稳定表达猪繁殖与呼吸综合征病毒NSP_2细胞株的建立及其对p65核转位影响的研究

Establishment of cell line stably expressing nonstructured protein 2 of porcine reproductive and respiratory syndrome virus

为建立一株稳定表达猪繁殖与呼吸综合征病毒(PRRSV)非结构蛋白2(NSP_2)的细胞株,本研究通过脂质体2000介导的方法将携带有PRRSV NSP_2基因序列的真核表达载体(pcDNA3.1-5'Flag-Nsp_2)瞬时转染于HEK293细胞,应用G418筛选、间接免疫荧光(IFA)与western blot方法进行鉴定,获得了1株稳定表达NSP_2的阳性细胞株,命名为NSP_2-11C1-HEK293;将TNF-α作用于该细胞株,采用IFA分析稳转细胞内NSP_2对p65入核的影响。IFA结果显示NSP_2主要在细胞浆中围绕细胞核表达,在细胞中的表达率达95%以上;western blot结果显示出现120 ku的蛋白条带,与预期相符。结果表明本研究获得了一株稳定表达NSP_2的阳性细胞株,在该稳转细胞内NSP_2对p65的入核并没有影响,这为进一步研究揭示PRRSV的致病机理奠定基础。

To establish a permissive cell line for porcine reproductive and respiratory syndrome vires （PRRSV）, the recombinant plasmid of pcDNA3.1-5＇Flag-Nsp2 was transfected into HEK293 cells and a cell line stably expressing the NSP2, named NSP2-11C1-HEK293, was generated using colony-purification selection in the present of 800 μg/mL G418. The expression of NSP~ in the cells was confirmed by indirect immunofluorescence assay （IFA） and western blot, indicating that porcine NSP2 was stably expressed in the HEK293 cell lines. The NSP2-11C1-HEK293 was treated with TNF-alpha and the effect on translocation of p65 was analyzed by IFA. The results showed that the NSP2 was 120ku mainly expressed in the HEK293 cytoplasm and rounded the nucleus. The rate of the NSP2 expression in the cell was over 95%. Indditionly, there was no effect on translocation of p65 from cytoplasm to nucleus by NSP2 in the NSP2-11C1-HEK293, which would facilitate further study on the mechanism of interaction between virus and host protein.