| 牡蛎抗菌肽Molluscidin的密码子优化、重组毕赤酵母表达及抑菌活性 |
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| 引用本文： | 谭强来,曾臻,许莉,杨彩娟,陆馨敏,丰艳,卢艺玲,罗家英,陈馨雨. 牡蛎抗菌肽Molluscidin的密码子优化、重组毕赤酵母表达及抑菌活性[J]. 食品工业科技, 2022, 43(3): 106-113. DOI: 10.13386/j.issn1002-0306.2021050019 |
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| 作者姓名： | 谭强来 曾臻 许莉 杨彩娟 陆馨敏 丰艳 卢艺玲 罗家英 陈馨雨 |
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| 作者单位： | 1.厦门医学院海洋生物医药资源福建省高校工程研究中心，福建厦门 3610232.厦门医学院天然化妆品福建省高校工程研究中心，福建厦门 361023 |
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| 基金项目： | 福建省卫生健康中青年骨干人才培养项目(2019-ZQNB-21);福建省中青年教师教育科研项目(JAT190849);海洋生物医药资源福建省高校工程研究中心(厦门医学院)开放课题(XMMC-MBS201901);天然化妆品福建省高校工程研究中心(厦门医学院)开放课题(XMMCNC201901);厦门医学院大学生创新创业训练计划项目(201912631029,202012631041,202112631012)。 |
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| 摘 要： | 目的:利用重组毕赤酵母高效表达抑菌活性较好的牡蛎抗菌肽Molluscidin.方法:按照毕赤酵母密码子偏好性优化合成牡蛎抗菌肽Molluscidin,利用生物信息学方法分析其基本理化性质,经EcoRⅠ和NotⅠ双酶切后与表达载体pPICZαA连接,构建重组质粒pPICZαA-CgMoCo,电转至毕赤酵母X-33,利用博...
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| 关 键 词： | 牡蛎 抗菌肽 毕赤酵母 重组表达 抑菌活性 Molluscidin |
| 收稿时间： | 2021-05-08 |
Optimization and Recombinant Expression of Antimicrobial Peptide Molluscidin in Pichia pastoris and Its Antibacterial Activity |
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| TAN Qianglai,ZENG Zhen,XU Li,YANG Caijuan,LU Xinmin,FENG Yan,LU Yiling,LUO Jiaying,CHEN Xinyu. Optimization and Recombinant Expression of Antimicrobial Peptide Molluscidin in Pichia pastoris and Its Antibacterial Activity[J]. Science and Technology of Food Industry, 2022, 43(3): 106-113. DOI: 10.13386/j.issn1002-0306.2021050019 |
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| Authors: | TAN Qianglai ZENG Zhen XU Li YANG Caijuan LU Xinmin FENG Yan LU Yiling LUO Jiaying CHEN Xinyu |
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| Affiliation: | 1.Engineering Research Center of Marine Biopharmaceutical Resource of Fujian Province, Xiamen Medical College, Xiamen 361023, China2.Engineering Research Center of Natural Cosmeceuticals College of Fujian Province, Xiamen Medical College, Xiamen 361023, China |
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| Abstract: | Objective: To express antimicrobial peptide Molluscidin with high antibacterial activity in recombinant Pichia pastoris. Methods: The optimized nucleotide sequence of Molluscidin were synthesized according to P. pastoris codon usage frequency. The physicochemical properties were analyzed by bioinformatics. The fragment was ligated to pPICZαA vector after digested with EcoR Ⅰ and Not Ⅰ. The recombinant expression vector was transformed into P. pastoris X-33 by electroporation. The recombinant strains were screened with Zeocin and identified by PCR. The recombinant Molluscidin was induced with methanol, and identified by SDS-PAGE and Western blot. The expression conditions were optimized by methanol concentration and culture time. The antibacterial activity was determined by disk diffusion test. Results: The optimized nucleotide sequence was obtained. Bioinformatics analysis showed that the predicted molecular weight was 6521.87 Da, the isoelectric point was 11.28, the estimated half-life in yeast was more than 20 h, and the peptide was classified as stable. The recombinant expression vector pPICZαA-CgMoCo was successfully constructed and transformed into P. pastoris. SDS-PAGE and Western blot demonstrated that one recombinant strain with high-level expression was obtained. The optimal expression conditions were 30 ℃, 250 r/min, 1.0% methanol for 48~72 h. Antimicrobial assay indicated that the culture medium supernatant containing recombinant Molluscidin had antibacterial activity against Gram-negative (i.e., Escherchia coli and Klebsiella pneumoniae) and Gram-positive (i.e., Staphylococcus aureus and Bacillus subtilis) bacteria. Conclusion: One recombinant P. pastoris X-33/pPICZαA-CgMoCo strain with high-level expression and antibacterial activity of recombinant Molluscidin is screened, which lays a foundation for its production and application, and provides a technical approach for the development of antimicrobial peptide from Mollusks. |
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| Keywords: | Crassostrea gigas antimicrobial peptide Pichia pastoris recombinant expression antibacterial activity Molluscidin |
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