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分离大肠癌患者外周血单个核细胞 (PBMC) ,在体外用灭活的大肠癌细胞再次致敏后 ,经EBV转化 ,然后用有限稀释法克隆分泌抗大肠癌细胞抗体的B细胞 ;多次PCR扩增和克隆其抗体可变区基因 (VH VLcDNA) ,并用编码 (Gly4Ser) 3 的互补序列连接成ScFvcDNA(VH linker VL) ,经酶切克隆入载体fUSE 5RF ,使之呈现于噬菌体表面。通过用 3轮肿瘤细胞和正常人PBMC淘选后 ,ELISA检测 80 %的单克隆噬菌体抗体显示了很强的阳性反应。以上结果初步说明 :联合应用体外致敏、EBV转化、PCR扩增和噬菌体呈现技术制备高亲和力全人源化的抗肿瘤抗体是可行的
Peripheral blood mononuclear cells (PBMCs) from patients with colorectal cancer were isolated and re-sensitized with inactivated colorectal cancer cells in vitro and transformed with EBV. B cells secreting anti-colorectal cancer cell antibodies were then cloned by limiting dilution; multiplex PCR. The antibody variable region gene (VH VL cDNA) was amplified and cloned, and was ligated with scFv cDNA (VH linker VL) using the complementary sequence encoding (Gly4Ser) 3 . The fragment was cloned into the vector fUSE 5RF and expressed on the phage surface. After panning with 3 rounds of tumor cells and normal human PBMCs, ELISA detection of 80% of monoclonal phage antibodies showed a strong positive reaction. The above results provide a preliminary explanation: It is feasible to combine the use of in vitro sensitization, EBV transformation, PCR amplification and phage display technology to prepare high-affinity fully humanized anti-tumor antibodies.