摘要
采用PCR技术，从原核表达质粒PUC19-VEGF165中获得上下游含有HindⅢ和BamH I酶切位点的目的基因VEGF165，与真核载体pEGFP-C1构建重组质粒pEGFP-VEGF165，观察其在内皮细胞中的表达。结果表明，带有绿色荧光蛋白报告基因的真核表达质粒pEGFP-VEGF165构建成功，并在PEI的介导下成功转染脐静脉内皮细胞（HUVEC），在荧光显微镜下可见强绿色荧光蛋白表达，ELISA检测证明VEGF在细胞上清中有效表达，RT—PCR证明了VEGF165mRNA水平上的有效表达。此研究结果将为再狭窄的基因治疗提供实验基础。
The VEGF165 gene segment with Hind Ⅲand BamHⅠ site was obtained from PUC19- VEGF165 by PCR, which was cloned into eukaryotic vector pEGFP-C1, and then its expression in HUVEC was observed. The results showed that plasmid pEGFP-VEGF165 with reporter gene pEGFP-C1 was correctly constructed and transfected into HUVEC by PEI. The green fluorescence protein expression was observed by fluorescence microscopy. VEGF was effectively expressed in the culture medium by ELISA and mRNA increased significantly by RT-PCR. This research can provide experimental foundation for the gene therapy of restenosis.
出处
《北京生物医学工程》
2009年第4期395-397,426,共4页
Beijing Biomedical Engineering
