结核分枝杆菌PPE59蛋白的表达、纯化和生物信息学分析  

Prokaryotic Expression,Purification and Bioinformatics Analysis of Mycobacterium tuberculosis PPE59 Protein

作　　者：王楚彤 袁美丽 魏婧 李敏英 许涛 汪洪涛 WANG Chutong;YUAN Meili;WEI Jing;LI Minying;XU Tao;WANG Hongtao(Laboratory Medicine Experimental Center,Laboratory Medicine College,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,安徽蚌埠233030 [2]蚌埠医学院检验医学院免疫学教研室 [3]蚌埠医学院检验医学院临床检验诊断学教研室 [4]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

出　　处：《长治医学院学报》2023年第1期1-7,共7页Journal of Changzhi Medical College

基　　金：安徽省自然科学基金项目(1908085MH252,2008085QH405);蚌埠医学院研究生科研创新计划项目(Byycx21008,Byycxz21016)。

摘　　要：目的:构建结核分枝杆菌pET28a-PPE59原核表达载体,异丙基-β-D半乳糖苷(IPTG)诱导表达和纯化获得PPE59蛋白;同时对PPE59蛋白进行生物信息学分析,预测其可能结构和功能。方法:以H37Rv基因组为模板扩增获得PPE59基因,利用同源重组将其克隆至原核表达载体pET28a,后转化至大肠杆菌表达菌株BL21(DE3)中,IPTG诱导表达,采用Ni~+-NTA亲和层析柱对PPE59蛋白进行纯化,获得重组PPE59蛋白并进行鉴定。使用Protparam、NetPhos 3.1 Servera、ABCpred、SYEPEITHI等软件对PPE59蛋白的理化性质、磷酸化位点和T、B细胞表位等进行预测和分析。结果:成功构建pET28a-PPE59重组载体,经诱导表达和纯化,成功获得PPE59蛋白;生物信息学预测发现,PPE59有2个开放阅读框,31个磷酸化位点,并含有多个T细胞和B细胞抗原表位。结论:成功构建pET28a-PPE59表达载体,获得高纯度PPE59蛋白,且被抗His-tag小鼠单抗特异性识别;PPE59具有大量的磷酸化位点及丰富的T、B细胞表位,可作为结核病疫苗的重要候选蛋白之一。Objective:To construct Mycobacterium tuberculosis pET28a-PPE59 prokaryotic expression vector,induce the expression of PPE59 by IPTG and purify the protein.Meanwhile,bioinformatics analysis of the PPE59 protein was performed to predict its possible structure and function.Methods:The PPE 59 gene was obtained by amplifying the H37Rv genome as a template,cloned into the prokaryotic expression vector pET28a by means of homologous recombination,and then transformed into E.coli expression strain BL21(DE3),and IPTG was used to induce expression.The recombinant PPE59 was purified by Ni+-NTA affinity chromatography colum and then identified.The physicochemical properties,phosphorylation sites and T and B cell epitopes of PPE59 protein were predicted and analyzed using Protparam,NetPhos 3.1 Servera,ABCpred,SYEPEITHI and other software.Results:The pET28a-PPE59 recombinant vector was successfully constructed,and the PPE59 protein was successfully obtained by induced expression and purification.Bioinformatics prediction revealed that PPE59 had 2 open reading frames,31 phosphorylation sites,and contained several T and B cell antigenic epitopes.Conclusion:The pET28a-PPE59 expression vector was successfully constructed,and the high purity PPE59 protein was obtained,which was specifically recognized by the anti-His-tag mouse monoclonal antibody.PPE59 has a large number of phosphorylation sites and abundant T and B cell epitopes,and can be one of the important candidate proteins for the tuberculosis vaccine.

关 键 词：结核分枝杆菌 PPE59蛋白 原核表达 生物信息学 

分 类 号：R378.911[医药卫生—病原生物学]