猪链球菌2型转座子文库构建及毒力基因筛选

Construction of type 2 transposon library of Streptococcus suis and screening of virulence genes

猪链球菌2型(Streptococcus suis serotype 2,SS2)毒力因子众多,致病性强弱与其毒力因子有关,但单个或多个毒力因子缺失对某些菌株的毒力影响并不明显,新的毒力因子仍有待发现。为挖掘新的毒力相关因子,用携带转座子TnYLB-1的穿梭载体pMar4S转化SS2 SC-19菌株,通过高温诱变构建SC-19菌株的突变体文库,筛选了450个转座子插入突变的单克隆。对随机挑选的18个突变体采用PCR验证,并通过大腊螟攻毒试验筛选毒力变化基因,利用反向PCR鉴定相关基因,通过同源重组方法构建SUT2基因敲除株、回补株,测定生长曲线,并进行小鼠毒力试验。结果表明,转座子TnYLB-1以单拷贝、随机方式插入到SC-19菌株的基因组;经过大腊螟攻毒试验筛选和反向PCR鉴定发现包含SUT2基因在内的8个毒力致弱基因。生长曲线测定表明,SUT2基因敲除株的生长速率比野生型菌株慢。小鼠致病性试验进一步表明,野生SC-19菌株小鼠组的死亡率为100%,SUT2基因敲除株小鼠组的存活率为100%。综上,本研究发现并鉴定了一个新的毒力相关因子SUT2,为进一步阐明SS2的致病机制奠定了基础。

Streptococcus suis serotype 2(SS2)has a large number of virulence factors,and its pathogenicity is related to its virulence factors.However,the influence of single or multiple deletions of the virulence of some strains is not obvious,and new virulence factors are still to be discovered.In order to explore new virulence related factors,SS2SC-19strain was transformed by the shuttle vector pMar4Scarrying transposon TnYLB-1and mutated at high temperature.A mutant library of SC-19strain was constructed and 450clones with transposon insertion mutations were screened.Eighteen randomly selected mutants were verified by PCR,and virulence variation genes were screened by challenge test of T.mellonis.Related genes were identified by reverse PCR,and SUT2 gene knockout and complement strains were constructed by homologous recombination method.The growth curves were measured,and mouse virulence was tested.The results showed that the transposon TnYLB-1was randomly inserted into the chromosome of SC-19strain in a single copy manner.Eight virulence weakening genes including SUT2gene were detected by challenge assay and reverse PCR.The growth curve of SUT2gene knockout showed that the growth rate of SUT2 gene knockout strain was slower than that of wild type.The pathogenicity test of mice further showed that the mortality rate of mice infected with wild SC-19strain was 100%,and the survival rate of mice infected with SUT2knockout strain was 100%.In conclusion,this study found and identified a new virulence related factor SUT2,which laid a foundation for further elucidating the pathogenesis of SS2.