DNA免疫制备抗人CD154单克隆抗体及其轻链可变区基因序列分析  

Preparation of Anti-hCD154 Monoclonal Antibody by DNA Immunization and Sequencing of Light Chain Variable(VL) Genes

作　　者：张志方[1] 张春艳[2] 

机构地区：[1]中山大学中山医学院生理学教研室,广东广州510089 [2]中山大学中山医学院免疫学教研室,广东广州510089

出　　处：《中山大学学报（自然科学版）》2002年第1期125-127,共3页Acta Scientiarum Naturalium Universitatis Sunyatseni

基　　金：广东省卫生厅基金资助项目 (A19970 98)

摘　　要：采用pcDNA3 1 hCD15 4重组质粒DNA免疫BALB c小鼠 ,制备抗人CD15 4单克隆抗体 ,得到了能分泌针对人T细胞表面CD15 4分子的特异性单抗的杂交瘤细胞株 (7E8)。通过RT_PCR扩增 7E8单抗轻链可变区基因 ,并对其进行克隆、测序、DNA序列分析和氨基酸推导 ,结果显示轻链可变区基因和氨基酸序列完全符合小鼠Ig轻链的结构特征 ,为制备基因工程抗体并应用于临床打下了基础。Anti-hCD154 monoclonal antibody (7E8) was prepared by pcDNA3.1/hCD154 immunization.Its specificity was verified by MIF and flow cytometry.The results show that McAb(7E8) can bound to CHO cells expressing the hCD154 and human T cells activated.Competition tests with standard anti-hCD154 McAb showed that McAb (7E8) could inhibit standard anti-hCD154 McAb binding CD154 molecules on human T cells.These results suggested that McAb (7E8) was a specificity McAb against hCD154 molecules.VL cDNA of McAb against hCD154 were amplified by RT-PCR from total cellular RNA of hybridoma cell 7E8 secreting specific McAb against hCD154 and was cloned into PUC18 vector.Sequence analysis of VL cDNA showed that VL cDNA of 7E8 McAb consists of 341bp encoding 113 amino acid residues.Comparing with mouse Ig database,the VL region is in agreement with the characterization of DNA sequence presented in the mouse Ig Vκ region.

关 键 词：人CD154 DNA免疫 单克隆抗体 基因克隆 跨膜糖蛋白 轻链可变区基因 序列分析 

分 类 号：R382.11[医药卫生—医学寄生虫学] Q785[医药卫生—基础医学]