奶牛隐性乳房炎中致病性大肠杆菌luxS及pfs基因表达及AI-2体外合成

Expression of luxS and pfs gene and biosynthesis AI-2 of pathogenic E.coli isolated from bovine subclinical mastitis in vitro

为了了解luxS基因和pfs基因变异情况,探索信号分子AI-2(Autoinducer-2,AI-2)体外合成的影响因素。本试验应用PCR法扩增了奶牛隐性乳房炎致病性大肠杆菌E285的luxS和pfs基因,经酶切后插入原核表达载体pET-30a(+)中,经PCR和酶切鉴定后进行序列测定及分析,构建luxS和pfs基因的重组表达质粒pET-luxS和pET-pfs,经诱导、纯化获得可溶性重组LuxS蛋白和Pfs蛋白,利用纯化的重组蛋白LuxS和Pfs体外合成AI-2分子。结果表明,扩增获得的luxS基因和pfs基因高度保守,与GenBank中已发表的核苷酸序列同源性均达到98%以上;两基因在原核表达系统中均获得了高效表达,利用获得重组蛋白LuxS和Pfs体外催化SAH,获得了AI-2分子,其浓度为200μmol/L,对其活性检测显示为阴性对照DH5α的61倍。提示奶牛隐性乳房炎致病性大肠杆菌E285的LuxS和Pfs蛋白在体外能催化SAH产生高活性的AI-2。为深入研究AI-2对E285的毒力基因表达、生物被膜形成的调控奠定基础。

In order to understand the variation of luxS gene and pfs gene,we anauped the factors affecting the synthesis of the signal molecule AI-2(Autoinducer-2,AI-2)in vitro.In this study,the luxS and pfs fragments of pathogenic E285 were amplified by PCR,and inserted into vector pET-30a(+)after digested by restriction enzyme.After PCR identification,restriction enzyme analysis and sequencing,the recombinant expression plasmid pET-luxS and pET-pfs were constructed successfully.The soluble recombinant LuxS and Pfs proteins were obtained and used to biosynthesize after inducted and purifiartion,and the purified proteins LuxS and Pfs were incubated with S-ribosylhomocysteine(SAH)to produce AI-2 in vitro.The results showed that the luxS gene and pfs gene both were highly conserved,the nucleotide homology was more than 98%compared with the nucleotide sequence published in Genbank.The luxS and pfs gene are highly expressed in prokaryotic expression system,and the SAH was catalyzed by purified recombinant proteins LuxS and Pfs and produced 200μmol/L AI-2,The AI-2 activity has detected by the V.harveyi bioluminescence assay was 352 times of that of the negative control DH5α.It is indicated that the LuxS and Pfs proteins of the pathogenic Escherichia coli E285 of bovine subclinical mastitis can catalyze SAH to produce highly activity AI-2 in vitro.