幽门螺旋杆菌重组抗原表位肽C-CagAL构建及其多克隆抗体特异性分析  

Construction of recombinant antigenic epitope peptide C-CagAL against Helicobacter pylori and specific analysis of its polyclonal antibody

作　　者：郭乐 周宁[3] 龚晓娟 王淑娥 狄银华 洪丹彤 张帆 刘宏鹏 刘昆梅[2,3] Le Guo;Ning Zhou;Xiaojuan Gong;Shu'e Wang;Yinhua Di;Dantong Hong;Fan Zhang;Hongpeng Liu;Liu Kunmei(Provincial Key Laboratory of Clinical and Pathogenic Microbiology,General Hospital of Ningxia Medical University,Yinchuan 750004,China;Breeding Base of State Key Levboratory for Craniocerebral Diseases,Ningxia Medical University,Yinchuan 750021,China;School of Clinical Medicine,Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学总医院,临床病原微生物省级重点实验室,宁夏银川750004 [2]宁夏医科大学颅脑疾病国家重点实验室培育基地,宁夏银川750021 [3]宁夏医科大学临床医学院,宁夏银川750004

出　　处：《生物技术》2018年第6期537-542,共6页Biotechnology

基　　金：国家自然科学基金项目(“靶向M细胞的乳酸菌表面展示投递体系LL-pSAM的构建及其在幽门螺旋杆菌多价表位疫苗中的实验研究”,No.31600744);宁夏高等学校科学研究项目(“以多表位肽FVpE及其特异性抗体为包被物的幽门螺旋杆菌胶体金检测试剂盒的研制”,No.NGY2017088);国家级大学生创新创业训练计划项目(“抗幽门螺旋杆菌感染的乳酸菌黏膜靶向疫苗LL-pSAM-FVpE的免疫学性质研究”,No.201810752010);自治区级大学生创新创业训练计划项目(“幽门螺旋杆菌四价黏附素重组表位肽在大肠杆菌中的表达与纯化及其免疫学性质研究”,No.201610752012);宁夏医科大学校级科研项目(“幽门螺旋杆菌四价黏附素多表位疫苗CFAdE的构建及其表达研究”,No.XM2015006)

摘　　要：[目的]制备幽门螺旋杆菌重组抗原表位肽C-CagAL,并分析其多克隆抗血清的特异性。[方法]用分子克隆技术构建重组表达质粒pETC-CagAL,将其转入大肠杆菌中表达,利用Ni-Agarose亲和层析纯化目的蛋白C-CagAL;免疫BALB/c小鼠,制备抗C-CagAL多克隆抗血清,通过ELISA方法分析抗血清的特异性。[结果]DNAstar等软件表明C-CagAL具有较好的抗原性,Linker易形成转角或β-折叠,为柔性结构;重组抗原C-CagAL经大肠杆菌表达,约56kDa,占菌体总蛋白的19.82%,经Ni-Agarose亲和层析纯化,获得纯度为97.5%的重组抗原C-CagAL;抗C-CagAL多克隆抗血清能够特异性识别CagA和CagL。[结论]获得了一种能够激发抗CagA和CagL特异性抗体的重组抗原表位肽C-CagAL,为进一步研究其预防幽门螺旋杆菌感染的免疫效果奠定了基础。[Objective] To construct recombinant antigenic epitope peptide C-CagAL against Helicobacter pylori and analyze the specificity of its polyclonal antiserum against C-CagAL.[Methods] The recombinant expression plasmid p ETC-CagAL was constructed by molecular cloning technology and transferred into Escherichia coli. Then,the interest protein C-CagAL was obtained by Ni-Agarose affinity chromatography. Polyclonal antisera were prepared from BALB/c mice immunized with the fusion protein CTB-CagAL. Finally,the specificity of polyclonal antisera was analysed by ELISA assay. [Results] The results from DNAstar and other software showed that C-CagAL had good antigenicity,and that linkers with flexible structure were easy to form corner or β – fold. Recombinant antigen C-CagAL was about 56 k Da and 19. 82% of total bacterial protein by expression in Escherichia coli. After purification with Ni-Agarose affinity chromatography,the recombinant antigen C-CagAL with a purity of 97. 5% was obtained. Moreover,polyclonal antiserum against C-CagAL can specifically recognize CagA and CagL.[Conclusion]A recombinant antigenic epitope peptide C-CagAL,which can excite specific antibodies against CagA and Ca-g L,was obtained. This work laid the foundation for further study on the immunological effect against Helicobacter pylori infection.

关 键 词：幽门螺旋杆菌 抗原表位肽 表达 CAGA CagL 特异性 

分 类 号：Q784[生物学—分子生物学] Q786