猪链球菌2型反应调控因子ReυS突变株的构建  

Construction of ReυS gene knock-out mutant of Streptococcus suis serotype 2

作　　者：鞠爱萍[1] 王长军[1] 李明[1] 程功[1] 郑峰[1] 潘秀珍[1] 陆承平[2] 唐家琪[1] 

机构地区：[1]南京军区军事医学研究所,江苏南京210002 [2]南京农业大学动物医学院

出　　处：《中华流行病学杂志》2008年第1期59-64,共6页Chinese Journal of Epidemiology

基　　金：基金项目：国家自然科学基金资助项目（30600533,30670105）;江苏省自然科学基金资助项目（BK2006014）

摘　　要：目的构建猪链球菌2型强毒株05ZYH33反应调控因子ReυS基因敲除突变体，研究基因敲除后对细菌基本生物学性状及对小鼠和猪的致病性的影响。方法构建中间为壮观霉素抗性基因，两侧为ReυS编码基因上、下游同源序列的基因敲除载体，通过同源重组和PCR法鉴定，获得ReυS编码基因完全被壮观霉素抗性基因替代的突变株。体外观察基因敲除后细菌的稳定性、生长曲线、菌落形态、溶血性、染色情况、镜下形态及蛋白表达等基本生物学性状有无发生明显改变。以10^8CFU野毒株和缺失株分别感染BALB／c小鼠和20日龄仔猪，观察致病性有无明显区别。结果PCR分析显示ReυS编码基因完全被壮观霉素抗性基因替代，基因敲除后细菌的基本生物学性状无明显改变。动物感染实验显示，RevS缺失株对小鼠的致病性显著减弱，但对其主要宿主猪的致病性未见显著改变。结论成功构建了猪链球菌2型强毒株05ZYH33反应调控因子ReυS基因敲除突变株，基因敲除后未明显影响细菌的基本生物学性状，但对小鼠和猪的致病性有所减弱。Objective To construct a gene knock-out mutant of response regulator named ReυS in Streptococcus suis serotype 2 virulent strain 05ZYH33, and to investigate the effects of its deletion on the biological characters of this pathogen and the pathogenesis to mice and piglets. Methods Recombinant gene knock-out vector consisting of Spc^r cassette was constructed and flanking was constructed consisting of Spc^r cassette with flanking homology regions to the RevS genes while the isogenic RevS-deficient mutant was screened by allelic replacement. The effects of RevS deletion on the basic biological characters of 05ZYH33 including growth stability, colonial morphology, haemolysis, Gram staining, growth curve and protein expression were examined in vitro. The mice and piglets were infected with 10^8 CFU wild virulent and mutant isolates. Results PCR analysis confirmed that the coding genes of RevS were replaced completely by Spc^r cassette and the basic biological characters of 05ZYH33 did not undergo any apparent change. Balb/c mice infection assay indicated that ReυS play a role in the pathogenesis of Streptococcus suis infections, while no remarkable difference was observed in the piglets＇ pathogenesis infection raters between mutant isolates A05ZYH33 and wild-type isolates 05ZYH33. Conclusion The mutant of Streptococcus suis 05ZYH33 response regulator was successfully constructed, while the mutation did not obviously affect the bacterial biological characters, while the knock-out mutant of ReυS was shown to be attenuated in pathogenesis to mice and piglets.

关 键 词：猪链球菌2型 反应调控因子 基因敲除 致病性 

分 类 号：S852.61[农业科学—基础兽医学]