鸭疫里默氏杆菌Mtan对底物SAH的催化活性

摘要

[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,pCold-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 Vibrio harveyi 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 LuxS (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 BB 170,demonstrating that recombinant RA-Mtan and LuxS 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.%[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pCold-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LuxS蛋白共同作用于底物时,可产生浓度为176.7 μmol/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LuxS 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.