小鼠抗人CD160分子单克隆抗体制备及其生物学功能初步分析

Preparation of a mouse anti-human monoclonal antibody and preliminary analysis of its biological function

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。

CD160 which is a glycosyl phosphatidyl inositol （GPI）-anchored protein is a new member of immunoglobulin super- family. 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 monoelonal antibody, human CD160 full-length eDNA was obtained from human whole blood eDNA library by RT-PCR and then was inserted into retroviral expressing vector pEGZ-Term. Then, we used this recombinant vector to transfeet L929 cell. Eventually, we obtain gene transfected cell lines that stably express human CD160. BALB/c mouse were immunized with CD160 transfeeted-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 seere- ted anti-CD160 mAb. T cell proliferation test showed that the mAb could recognize human T lymphoeytes from health donors and inhibit the proliferation of T lymphoeytes in vitro.