幽门螺杆菌双亚单位表位融合蛋白的构建、表达及鉴定

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.coli BL21(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western blotting、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 Hp HpaA-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.

Construction, expression, and identification of HUepi-LtB fusion

Objective To construct and prokaryotically express 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 expressed under IPTG induction. After purification 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 as 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. Conclusion The fusion protein HUepi-LTB was successfully constructed and well expressed in prokaryotic expression system, which may benefit the development of H. pylori vaccine.