嗜水气单胞菌cpxRA缺失株构建及生物学特性研究

为了探讨Cpx系统在嗜水气单胞菌生长及毒力等方面发挥的作用, 利用融合PCR和基因同源重组原理, 以自杀质粒pRE112为载体构建缺失57—1879 bp序列的cpxR-A基因簇突变株 Δcpx, 然后比较缺失株和野生株在生长、生物膜形成、应激耐受及毒力等生物学特性方面的差异。普通PCR及荧光定量PCR结果验证了突变株中cpxRA基因簇片段的部分缺失, 表明突变株构建成功; 生物学特性研究结果显示, 突变株在形态、生长、生物膜形成及毒力等方面与野生株没有显著差异, 两者主要在应对高渗透压、SDS (十二烷基磺酸钠)刺激及含有EDTA (乙二胺四乙酸二钠)或多黏菌素B环境表现不同。结果表明Cpx双组分系统在嗜水气单胞菌应对外界刺激方面扮演着重要角色, 但在毒力方面则可能处于次要地位。

CONSTRUCTION AND BIOLOGICAL CHARACTERISTICS ANALYSIS OF CPXRA MUTANT IN AEROMONAS HYDROPHILA

To investigate the roles of two Cpx component systems in the growth and virulence of Aeromonas hydrophila, we constructed the cpxRA gene cluster mutant Δcpx with the deletion of 57—1879 bp using suicide plasmid pRE112 as the vector. This procedure was based on fusion PCR and gene homologous recombination principles. Through electrophoresis and fluorescence quantitative PCR, the partial deletion of the cpxRA gene cluster in the mutant was confirmed. Then we compared the differences between mutant and wild strains in biological characteristics including growth, biofilm formation, stress tolerance and virulence. The results showed that the mutant had no significant difference with the wild strain in morphology, growth, biofilm formation and virulence. The main differences existed in the response to high osmotic pressure, SDS (Sodium dodecyl sulfate), EDTA (Ethylene Diamine Tetraacetic Acid) and polymyxin B stimulation. This study reveals that the Cpx system of Aeromonas hydrophila is involved in the response to external stimulus factors, and plays a relatively minor role in virulence.