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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 co-expression plasmid of vascular endothelial growth factor (VEGF) and Fas ligand (FasL) and identify its expression in cells. Methods According to the cDNA sequence of human VEGF165 and FasL, primers were designed and synthesized. The recombinant plasmids pCI VEGF165 and pCI FasL were obtained by amplification, ligation, digestion and insertion. Based on this, a recombinant plasmid pCI FasL IRES VEGF165 (pCI FIV) encoding FasL and VEGF165 was constructed. The expression plasmids (2 μg) were transfected into NIH 3T3 cells by lipofectamine. The expression of FasL and VEGF165 was detected by flow cytometry, ELISA and Western blot. Results Western blot analysis showed that recombinant plasmid pCI VEGF165 and pCI FIV could express VEGF165 with the same molecular weight as expected kD. ELISA examination of cell culture supernatant showed that both VEGF165 and pCI FIV secreted VEGF165. Flow cytometry showed that the positive rates of green fluorescence of NIH3T3 cells transfected with plasmid pCI FasL and pCI FIV were 98.0% and 96.5%, respectively. The positive rate of empty vector transfected cells was 3.2%. The results of ELISA showed that soluble FasL was found in the supernatant of NIH3T3 cells transfected with pCI FasL and pCI FIV. Conclusions The constructed recombinant plasmids of FasL and VEGF165 can successfully transfect cells and express VEGF165. At the same time, the expression of membrane-associated FasL and soluble FasL can be expressed, which lays the foundation for future gene therapy of intimal hyperplasia.