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ＲＴＰＣＲ 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ｐＧＥＭＴ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
出处
《细胞与分子免疫学杂志》
CAS
CSCD
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
关键词
Α毒素
单克隆抗体
基因克隆
序列分析
Clostridium perfringens type A α toxin monoclonal antibody gene cloning nucleotide sequencing
