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

目的:克隆人血管生成素相关家族蛋白-1 angioarrestin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FD domain,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1 473 bp的angioarrestin目的片段及其C-端560 bp FD domain,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.

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

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.