幽门螺杆菌双亚单位表位融合蛋白的构建、表达及鉴定  

Construction, expression, and identification of HUepi-LtB fusion

作　　者：周维英[1] 吴超[1] 石云[1] 张卫军[1] 邹全明[1] 

机构地区：[1]第三军医大学临床微生物学及免疫学教研室暨重庆市生物制药工程技术研究中心,重庆400038

出　　处：《免疫学杂志》2007年第2期121-125,130,共6页Immunological Journal

基　　金：国家"十五"重大科技专项(2003AA2Z3C64);国家自然科学基金项目(30400406)资助

摘　　要：目的构建和表达幽门螺杆菌(Helicobacter pylori,Hp)黏附素(HpaA)和尿素酶B亚单位(UreB)表位串联体(HUepi)与大肠杆菌不耐热肠毒素B亚单位(LTB)融合蛋白(HUepi-LTB),并对其进行纯化和鉴定。方法采用PCR技术分别扩增HpaA-UreB表位多肽编码基因HUepi和LTB的编码基因LtB,重叠延伸PCR将2段基因拼接起来,T-A克隆后构建融合基因表达质粒pET-22b(+)-HUepi-LtB,经酶切鉴定后转化E.coliBL21(DE3),IPTG诱导表达,采用阳离子和阴离子交换层析进行纯化,PAGE检测、N端测序和westernblotting、ELISA检测、GM1活性检测。结果成功构建表位串联体(HUepi)与LTB融合蛋白的高效表达质粒pET-22b(+)-HUepi-LtB,重组工程菌pET-22b(+)-HUepi-LtB/BL21经IPTG诱导目的蛋白表达率约28%,PAGE初步测定目的蛋白的相对分子质量(Mr)约20.7×103,破菌后电泳证实目的蛋白以包涵体形式表达,纯化后蛋白纯度达97%,免疫学鉴定该融合蛋白与兔抗LTB抗血清可以发生特异性的结合。结论HpHpaA-UreB表位串联体(HUepi)与LTB融合蛋白(HUepi-LTB)经基因克隆获得了较高的表达量,并初步显示了较好的免疫活性。为新一代Hp疫苗的研制奠定基础。Objective To construct and prokaryotically cexpress the fusion protein-the epitopes of HpaA and UreB of Helicobacter pylori （HUepi） and the recombinant heat- labile toxin B subunit （rLTB） of Escherichia coli, and to analyze the characteristics of the fusion protein. Methods The genes coding HUepi and LtB were amplified from plasmids pET-22b-Uepi and pET-11c-LtB respectively, and then were linked by overlap extension PCR （HUepi-LtB）. Recombinant plasmid pET-22b （ ＋ ）-HUepi-LtB obtained by T-A clone was transformed into E. coli BL21 （DE3）, and then expresaed under IPTG induction. After pmification with ion exchange chromatography, the expression protein was analyzed with Tris-tricine SDS-PAGE, Western blotting, ELISA essay, GM1 essay, and N-terminal aminoacid residual sequencing. Results The expression vector of HUepi-LtB fusion protein was constructed successfully. The expression rate of the fusion protein in Escherichia coli inclusion bodies was 28% ; the relative molecular weight of the expressed product was 20 700; the purity of the fusion protein was up to 97 % after ion exchange chromatography. Immunological investigation suggested that the fusion protein could react with rabbit-anti-LTB specifically. Canclusion The fusion protein HUepi-LTB was successfully constructed and well expressed in prokaryotic expression system, which may benefit the devclopment of H. pylori vaccine.

关 键 词：幽门螺杆菌 黏附素 尿素酶B亚单位 大肠杆菌不耐热毒素B亚单位 

分 类 号：R392[医药卫生—免疫学]