产气荚膜梭菌ε毒素基因的克隆、表达及其抗血清的制备

Cloning,expression of Clostridium perfringens epsilon-toxin gene and preparation of antisera

PCR扩增B型产气荚膜梭菌C58-2株ε毒素完整基因,并将其插入到pGEM-TEasy载体中,构建克隆载体pGEM-T-ε。对克隆载体pGEM-T-ε进行双酶切,将得到的918bp片段以正确的阅读框架定向克隆于pET-28a(+)中,然后将重组质粒转化进宿主菌BL21(DE3)中,在37℃1mmol/LIPTG诱导下该基因获得良好表达。经SDS-PAGE分析,其表达的蛋白约为36600,与预期值一致。Western-blotting试验显示,该重组蛋白可被D型产气荚膜梭菌血清识别,表明该重组蛋白具备天然毒素相似的反应原性。重组ε毒素蛋白在菌液上清、超声波裂解上清和包涵体中均有分布,表明重组蛋白可同时以胞外、周质和胞浆的形式表达,且以周质方式为主。重组蛋白在胰酶活化前后均可致死小鼠,活化后毒力可为原来的150倍。以重组ε毒素蛋白作为抗原免疫家兔制备血清,效价测定结果表明,重组ε毒素蛋白抗血清每毫升可中和30000个小鼠MLD。毒素-抗毒素中和试验和琼脂扩散试验均表明,该抗血清具有ε毒素特异性。

One pair of primers were designed and synthesized based on the epsilon-toxin gene of Clostriudm perfringens.The complete epsilon toxin gene fragment was amplified by polymerase chain reaction （PCR）,and then was cloned into pGEM-T Easy vector to construct pGEM-T-ε.Digested with EcoRⅠ and XhoⅠ,a fragment 918 bp was cloned into the expression plasmid vector pET-28a（＋）.The recombinant plasmid was transformed into the BL21 and induced to express by 1.0 mmol/L IPTG at 37℃.The expression product was identified by SDS-PAGE and found to be 36 600 as expected and confirmed by Western blotting with Clostriudm perfringens type D antisera,indicating similar reactivity with native epsilon toxin.Recombinant epsilon-toxin protein was simultaneously found in culture supernatant,postsonic supertanant and inclusion bodies.Recombinant epsilon-toxin protein in postsonic supertanant could make mice die,indicating epsilon-toxin activity.Toxin potency of the recombinant protein with 150 folds as before was achieved after trypsin activation.Upon immunization of rabbit with the recombinant protein,antisera with high antibody titre neutralizing 30 000 MLD toxin per 1 mL were prepared.Toxin-antitoxin neutralization test and agar diffusion assay showed that antisera of recombinant protein were specific for epsilon toxin.