结核分枝杆菌分子伴侣Acr2蛋白的原核表达及其功能分析  

Prokaryotic expression and functional analysis of molecular chaperone Acr2 protein of Mycobacterium tuberculosis

作　　者：张宗欣 赵纪元 孙红宾 ZHANG Zongxin;ZHAO Jiyuan;SUN Hongbin(School of Food and Biological Engineering,Zhengzhou University of Light Industry,Zhengzhou 450002,Henan Province,China)

机构地区：[1]郑州轻工业大学食品与生物工程学院,河南郑州450002

出　　处：《中国生物制品学杂志》2024年第1期37-42,共6页Chinese Journal of Biologicals

基　　金：河南省自然科学基金面上项目(212300410415)。

摘　　要：目的 在E.coli中表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)分子伴侣Acr2蛋白,并分析其功能。方法 将重组质粒pET-28a-Acr2转化感受态E.coli BL21(DE3),IPTG诱导表达His-Acr2蛋白,经Ni-NTA层析及SuperdexTM200 10/300 GL凝胶过滤层析纯化获得Acr2蛋白。将Acr2蛋白经热变性(100℃温浴15 min)及化学变性(8 mol/L尿素,37℃温浴4 h)后进行自发再折叠和再组装的复性处理,采用圆二色性光谱法和非变性SDS-PAGE检测变性及复性后Acr2蛋白的二级结构。通过底物结合试验检测Acr2蛋白在体外的分子伴侣功能。结果 纯化Acr2蛋白的相对分子质量约为232 000,纯度达90%以上,浓度约为2 mg/mL。Acr2蛋白经变性-复性处理后可恢复其天然二级结构,且在48℃条件下可与变性的苹果酸激酶(malate dehydrogenase,MDH)形成稳定复合物。结论 Acr2蛋白经变性-复性处理后可恢复其天然分子构象,且具有体外分子伴侣活性。本研究为制备具有天然活性的结核杆菌蛋白抗原提供了新策略。Objective To express the molecular chaperone Acr2 protein of Mycobacterium tuberculosis(Mtb)in E.coli and analyze the function.Methods The recombinant plasmid pET-28a-Acr2 was transformed into competent E.coli BL21(DE3),and induced by IPTG.The expressed His-Acr2 protein was purified by Ni-NTA chromatography and SuperdexTM200 10/300 GL gel filtration chromatography to obtain Acr2 protein.The Acr2 protein was refolded by spontaneous refolding and reassembly after thermal denaturation(100 ℃ for 15 min)and chemical denaturation(8 mol/L urea,37 ℃ for 4 h).The secondary structure of Acr2 protein before and after denaturation-renaturation was detected by circular dichroism spectroscopy and non-denaturing SDS-PAGE,and the molecular chaperone function of Acr2 protein in vitro was detected by substrate binding assay.Results The purified Acr2 protein had the relative molecular mass of about 232 000,the purity of over 90%,and the concentration of about 2 mg/mL,which recovered its natural secondary structure after denaturationrenaturation,and formed stable complexes with the denatured malate dehydrogenase(MDH)at 48 ℃.Conclusion The Acr2protein can restore its natural molecular conformation with molecular chaperone activity in vitro after denaturation-renaturation treatment,providing a new strategy for the preparation of Mtb protein antigen with natural activity.

关 键 词：结核分枝杆菌 小分子热休克蛋白 Acr2蛋白 变性 复性 分子伴侣 

分 类 号：R378.911[医药卫生—病原生物学]