表达抗α毒素单链抗体基因工程菌株的构建

THE CONSTRUCTION OF ENGINEERING STRAIN EXPRESSED ANTI αTOXIN ScFv GENES

将 2种抗A型产气荚膜梭菌α毒素单链抗体 (ScFv)基因ScFv 2E3和ScFv 1A8分别克隆至表达质粒pUC119,pET 2 0b ,pET 2 8a和pHOG2 1中 ,构建了重组质粒 ,分别转化至相应的受体菌JM10 5 ,BL2 1(DE3)和XL1 BLUE中 ,得到表达ScFv的重组菌株。ELISA和SDS PAGE分析检测表明 :经IPTG诱导后所表达的ScFv目的蛋白主要形成了包含体的形式 ,但也有少量ScFv存在于重组菌株的培养上清和胞周质中。经薄层扫描分析 :重组菌株XL1 BLUE (pHOG 2E3)的蛋白表达产物分别占菌体可溶性蛋白的 4% ,重组菌株XL1 BLUE (pHOG 2E3)的蛋白表达产物占菌体总蛋白的相对含量为 2 2 % ,其相对分子量约为 31ku。表达的ScFv蛋白不但具有中和卵磷脂酶的活性 。

ScFv genes for anti-alpha-toxin of Clostridium perfringens type a were inserted into plasmid pHOG21, pET28a, pUC119 which cleaved with Not I and Sfi I,Bam HI and Nco I respectively and expressed in JM109, XL1-BLUE, BL21 (DE3) respectively. After induced by IPTG, ELISA and SDS-PAGE analysis showed that the ScFv could express in soluble periplasmic supernatant, culture medium and was produced in the form of inclusion body. The expressed ScFv was highly produced up to 22% in the recombinant strain XL1-BLUE (pHOG-2E3) with molecular weight of 31 ku. The expressed ScFv could neutralize phospholipase C activities of alpha-toxin and provide protection to mice from the challenge of lethal dose of alpha-toxin from Clostridium perfringens type A. These showed that ScFv has potential valuable use in therapy of alpha-toxin toxicosis.