铜绿假单胞菌外毒素A原核表达载体的构建及鉴定

CONSTRUCTION AND IDENTIFICATION 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加以鉴定。[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。[结果]PET-32a-PEA原核表达质粒构建完成后,用限制性内切酶消化、PCR及DNA测序等多种方法进行鉴定,证实其构建成功。[结论]PET-32a-PEA原核表达质粒的成功构建,为进一步研究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。

[Objective] To perform gene cloning to the whole genome of PET-32a-PEA, and to construct anti identify the prokaryofic expression plasmid. [Methods] The DNA genome was extracted from genome of Pseudomonas aemginosa, file gene of PEA with signal peptide was amplified by primers of PCR designed. Then the PCR products were digested by Hind IH and EcoR ][ and connected to recombinate with prokaryotic expression plasmid PET-32a. [ Results] After plasmid construction of prokaryotic expression plasmid PET-32a-PEA. the PET-32a-PEA was digested with restnction enzyme, and identified with PCR and DNA sequencing, so as to confirm its consmmtion. [Conclusion] Successful construction of PEA in prokaryotie expression vector coold pr,wide basis for further study of genetic engineering vaccine and reconthmant immunotoxin for inmtune target therapy.