抗A型产气荚膜梭菌α毒素单链抗体基因的克隆、表达及其生物学活性(英文)

Molecular Cloning, Expression and Biological Activity of a Functional ScFv to the 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 (McAb)的杂交瘤细胞株 1A8中 ,分别扩增出抗体VH 和VL 基因 ,用linker (Gly4Ser) 3 基因 ,将VH 和VL 基因连接成单链抗体 (ScFv)基因 ,并将其克隆至pGEM T载体中 .经核苷酸序列分析证实 ,VH 和VL 基因及linker基因拼接正确 ,ScFv 1A8基因全长为 72 6bp ,编码2 4 2个氨基酸 ,VH 和VL 基因符合功能性重排的鼠抗体可变区基因特征 ,分别属于鼠免疫球蛋白重链Ⅱ (A)和轻链κⅣ家簇 .将ScFv 1A8基因克隆至表达载体pHOG2 1中 ,构建了重组质粒pHOG 1A8,然后转化至受体菌XL1 BLUE中 ,得到重组菌株XL1 BLUE (pHOG 1A8) .ELISA检测和SDS PAGE分析表明 :经IPTG诱导后所表达的目的蛋白存在于重组菌株XL1 BLUE (pHOG 1A8)的胞周质中 .经薄层扫描分析 :重组菌株XL1 BLUE(pHOG 1A8)的蛋白表达产物占菌体可溶性蛋白的 1 2 % ,其相对分子量约为 31kD .ScFv的生物学活性研究表明 ,ScFv蛋白不但具有中和磷脂酶C的活性 。

The heavy\|chain and light\|chain variable region genes of an anti alpha\|toxin hybridoma cell line (1A8) were amplified by RT\|PCR and joined by a flexible DNA linker encoding peptide (Gly\-4Ser)\-3 as a single\|chain Fv (ScFv) DNA fragment. The ScFv\|1A8 DNA fragment was cloned into a plasmid pGEM\|T and sequenced. The ScFv gene consists of 726 bp encoding 242 amino acid residues. Both V\-H and V\-L genes were confirmed as functionally rearranged mouse immunoglobulin variable region gene segments. The variable region gene segments of ScFv\|1A8 belong to mouse Ig heavy chain subgroup Ⅱ(A) and kappa light chain subgroup Ⅳ, respectively. Then the ScFv DNA fragment was cloned into expression vector pHOG21 and highly expressed in E. coli XL\|BLUE. The ScFv could be expressed in the soluble periplasmic supernatant and its molecular weight was 31 kD. The expressed ScFv could neutralize phospholipase C activities of alpha\|toxin and could provide protection for mice from the challenge of lethal dose of alpha\|toxin of Clostridium perfrigens type A. The results showed that the expressed ScFv had potential valuable use in alpha\|toxin toxicosis therapy.