鸭疫里默氏杆菌Mtan对底物SAH的催化活性  

Catalytic activity of Mtan catalyzing S-adenosylhomocysteine in Riemerella anatipestifer

作　　者：吴小卡 徐达[1,2] 荆雅玮 吕小龙[1] 胡剑刚 米荣升[1] 黄燕[1] 王成明[1,2] 陈兆国[1] 韩先干[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《微生物学通报》2017年第5期1165-1170,共6页Microbiology China

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)~~

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。[Objective] The sequences of pfs gene(encoding the Mtan protein, also known as Pfs) from different serotypes of Riemerella anatipestifer(RA) were analyzed, and catalytic activity of Mtan was studied. [Methods] The different serotypes of RA pfs gene were amplified by PCR and then the homology of nucleotide sequences was analyzed. The recombinant plasmid, p Cold-RA-pfs was constructed, and then expressed in BL21 and the recombinant protein RA-Mtan was purified. Furthermore, the activity of RA-Mtan catalyze S-adenosylhomocysteine(SAH) to produce Homocysteine(HCY) was evaluated by Ellman's assay, and the activity of AI-2 was detected by Vibrioharveyi reporter strain BB170. [Results] The sequence analysis of pfs indicated that the homology of different serotypes varied from 93.9% to 100%. The SDS-PAGE showed that RA-Mtan was soluble expression in BL21. Moreover, the result suggested that RA-Mtan and recombinant protein Lux S(from Avain pathogenic Escherichia coli) could catalyze SAH to produce 176.7 μmol/L HCY. The reaction products were able to induce luminescence of Vibrio harveyi BB170, demonstrating that recombinant RA-Mtan and Lux S synthesize AI-2 in vitro from SAH. [Conclusion] The RA pfs genes from different serotypes were highly conserved. The RA-Mtan can catalyze SAH to produce HCY, and produce AI-2 with biological activity as well. This study will contribute to further study of the roles of pfs in RA.

关 键 词：鸭疫里默氏杆菌 S-腺苷高半胱氨酸核苷酶 S-腺苷同型半胱氨酸 密度感应系统 自诱导物质AI-2 

分 类 号：S852.61[农业科学—基础兽医学]