2型猪链球菌cps2D基因敲除株的构建及其基本生物学特性的研究

Construction and biological characterization of a cps2D gene knockout strain of Streptococcus suis serotype 2

目的构建2型猪链球菌荚膜多糖基因簇中cps2D基因敲除株(Δcps2D),比较野毒株05ZYH33与敲除株Δcps2D基本生物学特性的差异。方法设计合成引物L1/L2、R1/R2、Spc1/Spc2,分别扩增cps2D基因上、下游同源臂L片段、R片段及Spc^(R)抗性基因S片段,将3个片段连接至pUC19线性载体上,构建敲除质粒pUC19::cps2D。将敲除质粒电转导入05ZYH33感受态细胞后,利用Spc^(R)抗性平板筛选敲除株,并通过组合PCR和RT-PCR进一步验证。比较野毒株05ZYH33和敲除株Δcps2D细菌生长特性、溶血活性、革兰染色、细菌链长、荚膜形态等方面的异同。结果L1/L2、R1/R2、Spc1/Spc2三组引物分别扩增出L(1030 bp)、R(1030 bp)以及S(1130 bp)基因片段;通过无缝克隆的方法成功构建出敲除质粒pUC19::cps2D;电转入感受态细胞后获得69个克隆,筛选出26个疑似敲除株;组合PCR以及RT-PCR筛选后获得cps2D基因敲除株Δcps2D;与野毒株05ZYH33相比,Δcps2D对数期生长变缓,链长显著变短,荚膜稀疏变薄。结论成功构建cps2D基因敲除株,为后续进一步研究其调控荚膜合成的作用机制奠定基础。

A cps2D gene knockout strain was constructed to study the effects of this gene on basic biological characteristics.The gene is located in the capsular polysaccharide locus of Streptococcus suis serotype 2.The cps2D upstream and downstream homology arms were amplified and named the L and R fragment respectively.The Spc^(R)resistance gene was amplified and named the S fragment.The three fragments were then ligated into the pUC19 linear vector to construct the knockout plasmid pUC19::cps2D,which was then electrotransformed into 05ZYH33 competent cells.We screened the knockout strains with Spc resistance plates and performed further verification with combined PCR and RT-PCR.We preliminarily assessed the differences in basic biological characteristics between 05ZYH33 andΔcps2D,including growth curve determination,hemolytic activity observation,Gram staining,bacterial chain length statistics and transmission electron microscopy observation.L(1030 bp),R(1030 bp)and S(1130 bp)gene fragments were amplified by the L1/L2,R1/R2,and Spc1/Spc2 sets of primers respectively.The knockout plasmid pUC19::cps2D was successfully constructed through In-fusion cloning.After electroporation into competent cells,69 clones were obtained,and 26 suspected knockout strains were screened.After combined PCR and RT-PCR screening,aΔcps2D gene knockout strain was obtained.Compared with 05ZYH33,Δcps2D grew slowly in the logarithmic phase,and its cell numbers per chain were significantly smaller.The capsule structure ofΔcps2D was also looser and thinner than that of 05ZYH33.In conclusion,we successfully constructed a cps2D gene knockout strain,thus laying a foundation for further research on its mechanism of regulating capsule synthesis.