摘要
目的建立能分泌人转铁蛋白受体(TfR)特异性抗体的杂交瘤细胞株,并对其分泌的抗体进行初步鉴定。方法将人肝癌细胞株HepG2分离纯化的TfR蛋白,采用常规法免疫BALB/C小鼠;采用间接ELISA法测定免疫血清抗体效价;采用常规聚乙二醇(PEG)介导法,将免疫小鼠脾脏细胞与小鼠骨髓瘤细胞株SP2/0融合,用含HAT培养基筛选杂交瘤细胞;采用间接ELISA和Cell-ELISA法筛选TfR抗体阳性的杂交瘤细胞;采用有限稀释法进行克隆和多次亚克隆以建立稳定分泌TfR抗体的杂交瘤细胞株;将杂交瘤细胞注射经石蜡油致敏的小鼠腹腔以产腹水抗体,采用硫酸-辛酸法纯化腹水抗体;采用秋水仙素处理,计数细胞染色体数以进行杂交瘤细胞鉴定;采用免疫荧光染色法和Western blot法初步鉴定抗体与细胞表面或细胞总蛋白中TfR的结合。结果提取纯化的人TfR蛋白免疫小鼠获得成功,其1∶5 000稀释的血清抗体光吸收值(A_(490))是对照小鼠血清(1∶200稀释)的16倍以上;细胞融合率为42.22%,其中TfR抗体阳性孔20个,抗体阳性率为13.16%;抗体阳性孔B6和孔E8中杂交瘤细胞经克隆和亚克隆后建立起稳定分泌抗体的细胞株;B6杂交瘤细胞染色体数量位于56~60条之间,应为B细胞和SP2/0细胞融合而来;腹水抗体染色使HepG2细胞表面出现明亮荧光,并且能使HepG2细胞提取的总蛋白电泳图谱在95 000处出现清晰条带,表明该杂交瘤细胞分泌的抗体能特异性结合TfR蛋白。结论分泌抗人TfR抗体的小鼠杂交瘤细胞株已被筛选和建立;其分泌的抗体能与HepG2细胞的TfR特异性结合。
Objective To establish mouse hybridoma cell lines producing human transferring receptor（ TfR）-specific monoclonal antibody（ mAb） and preliminarily characterize this antibody. Methods The TfR protein purified from HepG2 cells was used to immunize BALB/C mice and the titer of immune sera was measured by indirect ELISA. The splenic cells of immunized mice were fused with SP2/0 cells under mediation of PEG and the growth of hybridoma cells was supported by HAT or HT medium. Indirect ELISA and subsequent cell-ELISA were used to screen TfR positive hybridoma cells and limited dilution method was used to clone or subclone the hybridoma cells for establishment of stable cell lines producing TfRspecific mAb. The ascitic antibody was prepared and purified by caprylic acid-ammonium sulfate（ CA-AS）precipitation. Colchicine block method was used to identify the hybridoma cells. Immunofluorescent assay and western blot were used to check mAb specific binding to its target antigen. Results Mice were successfully immunized by purified TfR protein with fine antibody titer. The hybridoma productivity was 42.22%,of which 20 wells were positive for TfR-specific antibody with antibody positive rate of 13. 16%.Hybridoma cells in wells B6 and E8 were cloned and subcloned for establishment of stable cell lines secreting anti-TfR antibody. Chromosome number of the hybridoma cells was between 56 and 60,suggesting that the hybridoma cells were originated from splenic/SP2/0 fusion cells. The mAb positively stained HepG2 cells and bound to 95000 D protein in total proteins extract of HepG2 cells,indicating its specific targeting to TfR antigen. Conclusion The hybridoma cell lines producing TfR-specific mAb have been successfully established and this antibody shows binding activity to TfR protein.
出处
《广东药科大学学报》
CAS
2017年第3期403-407,共5页
Journal of Guangdong Pharmaceutical University
基金
教育部留学回国人员启动基金(44143006)
四川省教育厅自然科学重点项目(13ZA0220)
