A/E大肠杆菌疫苗动物模型的建立

An Animal Model of Attaching and Effacing E.coli Infection and Immunization

致病性和出血性大肠杆菌 (代表株 EPEC E2 34 8和 EHEC O15 7)是婴幼儿腹泻、出血性结肠炎和尿路感染综合征 (尿毒症 )的主要病原菌 ,它们同属于粘附脱落 (Attaching/ Effacing,A/ E)大肠杆菌群 ,具有许多共同的毒力基因 ,定位于致病岛 L EE(the locus of enterocyte effacement)上。本试验主要就 A/ E大肠杆菌致病岛 L EE的 2个主要调节基因 lux和 ler基因对细菌致病性和免疫原性的影响进行了研究。所使用的始发菌株为兔致病性大肠杆菌 RDEC- 1,根据同源重组的原理 ,利用自杀性载体 p CVD44 2技术 ,敲除了位于染色体上的 lux和 ler基因 ,构建了 lux和 ler基因缺失突变株 ,研究了这 2个基因对细菌生长、毒力因子表达的调控作用以及基因缺失突变株的致病性和免疫保护作用。家兔实验研究表明 ,lux基因缺失突变株仍然残存着部分致病作用 ,不足以成为理想的致弱疫苗 ;而 ler基因缺失突变株安全性好 ,具有良好的免疫保护作用 ,是理想的家兔致弱疫苗候选株。这些研究资料为人 A/ E大肠杆菌疫苗 ,尤其是 EHEC O15 7疫苗的研制指明了方向 ,并提供了技术路线。

Attaching and effacing (A/E) E.coli strains include human enteropathogens responsible for dehydrating infantile diarrhea,hemorrhagic colitis and the hemolytic uretic syndrome as well as for diarrheal disease in several animal species.All of these pathogens share highly-conserved virulence genes encoded on a 35 kb pathogenicity island(PAI) termed the loccus of enterocyte effacement (LEE).The luxS gene and ler (LEE encoded regulator) gene are up-regulators of many of the virulence genes of the LEE,including eae gene which encodes an adherence intimin,an important outer membrane protein of the type Ⅲ secretion system.For EHEC and EPEC,in vivo roles for luxS and ler in bacterial virulence have not been documented.The goals of this study were to genetically characterize luxS and ler of rabbit EPEC strain,RDEC-1, to determine the effects of deletion of luxS and ler on in vivo virulence.In order to determine whether deletion of the luxS or ler would attenuate the A/E strain while maintaining its immunogenicity,two kinds of internal deletion mutations in luxS and ler were respectively introduced into RDEC-1 using suicide delivery vector (pCVD442) technology.Although luxS deletion mutant of RDE-1 showed no release of hormone-like molecule(an auto-inducer),which increase in concentration as populations grow,it still partially maintained its virulence.The immunization with the ler mutant of RDE-1 and challenge with RDEC-1H19A,which is a RDEC-1 derivative constructed by transformation of phage H19A carrying stx gene,showed that immune inoculation with the ler mutant provided all immunized rabbits with complete protection against RDEC-1 carrying phage H19A.The experiment also demonstrates that the ler deletion mutant of RDEC-1 maintains its immunogenicity while being sufficiently decreased in virulence.Therefore,both of the ler deletion mutants are safe attenuated vaccine candidates against rabbit enteropathogenic E.coli.This study provides the important information not only for human attenuated vaccine against EHEC and EPEC,but also for animal vaccine against rabbit enteropathogenic E.coli.