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目的猪链球菌CovR是具有全局调控作用的负反应调控因子。文章采用染色质免疫共沉淀(chromatin immu-noprecipitation,ChIP)方法筛选转录调控因子CovR的靶基因,为进一步开展CovR调控机理研究提供条件。方法利用甲醛固定猪链球菌2型05ZYH33菌株活细胞,超声波破碎法将DNA随机断裂成100～500bp大小不同的片段,再利用CovR特异性单抗免疫沉淀该蛋白质-DNA片段解交联并分离纯化DNA。针对8个潜在的靶标基因启动子区设计引物,分别用设计的引物对免疫共沉淀的DNA样品进行PCR扩增,验证CovR与DNA的结合。结果最佳ChIP实验条件为1%甲醛交联DNA与蛋白质的复合物;功率80 W、工作10 s、间隔30 s、超声40次破碎该复合物,可得到100～500 bp大小不同的片段用于后续实验。PCR结果显示SSU1668基因和SSU1732基因启动子区扩增出阳性条带,另外6种基因的启动子区未扩增出阳性条带。结论 CovR抗体免疫沉淀得到的DNA片段中含有SSU1668基因和SSU1732基因启动子区,表明SSU1668基因和SSU1732基因是CovR的靶基因。
Objective Streptococcus suis CovR is a negative regulator of global regulation. In this study, the target gene of transcriptional regulator CovR was screened by chromatin immu-noprecipitation (ChIP), which provided conditions for the further study on the regulation mechanism of CovR. Methods Formaldehyde was used to immobilize live cells of Streptococcus suis type 2 05ZYH33 strain. The DNA was randomly fragmented into 100-500bp fragments by sonication. The protein-DNA fragments were immunoprecipitated using CovR-specific monoclonal antibody to separate and purify DNA. Primers were designed according to the promoter regions of eight potential target genes. Coimmunoprecipitated DNA samples were respectively amplified by PCR using designed primers to verify the binding of CovR to DNA. Results The optimal conditions of ChIP experiment were 1% formaldehyde cross-linked DNA and protein complex. The power of 80 W was 10 s for 30 s, and the complex was broken by sonicating 40 times to obtain fragments of 100 ~ 500 bp in size In the follow-up experiment. The results of PCR showed that the positive bands were amplified from the promoter region of SSU1668 and SSU1732 genes, and the positive bands were not amplified from the other six promoter regions. Conclusions The DNA fragment of CovR antibody immunoprecipitation contains the promoter regions of SSU1668 and SSU1732, indicating that SSU1668 and SSU1732 are the target genes of CovR.