PhoQ基因重组鼠伤寒沙门氏菌株的构建及毒力研究

应用PCR技术从鼠伤寒沙门氏菌基因组DNA中克隆phoQ基因片段，构建原核表达pUC18重组质粒，测定序列(GenBank登录号为DQ787014)，并转入鼠伤寒沙门氏菌，经异丙基硫代半乳糖苷(IPTG)诱导，进行高效表达。对重组菌株、野生菌株进行毒力检测对比实验，通过口腔注入45日龄健康无菌KM小鼠，测定其半数致死量(LD50)。结果发现：重组菌株与野生菌株的毒力存在显著差异，其半致死量分别为3.981×107 cf u/ mL and 5.012×102 cf u/ mL，PhoQ基因重组菌株的毒力远远低于非重组菌株。说明phoQ基因是调节鼠伤寒沙门氏菌致病机制中一个重要的调节因子。

Construction of a Salmonella Typhimurium PhoQ Gene recombinant strain and Detection their Virulence

PhoQ gene that was amplified by polymerase chain reaction(PCR)from Salmonella typhimurium genome DNA was cloned into a procaryotic expression plasmid pUC18, sequenced and GenBank accession No. is DQ787014. The identified recombinant plasmid was then used to transform into the competent Salmonella, and the positive clones were screened by PCR and restriction enzyme digestion.and phoQ recombinant protein was expressed by IPTG induced method. PhoQ gene expression was analyzed by SDS-PAGE. The stability of recombinant bacterial cultured in vivo was observed. It showed t hat recombinant bacterium could stably reproduction, growth and hand down from generation to generation in vivo. To investigate the effects of different virulence of the recombinant S. typhimurium and wildness S. typhimurium, which were tested in 45 days old asepsis' KM mice by nonnasality rejection. The data showed that the LD50 of the recombinant and wildness strains were 3.981×107 cf u/ mL and 5.012×102 cf u/ mL ,respectively. It suggests that the different virulence between the two strains were occurred, the wildness S. typhimurium was more virulent than the recombinant S. typhimurium. The result testified phoQ gene is one of the important regalatots in the virulence mechanism of S. typhimurium.