The CDNA Fragment Cloning Of Chicken β-defensin-8 And Its Expression In Pichia Pastoris

Beta-defensin is an antimicrobial peptide in poultry and showes activity of antimicrobial and function of enhances immunity and little is known about resistivity for pathogenic microrganism , it is one of the focus for many scholar. In order to investigate the high expression ofβ-defensins in Pichia Pastoris system ,the study mainly applied molecular biology and molecular cloning technology. The fragment of Gal-8 was cloned from the liver of Three Yellow Broiler .The recombiant plasmid of pGEM-T Easy-Gal-8 and pPIC3.5K-Gal-8 were constructed , and to lead the fusion protein GST-Gal-8 was presented. The results were showed as followed:1.Trizol Reagent was adopted to extract the RNA in the liver of Three Yellow Broiler. 28s and 18s RNA bands were found by 1.0% agarose gel electrophoresis, The OD values of RNA at wavelenghs of 260nm and 280nm were determinated by spectrophotometer , The ratios of OD260/ OD280 was 1.8. The results suggested that the purity of obtained RNA was high enough to satisfy the requirement of experiment.2. According to the reported cDNA gene sequence (DQ677639) of Gallinacin beta-defensin, two pairs of primers were designed , P1/P2 amplified Gal-8 gene and P1/P3 amplified the gene of mature peptide.RNA from the tissues was reverse transcribed into a first-strand cDNA with random primers. The objective gene fragment were cloned successfully from the liver of Three Yellow broiler, by reverse transcription-polymerase chain reaction(RT-PCR), then the produces purified and recovered ,then the cloning vector of pGEM-T Easy-Gal-8 was constructed. The recombinant plasmid that was extracted and sequenced was compared by BLAST of www.ncbi.com. Compared with gene fragments registered in GenBank , there was a difference in the 101,117 bp of the sequenced 201 bases of Three Yellow Broiler the highest degree of identity in nucleotide was 98.3%; The cDNA fragment coded 66 amino acid residues, comprised of signal peptide with 20 amino acid residues,propriece peptide with 5 amino acid residues and mature peptide with 41 amino acid residues. The mature peptide sequences, 123bp, were amplified by nested PCR from a pair of primer of P2/P3 and the mould of recombinant plasmid pGEM-T Easy-Gal-8 .3. After the expression vector pPIC3.5K and mature peptide sequence of Gal-8 gene being double enzyme cutted, they were connected to each other. Then positive recombinant plasmid was identified by PCR and double enzyme cutted respectively , linearized, purified, at last electrotransformated into the Pichia Pastoris. The amplified positive recombinant plasmid was induced with 1% alcohol, following SDS-PAGE to detect the expression information .The above results showed: the objective gene fragments of Gal-8 were cloned from the tissues of laboratory animals and constructed the cloning vector and expression vector of mature peptide successfully. The objective fusion protein was induced with alcohol. These results provides scientific guidance for development and application of antibiotic of defensin in animal husbandry.