猪链球菌2型强毒株sly基因敲除株构建及生物学特征分析

发布时间：2018-05-21 12:22

本文选题：猪链球菌型 + 溶血素　；参考：《生物技术》2017年01期

【摘要】：[目的]构建猪链球菌2型05ZYH33溶血素sly基因敲除突变株,比较突变株与野生株生物学特征。[方法]分别克隆sly上游序列L、下游序列R和壮观霉素抗性基因spcr,构建基因敲除质粒p UC18-LSR,并电转化入05ZYH33感受态细菌。用多重PCR、RT-PCR及Southern杂交对sly基因敲除突变株进行鉴定。比较突变株与野生株的生长速率和溶血现象。[结果]多重PCR和RT-PCR分别显示,野生株DNA和c DNA都能扩增出579 bp的sly内部片段,而突变株DNA和c DNA都未能扩增出此片段。Southern杂交显示,突变株中未见sly探针杂交条带。突变株生长速率较野毒株迟缓,溶血能力减弱,但依然存在。[结论]建立了高效稳定的电转化方法,成功构建出溶血能力减弱的双交换同源重组sly基因敲除突变株。
[Abstract]:[objective] to construct Streptococcus suis type 2 05ZYH33 hemolysin sly knockout mutant and compare the biological characteristics between the mutant and wild strain. [methods] the upstream sequence of sly, the downstream sequence R and the spectinomycin resistant gene spcrs were cloned, and the gene knockout plasmid pUC18-LSRR was constructed and transformed into 05ZYH33 receptive bacteria. Multiplex PCR RT-PCR and Southern hybridization were used to identify the sly gene knockout mutant. The growth rate and hemolysis of mutant and wild plant were compared. [results] Multiplex PCR and RT-PCR showed that both DNA and c DNA could amplify 579bp sly internal fragment, but DNA and c DNA could not amplify the fragment. Southern blotting showed that there was no sly probe hybridization band in the mutant. The growth rate of mutant strain was slower than that of wild strain, and the hemolysis ability was weakened, but still existed. [conclusion] an efficient and stable electroporation method was established, and a double exchange homologous recombinant sly knockout mutant with weakened hemolytic capacity was successfully constructed.
【作者单位】：扬州科技学院医学院;中国人民解放军南京军区军事医学研究所流行病室;
【基金】：国家自然科学基金-青年科学基金项目(“转录调节因子Rgg与环境因素对2型猪链球菌89K致病岛的交互调控研究”;No.31300119)
【分类号】：S852.611

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