血管内皮生长因子和Fas配体共表达质粒的构建及其在细胞中的表达

Co-expression plasmid construction of vascular endothelial growth factor and Fas ligand and detection of its expression in cultivated cells

目的 构建血管内皮生长因子 (VEGF)和Fas配体 (FasL)共表达质粒 ,并鉴定其在细胞中的表达情况。方法 根据人VEGF165和FasL的cDNA序列分别设计、合成引物 ,经扩增、连接、酶切、插入等得到重组表达质粒 pCI VEGF165和 pCI FasL。再以此为基础构建得到编码FasL和VEGF165的共表达质粒 pCI FasL IRES VEGF165 (简称pCI FIV )。用脂质体将各表达质粒(2 μg) 转染NIH 3T3细胞 ,用流式细胞术、ELISA和Western blot检测其FasL和VEGF165表达情况。结果 细胞裂解液的Western blot检测表明 ,重组质粒 pCI VEGF165和 pCI FIV能表达VEGF165 ,分子量与预期kD一致。细胞培养上清的ELISA检测表明 ,pCI VEGF165和pCI FIV均能分泌性表达VEGF165。流式细胞仪检测表明 ,质粒pCI FasL、pCI FIV稳定转染的NIH3T3细胞表面的绿色荧光阳性率分别为 98.0 %和 96.5 % ,而空载体转染细胞的阳性率为 3 .2 %。ELISA检测结果显示 ,pCI FasL、pCI FIV转染NIH3T3细胞培养上清中均存在可溶性FasL。 结论 成功构建的FasL和VEGF165共表达质粒可成功转染细胞并能分泌性表达VEGF165 ,同时可表达膜结合形式的FasL和可溶性FasL ,为将来血管内膜增生的基因治疗奠定了基础。

Objective To construct the co expression plasmid of vascular endothelial growth factor (VEGF) and Fas ligand (FasL) and detect its expression in cultivated cells.Methods The primers were designed and synthetized according to the cDNA sequences of human VEGF165 and FasL,then PCR amplication,connection,splicing and insertion were carried out and the recombinant plasmids of pCI VEGF165 and pCI FasL were obtained.The co expression plasmid encoding FasL and VEGF165 named pCI FasL IRES VEGF165 (simplified as pCI FIV) was successfully constructed.The constructed plasmids (2 μg) were transfected to NIH3T3 cell with lipofectamine and the expressions of FasL and VEGF165 were detected by flow cytometry,ELISA and western blot.Results Western blot of the cell splitting solution of pCI VEGF165 and pCI FIV detected the expression of VEGF165 with the estimated molecular weight,and ELISA detection of the supernatant of pCI VEGF165 and pCI FIV revealed a secretory expression of VEGF165.Flow cytometry found the green fluorescent staining cells of pCI FasL and pCI FIV stably transfected NIH3T3 were 98.0% and 96.5% respectively,while that of the empty vector was 3.2%.ELISA detected the soluble FasL expression in the supernatant of pCI FasL and pCI FIV transfected cells.Conclusion The FasL and VEGF co expression plasmid was successfully constructed and it could express secretory VEGF165 and membrane binding FasL and soluble FasL,which lay a foundation for future gene therapy of vascular intimal hyperplasia.