结核分枝杆菌PPE68蛋白的原核表达及纯化  

Prokaryotic Expression and Purification of PPE68 Protein of Mycobacterium tuberculosis

作　　者：佘茜[1,2] 徐蕾[1,2] 何永林[1,2] 靳志栋[1,2] 张丹[1,2] 杨春[1,2] 

机构地区：[1]重庆医科大学病原生物学教研室,重庆400016 [2]重庆医科大学神经科学研究中心,重庆400016

出　　处：《中国生物制品学杂志》2010年第12期1295-1298,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30771922;30901280);重庆市自然科学基金(2009BB5275)

摘　　要：目的构建结核分枝杆菌PPE68基因的原核表达质粒,并在大肠杆菌中进行表达和纯化。方法以结核分枝杆菌H37Rv基因组DNA为模板,PCR法扩增PPE68基因,将其克隆至pET-32a(+)载体中,构建重组原核表达质粒pET-32a(+)-PPE68,转化至大肠杆菌BL21(DE3)中,IPTG诱导表达。表达产物经SDS-PAGE及Western blot鉴定后,进行纯化。结果重组表达质粒pET-32a(+)-PPE68经PCR及双酶切鉴定构建正确,测序结果与GenBank中登录的PPE68基因序列一致。表达的Trx-PPE68融合蛋白相对分子质量约为57000,表达量约占菌体总蛋白的41%,可与结核分枝杆菌免疫小鼠血清发生特异性反应。纯化的重组蛋白纯度约为93%。结论已成功构建了结核分枝杆菌PPE68基因重组原核表达质粒pET-32a(+)-PPE68,原核表达并纯化了重组蛋白,为PPE68作为结核病特异性诊断抗原的开发及重组BCG疫苗的研制奠定了基础。Objective To construct a prokaryotic expression vector for PPE68 gene of Mycobacterium tuberculosis,express in E.coli and purify the expressed product.Methods PPE68 gene was amplified by PCR using the genomic DNA of M.tuberculosis H37Rv as a template and cloned into vector pET-32a（＋）.The constructed recombinant plasmid pET-32a（＋）-PPE68 was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot then purified.Results Recombinant plasmid pET-32a（＋）-PPE68 was constructed correctly as proved by PCR and restriction analysis,of which the sequencing result was consistent with that of PPE68 gene reported in GenBank.The expressed fusion protein TrxPPE68,with a relative molecular mass of about 57 000,contained about 41% of total somatic protein and showed specific reaction with the sera of mice immunized with M.tuberculosis.The purified recombinant protein reached a purity of about 93%.Conclusions Recombinant plasmid pET-32a（＋）-PPE68 was successfully constructed,and recombinant protein was expressed in prokaryotic cells and purified,which laid a foundation of developing PPE68 as a specific antigen for diagnosis of tuberculosis and preparing recombinant BCG vaccine.

关 键 词：结核分枝杆菌 PPE68 原核细胞 基因表达 纯化 

分 类 号：R378.91[医药卫生—病原生物学] Q78[医药卫生—基础医学]