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目的将铜绿假单胞菌外毒素A(Pseudomonas aeruginosa exotoxin A,PEA)基因突变为无毒性的ntPE基因,并在大肠埃希菌中进行表达。方法利用Primer Premier 5.0软件设计PEA基因引物及PEA基因第553位氨基酸缺失的突变引物,以铜绿假单胞菌(Pseudomonas aeruginosa,P.aeruginosa)基因组DNA为模板,PCR扩增PEA基因,插入pGEM-T载体中,构建重组克隆质粒pGEM-PEA,以其为模板,利用突变引物,扩增ntPE基因(无毒性PEA),插入pET-30a载体中,构建原核表达质粒pET-ntPE,转化大肠埃希菌BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE及Western blot分析。结果测序结果证明已成功获得突变基因;原核表达质粒经双酶切鉴定证明构建正确;表达的重组蛋白相对分子质量约69 000,主要以包涵体形式存在,表达量占菌体总蛋白的30%,可被小鼠抗PEA单克隆抗体特异性识别,具有较好的抗原性。结论已成功获得无毒性的PEA突变基因,为构建以PEA为蛋白佐剂的融合蛋白疫苗奠定了基础。
Objective To mutate Pseudomonas aeruginosa exotoxin A (PEA) gene into non-toxic ntPE gene and express it in Escherichia coli. Methods Primer Premier 5.0 software was used to design the PEA gene primer and PEA gene deletion mutant at position 553. The Pseudomonas aeruginosa genomic DNA was used as template to amplify PEA gene and inserted into pGEM- T vector, a recombinant cloning plasmid pGEM-PEA was constructed and used as a template to amplify ntPE gene (non-toxic PEA) by using the mutated primer and inserted into pET-30a vector to construct prokaryotic expression plasmid pET-ntPE, which was transformed into Escherichia coli The strain BL21 (DE3) and IPTG were induced to express. The expressed products were analyzed by SDS-PAGE and Western blot. Results The sequencing results proved that the mutant gene was successfully obtained. The prokaryotic expression plasmid was proved by double enzyme digestion, and the recombinant protein was constructed correctly. The relative molecular mass of the expressed recombinant protein was about 69 000, which mainly existed in the form of inclusion body. The expressed protein accounted for 30% , Which can be specifically recognized by mouse anti-PEA monoclonal antibody and has good antigenicity. Conclusion The non-toxic PEA mutant gene has been successfully obtained, which lays the foundation for constructing a fusion protein vaccine with PEA as adjuvant.