CAJ | 학술논문

目的构建结核分枝杆菌mpt51基因原核表达载体并进行表达、纯化.方法用PCR扩增结核分枝杆菌mpt51基因,并克隆入pTA2质粒.测序正确后.再亚克隆人pET30a(+)质粒,构建pET30a(+):mpt51重组体.结果以pET30a(+):mpt51转化BL21(DE3)后.经0.4 mmol/L异丙基硫代-β-D-半乳糖苷诱导,重组菌表达出相对分子质量为38 000的重组蛋白.SDS-PAGE分析显示,异丙基硫代-β-D-半乳糖苷诱导4小时重组蛋白的表达量最高.表达蛋白以包涵体形式存在于胞质中.表达量占全菌蛋白质的30%.经Ni-NTA树脂纯化,获得纯度为90%的重组蛋白.结论成功地构建原核表达载体pET30a(+):mpt51,并获得MPT51重组蛋白.为血清学诊断活动性结核病奠定了基础.
목적 구건결핵분지간균mpt51기인원핵표체재체병진행표체、순화.방법 용PCR확증결핵분지간균mpt51기인,병극륭입pTA2질립.측서정학후.재아극륭인pET30a(+)질립,구건pET30a(+):mpt51중조체.결과 이pET30a(+):mpt51전화BL21(DE3)후.경0.4 mmol/L이병기류대-β-D-반유당감유도,중조균표체출상대분자질량위38 000적중조단백.SDS-PAGE분석현시,이병기류대-β-D-반유당감유도4소시중조단백적표체량최고.표체단백이포함체형식존재우포질중.표체량점전균단백질적30%.경Ni-NTA수지순화,획득순도위90%적중조단백.결론 성공지구건원핵표체재체pET30a(+):mpt51,병획득MPT51중조단백.위혈청학진단활동성결핵병전정료기출.
Objective To construct,express and purify prokaryotic expression vector carrying mpt51 gene from Mycobacterium tuberculosis. Methods Mycobacterium tuberculosis mpt51 gene was amplified by PCR and then cloned into plasmid pTA2.After sequeneing,the gene was cloned into plasmid pET30a(+)to construct recombinant prokaryotic expression vector pET30a(+):mpt51.Results After transformation of the Escherichia coli and induction with 0.4 mmol/L isopropy-β-D-thiogalactoside(IPTG),recombinant target protein mpt51 with relative molecular mass 38 000 was expressed in pET30a(+):mpt51 system,and the expression of recombinant protein was maximum when induced with IPTG for 4 hours.The expressed protein existed in cytoplasm in cytoryctes form and amounted to 30% of total protein in Escherichia coli.The purity of the protein purified through the Ni-NTA resin reached 90%.Conclusions The prokaryotic expression vector pET30a(+):mpt51 is successfully constructed and the recombinant protein MPT51 is obtained,which lay the foundation for serological diagnosis of active tuberculosis.