结核分枝杆菌分泌蛋白MPT51在原核表达载体中的克隆、表达、纯化与鉴定  

Cloning, expression, purification and identification of secreted protein MPT51 from Mycobacterium tuberculosis

作　　者：王晓闻[1] 蔡宏[1] 朱玉贤[1] 

机构地区：[1]北京大学生命科学学院蛋白质工程及植物基因工程国家重点实验室,北京100871

出　　处：《中国人兽共患病学报》2006年第11期1031-1034,共4页Chinese Journal of Zoonoses

摘　　要：目的对结核分枝杆菌的一种分泌蛋白MPT51的基因进行克隆,表达,纯化与鉴定,为核酸疫苗的研制与应用打下基础。方法以结核杆菌H37Rv株基因组DNA为模板,用PCR法对编码MPT51蛋白的抗原基因进行扩增,产物克隆到表达载体pET22b中,构建了含mpt51的重组质粒,将此重组质粒先转化到E.coliXLI-Blue内提取质粒,酶切及测序鉴定,再转化入E.coliBL21(DE3)PLysS菌株内,以IPTG进行诱导后,破菌,离心,包涵体经尿素变性溶解后用His·Bind亲和层析柱纯化,透析复性,纯化后蛋白进行SDS-PAGE,通过电转移将胶中蛋白转到硝酸纤维素薄膜上后进行特异性免疫检测。结果电泳发现转化了重组质粒的菌株有蛋白表达,所表达的蛋白质相对分子质量为27800,抗体检测有特异条带,大小为27.8kDa。结论目的基因克隆到了表达载体内并证明表达,重组结核杆菌分泌蛋白MPT51的成功表达为核酸疫苗的研制与应用和免疫效应检测以及上述抗原、抗体的大规模制备打下基础。The purpose of this study is to clone, express, purify and identify the secreted protein MPT51 from Mycobacterial tuberculosis so as to provide foundation for development and applications of DNA vaccine in clinic practice. The gene encoding for protein MPT51 was amplified from M. tuberculosis H37Rv genomic DNA by using PCR technique. PCR product was cloned initially into the pET22b expression vector, then transformed into E. coli XLI-Blue strain. Clones containing the vectors were selected on LB-plus ampicillin（100μg/ml） plates, and plasmid DNA was extracted and digested with enzymes. Plasmids containing the right insertion were sequenced to confirm its identity and retransformed into E. coli BL21 （DE3）PLysS strain. Bacterial lysates prepared from 0.8mmol/L IPTG induced cultures were loaded directly onto SDS-PAGE. Upon IPTG induction, the recombinant pET22b-mpt51 indeed produced a new protein with an apparent MW of 27.8kDa. The protein was contained in the inclusion body after being ultrasonically broken and was purified by His · Bind Kit after being dissolved in the binding buffer containing 8mol/L urea. The denatured protein was renatured through dialysis. The purified protein on the SDS- PAGE gel was transferred to nitrocellulose membrane and detected with specific antibody. A specific protein was visualized on membrane at molecular mass 27.8kDa. In conclusion, we have obtained recombinant pET22b expression vector containing the gene coding for MPT51 protein, which has been expressed successfully. It will be useful for the detection of immunity effectiveness and preparation of the antigen and antibody of MPT51 protein in large scale.

关 键 词：结核分枝杆菌 分泌蛋白 重组质粒构建 表达与鉴定 

分 类 号：R392.7[医药卫生—免疫学]