幽门螺杆菌CagA蛋白N端片段的表达、纯化及其与磷脂酰丝氨酸的亲和力检测  

Expression and Purification of CagA Amino Terminal Fragment from Helicobacter pylori and Interaction Between CagA with Phosphatidylserine

作　　者：管张燕[1] 王芃[1] 陶好霞[1] 袁盛凌[1] 王艳春[1] 申严杰[1] 王令春[1] 刘纯杰[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2011年第5期631-635,共5页Letters in Biotechnology

基　　金：国家高技术研究发展计划重大项目(2006AA02A219);国家新药创制重大专项(2009ZX09301-002)

摘　　要：目的:表达和纯化幽门螺杆菌不同菌株的CagA蛋白N端片段,检测其与磷脂酰丝氨酸(PS)的相互作用及亲和力。方法:用PCR方法从幽门螺杆菌3个菌株中扩增出CagA蛋白N端基因,并连接到表达载体pET-28a上;转化大肠杆菌BL21,经IPTG诱导可溶性表达CagA蛋白N端880残基片段;经镍柱亲和纯化后,利用PLOA法检测CagA蛋白与PS的相互作用。结果:构建了3种幽门螺杆菌菌株cagA基因的原核表达质粒pET-28a/cagAJ99、pET-28a/cagA11637及pET-28a/cagASS1,并在大肠杆菌中获得可溶性表达,SDS-PAGE和Western印迹证实得到目标融合蛋白,亲和纯化得到高纯度CagA蛋白。PLOA结果表明,CagA蛋白与PS有明显的相互作用。结论:3种幽门螺杆菌菌株CagA蛋白与PS之间存在相互作用,且不同的CagA与PS有不同的亲和力。Objective： To express and purify the amino terminal fragment of CagA protein from three Helicobac-ter pylori strains and to investigate interaction between CagA protein with phosphatidylserine（PS）.Methods： The 5＇-terminal part of cagA gene was cloned by PCR from H.pylori 11637,J99 and SS1 separately,and subcloned into the prokaryotic expression vector pET-28a before being transformed into E.coli BL21 cells.Those recombinant His-CagA fusion proteins were expressed and then purified by His tag affinity chromatography.Then PLOA was per-formed to determine the interaction between CagA and PS.Results： Prokaryotic expression constructs were generat-ed and confirmed by DNA sequencing.Those CagA protein segments were successfully expressed in soluble form and highly purified proteins were obtained.The PLOA assay revealed a strong interaction between CagA protein with PS in vitro.Conclusion： Our results showed that CagA protein has the ability to bind PS in vitro,and the binding affinity varied in three detected H.pylori strains.

关 键 词：幽门螺杆菌 CAGA蛋白 磷脂酰丝氨酸 原核表达 纯化 

分 类 号：Q78[生物学—分子生物学]