小鼠抗人T淋巴细胞表面抗原噬菌体抗体库的构建及筛选

Construction and screening of mouse phage antibody libraries against the surface antigens of human T lymphocytes

目的 构建并筛选小鼠抗人静息 /活化T淋巴细胞表面抗原的噬菌体展示抗体库。方法 以人静息 /活化T淋巴细胞免疫Balb/c小鼠 ,取脾细胞 ,RT -PCR扩增VH 和VK 基因 ,拼接为ScFv ,插入噬菌粒载体转化大肠杆菌 ,构建了噬菌体展示抗体库 ,以人T淋巴细胞为靶抗原 ,对该库进行了 3轮淘洗 ,ELISA方法鉴定噬菌体抗体。结果 成功构建了最大实际库容量为 1 .75× 1 0 6的小鼠抗人静息T淋巴细胞表面抗原的噬菌体展示ScFv抗体库和小鼠抗人活化T淋巴细胞表面抗原的噬菌体展示ScFv抗体库。结论 所构建的抗体库可进一步用于对T淋巴细胞表面蛋白抗原的差异显示分析及构建基因工程抗体。

Objective To construct and screen the mouse phage display antibody libraries against the surface antigens of human resting / activated T lymphocytes.Methods V H and V K genes were amplified by RT PCR from spleen cells of Balb/c mice immunized with human resting / activated T lymphocytes. Phage display antibody libraries were expressed by Escherichia coli containing phagemid vector with assembled ScFv gene insertion. The libraries were panned with human T lymphocytes for 3 rounds and phage antibodies against T lymphocyte's surface antigens were assayed by ELISA.Results The mouse phage display ScFv antibody libraries against the surface antigens of human resting T lymphocytes and activated T lymphocytes were generated successfully. The maximal actual capacity of constructed antibody libraries was 1.75×10 6.Conclusion The constructed antibody libraries can be used in differential analysis of surface proteins on T lymphocyte and construction of genetically engineered antibody.