鸡AvBD6成熟肽在毕赤酵母中的表达及其抗菌活性分析-食品加工与安全专业论文.docxVIP

且其在500mg／L浓度下的溶血活性仅为2．44％。本研究为AvBD6的牛物合成及 且其在500mg／L浓度下的溶血活性仅为2．44％。本研究为AvBD6的牛物合成及 发酵生产和产业化应用提供了基础。 关键词：13一防御素6，毕赤酵母，抑菌活性，组成型表达 儿 万方数据 AbstractChicken Abstract Chicken 13-defensins(Avian beta-defensin)，a low molecule peptide which is rich in cysteine．Because of its special antibacterial mechanism，it’S possible to substitute the antibiotics and realize the industrialization． In this stud％the function of Avian beta-defensin-6(AvBD6)was investigated． Construct constitutive expression system of AvBD6 with glyceraldehyde··3··phosphate dehydrogenase constitutive expression vector(pGHKa)in Pichia pastoris．As a result， the recombinant protein of AvBD6 was obtained，and its antimicrobial activity Was employed． 1 Constructing the vector AvBD6 According to cDNA coding sequence of AvBD6 in NCBI(accession number： NM_00 1 00 1 1 93)and preference of Pichia pastoris，added the restriction site of EcoR I and six histidine in AvBD6 mature peptide gene 5’end，while TAA site and restriction sites of Not I in 3’end to designand synthesize the gene sequence of pUC57一AvBD6．The pUC57一AvBD 6pGHKaweredouble digested by EeoR I and Not I respectively,then the fragment was collected and connected to construct the recombinant plasmid pGHKa—AvBD6．The pGHK n—AvBD6 Was linearized after successively digestingby Sac I and Bgl II to remove Amp resistance gene．The linearized fragment was transformed into Pichia pastoris GS I 1 5，the positive transformants were identified by PCR，double digesting and sequencing． 2 Expression of recombinationAvBD6 in Pichia pastoris In order to choose the correct positive transformants，the pGHKa and pGHKa—AvBD6 were identified respectively．They were inoculated in5mLYPD broth． 28 oC，200×g to log phase．Then transformed to 50mL fermentation medium for 48h， the ratio of culture and medium was 1％(v／V)．Centrifuged at 40C，1 2000×g for 1 0 万方数据 min，the min，the supernatant collected was AcBD6 recombinant protein．The expression of AvBD6 was analyzed by Tricine-SDS—PAGE，the diffe