鸭疫里默氏杆菌Mtan对底物SAH的催化活性

发布时间：2018-06-01 13:31

本文选题：鸭疫里默氏杆菌 + S-腺苷高半胱氨酸核苷酶　；参考：《微生物学通报》2017年05期

【摘要】：【目的】分析鸭疫里默氏杆菌(Riemerella anatipestifer,RA)不同血清型pfs基因的序列差异,并开展其编码蛋白S-腺苷高半胱氨酸核苷酶(Mtan,又称Pfs)的催化活性研究。【方法】PCR扩增9株不同血清型RA的pfs基因,分析其核苷酸序列的同源性;构建该基因的重组表达载体p Cold-RA-pfs,表达、纯化RA的重组蛋白Mtan(RA-Mtan);测定RA-Mtan对底物S-腺苷同型半胱氨酸(S-adenosylhomocysteine,SAH)的催化活性,运用哈维弧菌报告菌株BB170检测催化底物的自诱导物2(Autoinducer-2,AI-2)活性。【结果】对RA的pfs序列分析结果表明,不同血清型RA的核苷酸一致性在93.9%-100%之间;SDS-PAGE检测结果表明,RA-Mtan呈可溶性表达;酶活测定表明RA-Mtan和禽致病性大肠杆菌(Avain pathogenic Escherichia coli,APEC)的Lux S蛋白共同作用于底物时,可产生浓度为176.7μmol/L的同型半胱氨酸(Homocysteine,HCY);AI-2活性检测结果表明,产生的AI-2具有生物学活性。【结论】RA不同血清型的pfs高度保守,RA pfs基因的编码产物RA-Mtan在体外具有催化SAH的活性,RA-Mtan和禽致病性大肠杆菌的Lux S蛋白共同作用于底物SAH时,能产生有活性的AI-2,为进一步研究pfs对RA的调控作用提供参考。
[Abstract]:[objective] to analyze the sequence differences of pfs gene in different serotypes of Riemerella anatipestifera (RIA). The catalytic activity of its encoding protein S- adenosine homocysteine ribosidase (Pfs) was studied. [methods] the pfs gene of 9 strains of RA with different serotype was amplified by PCR and the homology of nucleotide sequence was analyzed. The recombinant expression vector pCold-RA-pfsof the gene was constructed to express and purify the recombinant protein of RA, and the catalytic activity of RA-Mtan to the substrate S-adenosylhomocysteine (S-adenosylhomocysteine) was determined. BB170 was used to detect the activity of autoinducer 2AI-2. [results] the pfs sequence analysis of RA showed that the nucleotide identity of different serotypes of RA was between 93.9-100%. The results of enzyme activity test showed that the activity of homocysteine (176.7 渭 mol/L) could be produced when RA-Mtan and Lux S protein of avian pathogenic Escherichia coli (Avain pathogenic Escherichia coli) co-acted on the substrate. [conclusion] the encoding product of pfs highly conserved RA pfs gene RA-Mtan has catalytic activity of SAH and Lux S protein of avian pathogenic Escherichia coli on the substrate SAH in vitro. It can produce active AI-2, which provides a reference for further study on the regulation of RA by pfs.
【作者单位】：中国农业科学院上海兽医研究所;扬州大学兽医学院;
【基金】：国家自然科学基金项目(No.31572546,31370045) 上海市科技兴农重点攻关项目(No.G20150109)~~
【分类号】：S852.61

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