Study On The Expression Sheep Myeloid Antibacterial Peptides-29(SMAP-29) In Pichia Pastotis

Antibacterial peptides are widely existed in many creatures of nature, which are the important parts of innate immunity in organisms.They have attracted great attention for its advantages such as showed a strong antibacterial activity, has a broad antifungal spectrum, heat stability, and it is difficult to produce antimicrobial resistance and so on.Sheep myeloid antibacterial peptides-29 (SMAP-29) is a cathelicidin-derived peptide deduced from sheep myeloid mRNA by research groups of Bagella and Margherita Zanetti in 1995.It was confirmed to beα-helical, 29-amino-acid C-terminal peptide, which has the biological functions of broad spectrum anti-bacterial, anti-fungus and endotoxin-neutralizing.A gene encoding SMAP-29(sheep myeloid antibacterial peptides-29) mature peptide was synthesized according to the published SMAP-29 gene (L46854) sequence in GenBank and the biased codon usage of Pichia Pastoris. The synthesized gene was inserted into Pichia Pastoris expression vector pPIC3.5K and constructed intracellular expression plasmid pPIC3.5K-SMAP-29 for Pichia Pastoris. Then the plasmid was identified with restriction enzyme digestion, PCR and sequencing. Then the recombinated plasmid was transformed into Pichia pastoris GS115 by electroporation after lineared by SalI, and screened with G418 for high-copy recombination strain, and selected methanol utility plus phenotype with MM, MD & PCR. The recombination strain were induced in the medium containing methanol and detected by Tricine-SDS-PAGE analysis. The results showed that after induced with methanol for 2 days, the lytase gave an induced band of SMAP-29 near 3.2KD, which was identified by RT-PCR amplification of SMAP-29 mRNA extracted with Trizol in expressing yeast. The expression products purified with gel filtration chromatography were used for antibacterial assay. The recombinant SMAP-29 displayed obvious antibacterial activity against Staphylococcus aureus and Candida albicans. But it could not distinctly inhibit the growth of Escherichia coli JM109.