【摘要】 CD160分子是糖基磷脂酰肌醇(GPI)锚定蛋白,是免疫超家族新成员,与其特异性配体HVEM分子结合所提供的共刺激信号在T细胞的增殖,活化,细胞因子产生和B细胞增殖,活化,分泌抗体产生等方面发挥着重要的调节作用。目前,市面上效果较好的IgG亚型的CD160抗体并不多,通过常规的基因克隆技术,基因重组技术,小鼠免疫技术以及经典的B淋巴细胞杂交瘤技术,获得能持续稳定分泌特异性鼠抗人CD160分子的IgG亚型单克隆抗体的杂交瘤细胞株;采用westen blot和竞争结合抑制实验,对获得的杂交瘤细胞进行生物特性方面的鉴定;采用间接免疫荧光法和流式细胞术分析CD160在健康人PBMC中T细胞表面的表达;T细胞增殖实验表明,此株单抗对于T细胞的体外增殖具有抑制作用。为进一步研究CD160分子的生物学效应提供了新的工具。更多还原

【Abstract】 CD160which is a glycosyl phosphatidyl inositol(GPI)-anchored protein is a new member of immunoglobulin superfamily.The interaction of CD160 with its ligand HVEM generates co-stimulatory signals that are important in the activation,proliferation and cytokine production of T cells during the secondary immune responses.In order to produce mouse anti-human CD160 monoclonal antibody,human CD160full-length cDNA was obtained from human whole blood cDNA library by RT-PCR and then was inserted into retroviral expressing vector pEGZ-Term.Then,we used this recombinant vector to transfect L929 cell.Eventually,we obtain gene transfected cell lines that stably express human CD160.BALB/c mouse were immunized with CD160transfected-cell L929/CD160 that stably expressed human CD160 molecule.The spleen cells of immunized mice were fused with myeloma cells SP2/0,and then cultured in HAT selective medium.Hybridoma cells were selected with L929/CD160 cells by flow cytometry.Eventually,we obtained the hybridoma cell line named 5A7 which sustainably and stably secreted anti-CD160 mAb.T cell proliferation test showed that the mAb could recognize human T lymphocytes from health donors and inhibit the proliferation of T lymphocytes in vitro.更多还原