结核分枝杆菌GroES蛋白表达、纯化及其生物信息学分析  

Prokaryotic expression,purification and bioinformatics analysis of protein GroES from Mycobacterium tuberculosis

作　　者：郭方正 魏婧 宋亚敏 袁美丽 王楚彤 李柏青 汪洪涛 许涛 Guo Fangzheng;Wei Jing;Song Yamin;Yuan Meili;Wang Chutong;Li Baiqing;Wang Hongtao;Xu Tao(Experimental Center of Laboratory Medicine,School of Laboratory Medicine,Bengbu Medical University&Anhui Provincial Key Laboratory of Immunology Basis and Clinic in Chronic Disease,Bengbu,Anhui 233030,China;Teaching and Research Office of Immunology,School of Laboratory Medicine,Bengbu Medical University,Bengbu,Anhui 233030,China;Teaching and Research Office of Clinical Diagnosis,School of Laboratory Medicine,Bengbu Medical University,Bengbu,Anhui 233030,China)

机构地区：[1]蚌埠医学院检验医学院检验医学实验中心,慢性疾病免疫学基础与临床安徽省重点实验室,安徽蚌埠233000 [2]蚌埠医学院检验医学院免疫学教研室,安徽蚌埠233000 [3]蚌埠医学院检验医学院临床检验诊断学教研室,安徽蚌埠233000

出　　处：《齐齐哈尔医学院学报》2024年第4期306-313,共8页Journal of Qiqihar 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)。

摘　　要：目的 将结核分枝杆菌H37Ra株GroES蛋白进行原核表达与纯化,并对其进行生物信息学分析。方法 通过PCR扩增GroES基因并克隆至pET28a中,测序鉴定后将pET28a-GroES转化至大肠杆菌BL21(DE3)中,IPTG诱导GroES蛋白表达,SDS-PAGE分析表达产物,镍亲和层析柱纯化重组GroES蛋白,Western blot鉴定GroES的免疫反应性。应用ORF Finder、ProtParam、TMHMM Server 2.0、NetPhos 3.1 Server、NetNGlyc1.0 Server、SignalP 4.1 Server、BLAST、Softberry、PSORT、Vaxijen、SOPMA、SWISS-MODE、ABC-pred、IEDB、SYFPEITHI、STRING工具对GroES的结构和功能进行生物信息学预测。结果 成功构建重组pET28a-GroES重组体,用ITPG诱导后GroES在大肠杆菌以可溶性形式表达,纯化后GroES纯度达90%,Western blot证实GroES具有免疫反应性。生物信息学显示GroES蛋白由100个氨基酸组成,分子式为C478H776N124O147S1,等电点为4.62,为亲水性蛋白,有9个磷酸化位点,无跨膜区及信号肽序列,其编码基因MRA-3458全长303bp,含1个开放阅读框;GroES二级结构中,α-螺旋占5%,β-折叠占37%,β-转角占8%,无规则卷曲占50%;预测GroES具有多种与之相互作用的蛋白质和潜在的T、B细胞表位。结论 成功表达和纯化具有免疫反应性的重组GroES蛋白,并预测了GroES结构与功能,提示GroES可作为制备结核疫苗或药物的候选蛋白,为更深入理解结核分枝杆菌的感染免疫机制奠定基础。Objective To perform the prokaryotic expression and purify GroES protein of Mycobacterium tuberculosis(H37Ra),and to analyze its bioinformatics.Methods The GroES gene was amplified by PCR and cloned into pET28a.After sequencing,pET28a-GroES was transformed into E.coli BL21(DE3).The expression of GroES protein was induced by IPTG,and the expression product was analyzed by SDS-PAGE.Purification of recombinant GroES protein by nickel affinity chromatography.The immunoreactivity of GroES was determined by Western blot.Bioinformatics prediction tools(including ORF Finder,ProtParam,TMHMM Server 2.0,NetPhos 3.1 Server,NetNGlyc1.0 Server,SignalP 4.1 Server,BLAST,Softberry,PSORT,Vaxijen,SOPMA,SWISS-MODE,ABC-pred,IEDB,SYFPEITHI,STRING) were used to predict structure and function of GroES.Results The recombinant pET28a-GroES was successfully constructed.GroES were expressed in soluble form in E.coli after induced by ITPG.The purity of GroES reached 90% after purification,and the immunoreactivity of GroES was verified by Western blot.Bioinformatics analysis showed that GroES protein was composed of 100 amino acids,the molecular formula was C_(478)H_(776)N_(124)O_(147)S1,the isoelectric point was 4.62,it was a hydrophilic protein,there were 9 phosphorylation sites,without transmembrane region and signal peptide sequence,its encoding gene MRA-3458 was 303bp in length,including an open reading frame.In GroES secondary structure,α-helix accounted for 5%,β-folding accounted for 37%,β-turning accounted for 8%,random curling accounted for 50%.It was predicted that Gro ES had a variety of proteins interacting with it and potential T and B cell epitopes.Conclusions The immunoreactive recombinant Gro ES protein is successfully expressed and purified in E.coli.The structure and function of Gro ES is successfully predicted,suggesting that Gro ES could be used as a candidate protein for preparing tuberculosis vaccines or drugs,and it lays a foundation for further understanding of the immune mechanism of Mycobacterium tuberculosis infe

关 键 词：结核分枝杆菌 GROES 基因重组 生物信息学 

分 类 号：R310.34[医药卫生—基础医学]