产气荚膜梭菌α毒素的原核表达及纯化

Prokaryotic expression and purification of Clostridium perfringens alpha toxin

目的克隆产气荚膜梭菌α毒素(Clostridium perfringens alpha-toxin,CPA)全基因,在大肠埃希菌(E.coli)中表达并纯化α毒素。方法设计并合成CPA的全基因,插入原核表达载体pET-28a中,构建重组表达质粒pET-28aCPA,转化E.coli BL21(DE3),IPTG诱导表达,优化表达条件,并对重组蛋白进行纯化。结果双酶切和测序鉴定结果表明,CPA基因以正确的阅读框架克隆入pET-28a载体中;表达的重组蛋白相对分子质量约为43 000,主要以可溶性形式表达,最佳培养基为TB培养基,最佳诱导温度为37℃,诱导时间为3 h;纯化的重组蛋白纯度达95%以上,浓度为0.663 mg/ml,收率为5 mg/g湿菌。结论成功在E.coli中表达了CPA,纯化后的CPA纯度较高,为进一步研究其结构、功能、致病机制及制备抗α毒素人源单链抗体奠定了基础。

Objective To clone the whole gene of Clostridium perfringens alpha-toxin（CPA）,express in E. coli and purify the expressed product. Methods Whole CPA gene was designed and synthesized,and inserted into prokaryotic expression vector pET-28 a. The constructed recombinant plasmid pET-28a-CPA was transformed to E. coli BL21（DE3）for expression under induction of IPTG. The condition for expression was optimized, and the recombinant protein was purified. Results Restriction analysis and sequencing proved that the correct ORF of CPA gene was cloned into vector pET-28 a. The expressed recombinant protein,with a relative molecular mass of 43 000,mainly existed in a soluble form. The optimal medium,temperature for induction,and time for induction were TB medium,37 ℃ and 3 h respectively. The purity,concentration and recovery rate of purified recombinant protein were more than 95%,0. 663 mg / ml and 5 mg / g wet bacteria respectively. Conclusion CPA was successfully expressed in E. coli and reached a high purity after puri-fication,which laid a foundation of further study on the structure,function and pathogenic mechanism of CPA and preparation of anti-CPA single chain antibody from human origin.