全人源化抗结肠癌单链抗体基因的克隆和表达

Cloning and Expression of Human Anti-colonic Cancer Single-chain Fv Fragment

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。

We report a new strategy for the generation of human anticolon cancer monoclonal antibodies based on the molecular cloning and expression of immunoglobulin variable region cDNAs derived from peripheral blood mononuclear cells (PBMC) that were transformed by EBV.The immortalized B cells secreting tumor\|specific antibodies were identified by HRT\|18 cell ELISA and cloned by limiting dilution.Heavy\|and light\|chain V\-H\|C H1 (γ) and V\-κ\|C\-κ cDNAs were rescued from ELISA\|positive cells wells by RT\|PCR.V\-H and V\-κ were amplified by 2nd PCR and linked them together by 3rd PCR assembly with the use of a (Gly\-4Ser)\-3 linker.The ScFv cDNAs were then cloned into the fUSE 5 vector and displayed on phage.Phage clones were selected on HRT\|18 cells and normal human PBMC.ELISA tested phage clones randomly picked after each panning step.>80% of the clones showed a strong ELISA reaction,demonstrating the effectiveness of the panning procedure for selecting anticolon cancer antibodies.This approach offers an effective method by combining in vitro immunization, B\|cell expansion for enrichment of specific B\|lymphocytes,PCR gene cloning and phage display to make high\|affinity human anticolon cancer monoclonal antibody molecules.