铜绿假单胞菌外毒素A原核表达载体的构建及表达

Construction and expression of prokaryotic expression vector of pseudomonas aeruginosa exotoxin A gene

目的对铜绿假单胞菌外毒素A(PEA)全基因进行基因克隆,构建原核表达质粒并进行表达。方法从铜绿假单胞菌中提取基因组DNA,设计PCR引物扩增出带信号肽的PEA目的基因,经HindⅢ和EcoRⅠ消化后,与PET-32a载体进行连接重组。用限制性内切酶消化、PCR及DNA测序等方法鉴定构建成功后,IPTG诱导目的基因表达。结果SDS-PAGE电泳结果显示,在分子量78kD处有一条明显的新生蛋白带。表达的融合蛋白量约占细菌总蛋白的35%。结论PET-32a-PEA原核表达质粒的成功构建及表达,为进一步研究有效的基因工程疫苗和免疫导向治疗的基因重组毒素奠定了基础。

[Objective] To construct and express the PET-32a-PEA prokaryotic expression plasmid. [Methods] The full length gene of PEA from genome of Pseudomonas aeruginosa was amplified by PCR. Then the PCR product was digested with Hind Ⅲ and EcoR I and cloned into prokaryotic expression plasmid PET-32a. Restriction en-zyme digesting and DNA sequencing confirmed that the recombinant prokaryotic expression plasmid PET-32a-PEA had been constructed correctly. After induction with IPTG, the expressed protein was analyzed by SDS-PAGE . [Results] A new single protein band was found on SDS-PAGE, located at the corresponding sites of the Mr 78 kD. The recombinants expression the fusion protein at a level of about 35 % of total cell proteins. [Conclusion] Successful construction and expression of PEA in prokaryotic expression vector are basis for further study of genetic engineering vaccine and recombinant immunotoxin for immune target therapy.