Cloning Of Chicken Gal-8 CDNA And Its Expression In E.coli And Lactobacillus

Beta-defensin is an antimicrobial peptide 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 prokaryotic expression system and its antibacterial activity ,the study mainly applied molecular biology and molecular cloning technology. The fragment of Gal-8 was cloned from the liver of Three Yellow broile.The cloning vector of pGEM-T Easy-Gal-8,E.coli expression vector of pGEX-6P-1-Gal-8 and Lactobacillus expression vector of pNZ8048-Gal-8 were constructed , The objective fusion protein was induced with IPTG, Gal-8 was obtained by using affinity chromatography and the primary biologic activity of purified product was investigated. The results were showed as followed:1.Gal-8 cDNA CloningAccording to the reported cDNA gene sequence (DQ677639) of Gallinacin beta-defensin, three pairs of primers were designed , The P1/P2 amplified Gal-8 gene ,The P3/P4 and P5/P6 amplified the gene of mature peptide.Total RNA 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 111 bp of the sequenced 201 bases of Three Yellow Broiler the highest degree of identity in nucleotide was 99%; 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 of Gal-8 were amplified by nested PCR from two pairs of primers of P3/P4 and P5/P6, and the mould of recombinant plasmid pGEM-T Easy-Gal-8 .2. Gal-8 mature peptide gene expression in E.coliThe Gal-8 mature peptide gene ,which was amplified by P3/P4 ,was purified and double enzyme cutted ,then inserted into the plasmid pGEX-6P-1 vector which was also double enzyme cutted .The positive recombinant plasmid was identified by PCR. The positive recombinant plasmid was induce with IPTG, following SDS-PAGE detected the expression information. The result were showed: the fusion protein of GST-Gal-8 was found in the point of 30kD,which Can be found in the supernatant and the precipitation. The soluble fusion protein was best expressed at 25℃by 0.5mmoL IPTG after 4h.The soluble fusion protein which was in the supernatant accounted for about 19.1% of the total bacterial protein,to achive 0.2921mg/mL. The expressed fusion proteins were purified with immobilized glutathione affinity chromatography column,Gal-8 polypeptide was cutted from Fusion protein by the Prescission protease. The biological activity of expression production in vitro was detected by monlayer agar plate diffusion method it appeared activity of antimicrobial.3. Gal-8 mature peptide gene expression in LactobacillusThe Gal-8 mature peptide gene ,which was amplified by P5/P6 ,was purified and double enzyme cutted ,then inserted into the plasmid pNZ8048 vector which was also double enzyme cutted .The positive recombinant plasmid pNZ8048-Gal-8 was induced by Nisin , following Tricine -SDS-PAGE detected the expression information.There was`t the purpose of protein bands,It needs identified by Western bloting in the further.In this study, the Gal-8 gene were cloned from the tissues of chicken liwer and constructed the cloning vector,the E.coli expression vector and the Lactobacillus expression vector of mature peptide successfully. The objective fusion protein was induced with IPTG, Gal-8 mature peptide was obtained by using affinity chromatography. the bacteriostasis study revealed that purified fusion protein had antibacterial.Lactobacillus transformants was induced by Nisin,There was not detected the purpose of protein bands. The physical and chemical characteristics study and antibacterial activity study of chickenβdefensin Gal-8 will be continued based on the results of this research.