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| eIF5A基因敲除MARC-145多克隆细胞系的建立及其对PRRSV增殖的影响 |
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李华玮1,2,王旭英3,井汇源3,万博4,乔宏兴3,郭科威4,侯文静2
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| (1.河南牧业经济学院 食品与生物工程学院, 郑州 450046;2.河南牧业经济学院 畜产品质量安全技术研究院, 郑州 450046;3.河南牧业经济学院 动物医药学院, 郑州 450046;4.河南农业大学 动物医学院, 郑州 450046) |
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| 摘要: |
| 旨在利用CRISPR/Cas9系统构建敲除真核翻译起始因子5A(Eukaryotic translation initiation factor 5A，eIF5A)的MARC-145多克隆细胞系，并验证该细胞系对PRRSV感染的影响。针对eIF5A基因构建并筛选成功获得重组慢病毒，用重组慢病毒感染MARC-145细胞，嘌呤霉素及有限稀释法进行筛选，获得敲除eIF5A的MARC-145多克隆细胞系。对构建细胞系进行活力检测，T7E1核酸内切酶、Real-time PCR以及Western blot验证eIF5A体外敲除效率，病毒增殖试验验证该细胞系对PRRSV复制的影响。结果表明:1)细胞活性检测结果显示敲除eIF5A基因对细胞活性无显著影响；2)通过T7E1核酸内切酶、Real-time PCR以及Western blot表明eIF5A表达显著降低，并将此细胞系命名为MARC-145-△eIF5A细胞系；3)使用PRRSV HN07-1感染MARC-145-△eIF5A，体外试验证明敲除eIF5A对PRRSV的增殖有抑制作用。综上，本研究成功构建eIF5A基因敲除的MARC-145多克隆细胞系，体外敲除eIF5A显著抑制PRRSV增殖，这将为进一步探索eIF5A调控PRRSV复制的分子机制奠定基础。 |
| 关键词: 真核翻译起始因子5A PRRSV CRISPR/Cas9 MARC-145细胞 |
| DOI：10.11841/j.issn.1007-4333.2023.07.11 |
| 投稿时间：2022-02-22 |
| 基金项目:国家自然科学基金项目(31902284)；河南省科技研发计划联合基金(应用攻关类)(222103810022)；河南省科技攻关计划项目(232102110104)；河南省高等学校青年骨干教师培养计划项目(2020GGJS258)；河南牧业经济学院预防兽医重点学科(XJXK202202) |
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| Establishment of eIF5A gene knockout polyclonal MARC-145 cell line and its effect on PRRSV infection |
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Li Huawei1,2,Wang Xuying3,Jing Huiyuan3,Wan Bo4,Qiao Hongxing3,Guo Kewei4,Hou Wenjing2
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| (1.College of Food and Bioengineering, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;2.Institute of Animal Product Quality and Safety Technology, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;3.College of Veterinary Medicine, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;4.College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China) |
| Abstract: |
| The study was to construct MARC-145 polyclonal cell line with eIF5A(Eukaryotic translation initiation factor 5A)knockout using CRISPR/Cas9 system and to verify the effect of this cell line on PRRSV infection. In this study, for eIF5A gene, the recombinant lentivirus was successfully constructed and screened. MARC-145 cells were infected with the lentivirus and screened using puromycin though limited dilution method. Viability assays were performed on the transformed cells, T7E1 nucleic acid endonuclease, real-time PCR and western blot to verify the efficiency of eIF5A knockdown in vitro, and viral proliferation assays to verify the effect of this cell line on PRRSV replication. The results showed that: 1)eIF5A knockout had no effect on cell activity. 2)The expression of eIF5A was significantly decreased by T7E1 endonuclease, Real-time PCR and Western blot, and the cell line was named MARC-145-△eIF5A cell line. 3)In vitro assays demonstrated that knockout of eIF5A had an inhibitory effect on PRRSV proliferation. In conclusion, this study successfully constructed MARC-145 polyclonal cell lines with eIF5A gene knockout, and eIF5A knockout can significantly inhibit PRRSV proliferation in vitro, which lay a foundation for further exploration of the molecular mechanism of eIF5A regulation of PRRSV replication. |
| Key words: eIF5A PRRSV CRISPR/Cas9 MARC-145 cell |
