uPA基因真核表达载体的构建和鉴定

目的:构建人尿激酶型纤溶酶原激活物(uPA)基因真核表达载体,并转染人肝癌细胞,为进一步体外研究uPA在肝癌发生发展中的作用奠定基础.方法:从肝癌组织中提取RNA,通过RT-PCR的方式,获得基因uPA的全长cDNA,并克隆于含有HA2-标签(tag)的真核表达载体pCMV上,酶切鉴定,质粒测序鉴定,用pCMV-uPA-HA2进行细胞瞬时转染及WesternBlotting鉴定.结果:经过测序证实得到获得包含uPA基因的pCMV-HA2真核表达载体,瞬时转染细胞有蛋白表达.结论:克隆了人肝癌的uPA基因,成功构建真核表达载体.

CONSTRUCTION AND IDENTIFICATION OF EUKARYOTIC EXPRESSION PLASMID OF uPA GENE

Objective: To investigate the role of uPA in the development of hepatocellular carcinoma in vitro, we clone the coding sequence of human uPA gene, constract its eukaryotic expression plasmid,and transfect the human hepatocellular carcinoma cell lines. Methods: The total RNA was isolated from human hepatocellular carcinoma tissue, and the uPA gene was synthesized and amplified from the total RNA by RT-PCR, and then cloning to the eukaryotic expression plasmid pCMV with HA2-tag. It was verified by restriction endonuclease analysis and sequencing,transfesting instantaeously human hepatocellular carcinoma cell lines and identified by Western Blotting. Result: pCMV-HA2 eukaryotic expression plasmid containing uPA gene was successfully obtained, and the protein expression was detected. Conclusion: The human uPA gene was cloned, and its recombinant plasmid was constructed successfully.