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目的 研制单链抗体 ,以减少鼠源抗体的异源性 ,为寻找合理的导向治疗方案提供工具。方法 将经抗原刺激的BALB/C小鼠脾细胞与小鼠骨髓瘤细胞融合制备杂交瘤 ,用多聚酶链反应 (PCR)方法克隆其抗体可变区基因 ,并用重叠延伸拼接法 (SOE)构建相应的单链可变区抗体片段 (ScFv)基因。结果 获得杂交瘤细胞株D10及 1C8,酶联免疫吸附法及免疫组织化学法证实所分泌的抗体可分别与人膀胱癌细胞及丝裂霉素C特异性结合。获得两种抗体的可变区基因( 32 0bp及 34 0bp片段 )及ScFv基因 ( 75 0bp片段 )。 结论 应用PCR SOE方法可快速便捷地获得目的抗体的ScFv基因 ,为进一步研制双特异性ScFv提供了实验基础。
Objective To develop single chain antibody to reduce the heterogeneity of murine antibody and to provide a tool for finding a reasonable targeted therapy. Methods Hybridomas were prepared by fusing BALB / C mouse splenocytes with mouse myeloma cells. The variable region of the antibody was cloned by polymerase chain reaction (PCR) and the corresponding regions were constructed by overlap extension splicing (SOE) Of the single chain variable region antibody fragment (ScFv) gene. Results Hybridoma cell lines D10 and 1C8 were obtained. ELISA and immunohistochemistry confirmed that the secreted antibodies could specifically bind to human bladder cancer cells and mitomycin C, respectively. The variable region genes (32 0 bp and 34 0 bp fragments) and ScFv gene (75 bp fragment) of the two antibodies were obtained. Conclusion PCR SOE method can be used to obtain the ScFv gene of target antibody rapidly and conveniently, which provides the experimental basis for further development of bispecific ScFv.