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  

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

作　　者：李华玮[1] 王旭英 乔宏兴[2] 李新锋 姬向波[3] 郭科威 杨中元 LI Huawei;WANG Xuying;QIAO Hongxing;LI Xinfeng;JI Xiangbo;GUO Kewei;YANG Zhongyuan(Institute of Animal Product Quality and Safety Technology,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;Henan Key Laboratory of Innovation and Utilization of Unconventional Feed Resources,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China)

机构地区：[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

出　　处：《畜牧兽医学报》2023年第3期1169-1176,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：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)。

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。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.

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 

分 类 号：S852.659.6[农业科学—基础兽医学]