CD154 cDNA克隆、单克隆抗体制备及特异性鉴定

本研究目的是获得特异性抗人CD15 4单克隆抗体。利用特异引物 ,通过RT PCR从人外周血淋巴细胞中扩增出编码CD15 4分子的cDNA全长 ,将其克隆在谷胱甘肽巯基转移酶 (GST )融合蛋白表达载体pGEX4T 3中 ,转染大肠杆菌Jm10 9经诱导获得GST CD15 4表达。融合蛋白经分离纯化后 ,免疫Balb/c小鼠 ,应用杂交瘤技术 ,通过ELISA双筛 ,得到 2株抗CD15 4单抗 (1D3和 5H4)。其亚型分别是IgG2a和IgG1。又将CD15 4cDNA全长构建真核表达载体pcDNA3 CD15 4,并转染COS细胞 ,以G418筛选后 ,经RT PCR结果证实pcDNA3 CD15 4已成功转染COS细胞。所获单抗对转染CD15 4的COS细胞和刺激活化的人淋巴细胞有特异性结合反应 ,为进一步研究单抗功能奠定了实验基础。

Cloning and Preparation of Monoclonal Antibody Against Human CD154

The aim of this study is to prepare the monoclonal antibody (McAb) against human CD154 protein The full length of CD154 molecule was amplified from human peripheral blood lymphocytes via RT PCR and cloned into prokaryotic expressive vector PGEX4T 3 Fusion protein GST CD154 was expressed after IPTG induction and isolated with glutathione sepharose 4B since they are existing in soluble form Purified target protein was used for immunizing Balb/c mice to prepare McAb against CD154 Two hybridomas (1D3 and 5H4) secreting McAb to CD154 had been obtained through ELISA screening Meanwhile, COS cells were transfected with mammalian expression vector pcDNA3 CD154 and screened by G418 The transfected cells were confirmed by RT PCR, and the specificity of the McAb was furthering identified with immunohistochemistry stained on transfected COS cells and stimulated lymphocytes