Study On The Activity Of The Antibacterial Peptide Equine Hepcidin Expressing In Pichia Pastoris

Antibacterial peptides (ABP), which know as no poison, no remnant, no drug resistance, are a kind of new type of antibiotics with tremendous marketing developing potential. Hepcidin is a special antimicrobial peptide, rich in cysteine, firstly found from human blood in 2000; not only it has remarkably antimicrobial activity and innate immunity, but also is considered a key regulator of iron metabolism. Even more, hepcidin plays a pathological role in the anemia and inflammation. It was greatly noticed because of its important value of medical applications.According to the sequences published on GenBank and the cordon bias of Pichia pastoris, a DNA sequence (279bp) of Equine hepcidin was designed and synthesized. Then, the target gene was directly cloned into multiple cloning site of the Pichia pastoris expression vector pPICZαA to construct the recombinant plasmid. After transformed into JM109 host bacteria, the recombinant pPICZαA-hepcidin was confirmed by PCR detection. DNA sequencing revealed that the correct insert of hepcidin into pPICZαA was confirmed. The analysis by SDS-PAGE indicated that the expression of hepcidin had a molecular weight of 10kD.The recombination expression vector, pPICZαA-hepcidin, was linearized by Sal I and electroporated into yeast host strain X-33, which was expressed by methanol, then collected the upper liquid for purification the protein. In order to improve the expression, the high copy inserts were selected by the different dose of Zeocin and optimized the expression condition. The SDS-PAGE results show that, compared the different expression level of the different copy strains, the high-copy strain was increased the expression of hepcidin. The highest level of gene expression even came up to 77.9% of the overall protein in induced supernant. Thus the highest expression level of protein of interest was calculated as approximately 214.2 mg/L. The most optimum condition of expression: the yeast was cultured under 28℃for 60 hours, and methanol was added to keep its concentration up to 0.5% between 24h. The results of bacteriostatic experiment showed that different antimicrobial activity and spectrum was found with different copies. The protein, which was expressed by the unscreened strain, only had inhibitory activity against Bacillus subtilis. However, the high-copy strain expressed the protein, which displayed significant antibacterial activity against Staphylococcus aureus, Bacillus subtilis,Streptococcus agalactiae. The characterization of hepcidin protein: the suitable storage temperature was -20℃, and maintains the bacteria activity after heating 30min. Therefore, the trails provide a basis for veterinary drugs,feed additive supplement and develop the diagnostic reagent of equine anemia and inflammation.