抗幽门螺杆菌VacA和HpaA双特异性单克隆抗体的制备
发布时间：2019-06-20 15:54
【摘要】： 研究目的:通过细胞杂交-杂交瘤技术制备抗幽门螺杆菌细胞空泡毒素(VacA)和粘附素(HpaA)抗原的双特异性单克隆抗体,并初步鉴定其生物学、免疫学和理化特性。为幽门螺杆菌(H.pylori)感染的防治研究、诊断试剂盒的研制及探讨其致病机制奠定基础。 方法:自重组幽门螺杆菌细胞空泡毒素、粘附素(pQE30-V/H-DH5α)基因工程菌提取细胞空泡毒素(VacA)和粘附素(HpaA)的重组蛋白,12%SDS一PAGE电泳鉴定,以VacA- HpaA重组蛋白为免疫原,免疫BALB/c小鼠。取免疫BALB/c小鼠脾细胞与骨髓瘤细胞株SP2/0,用50%聚乙二醇(PEG)融合,HAT培养基选择培养出杂交瘤细胞。以纯化的rVacA为抗原,间接ELISA法初筛分泌抗VacA抗体的杂交瘤细胞,有限稀释法克隆化培养获得稳定分泌抗rVacA单抗的阳性杂交瘤细胞株。阳性杂交瘤细胞株再与血清HpaA高滴度的免疫BALB/c小鼠脾细胞用50%聚乙二醇(PEG)融合,HAT培养基选择培养出杂交瘤细胞,分别用纯化的rVacA、rHpaA为抗原,间接ELISA法筛选同时分泌抗VacA和HpaA抗体的杂交-杂交瘤细胞,有限稀释法克隆化培养获得稳定分泌抗rVacA和rHpaA双特异性单克隆抗体阳性的杂交-杂交瘤细胞株。阳性细胞株体外连续传30代、反复冻存复苏6次、液氮中冻存1.5个月后复苏,测定细胞株分泌的双特异性单克隆抗体的稳定性。做杂交-杂交瘤细胞的染色体计数并与骨髓瘤细胞系SP2/0和小鼠骨髓细胞的染色体计数进行比较。制备腹水型和上清型双特异性单克隆抗体,以饱和硫酸铵沉淀法和免疫亲和层析法进行纯化,Bradford法测定其蛋白浓度,Western blot检测其免疫特异性,b.v公司的抗体亚型检测试剂盒测定双特异性单克隆抗体的Ig类别,ELISA间接法测定其抗体效价及亲和常数。测定pH值对双特异性单克隆抗体稳定性影响实验,分别以0.05M pH2.2甘氨酸-盐酸缓冲液、0.05M pH9.6碳酸盐缓冲液和0.01M pH7.4磷酸盐缓冲液将腹水型双特异性单克隆抗体(以免疫前小鼠血清作阴性对照)作1:10稀释,4℃静置24h、48h后,用间接ELISA测定其0D450nm与阴性对照0D450nm的比值。 结果:自基因工程菌pQE30-V/H-DH5α中提取重组VacA- HpaA融合蛋白,12%SDS-PAGE鉴定后,作为免疫原成功免疫小鼠。经两次细胞融合,HAT选择培养,抗体检测筛选,获得37孔分泌同时抗rVacA和rHpaA双特异性单克隆抗体的融合细胞孔,经克隆化培养得到3株稳定分泌双特异性单克隆抗体的杂交-杂交瘤细胞株,命名为fB8、fC5和fE7,其分泌的双特异性单克隆抗体均可与纯化的rVacA或rHpaA蛋白特异性结合。fB8杂交-杂交瘤细胞株稳定性较好,体外连续传30代、反复冻存复苏6次、液氮中冻存1.5个月后复苏测定细胞株仍可稳定分泌双特异性单克隆抗体。fB8杂交-杂交瘤细胞染色体计数为127±5条,而小鼠脾细胞为40条,Sp2/0骨髓瘤细胞为64±3条。三株同时抗rVacA和rHpaA的双特异性单克隆抗体杂交-杂交瘤细胞(fB8、fC5和fE7)分泌的抗体均为IgG1。fB8腹水型双特异性单克隆抗体的效价为5.12×105,蛋白浓度为11.32 mg/ml(Bradford法)。pH值对双特异性单克隆抗体稳定性影响的实验结果显示,酸性和碱性都可使双特异性单克隆抗体稳定性下降。 结论:初步建立起三株分泌同时抗幽门螺杆菌细胞空泡毒素和粘附素双特异性单克隆抗体的杂交-杂交瘤细胞株.成功制备并纯化了抗VacA和HpaA的双特异性单克隆抗体,其效价较高,亲和力较强,特异性强,将为幽门螺杆菌感染的诊断、预防和治疗发挥重要作用。
[Abstract]:Objective: To prepare a bispecific monoclonal antibody against Helicobacter pylori (VacA) and adhesin (HpaA) antigen by cell hybridization-hybridoma technique, and to identify its biological, immunological and physical and chemical properties. To study the prevention and treatment of H. pylori infection, the development of the diagnostic kit and the foundation of its pathogenesis. Methods: The recombinant protein of the cell vacuolated toxin (VacA) and the adhesin (HpaA) was extracted from the recombinant H.pylori cell vacuolated toxin and the adhesin (pQE30-V/ H-DH5). The recombinant protein of the cell vacuolated toxin (VacA) and the adhesin (HpaA) was identified by SDS-PAGE. The recombinant protein of vacA-HpaA was used as the immunogen to immunize BALB/ c. C. The spleen cells of the immunized BALB/ c mice were fused with the myeloma cell strain SP2/0, fused with 50% polyethylene glycol (PEG), and the HAT medium selected to culture the hybridization. Tumor cells. The purified rVacuA is an antigen, and the indirect ELISA method is used for the primary screening of a hybridoma cell secreting anti-VacA antibody, and the positive hybridomas stably secrete anti-rVacuA monoclonal antibody are obtained by cloning and culturing by a limited dilution method. The positive hybridoma cell line and the immunized BALB/ c mouse spleen cells with high titer of the serum HpaA were fused with 50% polyethylene glycol (PEG), and the HAT medium was selected to culture the hybridoma cells, and the purified rVacuA and rHpaA were used respectively. Screening of hybrid-hybridoma cells with simultaneous secretion of anti-VacA and HpaA antibodies by indirect ELISA, and the hybrid-hybridomas that stably secrete anti-rVacA and rHpaA bispecific monoclonal antibodies are obtained by cloning and culturing in a limited dilution method. Cell strain. The positive cell line was continuously transmitted for 30 generations in vitro, the recovery was repeated for 6 times, and the recovery was performed in liquid nitrogen for 1.5 months, and the double-specific monoclonal antibody secreted by the cell line was determined. Stability. The chromosome counts of the hybrid-hybridoma cells were counted and the chromosomes of the myeloma cell line SP2/0 and the mouse bone marrow cells were counted Line comparison. Ascites and supernatant type bispecific monoclonal antibodies were prepared, and purified by saturated sulfuric acid precipitation method and immunoaffinity chromatography, and the protein concentration was determined by Bradford method. Western blot was used to detect its immunity. The Ig category of bispecific monoclonal antibody was determined by the antibody subtype detection kit of b. v, and the antibody titer and affinity of the two-specific monoclonal antibody were determined by ELISA. And the stability of the double-specificity monoclonal antibody is measured by measuring the pH value, and the stability of the double-specificity monoclonal antibody is measured with 0.05M pH2. 2glycine-hydrochloric acid, respectively. Ascites-type bispecific monoclonal antibody (negative control with the serum of the pre-immunized mouse) was used as 1:10 for buffer, 0.05M pH9.6 carbonate buffer and 0.01 M pH 7.4 phosphate buffer, and the 0 D450nm and negative control 0D450nm were determined by indirect ELISA after 24 h and 48 h at 4 鈩，
本文编号：2503345
[Abstract]:Objective: To prepare a bispecific monoclonal antibody against Helicobacter pylori (VacA) and adhesin (HpaA) antigen by cell hybridization-hybridoma technique, and to identify its biological, immunological and physical and chemical properties. To study the prevention and treatment of H. pylori infection, the development of the diagnostic kit and the foundation of its pathogenesis. Methods: The recombinant protein of the cell vacuolated toxin (VacA) and the adhesin (HpaA) was extracted from the recombinant H.pylori cell vacuolated toxin and the adhesin (pQE30-V/ H-DH5). The recombinant protein of the cell vacuolated toxin (VacA) and the adhesin (HpaA) was identified by SDS-PAGE. The recombinant protein of vacA-HpaA was used as the immunogen to immunize BALB/ c. C. The spleen cells of the immunized BALB/ c mice were fused with the myeloma cell strain SP2/0, fused with 50% polyethylene glycol (PEG), and the HAT medium selected to culture the hybridization. Tumor cells. The purified rVacuA is an antigen, and the indirect ELISA method is used for the primary screening of a hybridoma cell secreting anti-VacA antibody, and the positive hybridomas stably secrete anti-rVacuA monoclonal antibody are obtained by cloning and culturing by a limited dilution method. The positive hybridoma cell line and the immunized BALB/ c mouse spleen cells with high titer of the serum HpaA were fused with 50% polyethylene glycol (PEG), and the HAT medium was selected to culture the hybridoma cells, and the purified rVacuA and rHpaA were used respectively. Screening of hybrid-hybridoma cells with simultaneous secretion of anti-VacA and HpaA antibodies by indirect ELISA, and the hybrid-hybridomas that stably secrete anti-rVacA and rHpaA bispecific monoclonal antibodies are obtained by cloning and culturing in a limited dilution method. Cell strain. The positive cell line was continuously transmitted for 30 generations in vitro, the recovery was repeated for 6 times, and the recovery was performed in liquid nitrogen for 1.5 months, and the double-specific monoclonal antibody secreted by the cell line was determined. Stability. The chromosome counts of the hybrid-hybridoma cells were counted and the chromosomes of the myeloma cell line SP2/0 and the mouse bone marrow cells were counted Line comparison. Ascites and supernatant type bispecific monoclonal antibodies were prepared, and purified by saturated sulfuric acid precipitation method and immunoaffinity chromatography, and the protein concentration was determined by Bradford method. Western blot was used to detect its immunity. The Ig category of bispecific monoclonal antibody was determined by the antibody subtype detection kit of b. v, and the antibody titer and affinity of the two-specific monoclonal antibody were determined by ELISA. And the stability of the double-specificity monoclonal antibody is measured by measuring the pH value, and the stability of the double-specificity monoclonal antibody is measured with 0.05M pH2. 2glycine-hydrochloric acid, respectively. Ascites-type bispecific monoclonal antibody (negative control with the serum of the pre-immunized mouse) was used as 1:10 for buffer, 0.05M pH9.6 carbonate buffer and 0.01 M pH 7.4 phosphate buffer, and the 0 D450nm and negative control 0D450nm were determined by indirect ELISA after 24 h and 48 h at 4 鈩，
本文编号：2503345
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