抗A型产气荚膜梭菌α毒素单链抗体的基因克隆及核苷酸序列分析

Cloning and Nucleotide Sequencing of ScFv Gene for Anti Alpha Toxin of Clostridium Perfringens Type A

应用 RT-PCR技术 ,从分泌具有中和活性的抗 A型产气荚膜梭菌α毒素单克隆抗体 ( Mc Ab)的杂交瘤细胞 2 E3中 ,扩增出抗体 VH 和 VL 基因 ,用 linker( Gly4Ser) 3基因 ,将 VH 和 VL 基因连接成 Sc Fv基因 ,并将其克隆至 p GEM-T载体中。经核苷酸序列分析证实 ,VH、VL 基因和 linker基因拼接正确 ,基因全长为 72 6bp。经计算机分析 ,VH 和 VL 基因均为新发现的基因序列 ,符合功能性重排的鼠抗体可变区基因特征。VH 和VL 基因分别属于鼠免疫球蛋白重链 ( B)和轻链κ

The V H and V L genes were amplified from a hybridoma cell line 2E3 producing mouse McAb against alpha txoin of Clostridium perfringens by RT PCR. The V H and V L genes were connected through a flexible linker (Gly4Ser)3, and the V H linker V L (ScFv) fusion gene was cloned into a clone vector pGEM T. The ScFv gene was sequenced, analyzed by computer to be consist of 726 bp encoding 242 amino acid residues. Both V H and V L genes were confirmed as functionally rearranged mouse immunoglobulin variable region genes and appeared to be new genes. According Kabat classed method, McAb 2E3 V H gene segment and V L gene segment belong to the mouse Ig heavy chain subgroupⅡ(b) and κ chain subgroup Ⅲ respectively.