抗隐孢子虫子孢子表膜单链抗体基因的克隆和核苷酸序列分析

Cloning and Nucleotide Sequencing of ScFv Gene against Cryptosporidium parvum Sporozoite

应用 RT- PCR技术 ,从分泌具有抗隐孢子虫子孢子表膜单克隆抗体 (Mc Ab)的杂交瘤细胞 3D6中扩增出抗体VH 和 VL 基因 ,用 linker(Gly4 Ser) 3基因 ,将 VH 和 VL 基因连接成 Sc Fv基因 ,并将其克隆至 p MD- 18T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 0 bp。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 (A)和轻链 κ 家族。

The V H and V L gene were amplified from a hybirdoma cell line 3D6 producing mouse McAb against Cryptosporidium parvum sporozoite by RT-PCR.The V H and V L genes were connected through a flexible linker(Gly 4Ser) 3,and the V H -linker-V L (ScFv) fusion gene were cloned into a clone vector pMD-18T.The ScFv gene were sequenced,analyzed by computer to be consist of 720 bp encoding 240 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 3D6 V H gene segment and V L gene segment belong to the mouse Ig heavy chain subgroup Ⅱ(A) and κ chain subgroup Ⅲ respectively.