张丽君,徐沁,褚孟洋,李程飞,石菲,高原,孙喜庆,赵疆东,闫铭,王永春
ZHANG Lijun,XU Qin,CHU Mengyang,LI Chengfei,SHI Fei,GAO Yuan,SUN Xiqing,ZHAO Jiangdong,YAN Ming,WANG Yongchun

• 1:第四军医大学学员一旅
• 2:第四军医大学航空航天医学系航空航天生物动力学教研室
• 3:第四军医大学西京医院骨科

摘要(Abstract)：

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.

关键词(KeyWords)： 质粒;载体;内皮细胞;Rho相关卷曲螺旋形成蛋白激酶
plasmid;vector;endothelial cells;Rho associated coiled coil forming protein kinase

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81372130,81301681,81301581,81471817)

作者(Author): 张丽君,徐沁,褚孟洋,李程飞,石菲,高原,孙喜庆,赵疆东,闫铭,王永春
ZHANG Lijun,XU Qin,CHU Mengyang,LI Chengfei,SHI Fei,GAO Yuan,SUN Xiqing,ZHAO Jiangdong,YAN Ming,WANG Yongchun

DOI: 10.19367/j.cnki.1000-2707.2017.06.005

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