百日咳杆菌腺苷酸环化酶毒素基因的克隆及原核表达

Cloning and Prokaryotic Expression of Bordetella pertussis Adenylate Cyclase Toxin Gene

目的克隆百日咳杆菌腺苷酸环化酶毒素(CyaA,ACT)基因,表达并纯化重组CyaA蛋白。方法从百日咳杆菌CS株的基因组DNA中PCR扩增CyaA编码基因,克隆入载体pET30a,构建重组原核表达质粒pET30a/cyaA,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达。表达的重组蛋白经8mol/L尿素变性、透析复性、DEAE阴离子交换柱纯化后,采用Western blot法鉴定其反应原性。结果重组原核表达质粒pET30a/cyaA经PCR、双酶切及测序证明构建正确;表达的重组蛋白主要以包涵体形式存在,表达量约占菌体总蛋白的20%;纯化的重组蛋白纯度达90%左右,可与全细胞百日咳疫苗和无细胞百日咳疫苗免疫血清结合。结论已成功克隆了百日咳杆菌cyaA基因,并在大肠杆菌中表达了重组CyaA蛋白,为进一步开展CyaA的应用研究奠定了基础。

Objective To clone Bordetella pertussis adenylate cyclase toxin（CyaA,ACT）gene,and express and purify recombinant CyaA protein.Methods The gene encoding CyaA was amplified from genomic DNA of B.pertussis CS strain by PCR and cloned into vector pET30a.The constructed recombinant plasmid pET30a /cyaA was transformed to competent E.coli BL21（DE3） for expression under induction of IPTG.The expressed recombinant protein was de-naturalized with 8 mol /L urea,re-naturalized by dialysis,purified by DEAE anion exchange chromatography and identified for reactogenicity by Western blot.Results PCR,restriction analysis and sequencing proved that recombinant plasmid pET30a /cyaA was constructed correctly.The expressed recombinant protein mainly existed in a form of inclusion body,contained about 20% of total somatic protein,reached a purity of about 90% after purification and showed specific reactions with sera of mice immunized with whole cell and acellular pertussis vaccines.Conclusion B.pertussis cyaA gene was successfully cloned,and recombinant CyaA protein was expressed in E.coli,which laid a foundation of study on application of CyaA.