SP-TAT-Apoptin融合基因真核表达载体的构建和鉴定  

Construction and identification of eukaryotic expression vector of SP-TAT-Apoptin

作　　者：韩苏夏[1,2] 马瑾璐[1] 吕毅[1] 黄辰[3] 段康民[2] 

机构地区：[1]西安交通大学医学院第一附属医院肿瘤中心 [2]西北大学生命科学院分子微生物研究室,陕西西安710069 [3]西安交通大学医学院中心实验室,陕西西安710061

出　　处：《第四军医大学学报》2009年第1期20-23,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30672069)

摘　　要：目的:通过基因克隆构建表达SP-TAT-Apoptin融合基因的真核表达载体.方法:通过DNA重组技术,以pCD-NA3.1/Apoptin质粒为模板,进行PCR反应;将具有分泌功能的信号肽和能够携带核酸、蛋白和肽自由地通过细胞膜和核膜的蛋白转导域TAT序列连接于Apoptin基因5′端;PCR反应的产物连接于plenti6-V5-D-TOPO(真核表达载体.用限制性内切酶酶切分析和DNA序列分析鉴定重组结果;将重组的真核表达载体瞬时转染HUVEC细胞,用免疫荧光标记对转染后的细胞进行处理,观察重组蛋白的表达情况.结果:PCR产物大小符合要求,重组质粒plenti6-V5-D-TOPO(/SP-TAT-Ap-optin双酶切鉴定与预期一致,测序结果正确.且经激光共聚焦显微镜观察到重组蛋白的表达,表明重组表达载体已构建成功.结论:成功克隆出SP-TAT-Apoptin融合基因,并成功构建其真核细胞表达载体,为下一步研究SP-TAT-Apoptin融合基因对肿瘤的生长抑制作用的体内外研究奠定了基础.AIM： To construct and identify the eukaryotic expression vector of SP-TAT-Apoptin. METHODS： In this study, we designed a secretory protein by adding a secretory signal peptide （SP） to the N terminus of Transactivating Transcription （TAT）-Apoptin （SP-TAT-Apoptin）. PCR was used to amplify the Apoptin gene and to incorporate the TAT transduction domain and SP sequence upstream. The primers were designed based on the published sequences in GenBank. The SP-TAT-Apoptin cDNA cloned in pLenti6/V5-D- TOPO vector was confirmed by restriction enzyme digestion and by DNA sequencing. The human umbilical vein endothelial cells （HUVECs） were transfected with the plenti6/V5-D-TOPO/ SP- TAT-Apoptin plasmid. The expression of SP-TAT-Apoptin in HUVECs was confirmed by immunofluorescence microscopy. RESULTS： PCR products met the expected ones. Double restriction enzyme digestion displayed the expected fragments of plenti6-V5-D-TOPO/ SP- TAT-Apoptin. Sequencing analysis results were consistent with the reported one. The expression of the recombinant protein was confirmed by eonfocal microscopy. CONCLUSION： The eukaryotic expression vector of SP-TAT-Apoptin is successfully constructed. It provides foun- dation for research on inhibitory effects of SP-TAT-Apoptin on tumor growth.

关 键 词：信号肽 蛋白转导域 鸡贫血病毒 Apoptin基因 

分 类 号：R730.5[医药卫生—肿瘤]