出血性大肠杆菌O157基因缺失疫苗株的构建及其免疫  

Construction and Immunization of a Attenuated Enterohemorrhagic Escherichia coli O157

作　　者：刘军[1] 孙洋[1] 冯书章[1] 

机构地区：[1]军事医学科学院军事兽医研究所,长春130062

出　　处：《生物工程学报》2007年第2期211-217,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金资助(No.30270985)~~

摘　　要：出血性大肠杆菌O157感染是重要的新发食物源性传染病,主要致病特征之一是能引起人肠上皮细胞特征性的A/E损伤,A/E损伤主要是由LEE致病岛所编码的毒力因子所引起,ler是LEE致病岛毒力基因群的中心调节基因,对LEE致病岛所编码的毒力因子有正调控作用。O157:H7另一个毒力因子是由整合到染色体上的原噬菌体编码的Stx毒素。以O157:H786-24为始发菌株,利用自杀性质粒pCVD442和同源重组的原理构建了O157:H7的ler基因缺失突变菌株(缺失了ler基因中第73-351位的碱基,共279bp),并利用噬菌体消除技术筛选到消除了编码Stx的原噬菌体DNA的菌株,构建出了O157:H7ler/stx基因缺失突变弱毒菌株,并对该菌株的Vero细胞毒性、小鼠模型的安全性以及乳鼠的被动免疫保护作用进行了研究。结果表明,O157:H7ler/stx基因缺失突变菌株丧失了对Vero细胞的毒性作用,并丧失了对实验小鼠的致病性,具有良好的安全性。乳鼠被动免疫保护性实验表明,用该菌株免疫母鼠后,乳鼠通过吸吮母乳可以获得良好的被动免疫保护作用。因此本研究所构建的O157:H7ler/stx基因缺失突变弱毒菌株可作为预防EHEC O157:H7感染的疫苗候选株,为最终研究制出O157的基因工程菌苗奠定基础。Enterohemorrhagie Escherichia coll （EHEC） O157：H7 is an important pathogen. One of the important virulence traits of EHEC O157 ： H7 is the capacity to produce attaching and effacing （A/E） lesions on enterocyte. This property encoded by a pathogenicity island termed the locus of enteroeyte effacement （LEE）. LEE contains ler （LEE-encoded regulator） gene. The product of ler is a central up-regulator of many virulence genes of the LEE. Another important virulence faetor of EHEC O157 ： H7 is Shiga toxin （Stx）, encoded by a prophage integrated into the chromosome of O157：H7, In order to obtain an attenuated vaccine candidate, a ler deletion mutant of O157：H7 was constructed by use of suicide veetor pCVD442. Meanwhile, due to potential instability of the prophage carrying the stx gene, the prophage was cured with serial passages of bacteria and confirmed by PCR and DNA sequencing. A lerlstx deletion mutant of EHEC O157 ： H7 was constructed, termed as O157 ： H7（AlerlAstx ）. The cultural supernatant of O157 ler/stx deletion mutant was inoculated in vero cell culture, and the result indicating that O157 lerlstx deletion mutant lost the toxigenieity to vero cell. Test group and eontrol group of mice were orogstrieally inoculated with the O157 ler/lstx deletion mutant and the virulent strain O157：H7 EDL933, respectively. Mice were observed daily for clinical signs and weight changes. After inoculation of the deletion mutant, test group of mice （inoculated with O157 ：H7（AlerlAstx） ） gained weight normally and experienced no clinical signs. In contrast, control group of mice （inoculated with O157： H7） exhibited weight loss and all died in four days. In another experiment, pregnant mice were orally vaccinated by O157：H7（Aler/ Astx） twice at interval of 14 days. Subsequently, the suckling mice were orally challenged with O157：H7 EDL933 at 7 days of age. The results showed that 78.34% of the sucking mice born by vaccinated mice were survival and 12.73% of the sucking

关 键 词：出血性大肠杆菌O157 基因缺失突变 免疫接种 弱毒疫苗 

分 类 号：R392[医药卫生—免疫学]