人膜联蛋白A5重组质粒的构建及其在宫颈癌细胞系SiHa细胞中的转染与表达

CONSTRUCTION OF RECOMBINANT PLASMID PCDNA3.1-ANXA5 AND ITS TRANSFECTION AND OVEREXPRESSION IN SIHA CELLS

目的:构建膜联蛋白A5(ANXA5)的重组表达质粒,转染宫颈癌细胞并检测其表达。方法:自行设计引物,引入BamHI和XhoI的酶切位点,PCR法从pJLA503-ANXA5中获得ANXA5基因;将pcDNA3.1质粒用BamHI和XhoI双酶切后,用T4连接酶将其与ANXA5基因连接,并经测序证实基因序列正确后,用磷酸钙共沉淀法转染宫颈癌细胞系SiHa细胞,并用Western blot方法检测细胞中ANXA5蛋白的过表达。结果:重组质粒中ANXA5基因序列正确,转染重组质粒后的SiHa细胞过表达ANXA5蛋白。结论:成功构建了pcDNA3.1-ANXA5重组质粒,转染肿瘤细胞后可过表达ANXA5蛋白,为研究ANXA5在肿瘤细胞中的作用奠定了基础。

Objective：To construct the recombinant plasmid pcDNA3.1-ANXA5,transfect it into uterine cervical carcinoma cell line and detect its expression.Methods：Annexin A5 （ANXA5） gene containing BamHI and XhoI endoenzyme sites was obtained by PCR;Double enzyme digestion with BamHI and XhoI was conducted for pcDNA3.1 plasmid.The pcDNA3.1 plasmid and ANXA5 gene with the same endings were linked by T4 lingase.After the ANXA5 gene in the recombinant plasmid pcDNA3.1-ANXA5 was detected correct,the recombinant plasmid was transfected into uterine cervical carcinoma cell line SiHa cells and the overexpression of ANXA5 protein was detected by Western blot.Results：The sequence of ANXA5 gene in the recombinant ptasmid was proved correct and after being transfected into SiHa cells,the ANXA5 protein was overexpressed. Conclusions：The recombinant plasmid pcDNA3.1-ANXA5 is constructed successfully and the SiHa cells transfected with the plasmid can overexpress ANXA5 protein, which may facilitate the study on ANXA5 function in tumor cells.