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目的:制备和鉴定抗胰岛素样生长因子Ⅰ型受体(IGF-IR)单克隆抗体(mAb),为深入研究肿瘤细胞中IGF-IR功能及致病机制奠定基础。方法:用脂质体法将制备的重组慢病毒载体三质粒pWPXL-IGF-IR、PMD2G、PAX2共转染包装细胞293T,将上清病毒转染目的细胞NIH-3T3,通过West-ern blot检测稳转NIH-3T3细胞中IGF-IR的表达。将细胞株NIH3T3-IGF-IR免疫BALB/c小鼠,提取小鼠脾细胞与小鼠骨髓瘤细胞Sp2/0进行杂交融合,筛选出针对IGF-IR的特异性杂交瘤细胞株,Western blot鉴定单克隆细胞株的特异性。并检测其免疫球蛋白亚类及mAb效价。结果:通过细胞融合和克隆化,筛选出1株持续分泌抗IGF-IR mAb的杂交瘤细胞株9F3。该抗体能与IGF-IR特异性结合,经鉴定该抗体属于IgG1亚类。结论:成功地制备了抗IGF-IR mAb,为研究IGF-IR在肿瘤中的生物学功能奠定了基础。
OBJECTIVE: To prepare and identify mAbs against insulin-like growth factor type I receptor (IGF-IR) and lay a foundation for further study on the function and pathogenesis of IGF-IR in tumor cells. METHODS: The recombinant lentiviral vector pWPXL-IGF-IR, PMD2G and PAX2 were co-transfected into 293T packaging cells by lipofectamine. The supernatant virus was transfected into NIH-3T3 cells by West-ern blot Stable expression of IGF-IR in NIH-3T3 cells. BALB / c mice were immunized with cell line NIH3T3-IGF-IR, and splenocytes of mouse were extracted and hybridized with Sp2 / 0 of mouse myeloma cells. The specific hybridoma cell lines screened for IGF-IR were screened by Western blot Specificity of monoclonal cell lines. The immunoglobulin subclasses and mAb titers were tested. Results: A hybridoma cell line 9F3 that secreted anti-IGF-IR mAb was screened by cell fusion and cloning. This antibody specifically binds to IGF-IR and was identified as belonging to the IgGl subclass. Conclusion: The anti-IGF-IR mAb was successfully prepared, which laid the foundation for the study of the biological function of IGF-IR in tumor.