| 摘要: |
| 烟草(Nicotiana tabacum) 是一个被广泛地用于生产临床药用重组蛋白植物平台。不过，植物源药用蛋白糖基修饰与哺乳动物间差异一直没得到很好地解决限制了其应用，特别是由岩藻糖基转移酶催化的植物源蛋白的岩藻糖基化被广泛关注。本研究构建一个靶向α-(1,3)-岩藻糖基转移酶4（α-1,3-FUCOSYLTRANSFERASE 4，FUT4）基因第一外显子RNA干涉载体FUT4-RNAi。用农杆菌（Agrobacterium tumefaciens, EHA105）介导法转化烟草（cv. Yunyan 87），29株再生烟草中用PCR方法检测确认了17株阳性烟草植株；其中，11个株系（4#, 6#, 7#, 11#, 12#, 15#, 19#, 22#, 26#, 28#, and 29#）中FUT4 的mRNA累积比对照云烟87（cv. Yunyan 87）低25%以上。对FUT4转录表达最低4 个转基因株系（7#, 12#, 15#, and 29#）进行蛋白印迹（western blotting，WB）和酶联免疫分析（enzyme-linked immunosorbent assay ,ELISA）发现，FTU4蛋白的累积显著低于对照云烟87；甚至转基株系29#的FUT4蛋白含量仅为对照的13%。我们成功地开发了一个FUT4基因在mRNA和蛋白水平均下调表达烟草底盘，该研究为用植物系统生产人源化药用重组蛋白提供了重要基础。 |
| 关键词: 岩藻糖基转移酶 烟草 RNA干涉 药用蛋白 |
| DOI： |
| 分类号: |
| 基金项目:中国烟草公司重点研发项目（2021-150-110202102026） |
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| Creating a novel tobacco chassis with downregulated expression of α-1,3-fucosyltransferase 4 |
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Shan Xuemeng1, Muhammad Naeem1, Wang Yangzhong2, Han Rong1, Zhou Mengqian2, Zhao Weihua1, Zhao Lingxia1
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1.Shanghai Jiao Tong University;2.Shanghai Tobacco Group Co. Ltd.
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| Abstract: |
| Tobacco is a widely used platform for producing recombinant proteins for clinical applications. However, plant-derived therapeutic proteins with mammalian-like glycosylation modifications remain a challenge, particularly fucosylation mediated by fucosyltransferases (FUTs). In this study, an RNA interference (RNAi) plasmid targeting the first exon of α-1,3-fucosyltransferase 4 (FUT4) gene was constructed, named FUT4-RNAi. Using Agrobacterium-mediated transformation with EHA105 harboring the FUT4-RNAi plasmid, we obtained 29 regenerated tobacco lines, of which 17 were confirmed as putatively positive by PCR. The mRNA transcript accumulation of the FUT4 gene was significantly reduced in 16 of the 17 transgenic lines compared to the negative control, cv. Yunyan 87. Among these, 11 lines (4#, 6#, 7#, 11#, 12#, 15#, 19#, 22#, 26#, 28#, and 29#) showed FUT4 transcript levels below 25% of those in cv. Yunyan 87. Four lines (7#, 12#, 15#, and 29#) with the lowest mRNA levels were selected for further analysis by western blotting and enzyme-linked immunosorbent assay (ELISA). The results confirmed a significant decrease in FUT4 protein levels in these lines compared with that in cv. Yunyan 87, with line 29# showing less than 13% of the FUT4 protein content compared to cv. Yunyan 87. This study successfully developed a tobacco chassis with severely downregulated FUT4 expression, laying an important foundation for the production of human therapeutic proteins using a plant expression system. |
| Key words: Fucosyltransferase tobacco RNAi therapeutic proteins. |
