【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cRNAP β’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；cRNAP β’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；cRNAP β’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cRNAP β’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。更多还原

【Abstract】 Objective To study the interaction between transcriptional activator GrgA and RNA polymerase(RNAP)β’sub‐unit during the transcriptional regulation of Chlamydia trachomatis gene.Methods Preparation of chlamydia trachomatis RNA poly‐merase(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 cRNAP β’subunit was amplified by Poly‐merase chain reaction(PCR).The expression plasmid was constructed by homologous recombination,and β’subunits and deletion sub‐units were prepared by the same method.Expresstion of cRNAP β’subunit protein(including subunits and missing subunits):The con‐structed plasmid was transformed into ArcticExpress expressing bacteria,and the protein expression was identified by Coomasil bright blue staining method.Purification cRNAP β’subunits and their subunit proteins:The corresponding proteins were purified by im‐mobilized metal affinity chromatography(IMAC).In vitro Pulldown protein binding assay(pull-down),polyacrylamide gel electro‐phoresis(SDS-PAGE)and Western blot(WB)were used to identify a specific amino acid sequence of GrgA and cRNAP β’subunits.Results GrgA could bind to β’subunit of cRNAP.GrgA binds to the N-terminal(1-731)amino acid sequence of the β’subunit of cRNAP,but does not bind to the C-termina(l671-1396)amino acid sequence.GrgA binds to β’subuni(t1-240)and(181-417)amino acid sequences,and there is no binding with β’(360-598),β’(533-731)amino acid sequence.GrgA is bound to β’△（1-180)and β’△（241-417),but not to β’△（1-417).Conclusion GrgA can bind to β’subunit of cRNAP.There are two binding sites between cRNAP’s β’subunit and GrgA,which are located at positions 1-180and 241-417 of the β’subunit N terminal respectively.更多还原