Secreted Expression Of Gal-2 Mature Peptide CDNA In Pichia Pastoris And Study On Gal-2 Bacteriostatic Activity

The chicken antimicrobial peptide is a kind ofβ-defensin, it is an important component of innate immunity. These peptides not only have the function of broad-spectrum anti-microbial and enhance immune, but also have a connection with host's innate and adaptive systems, and thus it become a hot topic in biomedical research. In this study, we choseβ-defensin-Gal-2 of chicken, using molecular biology techniques, we successfully cloned the whole fragment of Gal-2 cDNA from the RNA of Green feet Ma chicken'lung. Then we built cloning vector(pMD18-T-Gal-2) and expression vector (pPIC9k-Gal -2), and we obtained supernatant with Gal-2 protein of induced yeast secreted. The product of biological activity of the supernatant were studied, as well as we studied the the biological activity of the supernatant. The specific content and results are as follows:1.According to the reported cDNA gene sequence (NM₂04992) of Gallinacin beta-defensin, two pairs of primers were designed, P3/P4 is to amplify the whole sequence of Gal-2 cDNA and P3/P4 with two restriction enzyme sites EcoR I and Not I is to amplify the sequence of mature peptide. We obtained the first strand cDNA from Total RNA by using random primer. Then we used the method of reverse transcription-polymerase chain reaction (RT-PCR) successfully cloned the whole fragment of Gal-2 cDNA from the RNA of Green feet Ma chicken'lung and purified fragment of the PCR product, then construct pMD18-T-Gal-2 cloning vector. The recombinant plasmid was extracted, then sequenced and compaired by BLAST of www.ncbi.com. The result displayed that Gal-2 cDNA of Green feet Ma chicken have total of 195 base pairs, and the homology of 100%. The cDNA fragment coded 64 amino acid residues,comprised of signal peptide with 20 amino acid residues, propriece peptide with 8 amino acid residues and mature peptide with 36 amino acid residues. The mature peptide sequences, 108bp, were amplified by nested PCR from a pair of primer of P3/P4 and the mould of recombinant plasmid pMD18-T-Gal-2.2.Connected Gal-2 mature peptide and the vector pPIC9k, we successfully construct the expression vector pPIC9k-Gal-2. The positive transformant which was identified by PCR was enriched, and the recombinant plasmid was extracted and sequenced, the blast result displayed that the homology of mature peptide was 100%. The expression vector pPIC9k-Gal-2 was linearized, and transformed into yeast strain GS115 by electroporation, and we got the positive transformant by histidine-deficient medium. The positive transformant was induced by methanol, and the the supernatant was freeze-dried and detected by SDS-PAGE protein electrophoresis. The results show that, the molecular weight of Gal-2 is about 3.9ku, it can be found in the supernatant of yeast and yeast secretion, the optimal induction time was 48h. Densitometric scanning analysis demonst rated that the expressed protein exaccounted for about 63.7% of the total secreted bacterial protein. The result of biological activity in vitro of expression product showed that The expression products had antimicrobial activity against Escherichia coli(CMCC44102), S.paratyphi B (CMCC50094) , Bacillus subtilis (CMCC63501) and Staphylococcus aureus (ATCC26113).The above results showed: the objective gene fragments were cloned from the lung of Green feet Ma chicken and constructed the cloning vector pMD18-T-Gal-2 and expression vector pPIC9k-Gal-2 successfully. Then we transferred the expression vector into Pichia Pastoris, the result of SDS-PAGE protein electrophoresis show that the induced supernatant contain the objective protein and the molecular weight of Gal-2 is about 3.9ku. The expression products had antimicrobial activity against Escherichia coli(CMCC44102), S.paratyphi B (CMCC50094) , Bacillus subtilis (CMCC63501) and Staphylococcus aureus (ATCC26113).