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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 clone the human Rho-related coiled-coil protein kinase (ROCK) gene and construct the eukaryotic expression vector pGV141-ROCK2 and observe its expression in human umbilical vein endothelial cells (HUVEC-C). Methods: The CDS region of ROCK2 gene was obtained by RT-PCR and ligated into pGV141 vector after digested by Xho I and Kpn I. The eukaryotic expression vector pGV141-ROCK2 was digested by PCR and double digestion After sequencing, it was transfected into HUVEC-C, and the expression of ROCK2 protein in HUVEC-C was detected by western blot. Results: The size of the target band detected by PCR and double enzyme digestion was the same as that of the target band. The sequencing results showed that the inserted ROCK2 gene sequence in the recombinant plasmid was completely identical with the coding region of Gen Bank. Compared with the untransfected recombinant plasmid Compared with HUVEC-C, the expression of ROCK2 protein in HUVECC transfected with pGV141-ROCK2 vector was significantly increased, the difference was statistically significant (P <0.05). CONCLUSION: The eukaryotic expression vector pGV141-ROCK2 was successfully constructed and the expression of ROCK2 protein was significantly increased after transfection into HUVEC-C in vitro.