干扰和稳定表达GP5对猪繁殖与呼吸综合征病毒感染早期复制的影响

doi:10.11843/j.issn.0366-6964.2018.06.015

摘要/Abstract

摘要：

为探讨猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）主要结构蛋白GP5对病毒复制的影响及其机制，利用PiggyBac Transposon载体系统构建表达GP5的重组质粒（pPB-GP5），转染Marc-145细胞通过嘌呤霉素抗性筛选和3次亚克隆，获得稳定表达GP5的Marc-145细胞，在确定GP5表达不影响细胞增殖的基础上，用PRRSV感染筛选的细胞，通过N基因拷贝数、N蛋白表达水平和病毒滴度检测确定GP5表达对病毒复制的影响，并采用ELISA方法检测培养上清中IFN-α、IFN-β、IFN-γ的水平；进一步针对PRRSV ORF5编码区设计合成3对特异性siRNA，转染Marc-145细胞6和12 h后以0.1 MOI病毒感染细胞，感染后24 h收集细胞，采用荧光定量PCR（qPCR）检测GP5 mRNA的表达，采用Western blot检测PRRSV N蛋白的表达。结果显示：经RT-PCR、Western blot和IFA检测，确定GP5在Marc-145细胞中获得稳定表达，且不影响细胞增殖，但可以在感染早期促进PRRSV在细胞中的复制，这种促进作用可能是通过下调IFN的表达发挥的；与阴性对照siRNA相比，3对特异性siRNA均能抑制PRRSV复制，其中100 nmol·L^-1的siRNA GP5-471的抑制效果最好。研究表明GP5在PRRSV复制中发挥着重要作用。

关键词: 猪繁殖与呼吸综合征病毒, GP5, 小干扰RNA, 稳定表达, 干扰素

Abstract:

To investigate the effect of GP5, the most important structural protein encoded by porcine reproductive and respiratory syndrome virus (PRRSV) ORF5, on virus replication and it's mechanism, a recombinant plasmid (named as pPB-GP5) was constructed using PiggyBac Transposon System Vectors and transfected into Marc-145 cells. Marc-145 cell line stably expressing GP5 was obtained by puromycin resistance screening and three times sub-cloning. GP5 was confirmed stable expression in Marc-145 cells by RT-PCR, Western blot and IFA detection and the cells was named as Marc-145-GP5. On the basis that GP5 expression does not affect cell proliferation, the effect of GP5 stable expression on virus replication was investigated by N gene copies, N protein expression, and viral titers in supernatant after PRRSV SD16 infecting Marc-145-GP5 cells. IFN-α, IFN-β, and IFN-γ protein levels were analyzed using monkey IFN ELISA kit. Then three pairs specific small interfering RNA (siRNA), siRNA GP5-471, siRNA GP5-404 and siRNA GP5-525, targeting ORF5 and one pair negative control siRNA, scrambled siRNA, were designed and synthesized. siRNAs were transfected into Marc-145 cells and then the cells were infected with 0.1 multiplicity of infection (MOI) PRRSV SD16 strain at 6 h or 12 h after transfection. The cells were collected at 24 h after infection and total RNA or protein were extracted. GP5 mRNA level was detected using real time PCR (qPCR) and N protein level was analyzed using Western blot, respectively. RT-PCR, Western bolt and IFA detection results showed that GP5 was stably expressed in Marc-145 cells and did not affect cell proliferation. But GP5 expression in Marc-145 cells promoted the replication of PRRSV in early infection time through down-regulating of IFN production. Transfection with specific siRNAs targeting ORF5 inhibited PRRSV replication and siRNA GP5-471, at a concentration of 100 nmol·L^-1, showed the most effective inhibition and had highly significant difference compared to negative control siRNA treated group. These findings suggest that GP5 plays a regulatory role in PRRSV replication.

Key words: porcine reproductive and respiratory syndrome virus, GP5, small interfering RNA (siRNA), stable expression, interferon

中图分类号:

S852.659.6

宋林林, 贾存瑜, 韩熙梦, 周恩民, 穆杨. 干扰和稳定表达GP5对猪繁殖与呼吸综合征病毒感染早期复制的影响[J]. 畜牧兽医学报, 2018, 49(6): 1231-1240.

SONG Lin-lin, JIA Cun-yu, HAN Xi-meng, ZHOU En-min, MU Yang. The Effect of Interfering and Stable Expression of GP5 on the Replication of Porcine Reproductive and Respiratory Syndrome Virus in Early Infection[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(6): 1231-1240.

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