猪繁殖与呼吸综合征病毒NSP9蛋白的亚细胞定位与功能预测

Subcellular Localization Analysis and Function Prediction of PRRSV NSP9 Protein

为了研究猪繁殖与呼吸综合征病毒(PRRSV)非结构蛋白9(NSP9)的亚细胞定位,并探索NSP9蛋白在PRRSV复制过程中的定位变化,首先预测NSP9蛋白的生物信息学功能,之后将含有NSP9基因的质粒转染Marc-145细胞,并接种PRRSV,通过间接免疫荧光方法检测NSP9蛋白的表达与定位情况。功能预测结果表明,NSP9蛋白内部存在双向核定位序列、酰胺化位点、N-糖基化位点、酪蛋白激酶磷酸化位点、N-豆蔻酰化位点、蛋白激酶C磷酸化位点、细胞结合位点、酪氨酸激酶磷酸化位点、伸展蛋白重复区域、RNA聚合酶结合区域。间接免疫荧光检测结果显示,PRRSV感染之初,NSP9蛋白定位在Marc-145细胞质中,围绕在细胞核附近,随PRRSV感染时间延长,其逐步往细胞核内转移,转移的面积也逐步增多。可见,PRRSV感染会引起NSP9蛋白向核内转移。

This study aimed to confirm the subcellular localization of NSP9 protein and verify whether PRRSV infection could affect its localization. The biological characters was predicted by softwares. Plasmids containing NSP9 gene were transfected into Marc-145 cells,and Marc-145 cells were infected withPRRSV. Indirect immunofluorescence assay( IFA) was used to detect its localization. The results of functional prediction showed that NSP9 protein had two-way nuclear localization sequence,amidation site,a casein kinase phosphorylation site,N-glycosylation site,N-myristoylation site,protein kinase C phosphorylation site,a cell binding site,a tyrosine kinase phosphorylation site,a stretch protein repeat region,and an RNA polymerase binding domain. The results of IFA showed that NSP9 protein was located in the cytoplasm in Marc-145 cells. After PRRSV infection,NSP9 protein gradually transfer to the nucleus,and the area also gradually increased. It demonstrated that PRRSV infection changed the localization of NSP9 protein.