摘要
为构建稳定表达猪繁殖与呼吸综合征病毒(PRRSV)受体CD163的HEK293细胞系,本研究采用RT-PCR方法从猪肺泡巨噬细胞(PAM)总RNA中扩增CD163基因并将其克隆至真核表达载体pc DNA3.1(+)中,构建重组质粒pc DNA-CD163。将该重组质粒转染HEK293细胞,并利用G418筛选、纯化,获得了稳定表达CD163受体蛋白的HEK293CD163细胞系。RT-PCR、流式细胞术及western blot检测结果表明,该细胞系在传代至15代后,CD163在HEK293细胞中仍然能够稳定表达。在该细胞系中接种PRRSV后进行间接免疫荧光及噬斑试验检测表明,病毒能够通过CD163感染HEK293CD163细胞并在其中增殖。该细胞系的建立为进一步研究PRRSV的侵入细胞和致病机制提供了病毒在体外增殖的易感细胞系。
To establish a permissive cell line for porcine reproductive and respiratory syndrome virus （PRRSV）, the CD163 gene was amplified by RT-PCR from porcine alveolar macrophages and cloned into the eukaryotic expressing vector pcDNA3.1 （＋）. The resultant recombinant plasmid was transfected into HEK293 cells and a cell line stably expressing the CD163, named HEK293CD163, was generated using colony-purification under the G418 selection. The expression of CD163 in the cells was confirmed by RT-PCR, flow cytometory and western blot, indicating that porcine CD163 was stably expressed in the HEK293CD163 cell lines. In addition, PRRSV was able to efficiently infect and replicate in the cell line detected by indirect immunofluorescence and plaque assays. These data demonstrated that the HEK293 cell line was converted into permissive cells to PRRSV by CD 163 expression, which would facilitate further study on the mechanism of virus entry and replication in vitro.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2015年第3期163-166,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31372416)
