摘要
目的探索荚膜多糖合成基因neuC与细菌唾液酸合成转化的关系。方法应用同源重组基因敲除方法构建筛选2型猪链球菌(SS2)强毒株05ZYH33荚膜多糖组分唾液酸合成相关基因neuC的缺失突变株;系统比较分析突变体与野生株的基本生物学特性的差异,小鼠和仔猪毒力试验分析neuC基因缺失对细菌毒力的影响。结果应用PCR检测和Southern杂交分析显示,neuC基因在2株转化重组体中被壮观霉素抗性基因所替代,表明neuC基因敲除突变体构建成功;突变体△neuC与野生株在菌落形态、染色特性和溶血活性方面无明显差异;突变体与2型特异性抗血清的凝集反应显著减弱;电镜观察显示突变体表面荚膜结构较野生株荚膜显著变薄,直径20～55nm,但质地更致密;小鼠和仔猪毒力试验显示,突变体毒力显著减弱。结论neuC是SS2荚膜多糖合成的重要结构基因,与细菌唾液酸的转化利用密切相关;neuC基因可能藉其在唾液酸转化中的作用影响SS2的基本生物学特性和细菌的侵袭致病能力。
Objective To construct and functionally analyze a mutant of the neuC gene encoding UDP-N-acetylglucosamine 2-epimerase of Streptococcus suis serotype 2（SS2）virulent strain 05ZYH33.Methods The neuC gene was knocked out based on the principle of homologous recombination.PCR analysis and Southern hybridization were used to identify the knockout.Transmission electron microscopy was employed to observe the morphology of the established strain.The virulence of the deficient strain was tested in piglet and murine models of infection.Results PCR analyses and Southern hybridization confirmed that the coding gene of neuC was replaced completely by an spcr cassette in the recombinant mutant,and thus a mutant △neuC was successfully constructed.Analysis of biological characteristics showed that there were no differences between the mutant and the wild type strain 05ZYH33 in terms of mycelial morphology,hemolytic activity,and staining properties.The agglutination reaction of the mutant △neuC differed significantly from that of the wild-type strain.Transmission electron microscopy showed that the capsule of mutant is thinner and more compact than that of the wild-type strain 05ZYH33.Results of an animal virulence assay confirmed that the mutant △neuC is avirulent.Conclusion These observations indicate that the sialic acid of capsular polysaccharide plays an important role in the pathogenesis and invasiveness of SS2.
出处
《中国病原生物学杂志》
CSCD
2010年第4期241-245,269,共6页
Journal of Pathogen Biology
基金
国家自然科学基金资助项目(No.30600533
No.30730081
No.30972638)
江苏省自然科学基金资助项目(No.BK2008066
No.BK2009042)
