尼罗罗非鱼Hepcidin抗菌肽在大肠杆菌中的融合表达及其抗菌活性

Hepcidin是一类分子量较小、富含半胱氨酸的阳离子抗菌肽,TH1-5则是从莫桑比克罗非鱼(Oreochromis mossambicus)中分离到的3种hepcidin cDNA序列中的1种;虽然化学合成的TH1-5的成熟肽显示了对若干细菌的抑菌活性,但通过重组DNA表达的此肽是否也具生物学活性则是未知的。本文参考莫桑比克罗非鱼hepcidin TH1-5的核苷酸序列,以尼罗罗非鱼(Oreochromis niloticus)的肝脏为基因克隆的材料,对其类似于hepcidin TH1-5的成熟肽(mTH)进行了重组DNA表达。在构建的重组表达质粒"pET-32a-mTH"中,mTH基因与携带有6×His-tag标签和肠激酶识别位点的trxA基因融合,25℃下,经1mmol/L IPTG诱导培养8h后,在E.coli BL21(DE3)中成功表达了"trxA-mTH"融合蛋白。经固化金属离子亲和层析(IMAC)纯化后的融合蛋白并不显示抑菌活性,但经肠激酶消化处理后,释放出的重组mTH显示了对革兰氏阳性的单增李斯特菌和金黄色葡萄球菌以及革兰氏阴性的大肠杆菌和铜绿假单胞菌的抑菌活性。

Hepcidin from Tilapia,Oreochromis niloticus:Its Fusion Expression in Escherichia coli and Antimicrobial Activity

Hepcidin(earlier named LEAP-1,liver-expressed antimicrobial peptide-1)is a small cysteine-rich cationic antimicrobial peptide produced predominantly in the liver.Some fish have multiple copies of hepcidin to defense against bacteria in complicated aquatic environment.TH1-5,one of the three different hepcidin cDNAs from tilapia(Oreochromis mossambicus),was deduced to be composed of 87 amino acid residues.Although its mature peptide chemically synthesized showed antibacterial activity against several bacteria,it is unknown whether this peptide expressed by recombinant DNA has biological activity.Thus,in the present study,a cDNA fragment encoding the mature peptide for TH1-5-liking(mTH)was cloned from tilapia(Oreochromis niloticus)liver by RT-PCR with reference to the cDNA sequence of tilapia(O.mossambicus)hepcidin TH1-5.Nucleotide sequencing of the PCR product revealed that this mTH fragment encoded a 22-amino acid mature peptide,and eight cysteine residues were observed at conserved positions.Subsequently,recombinant DNA expression of the mTH was performed using pET-32a(+)with trxA partner as expression plasmid and E.coli BL21(DE3)as host.In the recombinant expression plasmid "pET-32a-mTH",the mTH gene was fused with a trxA partner with a 6×His-tag and an enterokinase site.The fusion protein "trxA-mTH" was successfully expressed at 25 ℃ in E.coli BL21(DE3)by adding 1 mmol/L IPTG,and the percentage of the fusion protein in total cell proteins was the most high after an 8-h induction.The Tricine-SDS-PAGE showed that the fusion protein existed in both the supernatant and inclusion body after sonication and centrifugation.As determined by Tricine-SDS-PAGE scanning,over 50% of the fusion protein was soluble,and accounted for about 22.5% of the total cell proteins.However,the fusion protein purified from the supernatant by Immobilized Metal Affinity Chromatography(IMAC)was not biologically active.It is concluded that the cationic mTH might be neutralized by the anionic trxA(pI 4.67)or the cationic surface might be shaded by the fusion partner,so as to the mTH could not interact with the membrane of bacteria.Such conjecture is confirmed by the subsequent experiment.After the trxA partner was removed by enterokinase cleavage,the released recombinant mTH exhibited obvious antibacterial activity against Gram positive-Listeria monocytogenes and Staphylococcus aureus,and Gram negative-E.coli and Pseudomonas aeruginosa.