Cloning And Prokaryotic Expression Of Monopterus Albus Hepcidin ORF

Antimicrobial peptides (AMPs) are regarded as important components of the host innate immune system and play crucial roles in host defense against microbial invasion. Gene-encoded, ribosomal synthesized antimicrobial peptides are widely distributed in nature and the display strong antimicrobial activity against a broad range of microbes including Gram positive and Gram negative bacteria, protozoa, fungi and viruses. Several AMPs exhibit ant parasitic and anticancer properties.Specific primers were designed and synthesized according to Monopterus albus hepcidin gene ORF sequences(GenBank accession:GU997139)from the cDNA library, which was constructed by our lab. A treaty 300bp target gene fragment was amplified from total RNA of M.albus intestine tract by RT-PCR. The target gene cloned into pMD18-T vector and sequencing results showed that hepcidin gene ORF with exactly the same area. Further analysis of antimicrobial peptide protein sequence contains a signal peptide of 24 amino acids and pro-region part of 40 amino acids before the mature peptide of 26 amino acids, class of antimicrobial peptide hepcidin contains eight cysteine typical structure, the formation of four pairs of disulfide key; structure prediction shows that it belongs toβ-sheet type peptide. Homologous than are found with a variety of animals, M. albus hepcidin sequences are highly homologous with the similarity of Larimichthys crocea croaker up to 81%.Were designed with restriction enzyme BamH I and Hind III restriction sites of the specific upstream and downstream primers pMD18-T-hepcidin positive clone plasmid as a template, were subcloned M.albus peptide hepcidin gene complete ORF region (s-hepcidin) and without signal peptide hepcidin gene (n-hepcidin) coding sequence, and were inserted into prokaryotic expression vector pET32a (+), prokaryotic expression vector pET32a (+)-s-hepcidin and pET32a (+)-n-hepcidin. Were transformed into E. coli BL21 (DE3)pLysS, at 37℃,1mM IPTG induced for 4 hours, by SDS-PAGE electrophoresis analysis, pET32a (+)-s-hepcidin-positive recombinant strain did not express the desired protein; The pET32a (+)-n-hepcidin positive bacteria clearly express the recombinant fusion protein, the size of about 28KDa, and expected the same molecular weight fusion protein. On the pET32a (+)-n-hepcidin in the BL21 (DE3)pLysS expression in IPTG concentration and induction of expression of the time optimization, IPTG induction was found when the concentration of 0.1 mM expression level, and the best inducing time 4h. The optimal expression conditions, pET32a (+)-n-hepcidin fusion protein in BL21(DE3) pLysS strain for expression of soluble expression.