结核杆菌嵌合蛋白Ag856A2的原核表达、纯化、抗原性分析及其单克隆抗体的制备

发布时间：2018-03-31 22:28

  本文选题：结核分支杆菌　切入点：嵌合蛋白　出处：《苏州大学》2008年硕士论文

【摘要】： 目的构建结核杆菌嵌合蛋白Ag856A2的原核表达载体,进行原核表达、纯化和抗原性分析,制备该蛋白的单克隆抗体,并鉴定单抗的重要性质。方法以DNA疫苗HG856A为模板,经PCR扩增获得ag856a2基因,克隆至pET28a质粒得到原核表达质粒pET856A2;用该重组质粒转化大肠杆菌BL21(DE3),以IPTG诱导目的基因表达,通过SDS-PAGE鉴定其表达形式;用Ni2+柱亲和层析纯化嵌合蛋白Ag856A2;分别用Ag85A及ESAT-6的小鼠抗血清对纯化的蛋白Ag856A2进行Western Blotting分析其抗原性。用DNA疫苗HG856A和纯化的嵌合蛋白Ag856A2免疫BALB/C小鼠,取脾细胞和骨髓瘤细胞SP2/0融合,通过HAT培养基选择性培养,间接ELISA法筛选阳性克隆,用有限稀释法将阳性克隆单克隆化,再用间接ELISA法筛选、鉴定,最终建立稳定分泌单克隆抗体的杂交瘤细胞株;分别用间接ELISA法鉴定单克隆抗体的亚类、效价及所识别的抗原,用Western Blotting分析识别蛋白ESAT-6的单克隆抗体的特异性。结果成功构建了重组质粒pET856A2,嵌合蛋白Ag856A2以包涵体形式表达,约占菌体总蛋白的35%;经Ni2+柱亲和纯化的样品SDS-PAGE分析纯度在90%以上,Ag85A及ESAT-6抗血清均可与其发生特异性反应。融合后经筛选、克隆化得到6株抗Ag856A2的杂交瘤细胞,经鉴定有五株可同时与蛋白Ag856A2、Ag85A反应,为IgG1亚类;一株可同时与蛋白Ag856A2、ESAT-6反应,为IgG2b亚类,其培养上清与蛋白ESAT-6反应效价为6400,与蛋白Ag856A2反应效价为12800;该株单克隆抗体经Western Blotting鉴定可与蛋白Ag856A2、ESAT-6发生特异性反应。结论以包涵体形式表达的嵌合蛋白Ag856A2具有Ag85A及ESAT-6的抗原性;以其DNA疫苗和纯化蛋白免疫小鼠后,初步建立起一株分泌抗蛋白ESAT-6单克隆抗体的杂交瘤细胞株。本研究为进一步研究该嵌合蛋白在结核病亚单位疫苗中的应用打下了基础,为抗蛋白ESAT-6单克隆抗体在结核病早期诊断上的应用奠定了基础。
[Abstract]:Objective to construct the prokaryotic expression vector of the chimeric protein Ag856A2 of Mycobacterium tuberculosis for prokaryotic expression, purification and antigenicity analysis, to prepare the monoclonal antibody against the protein and to identify the important properties of the monoclonal antibody.Methods DNA vaccine HG856A was used as template, ag856a2 gene was amplified by PCR and cloned into pET28a plasmid to obtain prokaryotic expression plasmid pET856A2. The recombinant plasmid was transformed into E. coli BL21DE-3, and the target gene was induced by IPTG, and its expression form was identified by SDS-PAGE.The chimeric protein Ag856A2 was purified by Ni2 affinity chromatography and its antigenicity was analyzed by Western Blotting with the mouse antiserum of Ag85A and ESAT-6.BALB/C mice were immunized with DNA vaccine HG856A and purified chimeric protein Ag856A2. The spleen cells and myeloma cells SP2/0 were fused. The positive clones were screened by indirect ELISA method in HAT medium, and the positive clones were monoclonal by limited dilution method.Then indirect ELISA method was used to screen and identify the hybridoma cell lines secreting monoclonal antibodies stably, and the subclasses, titers and recognized antigens of monoclonal antibodies were identified by indirect ELISA method, respectively.The specificity of monoclonal antibody to recognize protein ESAT-6 was analyzed by Western Blotting.Results the recombinant plasmid pET856A2 was successfully constructed, and the chimeric protein Ag856A2 was expressed in the form of inclusion body, accounting for about 35% of the total bacterial protein, and the purity of the sample SDS-PAGE purified by Ni2 column affinity was more than 90%, and the specific reaction of Ag85A and ESAT-6 antiserum could be observed.After fusion, six hybridoma cells resistant to Ag856A2 were obtained. Five of them could react with the protein Ag856A2, Ag85A, and one could react with Ag856A2, ESAT-6, which were IgG2b subclasses at the same time, and five of them could react with Ag856A2A2Ag85A at the same time, and one of them could react with Ag856A2ESAT-6 at the same time.The supernatant reacted with protein ESAT-6 and protein Ag856A2 with the titer of 6400 and 12800.The monoclonal antibody was identified by Western Blotting to react specifically with protein Ag856A2ESAT-6.Conclusion the chimeric protein Ag856A2 expressed in the form of inclusion body has the antigenicity of Ag85A and ESAT-6. After immunizing mice with its DNA vaccine and purified protein, a hybridoma cell line secreting monoclonal antibody to protein ESAT-6 was established.This study laid a foundation for further study on the application of the chimeric protein in tuberculosis subunit vaccine and laid a foundation for the application of anti-protein ESAT-6 monoclonal antibody in the early diagnosis of tuberculosis.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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