抗人肺腺癌单抗5F-11噬菌体抗体库的构建及表达

The construction and expression of phage display library of anti human lung adenocarcinoma monoclonal antibody 5F-11

目的 构建抗人肺癌单抗 5F 11的噬菌体显示文库。方法 利用RT PCR方法从 5F 11杂交瘤细胞扩增抗体轻重链可变区基因 ,用连接子连接成为ScFv基因后克隆至噬菌体载体pCANTAB5E。以肺腺癌细胞系A2 为抗原进行 4轮筛选 ,得到富集的次级噬菌体表达文库。结果 制备了库容为 8× 10 7pfu/ml的噬菌体展示文库。随机抽取未经筛选的克隆进行酶切 ,酶切图谱呈多样性 ,富集后的酶切图谱则显示特异构型的噬菌体得到富集。检测 30个噬菌体克隆上清 ,其中 2 3个与A2 细胞有较强反应。结论 本研究获得了抗人肺腺癌单抗 5F 11的单链抗体 ,为拓展抗体的应用创造了条件。

Objective To construct and express a phage display library of anti human lung cancer monoclonal antibody 5F-11. Methods Immunoglobulin variable regions (VH,VL) were amplified from 5F-11 hybridrom by RT-PCR. ScFv genes consisting of VH DNA and VL DNA joined together by a linker DNA were cloned into a phage vector pCANTAB5E. After 4 rounds of screening with lung adenocarcinoma cell line A2 as antigen, an enriched secondary phage display library was obtained. Results A recombinant phage display library with total of 8×10 7 pfu/ml was established. Randomized clones from unselected library digested with BstNⅠ showed different patterns, however, those from selected library showed that phages with special pattern were enriched. Twenty-three out of 30 clones were found to respond strongly to A2 cell lines. Conclusion The ScFv of anti-lung adenocarcinoma monoclonal antibody 5F-11 can be successfully produced, which may be useful to widen the application of the antibody.