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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：1446 下载量：380
| 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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