牙龈卟啉单胞菌血凝集素黏附结构域ha2基因在大肠杆菌中的融合表达和纯化  

Construction of prokaryotic expression vector of ha2 gene from Porphyromonas gingivalis, fusion expression and purification in E.coli

作　　者：曾凤娇 杨琳 刘建国 田源 陈靖 白国辉 Zeng Fengjiao;Yang Lin;Liu Jianguo;Tian Yuan;Chen Jing;Bai Guohui(School of Graduate,Zunyi Medical University,Zunyi Guizhou 563099,China;Key Laboratory of Oral Disease of Higher Schools in Guizhou Province,Zunyi Guizhou 563099,China;Guiyang Hospital of Stomatological,Guiyang Guizhou 550002,China)

机构地区：[1]遵义医科大学研究生院,贵州遵义563099 [2]贵州省普通高等学校口腔疾病研究特色重点实验室,贵州遵义563099 [3]贵阳市口腔医院,贵州贵阳550002

出　　处：《遵义医学院学报》2019年第4期378-382,共5页Journal of Zunyi Medical University

基　　金：国家自然科学基金资助项目(NO:81560186);贵州省科技厅计划项目(NO:黔科合基础SY字[2019]1333);遵义市科技计划项目(NO:遵义市科合HZ字[2019]21);贵州省科学技术项目(NO:黔科合J字LKZ[2012]15)

摘　　要：目的在原核表达质粒pET21a-ha2成功构建的基础上,诱导目的基因在大肠杆菌中表达,进行表达产物的纯化及鉴定。方法原核表达质粒pET21a-ha2经PCR鉴定后,转入大肠杆菌Rosetta感受态细胞诱导筛选,经SDS-PAGE电泳检测,IPTG诱导表达。通过包涵体的变复性、Millipore超滤系统的滤浓缩、Ni-TA层析分离纯化HA2蛋白。结果原核表达质粒pET21a-ha2转化大肠杆菌经IPTG诱导后,成功表达HA2蛋白,大小约15 kD,与预计目的蛋白大小相同。HA2纯化蛋白浓度为0.449 mg/mL,经测序与NCBI蛋白数据库比对无突变。结论pET21a-ha2可在大肠杆菌中成功表达,并成功得到纯化的HA2蛋白。Objective Based on the successful construction of the prokaryotic expression plasmid pET21a-ha2,the target gene was induced to express in Escherichia coli and the expression product was purified and identified.Methods Prokaryotic expression plasmid pET21a-ha2 was identified by PCR and transformed into E.coli Rosetta competent cells for next induction and screening.The expression of fusion protein was induced by isopropylβ-D-1-thiogalactopyrano-side(IPTG)and detected by SDS-PAGE electrophoresis.HA2 protein was separated and purified through the denaturation and renaturation of inclusion body,filtration of Millipore Ultrafiltration system and Ni-NTA chromatography.Results The prokaryotic expression plasmid pET21a-ha2,transformed into E.coli and induced by IPTG,was successfully expressed HA2 protein at the size of 15 kD,the same size as the expected protein.HA2 protein was inclusion body protein,and the concentration was 0.449 mg/ml after purify.No mutation was found based on the NCBI protein database after sequencing.Conclusion pET21a-ha2 can be expressed in E.coli and the purified HA2 protein was successfully obtained.

关 键 词：原核表达质粒 ha2基因 包涵体 纯化 

分 类 号：R780.1[医药卫生—口腔医学]