首页|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

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

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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.

Clostridium thermocellumCthe_2401Veg proteinbioinformaticsporotein expression and purificationEscherichia coli

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

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贵州医科大学基础医学院微生物学教研室,贵州贵阳 550025

贵州省病原生物学特色重点实验室,贵州贵阳 550004

贵州省分子生物学重点实验室,贵州贵阳 550004

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

国家自然科学基金国家自然科学基金留学人才创新创业项目贵州省科技计划项目贵州省科技计划项目大学生创新训练计划

3176031832160015202208[2019]1441[2020]1Z067202210660057

2024

10.19367/j.cnki.2096-8388.2024.04.004

贵州医科大学学报
贵阳医学院

贵州医科大学学报

CSTPCD
影响因子：0.827
ISSN：2096-8388
年,卷(期)：2024.49(4)
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