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目的构建幽门螺杆菌IV型分泌系统组分中与CagA转运相关蛋白cagE(HP0544)、cagδ(HP0522)及cagβ(HP0524)编码基因重组质粒,在大肠埃希菌中表达并分析表达蛋白的免疫反应性。方法根据在线预测的3种蛋白信号肽及跨膜区特征,以标准菌株HP26695染色体DNA为模板扩增相关蛋白编码片段,与表达载体pET28a及pGEX4t-1连接后转入大肠埃希菌BL21(DE3)中诱导表达,分析蛋白表达状态,用Western blot检测表达蛋白与HP26695全菌蛋白免疫血清的反应。结果测序及飞行质谱鉴定表明成功构建CagE、Cagδ及Cagβ的表达载体,其中Cagδ可大量可溶表达,CagE及Cagβ主要以包涵体形式表达。Westernblot显示3种表达蛋白均能被HP26695全菌蛋白免疫血清识别,但Cagδ的免疫反应性较弱。结论成功获得HP26695的与CagA转运相关CagE、Cagδ及Cagβ重组蛋白,其中CagE和Cagβ具有较好的免疫反应性,是潜在的药物靶点和诊断生物标志物。
Objective To construct a recombinant plasmids encoding CagE (HP0544), cagδ (HP0522) and cagβ (HP0524) gene of Helicobacter pylori type IV secretion system and express in Escherichia coli and analyze the expressed protein immunoreactivity Sex. Methods According to the on-line prediction of three protein signal peptides and their transmembrane domain features, the HP26695 chromosomal DNA of the standard strain was used as a template to amplify the related protein coding fragment and ligated with the expression vectors pET28a and pGEX4t-1 and then transformed into Escherichia coli BL21 (DE3 ), And the protein expression status was analyzed. Western blot was used to detect the reaction of the expressed protein with the whole serum of HP26695. Results Sequencing and mass spectrometry identification showed that CagE, Cagδ and Cagβ were successfully constructed. Among them, Cagδ was soluble in a large amount and CagE and Cagβ were mainly expressed in inclusion bodies. Western blot showed that the three expressed proteins were all recognized by HP26695 whole-cell immune serum, but immunoreactivity of Cagδ was weak. Conclusion The recombinant proteins of CagE, Cagδ and Cagβ related to CagA transport of HP26695 were successfully obtained. Among them, CagE and Cagβ have good immunoreactivity and are potential drug targets and diagnostic biomarkers.