利用信息肽诱导和Cre/LoxP系统构建猪链球菌基因缺失株

Gene deletion of Streptococcus suis by combination of pheromone induction and Cre/LoxP system

为建立一种高效的猪链球菌(Streptococcus suis)基因缺失方法,本研究利用信息肽诱导DNA片段转化和Cre/LoxP系统去除抗性基因这两种技术,通过在S.suis 05ZYH33的ssu05_1921基因上、下游片段和壮观霉素抗性基因(spc)片段之间引入LoxP位点,采用信息肽GE9诱导构建的DNA片段转化S.suis并发生重组,经抗性筛选快速获得spc替代ssu05_1921基因的缺失株;进一步引入pSET6s/P_(tufA-cre)质粒表达Cre重组酶作用于LoxP位点,去除spc基因,产生无痕ssu05_1921基因缺失株,经测序和RT-PCR验证缺失正确。本研究建立的该方法简单、快捷、阳性率高,为构建S.suis基因缺失株、研究S.suis致病机制提供了新的选择。

In the present study, an efficient method for gene-deletion of Streptococcus suis was established by combination of recently reported pheromone-induced DNA fragment transformation and Cre/LoxP system. Briefly, a DNA fragment was prepared by fusion of the upstream fragment of ssu051921 gene of S.suis, the spectinomycin resistance(spc) marker gene and the downstream fragment of ssu051921 gene. During the fusion processing, LoxP sites were introduced sequentially at 5’ and 3’ ends of the spc gene. Next, the DNA fragment was transformed into S.suis 05 ZYH33 strain induced by the pheromone for recombination, resulting in ssu051921-deleted from the S.suis strain in which the ssu051921 gene was substituted by spc marker gene.Then, the spc marker gene flanked with LoxP sites was removed, leading to a marker-less ssu051921-deleted strain by introducing the Cre recombinase expressing recombinant plasmid of pSET6s/PtufA-cre. It was further verified that the ssu051921 gene was deleted by sequencing and RT-PCR. The results showed that this method is simple and rapid, with high positive rate. It provides a efficient alternative protocol for gene-deleted strain construction of S.suis, facilitatinge further pathogenesis study of the pathogen.