畜牧兽医学报2018，Vol.49Issue(6)：1231-1240,10.DOI:10.11843/j.issn.0366-6964.2018.06.015
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
The Effect of Interfering and Stable Expression of GP5 on the Replication of Porcine Reproductive and Respiratory Syndrome Virus in Early Infection
摘要
Abstract
To investigate the effect of GP5,the most important structural protein encoded by por-cine 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 Trans-poson 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 con-firmed 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 prolifera-tion,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 siR-NA GP5-525,targeting ORF5 and one pair negative control siRNA,scrambled siRNA,were de-signed and synthesized.siRNAs were transfected into Marc-145 cells and then the cells were in-fected with 0.1 multiplicity of infection(MOI)PRRSV SD16 strain at 6 h or 12 h after transfec-tion.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 expres-sion 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 trea-ted group.These findings suggest that GP5 plays a regulatory role in PRRSV replication.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Key words
porcine reproductive and respiratory syndrome virus/GP5/small interfering RNA (siRNA )/stable expression/interferon分类
农业科技引用本文复制引用
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):1231-1240,10.基金项目
国家自然科学基金青年项目(31201883) （31201883）
国家自然科学基金重点项目(31430084) （31430084）
