猪繁殖与呼吸综合征病毒非结构蛋白2分段真核表达及其细胞定位分析

EUKARYOTIC EEPRESSION AND SUBELLULAR LOCALIZATION OF NON-STRUCTUAL PROTEIN 2(NSP2) FROM PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS(PRRSV)

为探讨非结构蛋白2(non-structural protein 2,NSP2)在猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)复制过程中的作用及机制,本研究构建真核重组质粒pcDNA 3.1-5'Flag-NSP2-147-323,并瞬时转染至HEK 293T中,同时设置完整NSP2基因序列重组质粒作为对照,转染48 h后分别用不同的抗体进行间接免疫荧光监测。结果显示,转染对照组中细胞核周围可见特异性荧光,而转染真核重组质粒pcDNA 3.1-5'Flag-NSP2-147-323后,不仅在细胞核周围,在细胞核内也可以观察到特异性荧光。本研究成功构建真核重组质粒pcDNA 3.1-5'Flag-NSP2-147-323,并可在HEK 293T细胞中正确表达,为进一步研究NSP2在PRRSV复制过程中的作用提供依据和基础。

In order to investigate the role and mechanism of non-structural protein 2(NSP2)in the replication of porcine reproductive and respiratory syndrome virus(PRRSV),the eukaryotic recombinant plasmid pcDNA 3.1-5'Flag-NSP2-147-323 was constructed and transiently transfected HEK 293T.A complete NSP2 gene sequence recombinant plasmid served as a control.Localization of the expressed of NSP2 was examined after transfection of 48h in indirect immunofluorescence test using different antibodies.Results show that the specific fluorescence was visualized in nuclei of HEK 293T cells transfected the recombinant plasmid containing complete NSP2 gene sequences while bright staining was observed within and surrounding nuclei of the HEK 293T cells transfected with the eukaryotic recombinant plasmid pcDNA 3.1-5'Flag-NSP2-147-323.The construction of the eukaryotic recombinant plasmid pcDNA 3.1-5'Flag-NSP2-147-323 and expression in HEK 293T cells laid the solid foundation for further study of the role of NSP2 in PRRSV replication.