods Human MET （1-507, 1-587, 553-970, 553-1057, 1018-1390） coding regions were amplified from human HCC Hep G2 cDNA library, and they were inserted into prokaryotic expression vector pGEX-4T-2. The recombinant plasmids pGEX-4T-2- MET were transformed into E.coli BL21. The expressed products were purified and identified by SDS-PAGE and Western blot analysis. Results The DNA fragments of 1 524, 1 764, 1 257, 1 518 and 1 119 bp were successfully amplified by PCR from human HCC Hep G2 cDNA library, and they were cloned into pGEX-4T-2 and identified by sequencing. The recombinant proteins with size 82, 91, 72, 82, 67 kU were successfully induced and purified, and highly purified recombination protein GST-c-MET was obtained. Conclusion The recombinant proteins of GST-c -MET （1-507, 1-587, 553-970, 553-1057, 1018-1390） are obtained successfully, which lay a foundation for further research on MET and small molecule inhibitors.

关 键 词：肝细胞生长因子受体 原核表达 纯化 

分 类 号：Q78[生物学—分子生物学]