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目的:制备鼠抗人VSIG4分子单克隆抗体(mAb)及对其生物学特性进行鉴定。方法:以高表达膜型VSIG4分子的基因转染细胞L929/VSIG4L为免疫原,常规免疫BALB/c小鼠,采用杂交瘤技术,经流式细胞术检测和多次克隆化培养,筛选出特异分泌鼠抗人VSIG4分子的杂交瘤细胞株;采用间接免疫荧光法、Westernblot、竞争抑制试验及MTT增殖试验等对单抗的生物学特性进行鉴定。结果:获得2株持续、稳定分泌鼠抗人VSIG4mAb的杂交瘤细胞株,分别命名为9A7和9D5。对mAb生物学特性的研究结果表明,2株mAb均能识别巨噬细胞以及Jurkat、THP-1、H446等肿瘤细胞株表面的VSIG4分子。对T细胞增殖抑制的阻断实验显示,这2株抗体对VSIG4分子抑制T细胞增殖均有阻断作用。结论:成功地获得了2株鼠抗人VSIG4功能性mAb,为进一步研究VSIG4及其未知受体的生物学功能奠定了基础。
OBJECTIVE: To prepare murine anti-human VSIG4 monoclonal antibody (mAb) and identify its biological characteristics. METHODS: BALB / c mice were immunized with the gene-transfected L929 / VSIG4L cell line with high expression of membrane type VSIG4 gene. The hybridoma technique was used to detect the expression of membrane-specific VSIG4 gene by flow cytometry and multiple clonal culture. The hybridoma cell lines secreting murine anti-human VSIG4 molecules were identified. The biological characteristics of McAbs were identified by indirect immunofluorescence, Western blot, competitive inhibition assay and MTT proliferation assay. RESULTS: Two hybridoma cell lines that consistently and stably secreted mouse anti-human VSIG4 mAb were obtained and named as 9A7 and 9D5, respectively. The results of biological characteristics of mAb showed that both mAbs could recognize VSIG4 molecules on macrophages and Jurkat, THP-1, H446 and other tumor cell lines. The blocking experiments on the inhibition of T cell proliferation showed that the two antibodies blocked the VSIG4 molecule inhibiting T cell proliferation. Conclusion: Two mouse anti-human VSIG4 functional mAbs were successfully obtained, which laid the foundation for the further study on the biological function of VSIG4 and its unknown receptors.