| 摘要: |
| 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1473bp的angioarrestin目的片段及其C-端560bp 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。 |
| 关键词: 血管生成素1 angioarrestin 遗传载体 转染 |
| DOI：10.3724/SP.J.1008.2007.00134 |
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| 基金项目:上海市科学技术发展基金（024319115）. |
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| Construction and identification of eukaryotic expression vector of human angiopoietin-related protein-1 angioarrestin |
| SUN Ming-juan, WANG Liang-hua, DONG Xiao-yi, ZONG Ying, GAO Yun, JIAO Bing-hua |
| (Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences,Second Military Medical University, Shanghai 200433, China) |
| Abstract: |
| 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. |
| Key words: angiopoietin 1 angioarrestin genetic vectors transfection |
