猪链球菌2型二元信号转导系统ciaR基因敲除突变株的构建及生物学功能研究

目的构建猪链球菌2型强毒株05ZYH33二元信号转导系统1094HK/1095RR的反应调节因子ciaR基因敲除突变株(ΔciaR),并研究ΔciaR的生物学功能。方法和结果基于同源重组原理筛选ciaR基因缺失株,PCR分析及Southern杂交结果均显示突变株构建成功。通过检测OD600值绘制生长曲线,发现突变株在37℃和40℃其OD600值均明显低于野生株;与40mmol/L过氧化氢共作用15min后涂板计数,突变株存活率明显低于野生株;不同pH值的培养基中培养,发现在pH5.0的酸性环境中突变株OD600值明显低于野生株;野生株与突变株对BALB/c小鼠攻毒(约1×109CFU/只),各10只,两组16h后均死亡9只。结论成功获得05ZYH33ciaR基因敲除突变株;发现删除ciaR基因会导致细菌的生长繁殖能力减弱,抗氧化应激能力下降,在酸性环境生长存活能力下降;但对小鼠毒力无差别。

Construction and characterization of a two-component signal transduction system ciaR gene knock-out mutant of Streptococcus suis serotype 2

To construct ciaR gene deletion strain（AciaR）which encodes the response regulator of the two-component signal transduction system 1094HK/1095RR in Streptococcus suis serotype 2 virulent strain 05ZYH33, and to investigate biological functions of the △ciaR. Recombinant gene knock-out vector was constructed consisting of SpcRcassette with flanking homology regions to the ciaR gene,and the ciaR gene deletion mutant was screened by homologous recombination. The △ciaR mutant was confirmed by PCR analysis and Southern hybridization. The AciaR mutant was found to grow more slowly than the wild type strain at 37℃ and 40℃,as judged by OD600. The sensitivity of cells to peroxide was test by the exposure of aliquots of cultures to 40mmol/L H2O2 for 15min. Viable cells were counted by plating them onto THB agar plates and the results were expressed as percentage of survival. The AciaR strain was significantly more sensitive to peroxide than the wild type. No obvious differences were observed at different pHs except at pH5.0, in which the △ciaR mutant presented growth and survival defects. Nine of ten mice infected with the wild type or mutant（1×10^9CFU/mouse） died at 16hours post-infection. It is evident that lack of the ciaR gene reduces the proliferative ability of S.suis 2 05ZYH33, tolerance to the oxidative stresses and survival at acid en- vironment in vitro, but no differences of virulence to mice.