斑点叉尾鮰hepcidin抗菌肽在大肠杆菌中的融合表达

通过RT-PCR从斑点叉尾鮰肝脏中克隆编码hepcidin原前体肽的基因"pIH"。与参考序列相比,显示两个氨基酸残基的变异。通过对编码hepcidin原前体肽的基因添加EcoR I和HindⅢ酶切位点,选择含"trxA"融合头的pET32a(+)作为表达质粒、E.coliBL21(DE3)作为工程菌,成功构建"pET32a-pIH"原核表达系统。阳性克隆经1 mmol.L-1IPTG诱导、在37℃培养12 h后,表达的"trxA-pIH"融合蛋白70%以上可溶。Tricine-SDS-PAGE分析表明,细胞经超声破碎后的上清通过固化金属离子亲和层析(IMAC)纯化后,可获得高纯度的目的蛋白。

Fusion expression of Channel catfish hepcidin antimicrobial peptide in Escherichia coli

In the present study,the cDNA encoding hepcidin prepro-peptide was cloned from liver of Channel catfish by RT-PCR with reference to the known hepcidin full-length cDNA.This cDNA encoded the prepro-peptide consisting of 72 amino acid residues,which contained a propeptide of 47 residues and a mature peptide of 25 residues.It was noted that there are two mutational residues in this prepro-peptide comparing with the known reference sequence.When the channel catfish hepcidin was compared with those not only from poikilothermal fish,but also from homoiotherms,eight conserved cysteine residues occurred at their mature peptide regions.The cDNA fragment carrying EcoR I and Hind III sites encoding hepcidin prepro-peptide(named pIH) was inserted into pET32a(+) plasmid with the same restriction sites to construct a recombinant expression plasmid"pET32a-pIH".The fusion protein"trxA-pIH"was successfully expressed in E.coli BL21(DE3) at 37 o C after a 12-h induction with 1 mmol.L-1 IPTG,and about over 70% of"trxA-pIH"was soluble.Tricine-SDS-PAGE analysis demonstrated that the highly purified fusion protein was obtained from the supernatant after sonication by IMAC.