APEC毒力基因ygeG的原核表达载体构建、表达纯化及鉴定  

Prokaryotic Expression Vector Construction,Expression,Purification and Identification of the APEC Virulence Gene ygeG

作　　者：姜楠 郑倩倩 李倩文[1] 涂健[1] 宋祥军 邵颖[1] 刘红梅[1] 祁克宗[1] JIANG Nan;ZHENG Qianqian;LI Qianwen;TU Jian;SONG Xiangjun;SHAO Ying;LIU Hongmei;QI Kezong(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,China)

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036

出　　处：《华北农学报》2021年第5期184-190,共7页Acta Agriculturae Boreali-Sinica

基　　金：国家自然科学基金项目(31772707)。

摘　　要：兽医病理生物学与疫病防控安徽省重点实验室前期预测了新的ETT2伴侣分子YgeG,为了获得大量有活性的YgeG蛋白,对其功能进行鉴定,并筛选出与其相关的具有活性的ETT2分泌效应蛋白,将对目的蛋白原核表达条件进行优化。以禽致病性大肠杆菌菌株81(AE81)为模板对基因ygeG进行扩增,将扩增产物克隆至pGEX-6p-1原核表达载体,构建重组质粒pGEX-6p-1-ygeG,并进行测序鉴定。经鉴定正确后的重组质粒转化至大肠杆菌表达菌株BL21(DE3)进行IPTG诱导表达,并对重组蛋白的诱导条件进行优化。SDS-PAGE及Western Blot鉴定结果显示,本试验中表达的最佳条件为:IPTG终浓度为0.25 mmol/L,16℃诱导16 h。重组蛋白大小约为44 ku,在大肠杆菌BL21(DE3)感受态细胞中高效表达,主要以可溶性蛋白的形式存在,并且具有良好的免疫原性和反应原性。成功表达了AE81-YgeG蛋白,为该蛋白的结构和功能及ETT2的深入研究奠定基础,为禽大肠杆菌病的防治提供理论依据。Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control predicted a new ETT2 chaperone molecule YgeG in the early stage.To obtain a large number of active YgeG protein,identify its function,and screen out its related active ETT2 secretion effector protein,the prokaryotic expression conditions of the target protein were optimized.The ygeG gene was PCR amplified,with the APEC 81 strain(AE81)as the template.The amplified product was cloned into pGEX-6p-1 prokaryotic expression vector,and the recombinant plasmid pGEX-6p-1-ygeG was constructed and identified by sequencing.Then,the correctly identified recombinant plasmid was transformed into E.coli BL21(DE3),and was induced by IPTG.And optimizing the induction conditions of the recombinant protein to determine the optimal conditions for the expression of YgeG recombinant protein.The results of SDS-PACE and Western Blot showed that the optimal induction conditions were determined to be an induction temperature of 16℃,an induction time of 16 h,and IPTG final concentration of 0.25 mmol/L.The size of the recombinant protein was about 44 ku,which was highly expressed in BL21(DE3)and mainly in the form of soluble protein and has good immunoreactivity and antigenicity.The AE81-YgeG protein was successfully expressed,which laid the foundation for the further study of the structure and function of the protein and ETT2,and provided a theoretical basis for the prevention and treatment of avian colibacillosis.

关 键 词：大肠杆菌 ygeG ETT2 原核表达 蛋白纯化 

分 类 号：S852.3[农业科学—基础兽医学] Q78[农业科学—兽医学]