基因工程乳腺癌特异性人单链抗体的表达与特性

研究解决分泌人单抗的杂交瘤细胞系难于稳定持久分泌抗体的难题，制备单链抗体，使单抗分子小型化，为进一步研究其在肿瘤诊断和治疗中的应用作准备。从分泌抗乳腺癌人单抗的杂交瘤细胞CM－1总RNA中，利用RT－PCR技术分别扩增出人单抗重链可变区VH基因和轻链可变区VL基因，将扩增产物纯化后克隆于pGEM-T载体中，进行DNA测序和序列比较分析后，将两者共克隆于表达载体中诱导表达，利用斑点免疫印迹及竞争抑制法检测表达产物的抗原性。所克隆的CM－1人单抗重链可变区和轻链可变区基因片段，分别属于人免疫球蛋白IgM Ⅲ亚群，和鼠κ轻链V亚群，ⅩⅦ家族，用斑点免疫印迹法检测可见表达产物能与人乳腺癌细胞特异结合；人CM－1单克隆抗体对此单链抗体与人乳腺癌细胞的结合有竞争性抑制作用，抑制率为75．7％。结论：成功地制备了可特异结合乳腺癌细胞的CM－1单链抗体。

Expression and characterization of single chain antibody derived from a breast cancer specific human monoclonal antibody hybridoma CM-1 cell line

Human monoclonal antibody hybridoma derived single chain antibodies(scFvs) hold great potential in the cancer immune imaging diagnosis and target therapy. CM 1 is a breast cancer specific human monoclonal antibody hybridoma cell line. To obtain CM 1 scFv for diagnosis and treatment of breast cancer the total RNA of CM 1 was isolated ,the variable fragment regions of heavy strain and the light strain were amplified by RT PCR and then inserted into pGEM T vector. DNA sequencing showed that V H fragment originated from human subtype and the V L fragment from mouse subtype. The coding sequence was then subcloned into expression vector pWAI180. The recombinant plasmid was transformed into E.coli Gm2163, scFv expressed after induction with IPTG. CM 1 scFv bound specifically to breast cancer CAM cell line as demonstrated by Immune dot blot assay. Competitive ELISA showed that 75.0% binding activity of CM 1 ascite was inhibited by the CM 1 scFv.