一个通用的TNFHis温度诱导融合表达载体的构建  

Construction of TNF-Fusion Protein Expression Vector

作　　者：韩苇[1] 张英起[1] 颜真[1] 石继红[1] 

机构地区：[1]第四军医大学生物技术中心,西安市710032

出　　处：《药物生物技术》2002年第6期317-321,共5页Pharmaceutical Biotechnology

摘　　要：以pBVTNF为起始质粒 ,设计合成了 2个DNA片段 :一个含有起始码、TNF起始码后的 2个氨基酸序列和 6个组氨酸序列 ;另一个含有Thrombin和hydroxylamine裂解位点序列。将上述 2个合成DNA片段分别与pBVTNF载体重组 ,获得了一个通用的TNFHis表达载体。在该表达载体中 ,含有 6×His的纯化标签和Thrombin和hydroxylamine融合蛋白裂解位点。分别将IFN IL 11和TNF基因插入该表达载体的多克隆位点 ,42℃诱导表达后 ,经SDS PAGE分析 ,结果表明 ,所有插入基因都获得了较高水平的表达。薄层扫描结果证实 ,所有融合表达蛋白的表达量均占菌体总蛋白的 2 0 %以上。Westernblot检测显示 ,所有融合表达蛋白均可与抗TNF α单抗发生免疫反应 ,间接证明融合蛋白均获得了表达。TNFHisTNF和TNFHisIFN在温度诱导表达后 ,菌体裂解液经Ni NTAAgaroseBeads亲和柱纯化 ,纯化结果表明 ,TNFHis中的 6×His序列可与Ni NTAAgaroseBeads结合。A TNF fusion protien expression vector was constructed Two DNA sequences were synthesized One contained start coden, sequences of first two amino acid of TNF and 6×His The other included sequences of Thrombin and Hydroxylamine The two sequences were respectively inserted into pBVTNF vector The final recombinant vector was named TNFHis IFN,IL 11 and TNF genes were cloned into EcoRI site and PstI site of the vector After different recombinant bacteria were induced at 42℃ for 4～5 h, all of TNF fusion proteins were expressed in high level The results of gel density scanning showed that expression levels of the proteins were between 20%～25% of total bacteria proteins Western blot analysis result showed that all of TNF fusion proteins could react with anti TNF αantibody The recombinant bacteria of TNFHis was induced at 42℃ for 4h and the bacteria cells were lysed with 8 mol/L Urea Then TNFHis fusion proteins were purified with Ni NTA Agarose Beads The purification result demonstrated that the 6×His sequence of TNFHis could be associated with the beads

关 键 词：TNFHis 温度诱导 融合表达载体 构建 

分 类 号：Q78[生物学—分子生物学]