| Hepcidin is a novel small molecular antimicrobial peptide and a principle regulator of systemic iron homeostasis that is widely expressed in various fish. To prevent virus, bacterial (especially resistant bacteria) and parasitic diseases as a beneficial means of improving fish cultivation, the study focused on antimicrobial peptides as alternative antibiotic drugs and feed immune additives has become the hot research areas.A pair of degenerate primers F1/R1 were designed according to the reported nucleic acid sequence of fishes from NCBI/GeneBank using primer 5.0. Expression primers (F2, R2) were introduced to enzyme cut sites and terminal anticodon. The restriction enzyme EcoR I was introduced to forward primer F2 and Not I to the reverse primer R2. The cDNA that encodes hepcidin from Chinese sturgeon was cloned from blood total RNA with RT-PCR. The hepcidin from Chinese sturgeon was amplified from the recombinant plasmid with the expression primer. After the target gene and pPICZaA were double digested with EcoR I and Not I, respectively, the two DNA fragments were linked together with T4 ligase at 16℃over the night. Transfer recombinant expression plasmid into competent E. coli TOP 10 cell. Then the recombinant expression plasmid pPICZaA-hepcidin was sequenced by BGI corporation.The recombinant expression plasmid of pPICZaA-hepcidin was linearized by BstX I and then transformed into Pichia Pastoris GS115 by electroporation after purification. Colonies grew on the ZeocinTM selective culture medium. Colonies with high resistance were chosen by increasing the concentration of ZeoeinTM After culture incubation for 24 h using BMGY (contain glycerol), OD600 value reach 2-6. Induction by 0.8% methanol in BMMY culture medium at 250 rpm at 28℃, was followed by induction by pPICZaA. After 72 h, the expression supernatant fluid was collected by using 85% saturation ammonium sulfate enrichment yeast expression supernatant fluid. Antibacterial activity, antibacterial efficiency and thermal stability of yeast expression supernatant fluid was determined.The result is shown in the list below:1, This study successfully expressed antimicrobial peptide hepcidin from Chinese sturgeon in Pichia pastoris. The cDNA of hepcidin was 327 basepairs (bp). The target gene was correctly inserted into pPICZaA vector. The recombinant protein was shown inhibit Staphylococcus aureus, Escherichia coli DH5a, Streptococcus agalactiae and Aeromonas sobria in vitro. It was also shown to have good bactericidal effects on resistant strains of Aeromonas hydrophila. However, there is no significant inhibitory effects on Bacillus subtilis and no inhibitory effect on Edwardsiella tarda. Recombinant peptide hepcidin from Chinese sturgeon successfully express in Pichia Pastoris expression system.2, Antibacterial results show that the addition of 70μL hepcidin antimicrobial peptide can inhibit growth of 40μL E. coli DH5a containing the 260μL LB medium. Antibacterial effect is equivalent to 1μg ampicillin.3, The results of the thermal stability of recombination antimicrobial peptide hepcidin showed that the recombinant peptide hepcidin that had been boiled 10 min, and 30 min still exhibit antimicrobial activity. But compared to the recombination peptide hepcidin that was not boiled, antibacterial activity is reduced. After recombinant peptide hepcidin was boiled for 1 h, it had lost all antibacterial activity.The recombinant peptide hepcidin from Chinese sturgeon can be expressed successfully in Pichia pastoris and that this recombinant peptide has potential clinical value for the prevention and treatment of bacterial disease on sturgeon. |
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