VEGF165基因表达载体的构建和鉴定  

Construction and identification of the expression vector of the vascular epidermal growth factor 165 gene

作　　者：马彩虹[1] 贺立新[1] 曹玉珏[1] 李冬海[1] 

机构地区：[1]北京市右安门医院烧伤科,北京100069

出　　处：《感染．炎症．修复》2010年第3期143-146,共4页Infection Inflammation Repair

基　　金：北京市丰台区卫生局科研基金

摘　　要：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 study the transfeetion and expression of the pcDNA3.1 （＋）-vascular epidermal growth factor （VEGF） 165 in mammalian cells, and to lay the foundation for further research of the activity and the clinical application of the VEGF165. Methods： The VEGF165 gene was obtained by polymerase ehain reaction （PCR） and was eonstrueted into the expressive vector pcDNA3,1（＋）. The pcDNA3.1（＋） VEGF165 plasmid was transfected into NIH/3T3 cells by Lipofectamine 2000. The transfeeted cells were screened by antibiotic G418. The positive cells were cloned by limiting dilution. The expression of the VEGF165 protein was identified by sandwich enzyme-linked immunoadsorbent assay （ELISA）, SDS-PAGE and Western Blotting. Results： The expressive vector pcDNA3.1 （＋）-VEGF165 was successfully constructed and transfected into the NIH/3T3 eells. After screened by G418 and cloned by limiting dilution, the positive elones expressed the VEGF165 with higher level than the control cells transfeeted by pcDNA3,1 （＋） vector, their optical degree was 0. 345±0. 064 and 0. 114±0. 012 respectively （P〈0.01）. The expression of the VEGF165 protein in the NIH/3T3 cells was obtained by sandwich ELISA, SDS-PAGE and Western Blotting. Conclusion： The NIH/3T3 cells transfected by the peDNA3,1（＋）-VEGF165 plasmid could express the VEGF165 protein.

关 键 词：VEGF165 转染 筛选 单克隆化 表达 鉴定 

分 类 号：R392.11[医药卫生—免疫学]