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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 Clostridium perfringens alpha-toxin (CPA) gene and express and purify α-toxin in E.coli. Methods The whole gene of CPA was designed and synthesized. The recombinant plasmid was inserted into prokaryotic expression vector pET-28a. The recombinant plasmid pET-28aCPA was constructed and transformed into E. coli BL21 (DE3) for expression under the induction of IPTG. The recombinant protein was purified. Results The results of double enzyme digestion and sequencing showed that the CPA gene was cloned into pET-28a vector with the correct reading frame. The relative molecular mass of the expressed recombinant protein was about 43 000, which was mainly expressed in soluble form. The best culture medium was TB culture The optimum induction temperature was 37 ℃ and the induction time was 3 h. The purity of the purified recombinant protein was above 95% with a concentration of 0.663 mg / ml and the yield was 5 mg / g wet bacteria. Conclusions CPA was successfully expressed in E.coli. The purity of purified CPA was high, which laid the foundation for further study of its structure, function, pathogenesis and preparation of anti-alpha toxin human single chain antibody.