沙眼衣原体质粒调控基因的上游序列有广泛的ChxR结合位点

Recombinant Chlamydial Chx R Has Extensive DNA Binding Activities to Upstream Sequences of Plasmid-Regulated Genes in Vitro

目的:利用凝胶阻滞实验分析沙眼衣原体质粒调控基因的上游DNA序列与ChxR 的相互作用;方法:利用表达载体p ET-21b在大肠杆菌BL21(DE3)中重组表达ChxR ,重组蛋白的N端有T7标签、C端有6×His标签,用TALON金属亲和介质纯化重组蛋白;PCR扩增沙眼衣原体质粒调控基因的上游区约150 bp,连接p CR2.1载体,重组载体经单酶切,制备3'端生物素标记探针;用引物延伸实验确定glgA的转录起始位点,并合成5条glgA上游区的30 bp重叠探针;用凝胶阻滞实验分析ChxR 与以上探针的相互作用。结果:4个质粒调控基因的上游均有多个ChxR 结合位点,特别是glgA上游区至少有6个结合位点;glgA上游的ChxR 结合位点多位于启动子和启动子下游区。结论:ChxR 可能参与质粒调控基因的转录;在glgA转录中,ChxR 可能起转录抑制子的作用。

Objective：To identify and characterize interactions between upstreams of Chlamydia trachomatis plas-mid-regulated genes and ChxR by gel shift assays.Methods：Recombinant ChxR was expressed in E.coli BL21（DE3） by using p ET-21 b,and purified by using TALON metal affinity resin.About 150 bp upstream of four plas-mid-regulated genes were amplified and ligated into p CR2.1.Recombinant vectors were then digested and used forpreparing biotinylated probes.Transcription start site of glgA was determined by primer extension assay.Five 30bp-overlapping probes of glgA upstream were synthesized.Interactions between ChxR and probes above were analyzedby gel shift assays.Results：All upstreams of four plasmid-regulated genes have multiple ChxR binding sites.Most ChxR binding sites in glgA upstream located in its promoter and downstream regions.Conclusion：ChxR may be involved in transcriptions of plasmid-regulated genes.In glgA transcription,ChxR likely functions as atranscriptional repressor.