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Expression Of Porcine β-Defensin-1 Gene In Pichia Pastoris

Posted on:2010-02-05Degree:MasterType:Thesis
Country:ChinaCandidate:H Q RenFull Text:PDF
GTID:2120360278459732Subject:Clinical Veterinary Medicine
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Defensins was an important antimicrobial peptides generated by the organism during the defense reaction. It possessed broad-spectrum antimicrobial, including many microorganisms like bacteria, fungi, enveloped viruses. Especially mammal defensins not only had poisoned role on bacteria, fungi and enveloped viruses, but also had killing effect.on mycoplasma, chlamydia, treponema and some malignant cells. Antibacterial peptides didn't induce the resistance of bacteria because of its distinctive antibacterial mechanism, which suggest that it can be a potential substitute of traditional antibiotics.Porcineβdefensin-1(PBD-1) was an antibacterial peptide that played an important role in defense system of porcine. PBD-1 had a high expression in porcine respiratory and digestive tracts, liver and kidney etc. Lots of researches proved that PBD-1 had distinctive mechanism, it can be used as food preservatives and feed additives. Now with the further study on PBD-1, it would play more important role in animal breeding, illness prevention and quality of livestock products. In recent years, with the development of genetic engineering technology, it hoped to product considerable defensins through biological engineering. Because the production of natural defensins was very low, and the extraction step was complicated, and chemical synthesis needed high cost, so it was the best option of using genetic engineering technology to product defensins. The purpose of this study was to clone porcine tongue epithelium defensin by a traditional techniques and meanwhile to synthesize PBD-1 mature peptide artificially using the biased codon usage of Pichia pastoris. Then they were secreted expression through Pichia pastoris expression system in order to obtain higher activity defensins, which lay a foundation for the mass production of PBD-1 and animal husbandry.In this study, the fresh porcine tongue epithelium mucosa was obtained from healthy porcine. Then primers were designed according to the cDNA sequence of porcine defensin from GenBank and multiple clone sites on the expression vector pPIC9, containing EcoRⅠand NotⅠon the upstream and the downstream, respectively. Then using RT-PCR technology to amplify the cDNA sequence of PBD-1, and cloned to pGM-T vector and sequenced. The fragment of mature PBD-1gene was amplified from recombinant plasmid, whose size was about 151 bp.Then insert into the EcoR1 and Xho1 sites of Pichia pastoris expression vector pPIC9 to construct the recombinant expression vector pPIC9-PBD-1. The gene of antibacterial peptide PBD-1M with 159 bp was synthesized manually using the biased codon of Pichia pastoris. A Kex2 and Ste13 signal cleavage sites were added in the 5'end of antibacterial peptide genes to ensure expression product having a natural N-terminal, in addition, XhoⅠ和EcoRⅠwere added into both ends. The antibacterial peptide gene was cloned into the expression vector pPIC9 to construct the recombinant expression vector pPIC9-PBD-1M. pPIC9-PBD-1and pPIC9-PBD-1M were linearized by BglⅡ, and then transformed into GS115 by electroporation. The transformants were identified by PCR using the bacteriam liquid as the templates, and PCR test showed that about 70% transformants had been integrated into genome of Pichia pastoris. After optimization of the flask-shaking culture fermentation and supernatant was concentrated, the positive clone was expressed on the gel during Tricine-SDS-PAGE. According to Agarose Diffusion Assay, the expression products had antimicrobial activity against Bacillus subtilis and Staphylococcus aureus. Besides, PBD-1M had strong antimicrobial activity, which further confirmed an influence of the biased codon of yeast on the expression.
Keywords/Search Tags:PBD-1, codon optimization, Pichia pastoris, recombinant vector, antimicrobial activity
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