猪链球菌2型分选酶srtBCD基因敲除株的构建及其生物学特性分析  

Construction and in vitro assay of the sortase BCD geneknock-out mutant of Streptococcus suis 2

作　　者：陈红娜[1,2] 廖辉[1] 王长军[1,2] 潘秀珍[1] 唐家琪[1,2] 

机构地区：[1]南京军区军事医学研究所,南京210002 [2]南京医科大学基础医学院,南京210029

出　　处：《微生物学报》2011年第3期386-392,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金项目(30730081;30972638;81071317);江苏省自然科学基金资助项目(BK2009042;BK2010025;BK2010114;BK2010113);南京军区医学科技创新课题(09Z040;07Z045)~~

摘　　要：【目的】阐明分选酶srtBCD基因在猪链球菌2型致病过程中的作用。【方法】利用同源重组原理构建中间为壮观霉素、两侧为srtBCD基因上下游片段的重组质粒,将构建好的质粒电转化入猪链球菌感受态,筛选srtBCD缺失的突变株,并通过组合PCR和逆转录PCR对其进行验证。生物学功能实验研究srtBCD突变株和野毒株05Z33在生长速率、粘附、毒力等方面的差异。【结果】组合PCR和逆转录PCR结果均证实srtBCD突变株构建成功,体外实验结果显示srtBCD缺失后细菌的生长速率减慢,与Hep-2上皮细胞的粘附率明显降低,小鼠毒力实验数据表明突变株毒力无明显变化。【结论】猪链球菌2型srtBCD基因与细菌的粘附能力有关,为进一步研究猪链球菌2型的致病机理奠定基础。[Objective]Streptococcus suis 2 is an emerging zoonotic pathogen responsible for a wide range of life-threatening diseases in pigs and humans.In this study,we investigated the functionality of one of Streptococcus suis 2 sortases,known as the srtBCD.[Methods]To obtain the isogenic mutant srtBCD,the competent cells of 05ZYH33 were subjected to electrotrans formation with recombinant plasmid based on the principle of homologous recombination.The resulting mutant strains was further confirmed by a series of PCR and reverse transcription PCR.To better assess the role of srtBCD gene in the virulence of 05Z33,cell adherence assays and experimental infection of mice was adopted.[Results]A SrtBCD defective mutant of 05ZYH33 was found to be associated with growth curve upon cultivation in standard laboratory used in our in vitro assays.Furthermore,abolishment of the expression of srtBCD result in impaired interactions of S.suis with human laryngeal epithelial cell line.However,there is no differences when infection mice by the WT and mutant strain.[Conclusion]These results suggest that srtBCD are critical for the pathogen-host interaction of S.suis 2,but abolishment of srtBCD does not impair the full virulence of 05Z33.It is to expect that future study carried out with S.suis 2 to verification the conclusions.

关 键 词：猪链球菌2型 SORTASE BCD 基因敲除 粘附 毒力 

分 类 号：R378[医药卫生—病原生物学]