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利用基因工程方法将鼠源性抗CD3抗体HIT3a的可变区和人源抗体 (IgG)的完整的恒定区连接起来 ,构建全抗型抗CD3嵌合抗体 ,该型抗体具有较低的免疫源性可作为免疫抑制剂应用于器官移植 ,减少受体产生免疫排斥 ,提高移植器官的存活率。利用PCR方法从抗CD3ScFv重组噬菌体表达载体pCANTAB 5E上扩增抗CD3抗体的轻链和重链可变区 ,将轻链和重链可变区组装到含有人抗体 (IgG)恒定区的表达载体中 ,构建抗CD3嵌合抗体IgG的轻链和重链表达载体PKN10 0和PG1D10 5 ,并用脂质体法共转染CHO细胞。结果证明 ,抗CD3嵌合抗体的VL 和VH 与HIT3a抗体的VL 和VH 完全相符 ,ELISA和Westernblot检测结果证实转染细胞的培养上清中含有抗CD3嵌合抗体IgG的表达 ,表达产物能与Jurkat细胞结合 ,并能竞争性抑制HIT3a抗体和Jurkat细胞结合活性 ,3H TdR掺入实验表明 ,抗CD3嵌合抗体与亲代抗体HIT3a一样 ,具有促进外周血单核细胞增殖的作用。我室构建的全抗型抗CD3嵌合抗体分子表达载体可在CHO细胞中稳定表达 ,表达产物有较好生物活性 ,具有潜在的临床应用价值
A fully engineered anti-CD3 chimeric antibody was constructed by genetically engineering the variable region of murine anti-CD3 antibody HIT3a and the complete constant region of a human antibody (IgG), which antibody has a lower immunogenicity Sex can be used as an immunosuppressive agent in organ transplantation, reduce the recipient immune rejection, improve the survival rate of transplanted organs. The light and heavy chain variable regions of the anti-CD3 antibody were amplified by PCR from the anti-CD3 ScFv recombinant phage expression vector pCANTAB 5E and the light and heavy chain variable regions were assembled into an expression vector containing the constant region of a human antibody (IgG) , The light and heavy chain expression vectors PKN10 0 and PG1D10 5 of the anti-CD3 chimeric antibody IgG were constructed and co-transfected into CHO cells by lipofectamine. The results demonstrated that the VL and VH of the anti-CD3 chimeric antibody exactly matched the VL and VH of the HIT3a antibody. The results of ELISA and Western blot confirmed that the culture supernatant of the transfected cells contained the anti-CD3 chimeric antibody IgG, Jurkat cells and competitive inhibition of HIT3a and Jurkat cell binding activity, 3H TdR incorporation experiments showed that the anti-CD3 chimeric antibody and the parent antibody HIT3a, with the promotion of peripheral blood mononuclear cell proliferation. The anti-CD3 chimeric antibody molecule expression vector constructed in our laboratory can be stably expressed in CHO cells and the expressed product has good biological activity and has potential clinical application value