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鸡β-防御素6及其衍生物的真核表达与生物学活性研究

发表时间:2013-06-15  浏览量:1108  下载量:303
全部作者: 曹艳萍,马清泉,单安山
作者单位: 东北农业大学动物营养研究所
摘 要: 为研究二硫键对鸡β-防御素抑菌活性的影响,并实现鸡β-防御素6(avian-β-defensin 6,AvBD-6)的真核表达。将AvBD-6成熟区的全部半胱氨酸(Cys)去掉,获得衍生物AvBD-6-B. 根据毕赤酵母(Pichia pastoris)密码子偏爱性,设计了AvBD-6和AvBD-6-B的基因片段,插入到真核表达载体pGAPHαM的XhoⅠ和EcoRⅠ酶切位点之间,构建重组表达质粒。将重组表达载体用Bgl Ⅱ线性化回收大片段后电击转化毕赤酵母GS115,利用PCR筛选阳性克隆,并将阳性重组子接种于100 mL 酵母浸出粉胨葡萄糖(yeast extract peptone dextrose,YPD)(含有1%酪蛋白水解物)培养基中表达。Tricine-SDS-PAGE结果显示,AvBD-6和AvBD-6-B在毕赤酵母中得到了表达,表达量分别为114.9 mg/L和93.8 mg/L. 抑菌实验结果表明:二者对枯草芽孢杆菌(Bacillus subtilis)CMCC 63501的抑菌效果相同,而对金黄色葡萄球菌(Staphylococcus aureas)ATCC29213抑菌效果AvBD-6-B高于AvBD-6,说明去掉二硫键对鸡β-防御素的抑菌活性没有负面影响。研究为利用基因工程技术实现抗菌肽工业化生产提供了理论依据。
关 键 词: 动物营养学;鸡β-防御素6;二硫键;毕赤酵母;表达
Title: Eukaryotic expression and biological activity of avian β-defensin 6 and its derivative
Author: CAO Yanping, MA Qingquan, SHAN Anshan
Organization: Institute of Animal Nutrition, Northeast Agricultural University
Abstract: To explore the effect of disulfide bonds on antimicrobial activity of avian β-defensin and to obtain the eukaryotic expression of avian β-defensin 6 (AvBD-6), all the Cys residues in the maturation region of AvBD-6 were removed and then obtained the derivative AvBD-6-B. According to the codon bias of Picha pastoris, the genes of AvBD-6 and AvBD-6-B were designed and synthesized. Then, the recombinant plasmid was constructed through inserting AvBD-6 and AvBD-6-B genes into the restriction sites between XhoⅠ and EcoRⅠ of the eukaryotic expression vector pGAPHαM. The Bgl Ⅱ-linearized recombinant plasmids pGAPHαM-AvBD-6 and pGAPHαM-AvBD-6-B were transformed into P. pastoris GS115 by electroporation. The recombinant AvBD-6 and AvBD-6-B were expressed in 100 mL yeast extract peptone dextrose (YPD) (including 1% casein hydrolyzate). Tricine-SDS-PAGE demonstrated that both AvBD-6 and AvBD-6-B were expressed in P. pastoris, the concentration of recombinant AvBD-6 and AvBD-6-B reached 114.9 mg/L and 93.8 mg/L, respectively. The bacteriostatic experimental results showed that this two expression products exhibited the same antimicrobial activity against Bacillus subtilis CMCC 63501. However, for Staphylococcus aureus ATCC29213, the antimicrobial activity of AvBD-6-B was higher than AvBD-6, indicating that the disulfide bonds in AvBD-6 were not necessary for antimicrobial activity. This research provides a theoretical basis for achieving industrial production of antimicrobial peptides using genetic engineering technology.
Key words: animal nutrition; avian β-defensin 6; disulfide bonds; Pichia pastori; expression
发表期数: 2013年6月第11期
引用格式: 曹艳萍,马清泉,单安山. 鸡β-防御素6及其衍生物的真核表达与生物学活性研究[J]. 中国科技论文在线精品论文,2013,6(11):1070-1077.
 
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