畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (3): 1169-1176.doi: 10.11843/j.issn.0366-6964.2023.03.028

• 预防兽医 • 上一篇    下一篇

慢病毒载体介导的稳定表达eIF5A细胞系的建立及其对猪繁殖与呼吸综合征病毒增殖的影响

李华玮1, 王旭英2, 乔宏兴2, 李新锋1, 姬向波3, 郭科威4, 杨中元4   

  1. 1. 河南牧业经济学院畜产品质量安全技术研究院, 郑州 450046;
    2. 河南牧业经济学院动物医药学院, 郑州 450046;
    3. 河南牧业经济学院河南省非常规饲料资源创新利用重点实验室, 郑州 450046;
    4. 河南农业大学动物医学院, 郑州 450046
  • 收稿日期:2022-08-10 出版日期:2023-03-23 发布日期:2023-03-21
  • 作者简介:李华玮(1981-),女,河南郑州人,博士,副教授,主要从事动物病毒感染机制及相关诊断试剂研究,E-mail:centrosome@126.com
  • 基金资助:
    国家自然科学基金(31902284);河南省科技研发计划联合基金(应用攻关类)(222103810022);河南省高等学校青年骨干教师培养计划项目(2020GGJS258);河南牧业经济学院重点学科建设项目(XJXK202202)

Establishment of a Lentiviral Vector-mediated Stable Expression of eIF5A Cell Line and Its Effect on Porcine Reproductive and Respiratory Syndrome Virus Propagation

LI Huawei1, WANG Xuying2, QIAO Hongxing2, LI Xinfeng1, JI Xiangbo3, GUO Kewei4, YANG Zhongyuan4   

  1. 1. Institute of Animal Product Quality and Safety Technology, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;
    2. College of Veterinary Medicine, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;
    3. Henan Key Laboratory of Innovation and Utilization of Unconventional Feed Resources, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;
    4. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China
  • Received:2022-08-10 Online:2023-03-23 Published:2023-03-21

摘要: 本研究旨在通过慢病毒载体构建稳定表达真核翻译起始因子5A(eukaryotic translation initiation factor 5A,eIF5A)的MARC-145细胞系并验证该细胞系对PRRSV感染的影响。全基因合成eIF5A序列,双酶切后构建慢病毒表达载体pLV-CMV-MCS-EF1a-Puro。将该载体和慢病毒包装质粒psPAX2与包膜蛋白质粒pMD2.G共转染HEK293T细胞,包装得到能表达eIF5A的慢病毒。将慢病毒转导至MARC-145细胞,经过嘌呤霉素筛选、细胞有限稀释法获得能稳定表达eIF5A的MARC-145细胞系。MTS试验证实构建的细胞系细胞活力无显著变化,病毒TCID50测定、实时荧光定量PCR、蛋白质免疫印迹和间接免疫荧光技术检测eIF5A稳定表达对PRRSV滴度、PRRSV N基因及N蛋白表达的影响,发现MARC-145-eIF5A细胞系能显著促进PRRSV的增殖。本研究成功构建了稳定表达eIF5A的细胞系MARC-145-eIF5A,PRRSV感染试验证实体外稳定表达eIF5A可以促进PRRSV在MARC-145细胞的增殖。

关键词: 猪繁殖与呼吸综合征病毒, 慢病毒载体, 真核翻译起始因子5A, MARC-145细胞, 感染

Abstract: The purpose of this study was to construct a MARC-145 cell line with stable expression of eukaryotic translation initiation factor 5A (eIF5A) by means of a lentiviral vector system to provide a research tool. First, the target eIF5A sequence was synthesized and the lentivirus expression vector PLV-CMV-MCS-EF1A-PURO was constructed after double enzyme digestion. HEK293T cells were co-transfected with pLV-CMV-MCS-EF1a-Puro, the lentivirus packaging plasmid psPAX2, the envelope protein pMD2.G, and the lentivirus expressing eIF5A was packaged. The harvested lentivirus was transduced into MARC-145 cells. The MARC-145 cell line that could stable overexpress eIF5A was obtained and named as MARC-145-eIF5A cell line. MTS assay confirmed that the cell viability of the constructed cell line did not change significantly. Real-time PCR, Western blot and Indirect immune fluorescence assay were used to verify the gene and protein level of PRRSV N in the constructed cell lines and control cells, the results showed that both the gene and protein level of PRRSV N in MARC-145-eIF5A cell line were significantly increased. PRRSV propagation was significantly promoted in the MARC-145-eIF5A cell line compared with the control group. In this study, the MARC-145-eIF5A cell line which is stable over expression eIF5A protein was successfully constructed. PRRSV propagation experiment confirmed that stable expression of eIF5A could promote the PRRSV propagation in MARC-145 cells.

Key words: porcine reproductive and respiratory syndrome virus, lentiviral vector, eIF5A, MARC-145 cell, infection

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