结核分枝杆菌Ag85A在大肠埃希菌中的表达与纯化  

Recombinant Expression and Purification of Antigen 85A of Mycobacterium Tuberculosis(Mtb.)

作　　者：蒋天舒[1] 娄加陶[1] 周晔[1] 陈燕[1] 陈波[1] 谷明莉[1] 仲人前[1] 邓安梅[1] 

机构地区：[1]第二军医大学长征医院实验诊断科,全军临床免疫中心,上海200003

出　　处：《现代检验医学杂志》2008年第1期16-18,共3页Journal of Modern Laboratory Medicine

基　　金：国家自然科学基金(30471616);上海市重大科技攻关基金(04DZ19116);上海市重点科研支撑条件项目(051409012);上海市青年科技启明星计划(QMX01423);上海市重点基础研究项目(05JC14052)资助

摘　　要：目的在大肠埃希菌中表达、纯化结核分枝杆菌Ag85A蛋白,探讨Ag85A蛋白作为候选疫苗用于结核免疫预防和治疗的可行性。方法扩增人结核分枝杆菌Ag85A基因序列,将结核分枝杆菌Ag85A的全长cDNA插入到原核表达载体pGEX-4T-1中,构建成Ag85A重组质粒。将重组质粒转入大肠埃希菌BL21并以IPTG诱导表达,经SDS-PAGE和Western blot鉴定大肠埃希菌表达的重组蛋白。结果成功克隆了结核分枝杆菌Ag85A,获得了pGEX4T-Ag85A重组子,Ag85A在大肠埃希菌中实现了稳定表达,表达的蛋白条带大小约32kD,与预期结果相符。结论成功地对结核分枝杆菌免疫保护性抗原Ag85A进行了基因克隆与表达,为进一步研究其在结核病新型疫苗研制中的应用奠定了基础。Objective To express recombinant antigen 85A of Mycobacterium tuberculosis（Mtb. ） for development of novel TB vaccine. Methods The whole gene containing DNA sequence encoding antigen 85A was synthesized and used as a template to amplify the DNA sequence by polymerase chain reaction（PCR） for expressing recombinant Rv0309 protein,con- taining restriction sites （BamH Ⅰ ,Xho Ⅰ ） on 5＇ and 3＇ end respectively. The PCR product was inserted into expression vector pGEX-4T-1 and then the recombinant plasmid was transformed into E. Coli BL21,induced by isopropylthio-β-D- galactoside （IPTG）. Results The recombinant 32kD Ag85A protein was expressed and confirmed by SDS-PAGE and Western-blot. Conclusion The recombinant TB antigen 85A was expressed successfully.

关 键 词：结核分枝杆菌 Ag85A抗原 表达纯化 

分 类 号：Q78[生物学—分子生物学] R378.911[医药卫生—病原生物学]