pcDNA3.1/hVEGF165的构建及其在COS-7细胞中的表达

目的　克隆人血管内皮生长因子基因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奠定基础。方法　从胎儿心肌组织中提取总RNA,应用RT PCR方法获得hVEGF165基因,将其重组入T载体,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,WesternBlotting方法检测rhVEGF165的表达蛋白。结果　RT PCR方法从胎儿心肌组织获得正确的hVEGF165基因序列,成功构建pcDNA3·1 /hVEGF165且实现转染COS 7细胞的瞬时表达。结论　该实验构建的pcDNA3· 1 /hVEGF165转染真核细胞COS 7能够表达rhVEGF蛋白。

Construction of pcDNA3.1/hVEGF165 and its expression in COS-7 cells

Objective The aim of this study is to clone hVEGF165 gene to construct the expressional plasmid pcDNA3 1/VEGF165 and observe its expression in COS 7. Methods Human vascular endothelial growth factor gene was amplified by RT PCR method from fetal human myocardium tissue, and then clone into Tvector being identified by PCR and inserted into the expression plasmid pcDNA3 1 to construct the recombined plasmids that encoded VEGF165 cDNA. COS 7 cells were transfected mediated by liposome, then expressed protein was detected by Western Blotting. Results Exect gene setuence 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 can successfully express the hVEGF protein after eukaryotic cells COS 7 were transfected.