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

作　　者：李鹏[1,2] 于永慧 王涛[1] 何君[1] 朱虹[1] 端青[1] 宋立华[1] 

机构地区：[1]病原微生物生物安全国家重点实验室军事医学科学院微生物流行病研究所,北京100071 [2]解放军第264医院,山西太原030001

出　　处：《生物技术通讯》2015年第4期501-504,共4页Letters in Biotechnology

基　　金："十二五"农村领域国家科技计划(2012AA101302)

摘　　要：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.

关 键 词：沙眼衣原体 质粒 ChxR Pgp4 glgA 基因调控 

分 类 号：Q78[生物学—分子生物学]