稳定表达猪SAMHD1蛋白的MARC-145细胞系构建与鉴定

ESTABLISHMENT AND IDENTIFICATION OF A MARC-145 CELL LINE STABLY EXPRESSING PORCINE SAMHD1

SAMHD1作为宿主的天然免疫限制性因子,参与机体的天然免疫调控,在机体抗病毒感染过程中发挥重要的作用。为了建立稳定表达猪SAMHD1的细胞系,本研究将猪SAMHD1全长编码基因克隆至慢病毒载体pWPXL,构建重组质粒pWPXLpSAMHD1。将重组质粒与慢病毒包装质粒共转染293T细胞,获得重组慢病毒。将该慢病毒感染MARC-145细胞,经嘌呤霉素筛选后,获得表达猪SAMHD1基因的MARC-pSAMHD1细胞系。经检测发现,该细胞系传至10代,仍能稳定表达猪SAMHD1蛋白。该细胞系的建立为猪SAMHD1抗病毒特性和猪繁殖与呼吸综合征病毒致病机理等研究奠定了坚实的基础。

The sterile alpha motif and HD domain 1（SAMHD1） protein has been identified as a novel restriction factor that participates in processes of host innate immunity and plays an important role in antiviral infection. To establish a cell line stably expressing porcine SAMHD1, the CDS of porcine SAMHD1 gene was cloned into lentiviral vector pWPXL and transfected into 293 T cells together with the packaging plasmids to get the recombinant lentivirus. MARC-145 cells were infected with the recombinant lentivirus and screened by puromycin to establish the cell line expressing porcine SAMHD1. The porcine SAMHD1 gene was shown to be stably expressed in MARC-145 cells. The established cell line is a useful tool to explore the antiviral roles of porcine SAMHD1 and pathogenesis of Porcine reproductive and respiratory syndrome virus.