S.suis 2中国强毒株烯醇化酶Enolase基因的分子克隆及蛋白生物功能研究
发布时间：2018-10-31 21:28
【摘要】：对中国强毒株S.suis 2烯醇化酶Enolase进行克隆表达、定位分析、酶活性检测及免疫相关功能研究,探讨Enolase在S.suis 2致病中的作用。基于S.suis 2 05ZYH33全基因组测序,对Enolase编码基因进行生物信息学分析。构建pET32a∷eno重组表达质粒,转化入E.coli BL21中诱导表达,利用His-Beads和FPLC纯化后获得Enolase重组蛋白。对纯化后的Enolase进行酶活性检测,鉴定其糖代谢功能。然后利用FCM分析Enolase在S.suis 2的定位情况,最后通过外周血单核细胞MTT实验检测Enolase对PBMCs活性的影响。同源性分析发现S.suis 2 eno与多种细菌中eno高度同源。SignalP和TMHMM分析发现Enolase没有信号肽也没有跨膜区。eno分子克隆并测序显示长度为1 308 bp。重组质粒经诱导表达并纯化后,获得75 kD的Enolase蛋白。纯化的重组Enolase有将2-PEG转化成PEP的能力。FCM分析表明Enolase也存在于细菌表面。MTT测试表明Enolase能够引发PBMCs活性的下降。Enolase不仅在S.suis 2体内参与基础代谢活动,同时也存在于S.suis 2表面,可能通过破坏单核细胞参与感染过程。
[Abstract]:The Enolase of Chinese virulent strain S.suis _ 2 enolase was cloned and expressed, its localization, enzyme activity and immune-related function were studied to explore the role of Enolase in the pathogenesis of S.suis _ 2. Based on S.suis 2 05ZYH33 genome sequencing, the Enolase coding gene was analyzed by bioinformatics. The recombinant expression plasmid of pET32a: eno was constructed and transformed into E.coli BL21. The recombinant Enolase protein was purified by His-Beads and FPLC. The enzyme activity of purified Enolase was determined to identify its glucose metabolism function. Then FCM was used to analyze the localization of Enolase in S.suis 2. Finally, the effect of Enolase on PBMCs activity was detected by MTT assay in peripheral blood monocytes. Homology analysis showed that S.suis 2 eno was highly homologous to eno in many bacteria. SignalP and TMHMM analysis showed that Enolase had no signal peptide or transmembrane region. Eno molecular cloning and sequencing showed that the length of Enolase was 1 308 bp.. The recombinant plasmid was induced to express and purified, and 75 kD Enolase protein was obtained. The purified recombinant Enolase had the ability to convert 2-PEG to PEP. FCM analysis showed that Enolase also existed on the bacterial surface. MTT test showed that Enolase could induce the decrease of PBMCs activity. Enolase not only involved in basal metabolism in S.suis 2, but also in S.suis 2. It also exists on the surface of S.suis 2 and may be involved in the infection process by destroying monocytes.
【作者单位】： 扬州科技学院;中国人民解放军南京军区军事医学研究所;
【基金】：国家自然科学基金青年科学基金项目(31300119)
【分类号】：R378
[Abstract]:The Enolase of Chinese virulent strain S.suis _ 2 enolase was cloned and expressed, its localization, enzyme activity and immune-related function were studied to explore the role of Enolase in the pathogenesis of S.suis _ 2. Based on S.suis 2 05ZYH33 genome sequencing, the Enolase coding gene was analyzed by bioinformatics. The recombinant expression plasmid of pET32a: eno was constructed and transformed into E.coli BL21. The recombinant Enolase protein was purified by His-Beads and FPLC. The enzyme activity of purified Enolase was determined to identify its glucose metabolism function. Then FCM was used to analyze the localization of Enolase in S.suis 2. Finally, the effect of Enolase on PBMCs activity was detected by MTT assay in peripheral blood monocytes. Homology analysis showed that S.suis 2 eno was highly homologous to eno in many bacteria. SignalP and TMHMM analysis showed that Enolase had no signal peptide or transmembrane region. Eno molecular cloning and sequencing showed that the length of Enolase was 1 308 bp.. The recombinant plasmid was induced to express and purified, and 75 kD Enolase protein was obtained. The purified recombinant Enolase had the ability to convert 2-PEG to PEP. FCM analysis showed that Enolase also existed on the bacterial surface. MTT test showed that Enolase could induce the decrease of PBMCs activity. Enolase not only involved in basal metabolism in S.suis 2, but also in S.suis 2. It also exists on the surface of S.suis 2 and may be involved in the infection process by destroying monocytes.
【作者单位】： 扬州科技学院;中国人民解放军南京军区军事医学研究所;
【基金】：国家自然科学基金青年科学基金项目(31300119)
【分类号】：R378
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