同源重组法衣原体RNA聚合酶表达载体的构建和应用  

Construction and application of chlamydia RNA polymerase expression vector by using homologous recom-bination method

作　　者：石禹[1] 包小峰 SHI Yu;BAO Xiaofeng(Department of Pharmacy,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]南通大学附属南通第三医院药学部,南通226006 [2]南通大学药学院,南通226006

出　　处：《医学研究与战创伤救治》2025年第2期113-119,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：目的利用同源重组法构建衣原体RNA聚合酶(cRNAP)各亚基质粒,转化入大肠埃希菌中表达蛋白并探讨其在衣原体转录调控中的应用。方法根据Gen Bank中的基因序列设计特异性引物,聚合酶链式反应(PCR)扩增cRNAP各亚基的目的基因片段,对PCR产物进行纯化,质粒载体进行质粒抽提、质粒酶切、胶回收等处理。采用同源重组法将基因片段插入到目的载体进行片段融合构建cRNAP各亚基表达质粒,融合产物转化后用特异性引物进行PCR验证和测序。构建好的融合产物转化入大肠埃希菌化学感受态细胞表达蛋白。结果成功构建cRNAP各亚基质粒并成功表达cRNAP各亚基蛋白,研究了衣原体转录调节因子GrgA与cRNAP的直接相互作用是GrgA与cRNAP的α、β′亚基有结合,与β亚基无结合。两者可以作为衣原体感染治疗和预防的药物作用新靶点。结论同源重组法可用于构建cRNAP各亚基质粒并成功表达相应蛋白,并用于研究转录调节因子GrgA与cRNAP各亚基蛋白的结合位点,为衣原体感染治疗和预防药物作用新靶点的研究提供参考信息。Objective To construct each subunit plasmid of Chlamydia RNA polymerase(cRNAP)by using homologous recombination method,transform them into expression proteins in Escherichia coli,and explore their application in transcription regulation of chlamydia.Methods Specific primers were designed according to the gene sequences in GenBank,the target gene fragments of cRNAP each subunit plasmid were amplified by polymerase chain reaction(PCR),the PCR products were purified,and the plasmid vectors were processed by plasmid extraction,plasmid enzyme digestion and gel recovery.The gene fragments were inserted into the target vector by homologous recombination method for fragment fusionto to construct the cRNAP subunit expression plasmid.After the fusion products were transformed,PCR validation and sequencing were performed with specific primers.The constructed fusion product was transformed into the expression protein of Escherichia coli chemoreceptor cells.Results cRNAP subunit plasmids were successfully constructed and cRNAP subunit proteins were successfully expressed.It has studied the direct interaction between Chlamydia transcriptional regulatory factor GrgA and cRNAP is that GrgA is bound toαandβ'subunits of cRNAP,but not toβsubunits.Both can serve as new drug targets for the treatment and prevention of Chlamydia infection.Conclusion Homologous recombination method can be used to construct cRNAP subunit plasmids and successfully express corresponding proteins,and can be used to study the binding sites of transcription regulatory factor GrgA and cRNAP subunit proteins,providing reference information for the study of new drug targets for treatment and prevention of chlamydia infection.

关 键 词：同源重组法 衣原体 cRNAP 亚基 质粒载体 融合蛋白 

分 类 号：R374[医药卫生—病原生物学]