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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。
The purpose of this study was to obtain specific anti-human CD15 4 monoclonal antibody. The full-length cDNA encoding CD15 4 was amplified by RT PCR from human peripheral blood lymphocytes using specific primers and cloned into the glutathione S-transferase (GST) fusion protein expression vector pGEX4T 3, transfected E. coli JM109 was induced to express GST CD154. The fusion protein was isolated and purified, Balb / c mice were immunized, and two anti-CD154 monoclonal antibodies (1D3 and 5H4) were obtained by hybridization using hybridoma technology. Its subtypes are IgG2a and IgG1, respectively. The full-length CD15 4 cDNA was used to construct the eukaryotic expression vector pcDNA3 CD15 4 and transfected into COS cells. After screening with G418, it was confirmed by RT PCR that pcDNA3 CD15 4 was successfully transfected into COS cells. The McAbs obtained showed specific binding to CD154-transfected COS cells and stimulated activated human lymphocytes, which laid the foundation for further study of monoclonal antibody function.