抗A型产气荚膜梭菌α毒素mAb V_H和V_L基因的克隆与序列测定  

Cloning and sequencing of V_H and V_L genes of mAb 2E3 against α toxin from

作　　者：许崇波[1] 赵宝华[2] 马从林 朱平[1] 

机构地区：[1]解放军农牧大学军事兽医研究所,长春130062 [2]河北师范大学生物系

出　　处：《细胞与分子免疫学杂志》1999年第4期296-299,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：全军医药卫生基金!资助项目; No-98Q076

摘　　要：用提取的Ａ 型产气荚膜梭菌α毒素包涵体为免疫原， 制备了１ 株ｍＡｂ ２Ｅ３ 。其Ｉｇ 亚类为ＩｇＧ３， 细胞培养上清和腹水的ｍＡｂ 效价分别为１∶１０２４ 和１∶１０８ 。该ｍＡｂ ２Ｅ３ 可中和α毒素的磷脂酶Ｃ 活性和溶血活性， 对α毒素致死性腹腔攻击的小鼠具有良好的被动保护作用。另外， 应用ＲＴＰＣＲ 技术， 从杂交瘤细胞株２Ｅ３ 中， 扩增出ｍＡｂ ＶＨ 和ＶＬ基因， 分别克隆至载体ｐＧＥＭＴ中。序列分析证实， ＶＨ 基因为３５７ ｂｐ， 编码１１９ 个氨基酸；ＶＬ 基因为３２４ ｂｐ， 编码１０８ 个氨基酸。计算机分析表明，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的特征。ＶＨ 和ＶＬ 基因分别属于鼠免疫球蛋白重链Ⅱ（ Ｂ） 和轻链κⅢ家族。Monoclonal antibody(mAb) 2E3 against α toxin from Clostridium perfringens type A was prepared The subclass of mAb 2E3 was IgG3 ELISA titers of cell supernatant and ascites were 1:1024 and 1:10 8 respectively Particularly, the mAb 2E3 could neutralize phospholipase C and hemolytic activities of α-toxin, and could provide protection for mice from the lethal challenge of α toxin from Clostridium perfringens type A On the basis of obtaining mAb 2E3, V H and V L genes from a hybridoma cell line 2E3 were amplified by RT PCR, and were cloned into vector pGEM-T The sequences of V H and V L genes were analyzed by the computer The V H gene consists of 357bp encoding 119 amino acid residues, whereas 108 amino acid residues are encoded by V L gene of the 324 bp Both V H and V L genes were confirmed as functionally rearranged mouse immunoglobulin variable region genes and appeared to be new genes According to Kabat classification method, V H and V L gene segments belong to the Ig heavy chain subgroup Ⅱ(B) and κlight chain subgroup Ⅲ, respectively

关 键 词：Α毒素 单克隆抗体 基因克隆 序列分析 

分 类 号：Q78[生物学—分子生物学]