摘要
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 construct the single chain f ragment of variable region, which showed a much lower antigenictiy and may be a practical solution for target therapy of malignant tumor.Methods Hybridoma were generated by fusing the immunized Balb/c mice splenocytes and myeloma. The genes of variable chains were cloned with PCR. And the ScFv ge ne of the antibodies were constructed by using PCR-SOE(splicing by overlap ext ension).Results The established cell lines of D10 & 1C8 could secret anti J82 monoclonal antibody and anti Mitomycin C (MMC) monoclonal antibody. The activity and specificity of the antibodies were confirmed by ELIS A and histochemistry method. The genes of the variable chain (320 bp and 340 bp fragments)and the ScFv (750 bp fragment) of the two antibod ies were obtained.Conclusion The ScFv gene of the antibod ies against bladder cancer and MMC could be obtained conveniently and quickly by using PCR-SOE method, which may be helpful to the research on bi-specific ScF v anti bladder cancer-MMC.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2001年第4期361-363,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目 (39470 796)
