重组铜绿假单胞菌外毒素A在大肠杆菌中的表达

目的实现铜绿假单胞菌外毒素A(PEA)全基因的原核载体构建及融合性表达。方法用基因重组技术构建PET-32a-PEA重组体,并在大肠杆菌BL21中进行表达。结果酶切鉴定及测序结果表明PET32a载体上成功地插入了PEA全基因。用IPTG诱导目的基因表达,沸水浴变性后进行SDS-PAGE电泳,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%。结论成功地对PEA全基因进行了原核载体构建及表达,获得了稳定表达的工程菌,为进一步研出有效的基因工程疫苗和用于免疫导向治疗的重组毒素奠定了基础。

Expression of recombinant Pseudomonas aeruginosa exotoxin A in E.coli

The full length Pseudomonas aeruginosa exotoxin A gene was purified and cloned into expression vector pET-32a by using gene recombination technique.Then the recombinant plasmid PET-32a-PEA was transformed into E.coli BL21.After induction with IPTG,the expressed protein was analyzed by SDS-PAGE.A new single protein band was found on SDS-PAGE,located at the corresponding sites of the Mr 78 kD.The recombinant expression of the fusion protein was at a level of about 35 % of total cell proteins.It is concluded that the prokaryotic expression plasmid for Pseudomonas aeruginosa exotoxin A gene was successfully constructed and effectively expressed in E.coli.These results would provide basis for the further studies of genetic engineering vaccine and recombinant immunotoxin for immune target therapy.