Cloning of β-defensin Gal-2 gene in Sichuan Mountain dark-bone chickens and construction of its yeast expression vector

To construct the yeast expression vector for β defensin Gal-2 gene in the Mountain dark-bone chickens,the primers were designed according to the gene sequence of β defensin Gal-2 published in GenBank.RT-PCR was used to amplify the Gal-2 genes of the bone marrow cells from the Mountain dark-bone chickens.The PCR products were cloned into pMD18-T and transformed into E.coli.Subsequently,the positive plasmids were identified by PCR,restriction enzyme analysis,and sequencing.Bioinformatics analysis was also carried out by using DNAStar software.According to the sequence which encodes beta-defensin Gal-2 of chicken,a pair of primer was designed.The coding sequence of mature peptide was cloned from plasmid pMD18-T-Gal-2 by PCR and inserted downstream to signal peptide to construct recombinant plasmid pPICZa-A-Gal-2.The plasmid was transformed into E.coli,and the positive clones were selected and sequenced.After linearization,the plasmid pPICZa-A-Gal-2 was electro-transformed into Pichia pastoris GS115 strains.1% methanol was used to induce Gal-2 gene.At 120 h of expression,the products were purified preliminarily and identified by Tricine-SDS-PAGE electrophoresis.The results showed that: Gal-2 genes which contains complete cds were cloned successfully.The recombinant eukaryotic expression vector containing chicken Gal-2 gene was constructed successfully.It indicates that the Gal-2 gene of Mountain dark-bone chickens was expressed in yeast.