单增李斯特菌新疆临床分离株毒力基因的克隆和序列分析

Cloning and Analysis of Virulence Genes of Listerisa Monocytogenes in Xinjiang Clinical Strains

为了研究单增李斯特菌新疆羊源临床分离的4b血清型菌株(简称LM90)毒力基因的变异情况,参考Genbank上收录的单增李斯特菌4b血清型菌株F2365(GeneBank登陆号AE017262)的Ami、Auto、InlB、InlC、InlJ基因序列,分别设计引物,对LM90的毒力基因Ami、Auto、InlB、InlC、InlJ的最大开放阅读框进行PCR扩增,回收基因序列,连接到PMD19-T载体上进行克隆,筛选阳性菌进行序列的测定,将序列提交到NCBI上(登录号分别为KF826613、KF826614、KF826611、KF703749、KF826612),测序后用DNAMAN软件对序列进行分析。结果显示:LM90的毒力基因Ami、Auto、InlB、InlC、InlJ与F2365毒力基因Ami、Auto、InlB、InlC、InlJ的同源性分别为98.04%、96.53%、98.20%、96.41%、98.23%,氨基酸的同源性分别为99.73%、99.83%、98.67%、99.66%、99.76%,可见单增李斯特菌新疆临床分离株LM90毒力基因Ami、Auto、InlB,InlC、InlJ序列相对稳定。

The aim of this study was to investigate the variation of virulence genes （Ami,Auto,InlB,InlC and InlJ） of Listeria monocytogenes serotype 4b LM90 isolated from clinical diseased sheep in Xinjiang.The open reading frames （ORFs） of five genes were amplified by PCR with specific primers designed according to the nucleotide sequence of serotype 4b F2365 （accession number： AE017262）.The PCR products were purified and cloned into pMD19-T vector,and then positive colonies were screened and sequenced.These gene sequences of Ami,Auto,InlC and InlJ were submitted to GenBank （accession numbers：KF826613,KF826614,KF826611,KF703749,and KF826612,respectively） and analyzed by DNAMAN.Alignment results indicated that the homology of the five virulence genes was 98.04%,96.53%,98.20%,96.41%,and 98.23% at nucleotide sequence level,and 99.73%,99.83%,98.67%,99.66% or 99.76% at amino acid sequence level between LM90 and F2365, respectively.Our research results revealed that the five tested virulence genes of Listeria monocytogenes LM90 are relatively stable.