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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 clone the human hepatocyte growth factor receptor (MET) gene and construct a prokaryotic expression vector of different domains (1-507, 1-577, 553-970, 553-1057 and 1018-1390) And purify the corresponding protein, which provides a basis for screening small molecule inhibitors based on different domains of MET. Methods The human MET gene (1-507, 1-587, 553-970, 553-1057 and 1018-1390) was amplified from the Hep G2 cDNA library of human hepatocellular carcinoma cell line by PCR and cloned into pGEX -4T-2 vector and expressed in Escherichia coli BL21, the prokaryotic expression product was purified and the expression and purification products were identified by SDS-PAGE and Western blot. Results The DNA fragments of about 1 524 bp, 1 764 bp, 1 257 bp, 1 518 bp and 1 119 bp, respectively, were amplified from Hep G2 cDNA library of human hepatocellular carcinoma cell line and were successfully constructed in pGEX-4T- 2 vector, and the sequence was exactly the same as the target sequence. The target protein with relative molecular masses of about 82 kU, 91 kU, 72 kU, 82 kU and 67 kU was induced and expressed in BL21. After purification, the target protein with high purity Recombinant protein GST-c-MET. Conclusion The recombinant protein GST-c-MET (1-507, 1-587, 553-970, 553-1057 and 1018-1390) in different domains of MET was successfully obtained, which lays the foundation for further screening of small molecule inhibitor of MET basis.