结核分枝杆菌GroEL2蛋白表达、纯化及生物信息学分析  

Expression,purification,and bioinformatics analysis of Mycobacterium tuberculosis GroEL2 protein

作　　者：宋亚敏 魏婧 郭方正 李柏青 许涛[1,2,4] 汪洪涛 SONG Yamin;WEI Jing;GUO Fangzheng;LI Baiqing;XU Tao;WANG Hongtao(Laboratory Center of Laboratory Medicine,College of Laboratory Medicine,Bengbu Medical College,Bengbu 233030,China;Anhui Key Laboratory of Basic and Clinical Immunology of Chronic Diseases,Bengbu Medical College;Department of Immunology,College of Laboratory Medicine,Bengbu Medical College;Department of Clinical Laboratory Diagnostics,College of Laboratory Medicine,Bengbu Medical College)

机构地区：[1]蚌埠医学院检验医学院检验医学实验中心,蚌埠233030 [2]蚌埠医学院慢性疾病免疫学基础与临床安徽省重点实验室 [3]蚌埠医学院检验医学院免疫学教研室 [4]蚌埠医学院检验医学院临床检验诊断学教研室

出　　处：《山西医科大学学报》2023年第12期1591-1599,共9页Journal of Shanxi Medical University

基　　金：安徽省自然科学基金项目(1908085MH252,2008085QH405);慢性疾病免疫学基础与临床安徽省重点实验室开放课题基金项目(KLICD-2002-Z3);呼吸系病临床基础安徽省重点实验室开放课题基金项目(HX2022-Z02);蚌埠医学院“512人才培育计划”项目(by51201309);蚌埠医学院研究生科研创新计划项目(Byycx22013,Byycx23078)。

摘　　要：目的克隆、表达和纯化结核分枝杆菌H37Ra株GroEL2蛋白,并进行生物信息学分析。方法以结核分枝杆菌H37Ra株基因组DNA为模板,PCR扩增GroEL2基因,克隆至pET-28a载体中,构建重组pET-28a-GroEL2载体,将其转化至BL21(DE3)菌株中,在异丙基硫代半乳糖苷(isopropylβ-D-thiogalactoside,IPTG)诱导表达后,利用十二烷基硫酸钠-聚丙烯酰胺(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)电泳分析表达产物,镍-亚氨基二乙酸(Ni-IDA)亲和层析柱纯化重组GroEL2蛋白,Western blot鉴定GroEL2的表达效果。利用生物信息学方法对GroEL2蛋白的理化性质、蛋白结构、抗原表位等进行预测。结果成功构建重组pET-28a-GroEL2表达载体,在大肠杆菌中以可溶形式表达GroEL2蛋白。Ni-IDA亲和层析柱纯化重组GroEL2蛋白,纯度达90%以上。生物信息学分析显示其能与多种蛋白相互作用,且存在多个潜在的T细胞、B细胞抗原表位。结论重组GroEL2蛋白具有良好的免疫反应性,可能是结核病新型疫苗和诊断方法开发的新靶点。Objective To clone,express,and purify the GroEL2 protein of Mycobacterium tuberculosis strain H37Ra and perform its bioinformatics analysis.Methods GroEL2 gene was amplified with the genomic DNA of Mycobacterium tuberculosis strain H37Ra as the template by PCR,and then cloned into the pET-28a vector to construct a recombinant pET-28a-GroEL2 vector,which was transformed into BL21(DE3)strain and expressed in an isopropylβ-D-thiogalactoside(IPTG).The expression products were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),the recombinant GroEL2 protein was purified by nickel-iminodiacetic acid(Ni-IDA)affinity chromatography column,and the expression efficacy of GroEL2 was identified by Western blot.Bioinformatics methods were used to predict the physicochemical properties,the protein structure,and the antigenic epitopes of GroEL2 protein.Results The recombinant pET-28a-GroEL2 expression vector was successfully constructed and the GroEL2 protein was expressed in soluble form in E.coli.The recombinant GroEL2 protein was purified by Ni-IDA affinity chromatography column with a purity of over 90%.Bioinformatics analysis showed that it could interact with a variety of proteins and there were several potential T and B cell antigenic epitopes.Conclusion The recombinant GroEL2 protein has good immunoreactivity and can be used as a new target for the development of novel vaccines and the diagnosis for tuberculosis.

关 键 词：结核分枝杆菌 GroEL2蛋白 原核表达 蛋白纯化 生物信息学 

分 类 号：R52[医药卫生—内科学]