结核分枝杆菌PPE68蛋白稳定片段的原核表达及纯化  

Prokaryotic expression and purification of a stabilized fragment of Mycobacterium tuberculosis PPE68 protein

作　　者：程龙云 谢荣现 李丽 袁西露 祝姚姚 刘晓宇 李冰清 CHENG Longyun;XIE Rongxian;LI Li;YUAN Xilu;ZHU Yaoyao;LIU Xiaoyu;LI Bingqing(Department of Pathogen Biology,School of Clinical and Basic Medicine,Shandong First Medical University&Shandong Academy of Medical Sciences,Jinan 250000,China)

机构地区：[1]山东第一医科大学(山东省医学科学院)临床与基础医学院(基础医学研究所)病原生物学系,山东济南250000

出　　处：《中国病原生物学杂志》2025年第3期316-320,共5页Journal of Pathogen Biology

基　　金：国家自然科学青年基金项目(No.32300019)。

摘　　要：目的了解结核分枝杆菌PPE68稳定片段,对稳定片段蛋白进行原核表达及纯化,为PPE68的结构解析奠定基础。方法使用Expasy-ProParam、MEME等生信网站对PPE68的基本性状和序列进化保守性进行分析;使用双酶切方法,PCR扩增PPE68基因,并克隆到pGL01载体上;使用大肠埃希菌BL21(DE3)进行PPE68片段蛋白的原核表达,在IPTG(isopropylβ-D-thiogalactoside)诱导表达后,利用SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)电泳分析表达产物并使用镍离子亲和层析及凝胶过滤层析的方法对表达产物进行体外纯化。后通过胰蛋白酶切实验、质谱分析确定PPE68最稳定片段。结果生物信息学分析初步确定了PPE68可能的稳定片段为第7-354个氨基酸并成功构建pGL01-PPE68(7-354aa)表达载体,蛋白表达纯化后获得2.4mg/mL的PPE68(7-354aa)片段蛋白但纯度较低;针对纯化的PPE68(7-354aa)蛋白较杂问题进行胰蛋白酶切和质谱分析找到稳定片段,为PPE68的第7-180个氨基酸,构建质粒后在大肠埃希菌中能稳定表达并获得2.9mg/mL的纯化蛋白,蛋白纯度可达95%以上。结论成功找到结核分枝杆菌PPE68的稳定氨基酸序列,为未来PPE68蛋白晶体结构解析以及进一步结核病诊断和疫苗开发奠定基础。Objective To investigate the stable fragment of Mycobacterium tuberculosis PPE68,prokaryotic expression and purification of the stable fragment protein were performed,laying the foundation for structural analysis of PPE68.Methods The fundamental characteristics and evolutionary sequence conservation of PPE68 were examined utilizing Expasy-ProParam,MEME,and various other bioinformatics analysis websites.The PPE68 gene was subsequently amplified via PCR and cloned into the pGLol vector through a double digestion technique.Prokaryotic expression of the PPE68 fragment protein was conducted in Escherichia coli BL21(DE3).Following induction with IPTG(isopropylβ-D-thiogalactoside),the expressed products were analyzed by SDS-PAGE(sodium dodecyl sulfatepolyacrylamide gel electrophoresis).Subsequently,the expressed products were purified in vitro using nickel ion affinity chromatography and gel filtration chromatography.Finally,trypsin digestion experiments and mass spectrometry analysis were performed to identify the most stable fragment of PPE68.Results Bioinformatics analysis preliminarily identified the potential stable fragment of PPE68 as the amino acids 7 to 354.The expression vector pGL01-PPE68(7-354 aa)was successfully constructed.The purification of protein expression resulted in a concentration of 2.4 mg/mL for the PPE68(7-354aa)fragment protein;however,the purity was suboptimal.To address the issue of heterogeneity in the purified PPE68(7-354aa)protein,we employed trypsin digestion followed by mass spectrometry analysis.This approach allowed us to identify the stable fragment corresponding to PPE68 as 7-180 amino acids.After constructing the plasmid,stable expression was achieved in Escherichia coli,resulting in a purified protein concentration of 2.9 mg/mL with a purity exceeding 95%.ConclusionThe stable amino acid sequence of Mycobacterium tuberculosis PPE68 was successfully found,which laid a foundation for the crystal structure analysis of PPE68 protein in the future,as well as further tuberculosis diagnos

关 键 词：结核分枝杆菌 PPE68 原核表达 蛋白纯化 

分 类 号：R37[医药卫生—病原生物学]