致病性嗜水气单胞菌气溶素基因的克隆与高效表达

Cloning and Expression of Aerolysin Gene of Pathogenic Aeromonas hydrophil

根据 Gen Bank中致病性嗜水气单胞菌气溶素 (Aer)基因的序列设计引物 ,以国内嗜水气单胞菌分离株为模板 ,扩增出 Aer基因的全长序列。经 T载体克隆和序列测定 ,证实克隆了国内分离株的不含信号肽的 Aer全长基因。将该基因以正确的读码框架与表达载体 p ET2 8b连接 ,经 IPTG诱导、SDS- PAGE检测 ,外源基因获得高效表达。诱导 4 h的培养物中 ,融合蛋白的表达占菌体总蛋白含量的 4 8.5 7%。 Western-印迹结果显示 ,利用纯化包涵体制备的兔抗血清可以很好地识别嗜水气单胞菌产生的天然毒素 。

Aerolysin(Aer) gene without signal peptide of Aeromonas hydrophila strain AhCS02 isolated in Jilin,China was amplified by PCR.After being cloned into T-vector and sequenced,the Aer gene was cloned into prokaryotic expression vector pET28b in correct reading-frame.The recombinant pAB46 was then transformed into host strain E.coli BL21,the fusion protein was expressed at high level,amounting to 48.57% of the total protein of the IPTG induced by E.coli BL21 after 4 hours.Western blot shows the rat antibodies rasied by the purified recombinant fusion protein can recognize the natural aerolysin peptide produced by Aeromonas hydrophila strain AhCS02 and also the antibodies rasied by natural aerolysin can recognize the purified recombinant fusion protein.