人肺癌相关基因HLCDG1在大肠杆菌中的表达

Expression of GST-human lung carcinoma deleted gene

目的构建新克隆的肺癌抑瘤基因候选者HLCDG1的融合表达质粒pGEX-6P-1/HLCDG1并在原核表达系统中表达。方法采用多聚酶链式反应(PCR)扩增HLCDG1基因蛋白编码序列,将该片断插入原核表达载体pGEX-6P-1中,转化大肠杆菌BL-21扩增。构建的融合表达质粒经酶切和测序鉴定后,经IPTG诱导表达,SDS-PAGE后考马斯亮蓝染色和W estern b lot鉴定实验结果。结果构建的融合表达质粒酶切和测序鉴定阅读框架正确,导入大肠杆菌中经IPTG诱导出一条分子量约为46kD的新蛋白带,主要存在于细菌沉淀中,W estern b lot鉴定其为GST-HLCDG1融合蛋白。结论HLCDG1基因编码的蛋白质能够在原核细胞中有效表达,为进一步研究该蛋白质的结构和功能奠定研究基础。

Objective To construct the recombinant plasmid pGEX-6p-1/human lung carcinoma deleted gene 1 （ HLCDG1 ） and to investigate the expression of HLCDG1 protein in Escherichia coli. Methods The coding region of HLCDG1 gene was obtained by polymerase chain reaction （PCR）. A fusion protein expression vector pGEX-6p-1/HLCDG1 was constructed by inserting fragment of the code region of HLCDG1 gene into the fusion protein expression vector pGEX-6p-1. The coding region of HLCDG1 gene was identified by endonuclease digest and DNA sequencing, The recombinant plasmid was transformed into Escherichia coli. The expression of GST-HLCDG1 fusion protein was induced by isopropy-13-D-thiogalactoside（IPTG） and confirmed by SDS-PAGE and western blot, Results The coding region of HL- CDG1 gene was correctly inserted into the pGEX-6p-1 vector and the pGEX-6p-1/HLCDG1 was constructed. After induction with IPTG, a new protein band of 46 kDa was detected on SDS-PAGE in the cells transformed with recombinant plasmid and identified by western blot, The GST-HLCDG1 fusion protein was mostly existed in the precipitation of broken bacteria. Conclusion The HLCDG1 protein can be expressed in prokaryotic expression system, which lays foundation for the further structural and functional research of the protein.