| 以莱茵衣藻为载体表达外源抗菌肽的研究 |
| Expression of Exogenous Antimicrobial Peptides in Chlamydomonas reinhardtii |
| 投稿时间：2020-03-30 修订日期：2021-05-24 |
| DOI：10.15928/j.1674-3075.202003300080 |
| 中文关键词:抗菌肽 莱茵衣藻 基因表达 载体 |
| 英文关键词:antimicrobial peptide Chlamydomonas reinhardtii gene expression carrier |
| 基金项目:国家水体污染控制与治理重大科技专项（2017ZX07203001）和国家载人航天工程空间站任务空间应用项目（YYWT-0801-EXP-09） |
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| 中文摘要: |
| 将黑水虻抗菌肽基因sarcotoxin3转入到莱茵衣藻细胞中，以期实现抗菌肽活性片段的大规模表达生产，为将来完成抗菌肽的临床测试及水产应用奠定实验基础。以质粒PHK85为骨架，插入荧光素酶基因和黑水虻抗菌肽sarcotoxin3基因，用DNA连接酶将质粒连接，构成抗菌肽质粒。抗菌肽质粒经克隆、提取、酶切线性化、衣藻玻璃珠转化、荧光素酶活性筛选和抑菌实验，结果表明：经过测序进一步确认质粒成功构建，碱基序列与所设计质粒序列一致，质粒浓度472.8ng/μL纯度（A260/A280）1.93，7d后每个巴龙霉素平板长有单克隆约200个，大多数单克隆荧光素酶活力值可高达106，少数单克隆荧光值甚至可达108；藻株在26kDa附近出现了特异信号，含有抗菌肽的蛋白对大肠杆菌DH5α有一定的抑制作用。莱茵衣藻可以作为抗菌肽外源表达的一种载体，为抗菌肽的大量生产提供了一种可能。 |
| 英文摘要: |
| Antimicrobial peptides are small peptides found widely distributed in nature. As part of the innate immune system, they provide the first line of defense against pathogens. While antimicrobial peptides have excellent potential as an antibiotic, scaling up production has limited application. Chlamydomonas reinhardtii, known as the "green yeast", is a single-celled eukaryotic green algae and a good bioreactor that integrates multiple advantages. In this study, we transferred the antibacterial peptide gene sarcotoxin3 from Hermetia illucens into C. reinhardtii cells to realize large-scale expression and production of active antibacterial peptide fragments. The objective was to lay an experimental foundation for completion of clinical testing and future aquatic production of antibacterial peptides. Using plasmid pHK85 as the lattice, the genes for luciferase gene and Hermetiaillucens peptide sarcotoxin3 were inserted into the plasmid, and DNA ligase was used to ligate the plasmid to form the antimicrobial peptide. The C. reinhardtii plasmid was cloned on glass beads and the antimicrobial peptide produced was then extracted and linearized using a restriction enzyme. Finally, luciferase activity was screened and the antibacterial activity was tested. Sequencing was used to further confirm that the plasmid was successfully constructed and the base sequence was consistent with that of the designed plasmid sequence. The plasmid concentration was 472.8ng/μL and the purity (A260/A280) was 1.93. After 7 days, each paromomycin plate had about 200 single clones. The fluorescence of the monoclonal luciferase was as high as 106 and, in a few cases, reached 108. The algae strain showed a specific signal near 26kDa, and the protein containing antibacterial peptides had an inhibitory effect on E. coli DH5α. Our results show that C. reinhardtii can be used as a carrier for the exogenous expression of antimicrobial peptides, providing a possible means for mass producing antimicrobial peptides. |
| 赵艳苹,李小燕,彭婷婷,黄开耀,王高鸿.2021.以莱茵衣藻为载体表达外源抗菌肽的研究[J].水生态学杂志,42(3):106-113. |
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