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Study on the Activity of the Antibacterial Peptide Equine Hepcidin Expressing in Pichia Pastoris

Author: SongZuo
Tutor: FuXiaoPing；WuPeiXing
School: Gansu Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Antimicrobial peptides Hepcidin Pichia expression system High copy sieve
CLC: S852.4
Type: Master's thesis
Year: 2010
Downloads: 64
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Abstract


Antimicrobial peptides (antibacterial peptides, ABPs) are a class of non-toxic , no residue , no drug resistance , has great potential for development of new antibacterial drugs . Hepcidin (hepatic antimicrobial activity) 2000 found a cysteine-rich antimicrobial peptides synthesized in the liver , inhibit bacterial and fungal growth in the body and other antimicrobial peptides involved in host 's natural defense . , Hepcidin is an extremely important iron - regulating hormone , a very close relationship between inflammation and anemia and other diseases with the body , the medical value of concern . According to the GenBank horse is a the antimicrobial peptide Hepcidin amino acid sequence , with reference to the Pasteur yeast Pichia (Pichia pastoris) codon preference , the design and synthesis of full-length 279bp of Hepcidin gene . This gene was inserted in the secretion expression vector pPICZαA , build the recombinant expression the plasmid pPICZaA - of hepcidin transformation of competent cells JM109 , DNA sequence analysis showed that the design of synthetic gene fragment was correctly inserted into the vector pPICZaA successfully constructed a recombinant expression vector pPICZαA-hepcidin . SDS-PAGE electrophoresis Hepcidin protein molecular weight of about 10kD . Was transformed into the the recipient strain Pichia pastoris X-33 . The positive recombinant yeast by methanol induction , optimization of expression conditions , screened by Zeocin resistance the high copy restructuring yeast . Supernatants were collected by centrifugation , purified and identified , SDS - PAGE electrophoresis determination that the level of expression of the recombinant protein , protein expression was significantly higher than the high-copy strains unfiltered strains , recombinant protein accounted for 77.9% of the total protein of the supernatant maximum expression amount of up to 214.2 mg / L. Expression of the best conditions : 28 ℃ shaking culture 96h, 24h add methanol to a final concentration of 0.5% . Antibacterial experiments showed that the different copy numbers of strains antibacterial activity and antibacterial spectrum , unfiltered expression of protein in Bacillus subtilis only antibacterial activity , high copy number screening strains of Bacillus subtilis , Staphylococcus aureus , Streptococcus agalactiae have antibacterial activity . The physical and chemical characterization of protein activity remains stored at -20 ℃ longest protein expression heated at high temperature for 30 min , maintained antibacterial activity . Provide the basis for future antimicrobial peptide Hepcidin in the field of veterinary drugs , feed additives , anemia disease diagnosis .

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Basic Veterinary Science > Animal Immunology
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