染色质免疫共沉淀法对猪链球菌反应调控因子CovR靶基因的筛选

Identification of the targets of CovR by chromatin immunoprecipitation

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 CovR plays a very important role in regulating the expression of virulence factors and inducing the patho- genicity of streptococcus suis . This study aimed to further investigate the regulating mechanism of CovR using chromatin immunoprecipita- tion to screen and identify the downstream targets of CorR. Methods Living ceils of streptococcus suis serotype 2 05ZYH33 were fixed with formaldehyde, sonicated to shear the DNA into 100 -500 bp fragments and then immunoprecipitated with the anti-CovR monoclonal antibody. The DNA fragments were extracted and purified, and the primers of 8 potential target gene promoter regions were designed. Specific amplification of DNA regions by each primer set was checked by PCR reaction using the purified DNA as the template. Results The optimal formaldehyde concentration to achieve efficient and reversible cross-linking turned out to be 1%. DNA fragments of 100 -500 bp were obtained using 40 rounds of 10 seconds each at the power of 60W. The CovR binding sequences on the promoters of the gene SSU1668 and SSU1732 were found in the anti-CovR monoclonal antibody immunoprecipitated DNA fragments, while another 6 gene pro- moter regions showed no positive bands. Conclusion SSU1668 and SSU1732 gene promoter regions exist in the anti-CovR monoclonal antibody immunoprecipitated DNA fragments, which shows that the SSU1668 and SSU1732 genes are CovR target genes.