Molecular Cloning And Expression Analysisof Antibacterial Peptide Hepcidin Gene In Qihe Crucian Carp Carassius Auratus

Antibacterial peptide is encoded by a specific gene in biological cells, which can produce a kind of broad-spectrum antibacterial peptides induced by external conditions. In recent years, a new type of antibacterial peptide hepcidin, as a small molecule cationic polypeptide, was found to be widely existed in fish, which wasmainly specific expressed in liver. It can kill various viruses, bacteria, fungi and protozoa, which is one of the important effector molecules in natural immune, and is a kind of signal molecule, involved in iron metabolism.According to the known antibacterial peptide hepcidin gene in GenBank, degenerate primers were designed based on the conservative sequence. The full length of hepcidin cDNA sequence in Qihe crucian carp Carassius auratus was amplified by RT-PCR and RACE methods.The full length of hepcidin cDNA was 708 bp, containing the open reading frame(ORF) 258 bp, 5’ untranslated region(5’ UTR) 94 bp and 3’ untranslation section(3’ UTR) 356 bp, and there was a poly A tail signal(AATAAA) and two mRNA unstable motif(ATTTA) in 3’ UTR. 258 bp hepcidin ORF encoded a mature peptide precursor of 85 amino acids, which contained signal peptide(24 amino acid residues), precursor peptide(36 amino acid residues) and mature peptide(25 amino acid residues). Mature peptide contained eight conservative cysteine residues, a single hairpin structure was formed through four disulfide bond, there is a special disulfide bond around the corner, may be associated with antimicrobial activity of hepcidin directly. Sequence analysis showed the high homology with the reported sequences in the other fish. Evolution analysis of translated peptide of hepcidin also showed the high homology with the other reported hepcidins. Based on the phylogenetics tree constructed by gene hepecidin, Qihe C. auratus showed the closest relationship with common carp.Using the method of RT- qPCR, the changes of hepcidin mRNA levels in various tissues in healthy fish and fish stimulated by Aeromonas hydrophila and LPS were detected. Result showed that the highest level of hepcidin mRNA expression was in the liver, followed by the spleen and kidney, and expression level was lower in gill and kidney, and further lower in heart, intestines, muscles, brain and blood. After C. auratus were injected with A. hydrophila and LPS respectively, the changes of hepcidin mRNA levels, with the extension of time, were detected in gill, kindey, head kindey, spleen, and liver. Results showed a similar change pattern that mRNA levels generally rose firstly and then decreased, but in different tissues or organs, the expression patterns were also different. In fish injected with LPS, hepcidin mRNA level got to the peak in head kidney, kidney, and spleen at 6 h after injection being higher more than 100 times than in the control. Hepcidin mRNA level in gill reached the highest at 12 h after stimulation, at about 60 times to the control. With regard to hepcidin mRNA level in liver, it began to rise at 3 h after injection, and reached the highest at 24 h, at about 30 times to the control, then slowly fell. In fish Injected with A. hydrophila, hepcidin mRNA expression level reached the highest in spleen at 6 h, at 150 times to the control, and got to the highest in gill, kidney, and head kidney at 12 h, at about 80 times to the control in gill and kidney, and at 150 times in head kidney. It reached the highest in liver at 24 h, which was about 18 times to the control.In the control, the fish were intraperitoneal injected with 0.75% saline, the hepcidin mRNA level showed slight change.Prokaryotic expression vector pET32a(+) – hepcidin was constructed and translated into the E. coli BL-21, induced expression by IPTG, and expression product was detected using the SDS-PAGE electrophoresised. Result showed that a specific band about 21 k D molecular weight was observed in the supernatant of bacterial lysis solution and inclusion bodies precipitation which was similar to the expected molecular weight of hepcidin fusion protein(21.72 kD). The highest expression level after IPTG induction was at about 8 h, which provided the reference basis for following experiments of protein purification and the antibacterial activity detection.