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目的:构建真核表达载体pcDNA3.1(+)-VEGF165并在哺乳动物细胞中实现目的基因的特异表达,为重组VEGF165进一步的生物学活性和临床研究奠定基础。方法:采用分子生物学方法将扩增的VEGF165基因克隆入真核表达载体pcDNA3.1(+),在脂质体介导下将重组质粒转染小鼠胚胎成纤维细胞NIH/3T3,以抗生素培养基筛选抗性细胞,并对抗性细胞进行单克隆化,采用双抗夹心酶联免疫吸附(ELISA)、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western Blotting等方法对目的蛋白在细胞中的特异表达进行分析鉴定。结果:完成真核表达载体pcDNA3.1(+)-VEGF165的构建,并成功转染NIH/3T3细胞,筛选到一批G418抗性的阳性细胞,单克隆化的阳性细胞(吸光度值0.345±0.064)与空载体转染(吸光度值0.114±0.012)的对照比较,有显著的VEGF165表达(P<0.01),SDS-PAGE和Western Blotting检测到VEGF165在扩大培养的克隆细胞中的特异表达。结论:应用pcDNA3.1(+)-VEGF165载体转染的NIH/3T3细胞可高效表达具生物学活性的VEGF165重组蛋白。
OBJECTIVE: To construct the eukaryotic expression vector pcDNA3.1 (+) - VEGF165 and to express the target gene in mammalian cells, which will lay the foundation for the further biological activity and clinical research of recombinant VEGF165. METHODS: The amplified VEGF165 gene was cloned into the eukaryotic expression vector pcDNA3.1 (+) by molecular biology method. The recombinant plasmid was transfected into NIH / 3T3 mouse embryonic fibroblasts by liposome, The medium was screened for the resistant cells and the resistant cells were monoclonated and analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western Blotting Methods The specific expression of the target protein in cells was analyzed and identified. Results: The eukaryotic expression vector pcDNA3.1 (+) - VEGF165 was constructed and successfully transfected into NIH / 3T3 cells. A number of G418 positive cells and monoclonal positive cells (absorbance 0.345 ± 0.064 ) Was significantly higher than that of blank vector (absorbance 0.114 ± 0.012) (P <0.01). The specific expression of VEGF165 in the expanded clonal cells was detected by SDS-PAGE and Western Blotting. CONCLUSION: NIH / 3T3 cells transfected with pcDNA3.1 (+) - VEGF165 can efficiently express VEGF165 recombinant protein with biological activity.