人肝细胞生长因子受体原核载体的构建表达及纯化

摘要

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.%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pGEX-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pGEX-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.