【摘要】：對中國強毒株S.suis 2烯醇化酶Enolase進行克隆表達、定位分析、酶活性檢測及免疫相關功能研究,探討Enolase在S.suis 2致病中的作用�；赟.suis 2 05ZYH33全基因組測序,對Enolase編碼基因進行生物信息學分析。構建pET32a∷eno重組表達質粒,轉化入E.coli BL21中誘導表達,利用His-Beads和FPLC純化后獲得Enolase重組蛋白。對純化后的Enolase進行酶活性檢測,鑒定其糖代謝功能。然后利用FCM分析Enolase在S.suis 2的定位情況,最后通過外周血單核細胞MTT實驗檢測Enolase對PBMCs活性的影響。同源性分析發(fā)現(xiàn)S.suis 2 eno與多種細菌中eno高度同源。SignalP和TMHMM分析發(fā)現(xiàn)Enolase沒有信號肽也沒有跨膜區(qū)。eno分子克隆并測序顯示長度為1 308 bp。重組質粒經誘導表達并純化后,獲得75 kD的Enolase蛋白。純化的重組Enolase有將2-PEG轉化成PEP的能力。FCM分析表明Enolase也存在于細菌表面。MTT測試表明Enolase能夠引發(fā)PBMCs活性的下降。Enolase不僅在S.suis 2體內參與基礎代謝活動,同時也存在于S.suis 2表面,可能通過破壞單核細胞參與感染過程。
[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.
【作者單位】： 揚州科技學院;中國人民解放軍南京軍區(qū)軍事醫(yī)學研究所;
【基金】：國家自然科學基金青年科學基金項目(31300119)
【分類號】：R378

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