Cloning, Expression And Biological Activity Analysis Of Hepcidin From The Blunt Snout Bream(Megalobrama Amlycephala)

Hepcidins, name as Iron-Regulatory Proteins, are antimicrobial peptides that play important roles including antimicrobial, antivirus and antitumor in innate immune system against pathogenic microbes invading. Hepcidins are also be factors involved in the regulation of iron.The cDNA that encodes hepcidin of liver in blunt snout bream (Megalobrama amlycephala) was cloned by RT-PCR. Recombinant expression plasmids, pGEX-KG-Hepcidin and pPICZaA-Hepcidin, were constructed by gene recombination technology. The recombinant plasmids were transfromed into Escherichia coli and Pichia pastoris, respectively. Recombinant proteins were inducibled. The blunt snout breams were injected Aeromonas hydrophila intraperitoneally, the liver, spleen, head kidney, intestine and blood were collected after challenging4h,8h,12h,24h,48h and72h. The mRNA expression of hepcidin in liver, spleen, head kidney and intestine were measured, and measured the iron content in serum and liver.The results were as follows:1. Sequencing result showed the hepcidin ORF nucleotide sequence was282bp in length, encoding a prepropeptide of93amino acids (aa). Three domains are proposed for preprohepcidin:signal peptide (24aa), prodomain (44aa) and mature peptide (25aa), respectively. The hypothetical iron regulatory sequence Q-S/I-H-L/I-S/A-L in the N-terminal region of mature peptide, and the C-terminal region possessed a typical arrangement of eight conserved cysteine residues.2. Recombinant expression plasmid, pGEX-KG-Hepcidin, was transformed into BL21(DE3). A fusion protein was observed on SDS-PAGE gel, and was found in both inclusion bodies and soluble protein. Recombinant expression plasmid, pPICZaA-Hepcidin, was transformed into GS115, and recombinant protein was induced. Bioactivity analysis showed that the recombinant protein had antimicrobial activity against Aeromonas hydrophila and Staphylococcus aureus.3. The hepcidin basal tissue expression analysis indicated that the levels of hepcidin amplification products were relatively high in liver, medium in spleen and head kidney. After the challenge, the expression of hepcidin increased firstly, and then decreased. The level of serum iron decreased, meanwhile, the level of tissue iron decreased firstly and then increased. It was predicted that the low level of serum iron reduce the recyclable iron for self-defence after the challenge.