hVEGF165基因克隆及其在COS-7细胞中的表达  

hVEGF165 Gene Clone and Its Expression in COS-7 Cells

作　　者：葛建军[1] 周正春[1] 陈兵[2] 张胜权[2] 罗欣[2] 徐从贞[2] 汪思应[3] 

机构地区：[1]安徽医科大学第一附属医院心血管外科,合肥230022 [2]安徽医科大学生物化学与分子生物学教研室,合肥230022 [3]安徽医科大学病理生理学教研室,合肥230022

出　　处：《中国胸心血管外科临床杂志》2005年第3期185-188,共4页Chinese Journal of Clinical Thoracic and Cardiovascular Surgery

基　　金：安徽省自然科学基金资助项目(03043707)~~

摘　　要：目的克隆人血管内皮生长因子(hVEGF)基因VEGF165片段,构建pcDNA3.1/hVEGF165,观察其在COS-7细胞中的表达,为基因治疗缺血性心脏病的研究奠定基础。方法从合法引产的胎儿心肌组织中提取总核糖核酸(RNA),应用逆转录-聚合酶链反应(RT-PCR)方法获得hVEGF165基因,将其重组入T载体,聚合酶链反应(PCR)法鉴定并测序,双酶切后克隆入真核表达载体pcDNA3.1/myc-his-B中,构建pcDNA3.1/hVEGF165重组体。用脂质体介导将其转染COS-7细胞,蛋白印迹(Westernblotting)法检测rhVEGF165的表达蛋白。结果用RT-PCR方法从胎儿心肌组织中获得了正确的hVEGF165基因序列,并成功构建pcDNA3.1/hVEGF165,且实现转染COS-7细胞的瞬时表达。结论构建的pcDNA3.1/hVEGF165转染真核细胞COS-7能够表达rhVEGF蛋白。Objective To study clone human vascular endothelial growth factor gene165(hVEGF165) to construct the recombined plasmid pcDNA 3.1/hVEGF165 and observe its expression in COS-7. Methods hVEGF gene was amplified by reverse transcription polymerase chain reaction(RT-PCR) method from fetal human myocardium tissue, was then loned into T-vector; identified by polymerase chain reaction(PCR); and was inserted into the expression plasmid pcDNA3.1 to construct the recombined plasmids that encoding VEGF165 comlementary DNA(cDNA). COS-7 cells were transfected mediated by liposome, then expressed protein was detected by Western blotting. Results Exact gene order of hVEGF165 was obtained from the fetal human myocardium tissue by RT-PCR; pcDNA3.1/VEGF165 was constructed, and transient expression was going after transfecting COS-7 cell. Conclusion The recombined plasmids we constructed could successfully express the hVEGF protein after eukaryotic cells COS-7 were transfected.

关 键 词：COS-7细胞 逆转录-聚合酶链反应(RT-PCR) 真核表达载体PCDNA3.1 基因克隆 聚合酶链反应(PCR) 人血管内皮生长因子 VEGF165基因 rhVEGF165 RT-PCR方法 COS7细胞 缺血性心脏病 VEGF蛋白 心肌组织 脂质体介导 基因治疗 核糖核酸 

分 类 号：R541[医药卫生—心血管疾病]