Construction Of Recombinant Yeast Expressing Porcine β-Defensin-2 Mature Peptide

Defensins is an important cationic antimicrobial peptides in the family of antimicrobial peptides. They widely exist in animals and plants and play an important role in natural immunity. Defensins are broad spectrum antibiotic, they can inhibit or kill bacteria, fungi, viruses and other pathogens, besides, mammals' defensins also kill mycoplasma, chlamydia, spirochetes, and some tumor. Defensins can also regulate the immune system,play an important role in resistance to infection.Porcineβ-defensin-2 (pBD-2) is an important porcine antimicrobial peptide. It mainly come from epithelial tissue, widely distributed in the skin, thymus, spleen, liver, tongue, respiratory tract, gastrointestinal tract and other organs.Porcineβ-defensin 2 mature peptide is composed of 37 amino acids and rich in arginine and six conservative cysteines. These positively charged amino acid and three pairs of intramolecular disulfide bonds formed by six conservative cysteines give the porcineβ-defensin-2 mature peptide which have stable.activity and form resistance uneasily, a unique mechanism of action. As the presence of intramolecular disulfide bonds to porcine P-defensin-2 mature peptide has a great influence on its activity, the traditional Prokaryotic expression system have no post-translational modification to the expressed product and the toxicity from the product to the host bacteria will also effect the expression level. In contrast, Eukaryotic yeast expression system has many advantages, such as a more effective post-translational modification and the high level of expression. In recent years, Eukaryotic yeast expression system has been successfully expressed many defensins. Considering the low level of pBD-2 released by the body, the complicated extraction procedur, and the high cost of chemosynthesis, genetic engineering method seems to be a convienent way to acquired pBD-2 in large amount.This experiment based on the published sequence of pBD-2 mature peptide in GeneBank, considered yeast codon bias, designed three primers and amplified using PCR technology to get DNA sequence of pBD-2 mature peptide. One ends of the resulting sequence is added EcoRⅠrestriction site, the other NotⅠ, then clone the resulting sequence to the secretion expression vector pPIC9K and pPIC9k-GST recombinant vectors which is constructed, and obtain pPIC9K-pBD-2 and pPIC9k-GST-pBD-2 recombinant vector. Positive clones identificated correct is selected, and the pPIC9K-pBD-2 and pPIC9k-GST-pBD-2 recombinant vector are linearized with enzyme SalⅠ, then transformed them into Pichia pastoris KM71 cells by electrotransformation technology. Through screening on a specific medium and PCR detection, porcine P-defensin-2 mature peptide gene fragments is correctly integrated into genome chromosome of the yeast KM71, which is stable Pichia pastoris strains expressing porcineβ-defensin-2 mature peptide. By adjusting the conditions of expression continuously such as pH, temperature etc. Screened yeast clones are induced by methanol and porcineβ-defensin-2 mature peptide is expressed in the medium BMMY.