人宫颈癌HPC2基因的克隆及其原核和真核表达载体的构建

Cloning of human cervical carcinoma HPC2 gene and construction of its prokaryotic and eukaryotic expression vectors

目的:构建人宫颈癌HPC2基因的原核及真核表达载体,进一步研究该基因在宫颈癌发生中的作用。方法:从人宫颈癌HeLa细胞中提取总RNA。用RT-PCR方法扩增出人宫颈癌HPC2全序列基因,插入pT7 b lue载体。酶切鉴定及序列分析后,构建人HPC2基因的原核及真核表达载体。结果:成功扩增出人宫颈癌HPC2基因;并构建pGEX4T1-HPC2原核表达载体及pCMV-F lag-HPC2真核表达载体。结论:人HPC2基因的原核及真核表达载体的构建为深入研究HPC2基因与宫颈癌的发生奠定了基础。

Objective：To obtain entirely coding human cervical carcinoma HPC2 gene and construct its prokaryotic and eukaryotic expression vectors. Methods： With total RNA extracted from human HeLa cell as template, HPC2 gene was amplified by RT - PCR with the designed primers based on the public sequence of GeneBank, and then was inserted into pT7 blue vector. The recombinant plasmid pMD18 -T- HPC2 was identified by restriction enzyme analysis and DNA sequencing. Digested by restriction endonuclease, and cloned HPC2 into muticlone sites of the prokaryofic and eukaryofic expression vector pGEX4T1 and pCMV2 -Flag, respectively. Results： The entirely coding human cervical carcinoma HPC2 gene was cloned. Recombinant pGEX4T1 - HPC2 and pCMV2 - Flag- HPC2 were successfully constructed. Conclusion： The successfully constructed pGEX4T1 -HPC2 and pCMV2 - Flag - HPC2 would provide a basis for a further study of the relationship between the cervical carcinoma and HPC2 gene.