pGV141-ROCK2真核表达载体的构建及其在血管内皮细胞中的表达

Construction of Eukaryotic Expression Plasmid pGV141-ROCK2 and Expression in Vascular Endothelial Cells

目的:克隆人Rho相关卷曲螺旋形成蛋白激酶(ROCK)基因,构建pGV141-ROCK2真核表达载体,并观察其在人脐静脉内皮细胞(HUVEC-C)中的表达。方法:以RT-PCR的方法获得ROCK2基因的CDS区,经Xho I、Kpn I双酶切后,连接到pGV141载体中;将构建的pGV141-ROCK2真核表达载体,经PCR、双酶切和测序鉴定后,将其转染入HUVEC-C中;采用western blot法检测转染后HUVEC-C中ROCK2蛋白的表达情况。结果:PCR法、双酶切法检测到的目标条带与目的条带大小一致,测序结果显示重组质粒中插入的ROCK2基因序列与Gen Bank编码上的编码区完全一致;与未转染重组质粒的HUVEC-C比较,转染pGV141-ROCK2载体的HUVECC中ROCK2蛋白的表达显著增高,差异有统计学意义(P<0.05)。结论:成功构建pGV141-ROCK2真核表达载体,体外转染入HUVEC-C后可使ROCK2蛋白表达显著增加。

Objective： To construct the eukaryotic expression plasmid pgv141-rock2 and observe the expression in vascular endothelial cells. Methods： The CDS region of ROCK2 gene was obtained from HUVEC by RT-PCR. The product was cloned into GV141 vector following by Xho I/Kpn I digestion.The recombinant plasmid p GV141-ROCK2 was confirmed by restrictive enzyme digestion,PCR and DNA sequencing. Finally p GV141-ROCK2 was transfected into HUVEC and the expression of ROCK2 was detected by western blot. Result： p GV141-ROCK2 was proved to be successfully constructed by restrictive enzyme digestion,PCR and DNA sequencing. The expression of ROCK2 protein in p GV141-ROCK2 transfection group was significantly higher than that in con-transfected group,difference was statistically significant（ P〈0. 05）. Conclusion： The study successfully constructed eukaryotic expression plasmid pgv141-ROCK2,vitro transfected HUVEC-C could significantly increase ROCK2 protein expression.