ATP5B原核表达和单克隆抗体的制备及其鉴定

摘要

目的:制备高纯度ATP合酶β亚基 (ATP5B) 单克隆抗体并进行鉴定, 为其后续研究奠定基础.方法:应用PCR法扩增ATP5B基因, 克隆入pET28a载体中并转入大肠杆菌BL21 (DE3) .异丙基-β-D-硫代吡喃半乳糖苷 (IPTG) 诱导蛋白表达并用镍亲和层析柱纯化融合蛋白, SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 检测蛋白纯度.用纯化的融合蛋白免疫3只雌性Balb/C小鼠, 取小鼠尾静脉血, 间接酶联免疫吸附法 (ELISA法) 检测血清中ATP5B抗体效价.取血清效价最高的免疫小鼠脾细胞与小鼠骨髓瘤SP2/0细胞混合培养建立杂交瘤细胞, 采用间接ELISA法筛选融合细胞并行单克隆化培养, 对阳性细胞进行核型分析.取12周龄Balb/C小鼠, 腹腔注射杂交瘤细胞制备腹水模型, 收集腹水, 间接ELISA法检测效价, SDS-PAGE检测抗体纯度, 应用ELISA法检测抗体亚型.结果:PCR扩增后得到1 455bp的特异性条带, 连接pET28a空载体获得重组pET28a/ATP5B载体.IPTG诱导转化的菌液表达目的蛋白, SDS-PAGE检测, 在相对分子质量为51 000处出现明显的诱导蛋白条带.间接ELISA法检测免疫小鼠静脉血, 血清效价最高达1:64 000.杂交瘤细胞染色体核型分析, 染色体总数约为骨髓瘤细胞与正常小鼠脾细胞之和.采用杂交瘤细胞株制备小鼠腹水, 抗体最高效价为1:240 000, 杂交瘤细胞产生的单克隆抗体的亚型为IgG1.结论:通过克隆、表达和纯化重组蛋白, 成功制备出抗ATP5B蛋白单克隆抗体.%Objective:To construct and identify the monoclonal antibody of ATP synthase beta subunit (ATP5B) with high purity, and to lay foundation for further study.Methods:The ATP5Bgene was amplified by PCR and cloned into the pET28avector and transformed into E.coli BL21 (DE3) .The protein expression was induced by IPTG and then the fusion protein was purified by nickel affinity chromatography column.The protein purity was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .Three female Balb/C mice were immunized with purified fusion protein and the tail vein blood was taken to detect the titer of ATP5Bantibody by indirect enzyme-linked immunosorbent assay (ELISA) .The spleen cells from the immunized mice with the highest serum titer were mixed with the SP2/0cells to establish the hybridoma cells and the fused cells were screened by indirect ELISA and monoclonally cultured.Karyotype analysis were performed in the positive cells.The hybridoma cells were intraperitoneally injected into 12weeks old BALB/C mice to estabilish the ascites models.The titer of ascites was detected by indirect ELISA.The purity of the antibody was detected by SDS-PAGE.The antibody subtype was detected by ELISA.Results:After PCR amplification, a specific band of 1 455bp was obtained, and the pET28aempty vector was ligated to obtain a recombinant pET28a/ATP5Bvector.The target protein was expressed in the IPTG-induced bacteria solution;the SDS-PAGE results showed that the protein band was found at51 000.The indirect ELISA results showed that the serum titer of the venous blood of immunized mice was up to1:64 000.In karyotype analysis, the total number of chromosomes in hybridoma cells was about the sum of myeloma cells and normal mouse spleen cells.The mouse ascites was prepared with the hybridoma cell line, and the highest titer of the antibody was 1:240 000.The subtype of the monoclonal antibody produced by the hybridoma cells was IgG1.Conclusion:The monoclonal antibody against ATP5Bprotein is successfully prepared by cloning, expressing and purifying the recombinant protein.