志贺氏菌样毒素Stx2e缺失突变株的构建及部分生物学特性的分析

CONSTRUCTION OF STX2E GENE DELETION MUTANTS FROM ESCHERICHIA COLI AND ANALYSIS OF SOME BIOLOGICAL PROPERTIES

水肿病是能由产志贺样毒素型大肠杆菌产生的Stx2e毒素引起的一种细菌传染病,对断奶仔猪有较高的致死率,常造成较大的经济。当前,Stx2e毒素影响病原菌与靶细胞之间的相互作用机制不明确,Stx2e基因缺失株作为后选疫苗的潜力有待确认。本研究中我们利用Red重组系统构建了大肠杆菌1522(serotype O139:K12:H1)和1525(serotype O157:H19)的Stx2e基因缺失株,并通过PCR检验,DNA测序手段以及Vero细胞毒性试验在细胞水平加以验证。研究发现,在同样的培养条件下,1522△Stx2e株的生长特性与原型株相比没有差异,其分解发酵糖类和氨基酸的能力也未发生改变,但是相较野生株,Stx2e基因缺失株粘附猪肠上皮细胞IPEC-J2的能力大幅度降低,可达30%。将表达K99菌毛操纵子基因克隆入1522△Stx2e中,该重组菌能够同时表达K99菌毛和自身的F18菌毛基因,且该重组株的粘附IPEC-J2细胞能力相较原型株增强近1倍。

Edema disease, mainly due to Stx2e toxin produced by shiga-like toxigenic Escherichia coli （SLTEC） of some serotypes, usually causes severe mortality in postwean pigs and great economic loss to swine industry. The underlying mechanism of Stx2e toxin during interaction between the pathogen and host cells is still unclear. The possibility of using Stx2e knocked out mutants as vaccine candidates also requires further evaluation. Here, we generated Stx2e gene deletion mutants from the parent strains 1522 （serotype O139：K12：H1） and 1525 （serotype O157：H19） with λ, Red-based recombination system based on the original sequences ofStx2e gene clusters in the GenBank and then confirmed the mutant 1522 A Stx2e in PCR, sequencing and cytotoxicity in Vero cells. The 1522 A Stx2e mutant showed similar growth and fermentation to some tested sugars and amino acids but decreased adhesion to IPEC-J2 cell by 30% as compared to wild type. After K99 gene cluster was cloned into mutant 1522 A Stx2e, it expressed exogenous K99 fimbriae and showed twice adhesion to IPEC-J2 cell as compared with parent isolate.