结核分枝杆菌MPT64在毕赤酵母中的分泌表达  

Secretive expression of Mycobacterium tuberculosis MPT64 in Pichia pastories

作　　者：熊志红[1] 高新[2] 李国利[1] 庄玉辉[1] 

机构地区：[1]解放军总医院第二附属医院结核病研究所,北京100091 [2]北京军事医学科学院,100850

出　　处：《临床肺科杂志》2008年第12期1593-1595,共3页Journal of Clinical Pulmonary Medicine

摘　　要：目的克隆结核分枝杆菌MPT64基因全长,构建毕赤酵母分泌型表达载体,获得表达MPT64的重组毕赤酵母工程菌。方法利用PCR技术扩增MPT64基因克隆到毕赤酵母分泌表达载体pPICZαA中;将正确的表达载体线性化后,电击导入到毕赤酵母GS115中,ZeocinTM抗性筛选正确的重组子;SDS-PAGE及Western-blot鉴定分泌表达MPT64的重组毕赤酵母。结果克隆获得了正确的MPT64酵母分泌表达载体,并获得了相应的重组酵母株,该重组子经甲醇诱导培养后能分泌大小约30kDa的蛋白,该蛋白能特异地与His抗体结合。结论本实验获得了酵母重组的结核杆菌MPT64蛋白,为探讨其生物学性能和临床上的应用奠定了实验基础。Objective To clone the Mycobacterium tuberculosis MPT64 into Pichia pastories secretive expression vector and gain the recombinant yeast. Methods Standard PCR was used to amplify the MPT64 genes. The amplified genes were cloned into the yeast secretive expression vector pPICZαA, generating pPICZα-MPT64. The recombinant plasmid was integrated into Pichia pastoris GS115 by electmporation and the recombinants were selected by plating cells on YPD/ZeocineTM plates;the expressed product was induced with methanol for 4 to 6 days and then. identified by SDS-PAGE and Western blotting. Results The result of restriction enzyme digestion and nueleotide sequencing confirmed that the recombinant plasmids were correct. A recombinant protein with a molecular size of approximately 30 kDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed supematant was able to bind with anti-His monoclonal antibody. Conclusion The recombinant MPT64 proteins were successfully expressed, which laid foundation for further biological study and clinical trial of MPT64.

关 键 词：结核分枝杆菌 MPT64 毕赤酵母 分泌表达 

分 类 号：R346[医药卫生—基础医学]