pET28a-PTD-Ag85B质粒构建及原核表达条件的优化  

Plasmid construction and optimization of Prokaryotic expression for pET28a-PTD-Ag85B

作　　者：杜昭弘 王婉[1] 戴京京 何江[1] 王文洋[1] 杨小康[1] 邢应如[2] 尤其[1] 穆敏[1] 胡东[1,3] 吴静[1,3] 张荣波[1,3] 

机构地区：[1]安徽理工大学医学院免疫与检验教研室,淮南232001 [2]安徽理工大学附属肿瘤医院检验科 [3]免疫与感染研究所

出　　处：《齐齐哈尔医学院学报》2015年第14期2029-2032,共4页Journal of Qiqihar Medical University

摘　　要：目的构建p ET28a-PTD-Ag85B原核表达载体,并对其在E.coli BL21(DE3)中表达条件进行优化。方法将TAT-PTD序列插入到用限制性内切酶Nhe I和Bam H I双酶切的p ET28a质粒中,获得p ET28a-PTD重组质粒;RT-PCR法扩增Ag85B基因并将其克隆至p ET28a-PTD质粒中,获得p ET28a-PTDAg85B重组质粒。将重组质粒p ET28a-PTD-Ag85B转化至大肠杆菌,异丙基硫代半乳糖苷(IPTG)诱导PTD-Ag85B融合蛋白表达,聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定表达产物。筛选最适诱导剂浓度、诱导温度和时间,尿素复性包涵体,并用His-tag离子亲和层析柱分离纯化融合蛋白。结果成功构建p ET28aPTD-Ag85B重组质粒,在E.coli BL21中能高效表达PTD-Ag85B融合蛋白。结论成功表达及纯化了PTD-Ag85B蛋白,为获得大量PTD-Ag85B融合蛋白提供了技术储备。Objective To construct p ET28a-PTD-Ag85 B prokaryotic expression vector,and to explore the optimization of expression conditions in E. coli BL21. Methods Using restriction enzymes Nhe I and Bam H I to digest plasmid p ET28 a and then insert the TAT-PTD sequence into it to obtain p ET28a-PTD recombinant plasmid,using RT-PCR for amplification of Ag85 B gene,and then cloning to p ET28a-PTD plasmid to obtain p ET28a-PTD-Ag85 B recombinant plasmid. Conversion p ET28a-PTD-Ag85 B recombinant plasmid to E. coli,isopropyl thiogalactoside（ IPTG） induces PTD-Ag85 B fusion protein expression, using polyacrylamide gel electrophoresis（ SDS-PAGE） identify the expression product. Screening optimal inducer concentration,the optimum induction temperature and induction time,refolding inclusion by urea,separation and purification the fusion protein with a His-tag ion affinity chromatography. Results The recombinant plasmid p ET28a-PTD-Ag85 B was successfully constructed, could highly express PTD-Ag85 B fusion protein in E. coli BL21（ DE3）.Conclusions Successful expression and purification the PTD-Ag85 B proteins,in order to obtain a large number of PTD-Ag85 B fusion protein provides technical reserves.

关 键 词：AG85B TAT-PTD 原核表达 融合蛋白 

分 类 号：R394[医药卫生—医学遗传学]