人血管生成素相关家族蛋白-1 angioarrestin基因真核表达载体的构建与鉴定  

Construction and identification of eukaryotic expression vector of human angiopoietin-related protein-1 angioarrestin

作　　者：孙铭娟[1] 王梁华[1] 董晓毅[1] 宗英[1] 高云[1] 焦炳华[1] 

机构地区：[1]第二军医大学基础部生物化学与分子生物学教研室,上海200433

出　　处：《第二军医大学学报》2007年第2期134-138,共5页Academic Journal of Second Military Medical University

基　　金：上海市科学技术发展基金(024319115).~~

摘　　要：目的克隆人血管生成素相关家族蛋白-1angioarrestin基因,构建angioarrestin基因的真核表达载体。方法从人肝cDNA文库中经PCR扩增出angioarrestin基因及其C-端FDdomain,经纯化、回收目的片段,将其插入克隆载体pcDNA3.1/His-Myc(-)B,构建重组质粒pcDNA3.1-ARP和pcDNA3.1-FD,稳定转染大细胞肺癌NCI-H460细胞,RT-PCR和Western印迹法鉴定。结果从人肝cDNA文库中扩增出1473bp的angioarrestin目的片段及其C-端560bpFDdomain,构建的重组质粒pcDNA3.1-ARP和pcDNA3.1-FD经PCR及酶切鉴定与预期相符,经RT-PCR和Western印迹法确定稳定转染NCI-H460细胞成功。结论成功构建了angioarrestin和C-FD基因的真核表达重组质粒并转染NCI-H460细胞,为angioarrestin抗肿瘤血管形成作用机制的研究奠定了初步基础。Objective： To clone human angiopoietin-related protein-1 angioarrestin genes and construct their recombinant eukaryotic expression vector. Methods： Full length sequence of angioarrestin gene and its C-FD domain were amplified from human liver cDNA library by PCR and were subsequently inserted into the eukaryotic expression vector pcDNA3. 1/His-Myc （-）B. Then angioarrestin and FD recombinant plasmids were stablely transfected in NCI-H460 cell line. The positive clones were identified by RT-PCR and Western blot. Results： Full length fragment of angioarrestin gene （1 473 bp） and C-FD domain （560 bp） were successfully amplified from human liver cDNA library by PCR. The pcDNA3. 1-ARP and pcDNA3. 1-FD recombinant plasmids were also constructed successfully as identified by PCR and enzyme digestion. RT-PCR and Western blot showed the expression of target mRNA and protein in NCI-H460 cells. Conclusion： The eukaryotic expression vectors of angioarrestin and C-FD gene have been successfully constructed and expressed in NCI-H460 cells, which pave a way for further study on the anti-angiogenesis function of angioarrestin in cancer.

关 键 词：血管生成素1 ANGIOARRESTIN 遗传载体 转染 

分 类 号：Q782[生物学—分子生物学]