抗CD3基因工程嵌合抗体IgG在哺乳动物细胞中的表达和活性测定

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CD3 ScFv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PKN100和PG1D105，并用脂质体法共转染CHO细胞。结果证明，抗CD₃嵌合抗体的V_L和V_H与HIT3a抗体的V_L和V_H完全相符，ELISA和Western blot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。

Expression of Chimeric Anti-CD3 IgG Antibody in Mammalian Cells and Analysis of Its Biological Activity

The anti CD3 antibody can improve success rate of organs transplant. HIT3a, a mouse anti CD3 antibody, was chimerized by using gene engineering methods to decrease its immunogenity. The anti CD3 genes, heavy chain and light chain, were cloned using PCR from the vector pCANTAB 5E containing anti CD3 scFv gene fragment, and two PCR fragments were recombined into the expression vector pKN100 with human antibody light constant domain and pG1D105 with human antibody heavy constant domain, respectively. The two vectors were co transfected into CHO cells using liposome. The anti CD3 antibody was detected by ELISA and Western blot assay in supernatant of transfected CHO cells culture. The primary results of competitive assays by FACS showed that anti CD3 antibody could partially block the sites through which parent antibody(HIT3a) bind to CD3 + Jurkat cells. The result of 3H TdR incorporation showed that the chimeric anti CD3 antibody could stimulated proliferation of peripheral blood mononuclear cells(PBMC) as the parent antibody. In this thesis, the results of some experiments indicated that the chimeric anti CD3 antibody expressed in CHO cells was an antibody with native biological activity, and it is possible to apply to in clinic in the future.