抗人纤维蛋白单克隆抗体重链和轻链可变区基因克隆和表达

Cloning and expression of heavy and light chain variable region genes of monoclonal antibody specific for human fibrin

目的：为了获得能与人纤维蛋白结合的最小的活性分子。方法：采用逆转录－多聚酶链反应技术从分泌抗人纤维蛋白单克隆抗体的８Ｅ５杂交瘤细胞中分离出免疫球蛋白重链和轻链可变区基因（ＶＨ和ＶＫ）。通过基因重组构建了ＶＨ－ＶＫ的表达载体ｐＯＰＥ５１－８Ｅ５。转化大肠杆菌ＪＭ１０９后用异丙基－β－Ｄ－硫代半乳糖苷诱导表达。结果和结论：经聚丙烯酰胺电泳和免疫杂交证明产物表达于细菌的壁膜间隙和包涵体中，表达产物为分子量３００００的单链抗体分子，具有特异性识别和结合人纤维蛋白的活性。表达产量为转化菌总蛋白的２８．９％。

Objective:To obtain the minimum molecule having activity to bind human fibrin.Methods: The immunoglobulin heavy and light chain variable region (V H and V κ) genes were isolated from 8E5 hybridoma cells,which secreted a monoclonal antibody against human fibrin,by RT PCR.An expression vector pOPE51 8E5 was constructed for the recombinant V H V κ expression.The transformed E.Coli JM 109 cells were propagated and induced by IPTG.Results and Conclusion:Expression product was found in the periplasmic space and inclusion bodies by SDS PAGE and immunobloting.It was a 30000 single chain fragment(scFv) with antigen binding specificity.The yield of the scFv was 28.9% of the total bacterial proteins.