沙眼衣原体转录激活因子GrgA与RNA聚合酶β'亚基的相互作用  

Interaction of Chlamydia trachomatis transcriptional activator GrgA with RNA polymeraseβ'subunit

作　　者：石禹[1] 包小峰 窦志华[1] SHI Yu;BAO Xiaofeng;DOU Zhihua(Department of Pharmacy,Nantong Third People’s Hospital Affiliated Nantong Hospital 3 of Nantong University,Nantong 226006,Jiangsu,China;School of Pharmacology,Nantong University,Nantong 226006,Jiangsu,China)

机构地区：[1]南通市第三人民医院,南通大学附属南通第三医院药学部,南通226006 [2]南通大学医学院,南通226006

出　　处：《医学研究与战创伤救治》2024年第9期897-903,共7页Journal of Medical Research & Combat Trauma Care

基　　金：国家自然科学基金(31370209);南通市卫生健康委员会面上指令课题(MS2022064)。

摘　　要：目的研究沙眼衣原体基因转录调控过程中转录激活因子GrgA与RNA聚合酶(RNAP)β'亚基相互作用的关系。方法制备沙眼衣原体RNA聚合酶(cRNAP)NH-β'(N末端携带His标签)质粒:根据GenBank中的基因序列设计特异性引物,以衣原体基因组DNA(gDNA)为模板,聚合酶链式反应技术(PCR)扩增cRNAPβ'亚基的基因片段;采用同源重组法将基因片段插入到目的载体上构建表达质粒,同样方法制备β'亚单位和缺失亚单位质粒;cRNAPβ'亚基蛋白(包括亚单位和缺失亚单位)的表达:将构建的质粒转化ArcticExpress表达菌中,采用考马斯亮蓝染色法鉴定蛋白表达情况;cRNAPβ'亚基及其亚单位蛋白的纯化:采用固定化离子亲和层析法(IMAC)纯化得到相应蛋白;借助体外蛋白结合实验(Pull-down技术)、聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白质印迹法(Western blot)分析鉴定出GrgA与cRNAPβ'亚基两者间相互结合的较为详细的某一段氨基酸序列。结果GrgA能与cRNAP的β'亚基结合;GrgA与cRNAP的β'亚基N端的(1-731)氨基酸序列有结合,与C端的(671-1396)氨基酸序列无结合。GrgA与β'亚基的(1-240)和(181-417)氨基酸序列有结合;与β'(360-598)、β'(533-731)氨基酸序列无结合。GrgA与β'△(1-180)和β'△(241-417)有结合,与β'△(1-417)无结合。结论GrgA能与cRNAP的β'亚基结合;cRNAP的β'亚基与GrgA的结合存在2个结合位点,分别位于β'亚基N末端的1-180和241-417位氨基酸序列。Objective To study the interaction between transcriptional activator GrgA and RNA polymerase(RNAP)β′subunit during the transcriptional regulation of Chlamydia trachomatis gene.Methods Preparation of chlamydia trachomatis RNA polymerase(cRNAP)NH-β′(N-terminal with His tag)plasmid:Specific primers were designed according to gene sequences in GenBank,chlamydia genomic DNA(gene DNA,gDNA)was used as template,and the gene fragment of cRNAPβ′subunit was amplified by Polymerase chain reaction(PCR).The expression plasmid was constructed by homologous recombination,andβ′subunits and deletion subunits were prepared by the same method.Expresstion of cRNAPβ′subunit protein(including subunits and missing subunits):The constructed plasmid was transformed into ArcticExpress expressing bacteria,and the protein expression was identified by Coomasil bright blue staining method.Purification cRNAPβ′subunits and their subunit proteins:The corresponding proteins were purified by immobilized metal affinity chromatography(IMAC).In vitro Pulldown protein binding assay(pull-down),polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot(WB)were used to identify a specific amino acid sequence of GrgA and cRNAPβ′subunits.Results GrgA could bind toβ′subunit of cRNAP.GrgA binds to the N-terminal(1-731)amino acid sequence of theβ′subunit of cRNAP,but does not bind to the C-terminal(671-1396)amino acid sequence.GrgA binds toβ′subunit(1-240)and(181-417)amino acid sequences,and there is no binding withβ′(360-598),β′(533-731)amino acid sequence.GrgA is bound toβ′△(1-180)andβ′△(241-417),but not toβ′△(1-417).Conclusion GrgA can bind toβ′subunit of cRNAP.There are two binding sites between cRNAP′sβ′subunit and GrgA,which are located at positions 1-180 and 241-417 of theβ′subunit N terminal respectively.

关 键 词：沙眼衣原体 cRNAP GrgA β'亚基 相互作用 融合蛋白 

分 类 号：R374.1[医药卫生—病原生物学]