热纤梭菌Cthe_2401蛋白功能预测及在大肠杆菌中的克隆表达与纯化  

Functional prediction of Clostridium thermocellum Cthe_2401 protein and clonal expression and purification in Escherichia coli

作　　者：崔古贞 管玉祝 王鑫鑫 花登雄 吴道艳 吴晓娟 钟欣言 洪伟 陈峥宏 CUI Guzhen;GUAN Yuzhu;WANG Xinxin;HUA Dengxiong;WU Daoyan;WU Xiaojuan;ZHONG Xinyan;HONG Wei;CHEN Zhenghong(Department of Microbiology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Medical microbiology and parasitology of Education Department of Guizhou,Guiyang 550004,Guizhou,China;Key Laboratory of Molecular Biology of Guizhou,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院微生物学教研室,贵州贵阳550025 [2]贵州省病原生物学特色重点实验室,贵州贵阳550004 [3]贵州省分子生物学重点实验室,贵州贵阳550004

出　　处：《贵州医科大学学报》2024年第4期501-507,共7页Journal of Guizhou Medical University

基　　金：国家自然科学基金(31760318,32160015);留学人才创新创业项目(202208);贵州省科技计划项目(〔2019〕1441、〔2020〕1Z067);大学生创新训练计划(202210660057)。

摘　　要：目的利用生信手段预测Cthe_2401蛋白的特性与功能,并在大肠杆菌中进行体外克隆、表达与纯化。方法利用国家生物技术信息中心数据库(NCBI)、蛋白保守结构域数据库(CDD)分析Cthe_2401蛋白的序列和进化特征,采用SWISS-MODEL在线软件进行同源建模并预测该蛋白的结构与功能;利用聚合酶链式反应(PCR)扩增Cthe_2401基因,并与pET28a载体链接构建pET28a-Cthe_2401表达质粒,转化大肠杆菌后利用异丙基硫代半乳糖(IPTG)诱导Cthe_2401蛋白表达;利用50%硫酸铵沉淀、Ni-NTA柱以及SephadexG75葡聚糖凝胶柱纯化Cthe_2401蛋白。结果生物信息学预测表明,Cthe_2401蛋白属于Veg蛋白家族,可能与生物膜的发生和孢子形成有关;Cthe_2401蛋白成功在大肠杆菌克隆与表达,体外纯化获得约10 ku大小的蛋白条带,与预期结果一致。结论成功预测Cthe_2401蛋白的功能,成功获得Cthe_2401蛋白在大肠杆菌中的克隆、表达与纯化。Objective To predict the characteristics and function of Cthe_2401 protein,and perform the cloning,expression and purification of Escherichia coli in vitro.Methods The sequence and evolutionary characteristics of Cthe_2401 protein were analyzed by the National Center for Biotechnology Information database(NCBI)and protein Conserved Domain database(CDD),and SWISS-MODEL online software was used to conduct homology modeling and predict the structure and function of the protein.Cthe_2401 gene was amplified by polymerase chain reaction(PCR),and the pET28a-Cthe_2401 expression plasmid was constructed with the pET28a vector.Cthe_2401 protein expression was induced by using isopropylthioate galactose(IPTG)after transformation of E.coli.Cthe_2401 protein was purified using 50% ammonium sulfate precipitation,Ni-NTA column as well as SephadexG75 dextran gel column.Results Bioinformatic predictions suggest that the Cthe_2401 protein belongs to the Veg protein family and may be related to biofilm occurrence and sporulation.Cthe_2401 protein was successfully cloned and expressed in E.coli and purified in vitro to obtain protein bands of about 10 ku in size,consistent with the expected results.Conclusion The function of Cthe_2401 protein is successfully predicted,and the cloning,expression and purification of Cthe_2401 protein in E.coli are successfully obtained.

关 键 词：热纤梭菌 Cthe_2401 Veg蛋白 生物信息学 蛋白表达与纯化 大肠杆菌 

分 类 号：R33[医药卫生—人体生理学] Q786[医药卫生—基础医学]