Rap2c基因真核表达质粒的构建与表达

目的：为了探讨Rap2c基因与肺癌发生的关系，克隆人Ras家族小G蛋白Rap2c的cDNA，构建其真核表达质粒并在人肺癌细胞株A549和H1299中表达。方法人骨肉瘤细胞株U2OS提取细胞总RNA，经逆转录聚合酶链反应（RT-PCR）逆转录成cDNA，PCR扩增Rap2c，酶切后插入pcDNA3．1（＋）构建真核表达质粒 pcD-NA3．1（＋）-Rap2c，采用酶切及测序鉴定。重组质粒转染A 549和H 1299细胞，Western blot 检测其目的基因表达。结果双酶切及测序结果显示重组质粒pcDNA3．1（＋）-Rap2c成功构建，Werstern blot 检测到 A549和H 1299细胞有相应蛋白表达。结论成功构建人Rap2c基因真核表达质粒，为后续研究奠定了基础。

C onstruction and expression of eukaryotic expression plasmid Rap2 c

Objective To clone Rap2c cDNA, which belongs to human Ras -related small G protein superfamily , then to construct its eukaryotic expression vector and detect its expression in A 549 and H1299 cell lines so as to explore the relationship between Rap2c gene and the pathogenesis of lung cancer .Methods Total RNA of human osteosarcoma cells U2OS was extracted and reversed into cDNA by RT -PCR.Then,Rap 2c gene was amplified by PCR and inserted into pcDNA3.1 （ ＋）.The reconstructed plasmid was identified by restricted enzyme digestion and sequencing .pcD-NA31. （＋）-Rap2c was transfected into A549 and H1299 cells, the expression of Rap2c was detected by Western blot.Results The results of enzyme digestion and sequencing showed that the coding sequence of pcDNA 3.1 （ ＋） -Rap2c was right and was inserted into the vector correctly .The results of Western blot showed that A 549 and H1299 cells were transfected successfully .Conclusion The plasmid pcDNA31.（＋）-Rap2c was constructed successfully and laid basis for subsequent studies .