幽门螺杆菌双亚单位表位疫苗的构建及免疫原性研究

目的构建幽门螺杆菌(Helicobacter pylori,Hp)黏附素(HpaA)和尿素酶B亚单位(UreB)双亚基多表位疫苗,并对其免疫原性进行研究。方法设计引物采用PCR法将黏附素(HpaA)的一个B细胞表位与尿素酶B亚单位的三个TH表位及一个B细胞表位串联起来(HUepi),表位之间用两个赖氨酸(KK)间隔,T-A克隆后构建融合基因表达质粒pET-22b( )-HUepi,在大肠杆菌BL21(DE3)中表达,重组蛋白采用阳离子和疏水层析进行纯化,经鉴定后皮下免疫BALB/c小鼠,检测细胞免疫应答及体液免疫应答。结果PCR法扩增出了约233bp的目的片段;原核表达质粒pET-22b( )-HUepi经酶切及测序鉴定,目的基因序列与设计序列一致;重组蛋白HUepi表达率约20.0%,PAGE初步测定目的蛋白的相对分子质量(Mr)约8.38×103Dr,破菌后电泳证实目的蛋白以可溶形式表达,纯化后蛋白纯度大于97.6%,经N端测序证实为设计的表位疫苗蛋白,TH表位多肽、表位疫苗蛋白及rUreB均能够刺激表位疫苗致敏的小鼠淋巴细胞增殖(SI>2),并且此疫苗能够刺激机体产生特异性抗体。结论Hp HpaA、UreB双亚基表位疫苗HUepi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原性。为新一代Hp疫苗的研制奠定实验基础。

Construction and prokaryotic expression of the double unit epitope vaccine of Helicobactor pylori and its immunogenicity

To construct and prokaryotically express the epitope vaccine of HpaA and UreB of Helicobacter pylori and to investigate its immunogenicity in BALB/c mice. the coding gene for epitopes of HpaA and UreB(HUepi)were amplified from plasmids pET-22b-Uepi ,after T-A cloning the gene was cloned into expression vector pET-22b ( ) , and expressed in Escherichia coli BL21(DE3) . BALB/c mice was immunized by the purified and identified protein subcutaneously and then the cellular and humoral immune response was detected. The gene of HUepi was successfully cloned into pET-22b ( ),the result of the nucleotide sequencing was identical to that of the molecular design. The recombinant protein expression rate was about 20.0% and tris-tricine SDS-PAGE assay showed that the molecular weight was 8.38kD. The purity of recombinant protein was up to 97.6 % after cation exchange chromatography and hydrophobic interaction chromatography.The result of the N-terminal amino acid residual sequencing was identical to that of the molecular design. It is evident that recombinant protein can elicit strong cellular and humoral immune responses. The epitope vaccine was proved to be successful this providing experimental basis a potential candidate of therapeutic and preventive vaccine of Helicobacter pylori.