产气荚膜梭菌ε毒素基因的克隆、表达及其抗血清的制备

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。

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

One pair of primers were designed and synthesized based on the epsilon-toxin gene of Clostriudm perfrin-gens. 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 EcoR Ⅰ and Xho Ⅰ ,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,in-dicating 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 su-pertanant 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,anti-sera 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.