抗CPAα毒素单链抗体基因的克隆和表达及其生物学特性研究

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 (CPA)α毒素单克隆抗体的杂交瘤细胞中 ,扩增出单链抗体 (ScFv)基因 ,并将其定向克隆于表达载体 pHOG2 1中 ,构建重组表达载体 pHOG 2E3,转化至大肠杆菌XL1 Blue中 ,筛选出表达菌株XL1 Blue(pHOG 2E3)。SDS PAGE分析结果表明 ,在 2 0℃用IPTG诱导培养时 ,表达的ScFv蛋白占菌体总蛋白的 2 5 %,ScFv蛋白主要以包涵体的形式存在 ,但在胞周质和培养上清中也能检测到ScFv蛋白 ,其中在胞周质中表达的ScFv蛋白占菌体可溶性蛋白的 4 %。生物学试验结果表明 ,ScFv基因表达产物不仅能够中和α毒素的磷酯酶C活性 ,而且对攻击致死剂量α毒素的小鼠产生良好的被动保护作用

Cloning and expression of ScFv gene against alpha-toxin of clostridium perfringens type A

The ScFv gene from a hybridoma cell line producing mouse McAb against alpha toxin of Clostridium perfringens type A were amplified by RT-PCR and was cloned into a vector pGEM T. The ScFv gene consists of 726bp encoding 242 amino acid residues.Then the ScFv gene was amplified inserted the expression vector pHOG21 and transformed into E coli XL1 BLUE.The ScFv protein was highly expressed in recombinant strain XL1 BLUE (pHOG 2E3) and the expression level of the ScFv was about 25% of total bacteria protein by SDS PAGE. The expressed products were consist of insoluble inclusion bodies and soluble protein. 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.