人Heparanase基因真核和原核表达载体的构建及融合蛋白的表达

Construction of eukaryotic and prokaryotic expression vector of human heparanase gene and expression of heparanase fusion protein

目的 :构建人Heparanase基因的真核、原核表达载体 ,大肠杆菌表达其融合蛋白 .方法 :采用反转录 聚合酶链反应从人肝癌细胞株HepG2cDNA中 ,分别扩增出Heparanase编码基因 ,用限制性内切酶BamHI消化后 ,插入真核表达载体pcDNA3.1中 ,经酶切鉴定与测序证实后 ,连接成包括完整的人Heparanase基因ORF区的真核表达载体 .以亚克隆法构建于原核表达载体pRSET的相应酶切位点 ,转化大肠杆菌BL2 1菌株 ,异丙基 β D硫代半乳糖苷 (IPTG)诱导产生融合蛋白 .结果 :构建的人Heparanase基因表达载体经序列测定证实 ,与GenBank登录结果完全一致 ;双酶切鉴定证实 ,克隆基因正确插入载体pcDNA3.1及pRSET ;SDS PAGE证实融合蛋白表达成功 .结论 :成功构建了人Heparanase基因真核、原核表达载体 ,成功正确表达了

AIM: To construct a eukaryotic and prokaryotic expression vector of human heparanase gene and to express heparanase fusion protein. METHODS: Anterior segment and posterior segment of heparanase gene were amplified by reverse polymerase chain reaction from cDNA of Hep G2 respectively. The heparanase gene was digested by Bam H I and inserted into eukaryotic expression vector pcDNA3.1. Thus an integrated human heparanase gene was obtained. Then it was subcloned into prokaryotic expression vector pRSET and transformed into E.coli BL21. A fusion protein was expressed by IPTG induction. RESULTS: The eukaryotic expression vector encoding human heparanase was identical with the result in GenBank and was accurately inserted into the vector pcDNA3.1 and pRSET, which was confirmed by restriction endonucleased digestion. SDS PAGE indicated that the expression of 6His/Heparanase was successful. CONCLUSION: A eukaryotic and prokaryotic expression vector of human heparanase has been successfully constructed and 6His/Heparanase has been correctly expressed.