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

doi:10.13560/j.cnki.biotech.bull.1985.2017.04.029

摘要/Abstract

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。

关键词: 猪链球菌2型（S. suis 2）, 烯醇化酶, 酶活性, 流式细胞术, 外周血单核细胞

Abstract: To investigate the role of enolase in the pathogenesis of highly virulent strains from Streptococcus suis serotype 2,its molecular cloning,protein location analysis,enzymatic activity assay,and immune related function were studied. Based on the whole genome sequencing of S. suis 2 05ZYH33,bioinformatics of gene eno encoding enolase was analyzed. The recombinant expression plasmid pET30a∷eno was transformed into Escherichia coli BL21 competent cells,and the expression was induced. Recombinant protein enolase was obtained by the purification with His-Beads and FPLC. Then the enzyme activity of enolase was detected,and its basal metabolic function was identified. Further,FCM was used to analyze the localization of enolase in S. suis 2. Finally,the effect of enolase on the activity of peripheral blood monouclear cells（PBMCs）was detected by MTT test. Homology analysis showed a highly homologous of eno with that in many others. SignalP and TMHMM analysis revealed that enolase had neither signal peptide sequence nor transmembrane domain structure. Molecular cloning and sequencing illustrated that the eno fragment length was 1 308 bp. The 75 kD protein was acquired after the recombinant plasmid was induced to express and purification. Enzyme activity assay showed that the purified recombinant enolase had the ability to convert 2-PEG into PEP. FCM analysis demonstrated that enolase also existed on the surface of bacteria. The results of MTT test showed that enolase resulted in the decrease of PBMCs activity. In conclusion,enolase is not only involved in the activities of glucose metabolism in S. suis 2,but also exists on its surface,probably involving in the infection process by destroying the mononuclear cells.

Key words: Streptococcus suis serotype 2（S. suis 2）, enolase, hemolytic activity, flow cytometry, peripheral blood monouclear cells

孙雯, 郑峰. 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[J]. 生物技术通报, 2017, 33(4): 222-230.

SUN Wen, ZHENG Feng. Molecular Cloning of Gene for Enolase in Highly Virulent Strains from Streptococcus suis serotype 2 and Its Protein Biological Function[J]. Biotechnology Bulletin, 2017, 33(4): 222-230.

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