目的 构建真核表达载体pcDNA3.1(+)-血管内皮生长因子(VEGF)165并在哺乳动物细胞中实现目的 基因的表达,对目的 蛋白进行鉴定和生物学活性分析.方法 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至哺乳动物细胞NIH 3T3并进行抗生素压力筛选,采用双抗夹心ELISA、SDS-PAGE和免疫印迹方法对目的 蛋白在转染细胞中的特异表达进行鉴定,在血管内皮细胞ECV304上对表达产物的细胞结合活性和促增殖活性进行分析.结果 构建的真核表达载体pcDNA3.1(+)-VEGF165成功转染NIH 3T3,获得加压筛选的重组细胞,目的 基因得到稳定表达,目的 蛋白能与血管内皮细胞ECV304特异结合,并具有促内皮细胞增殖活性.结论 应用pcDNA3.1(+)-VEGF165载体转染的NIH 3T3细胞可稳定表达具生物学活性的VEGF165重组蛋白.%Objective To study VEGF165 gene expression and the artivity of the product in mammalian cells transfected by a recomhinant plasmid pcDNA3. 1( + ) - VECF165. Methods VEGF165 gene was ohtained by PCR and was constructed into the expression vector pcDNA3. 1 ( + ). The pcDNA3. 1( + ) - VEGF165 plasmid was transfected into NIH 3T3 cells by Lipofertamine 2000. The positive clones were screened by G418. The expressed product was identified by sandwich ELISA. SDS - PACE and Western blotting. The biological activities of the product were analyzed by cell ELISA and MTT assay. Results The expression vector pcDNA3 . 1( + ) - VEGF165 was successfully constructed and transfected into NIH 3T3 cells. After screened by G418 , the resistant cells were cultured and passaged. The VEGF165 gene expressed in the recombinant NIH 3T3 cells. The profluct could bind to the receptor cells ECV304 and stimulate the proliferation of ECV304. Conclusion NIH 3T3 cells transfected by the pcDNA3. 1( + ) - VECF165 plasmid can express active VEGF165 protein.
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