绿脓杆菌外毒素A单链抗体基因的制备及其克隆的研究

Preparation of the Anti-PEA Monoclone Antibody and the Cloning of Its ScFv

为制备中和活性较高的单抗细胞2F6的单链抗体(ScFv)基因,以绿脓杆菌外毒素A(PEA)类毒素为免疫原,用单克隆抗体技术制备可分泌具有中和活力单克隆抗体的杂交瘤细胞,再应用RT-PCR从其中一株杂交瘤细胞中扩增出抗体V_H和V_L基因,用Linker(G4S)3将V_H和V_L基因连接成ScFv基因,并将其克隆至pGEM-T载体中。经核酸序列分析证实,V_H、V_L和Linker基因连接正确,基因全长729bp。经计算机分析V_H、V_L基因符合功能性重排的鼠抗体可变区基因的特征。V_H和V_L基因分别属于鼠免疫球蛋白重链Ⅱ(A)和轻链κⅥ家簇。

To get the single chain variable fragment antibody (ScFv) gene of active higher the monoclonal antibody cell 2F6 with higher neutralizing activity, the hybridoma cell lines 4D5.2F6 and7G3,which could secrete anti-PEA monoclone antibody were prepared. The VH.VL gene were amplified from the cell line 2F6 by RT-PCR. The VH and VL gene were connected through a flexible linker (Gly4Ser)3, and the VH-Linker-VL (ScFv) fusion gene was cloned into a vector pGEM-T. The ScFv gene was sequenced. analyzed by computer and the results were showed that it was consist of 729bp .encoding 243 amino acid residues. Both VH and VL were confirmed as functionally rearranged mouse immunoglobulin variable region genes and appeared to be new genes'. According Kabat classed method. McAb 2F6 VH gene segment and VL gene segment belong to the mouse Ig heavy chain subgroup Ⅱ (A) and κ chain Ⅵ subgroup respectively.