人pot1基因cDNA的克隆及其在HeLa细胞中的表达

Cloning and eukaryotic expression of hpot1 cDNA in HeLa cells

目的:克隆人端粒保护蛋白(hum an protection of telom eres 1,p ot1)基因cDNA全编码区,构建成真核表达质粒并实现在H eL a细胞中的表达。方法:RT-PCR法扩增出H eL a细胞hp ot1基因cDNA编码区全序列,定向克隆至真核表达载体pcDNA 3,阳性重组质粒经插入片段测序验证;重组的pcDNA 3-hp ot1质粒瞬时转染H eL a细胞,RT-PCR和EM SA法(电泳迁移率改变分析)检测hp ot1基因的表达水平。结果:来自H ela细胞的hp ot1基因cDNA编码区全序列构建成的真核表达质粒pcDNA 3-hp ot1,经测序分析序列和ORF正确;该重组质粒转染在H eL a细胞48h后,hp ot1基因表达水平增高。结论:真核表达载体pcDNA 3-hp ot1构建成功,并实现了hp ot1在H ela细胞中的表达。

Objective： To clone the whole length of coding sequence of hpot1 （human protection of telomeres 1） cDNA, and construct an eukaryotic expression vector containing hpot1 to study the expression of hpot1 in HeLa cells. Methods： hpot1 cDNA was amplified with RT-PCR. directionally cloned into pcDNA3 plasmid and then confirmed by DNA sequencing; the recombinant pcDNA3-hpot1 plasmid was instantly transfected into HeLa cells; the expression of hpot1 was examined by RT-PCR and EMSA. Results： The recombinant pcDNA3-hpot1 plasmid was successfully constructed and the whole coding sequence and ORF of hpot1 extracted from the transfected Hela cells were proven to be correct. The transfected recombinant hpot1 plasmids was highly expressed in Hela cells after the transfection of 48 hours. Conclusion： The recombinant pcDNA3-hpot1 plasmid was successfully constructed and could be expressed in Hela cells.