铜绿假单胞菌外毒素A基因的克隆与原核表达

为建立铜绿假单胞菌(Pseudomonas aeruginosa)外毒素A(Exotoxin A,ETA)的原核表达方法,采用PCR技术扩eta基因,将其插入pET-28a载体构建重组质粒pET-28a-eta,并对重组质粒进行双酶切、PCR及测序鉴定。转入BL21(DE3)中诱导表达,检测目的蛋白大小及反应原性,然后进行表达条件的优化及其表达形式检测。结果表明,所扩增eta基因大小为1917 bp,与GeneBank参比序列一致性在98.70%;蛋白分析表明,rETA大小为74 KDa,具有和天然毒素相似的反应原性。目的基因在37℃、0.6 mmol/LIPTG诱导3 h获得最佳表达。该蛋白在超声波裂解上清和包涵体中均有分布,但主要以包涵体为主。该研究为进一步开展ETA的批量纯化提供了前提条件。

Cloning and Prokaryotic Expression of Pseudomonas Aeruginosa Exotoxin A Gene

To establish the method for prokaryotic expression of Pseudomonas aeruginosa exotoxin A(eta) gene,the eta gene was amplified by PCR and be inserted into vector pET-28a form a recombinant vector pET-28a-eta.After identified by PCR,restriction enzyme digestion and sequencing methods,the recombinant plasmid was transformed into BL21(DE3) and induced to express by IPTG.The size and reactionogenicity of the target protein was detected,then its expression conditions was optimized and expression form was also detected.The results showed that the eta gene was 1917 bp and was 98.70% in homology with reference sequence of GeneBank.The target protein was found to be about 74 KDa as expected and showed similar reactionogenicity to native exotoxin A,the eta which expressed 3 h at 37 ℃ in 0.6 mmol/L IPTG had the optimal expression and distributed both in ultrasonic lysis supernatant and inclusion bodies,but mainly in inclusion bodies.The research provided the foundations for batch purification of ETA.