通过改进Red重组方法快速构建肠炎沙门菌毒力岛SPI-1缺失株

Quick construction of SPI-1 deleted Salmonella enterica by the improved Red homologous recombination system

沙门菌依赖毒力岛SPI-1编码的Ⅲ型分泌系统在侵染宿主肠道上皮细胞中起重要作用。为制备沙门菌毒力岛SPI-1缺失株,本研究利用PCR扩增毒力岛SPI-1上下游同源臂和含有氯霉素抗性基因片段,连接p ET-28a构建打靶质粒,采用电击法将其转入含有p KD46a质粒的肠炎沙门菌中。经L-阿拉伯糖诱导同源重组后,鉴定阳性重组子,转化诱导产生重组酶FLp的p CP20质粒,从而删除氯霉素抗性基因,通过PCR方法鉴定缺失株,命名为SM6ΔSPI-1。连续培养鉴定表明缺失株具有良好的遗传稳定性。生长特性研究显示缺失株生长速率高于野生菌株。本研究利用改进的Red重组系统通过一步法构建了肠炎沙门菌毒力岛SPI-1缺失株,为沙门菌毒力岛的功能研究与基因工程减毒疫苗研制奠定了基础。

The Salmonella pathogenicity island 1 （SPI-1） encoded type Ⅲ secretion system is attributed to the infection of intestinal epithelial cells.In order to construct of △SPI-1 mutant of Salmonella enterica,the chloramphenicol resistant gene flanked by homologous sequences of SPI-1 was amplified by PCR and ligated into pET-28a to construct targeting plasmid of pKD46a,and transformed into S.enterica by electric shock.After induced by L-arabinose,the positive recombinant S.enterica were identified by PCR and the final mutant （designated SM6△SPI-1） was identify by PCR in which the chloramphenicol resistant gene was eliminated by pCP20 inducing recombinase FLP mediated recombination events.In addition,the mutant strain was stable after continuous subculture in LB,indicating high genetic stability of SM6△SPI-1 and the growth rate of mutant strain was higher than that of the parental strain.The SM6△SPI-1 mutant was successfully constructed by the one-step method using improved Red homologous recombination system,which provided a certain basis for further functional research of SPI-1 and development of genetic attenuated vaccine.