Huang Yanyan, Guo Rui, Zhang Yu, Zhang Xinyu, Xia Xiaoli, Sun Huaichang
College of Veterinary Medicine, Hangzhou University, Yangzhou 225009, China.
Wei Sheng Wu Xue Bao. 2013 May 4;53(5):507-14.
In order to study the role of porcine CD151 in infection of porcine cells by porcine reproductive and respiratory syndrome virus (PRRSV), we established a porcine CD151 transgenic PK-15 cell line.
The full-length complementary DNA (cDNA) for porcine CD151 was amplified from porcine alveolar macrophages by reverse transcription-polymerase chain reaction (RT-PCR), and subcloned into eukaryotic expression vector pcDNA3. The recombinant vector pcDNA-CD151 was transfected into PK-15 cells and the transgenic cell line was generated after G418 selection. Transcription of the CD151 cDNA in transgenic cell line was detected by RT-PCR and immunofluorescence. The cell line, together with control cell lines PK-15, 3D4-CD163 and MARC-145, was infected with PRRSV, and the viral RNA genome or antigens in the infected cells was detected by RT-PCR or immunofluorescence. At different time points post-infection, the virus was harvested and titrated on MARC-145 cells.
The expected size of porcine CD151 cDNA was amplified with a sequence identity of 99.7% to the published sequence. From the pcDNA-CD151-transfected cell culture, a transgenic cell line PK15-CD151 was generated and correct expression of porcine CD151 was confirmed. After PRRSV infection, the viral RNA genome and antigens were detected in the cell-line. Although apparent cytopathic effect was not observed in the virally infected cell line, the infectious virus with a high viral title was detected. The cell line had been passed for more than 30 generations and no significant difference in viral title was observed among generations 10, 20 and 30 after PRRSV infection.
Transfection of non-permissive PK-15 cells with porcine CD151 cDNA conferred the susceptibility to PRRSV infection, indicating an important role of the porcine CD151 in PRRSV infection of porcine cells.
为研究猪CD151在猪繁殖与呼吸综合征病毒（PRRSV）感染猪细胞中的作用，我们建立了猪CD151转基因PK - 15细胞系。
通过逆转录 - 聚合酶链反应（RT - PCR）从猪肺泡巨噬细胞中扩增猪CD151的全长互补DNA（cDNA），并亚克隆到真核表达载体pcDNA3中。将重组载体pcDNA - CD151转染到PK - 15细胞中，经G418筛选后获得转基因细胞系。通过RT - PCR和免疫荧光检测转基因细胞系中CD151 cDNA的转录情况。该细胞系与对照细胞系PK - 15、3D4 - CD163和MARC - 145一起感染PRRSV，通过RT - PCR或免疫荧光检测感染细胞中的病毒RNA基因组或抗原。在感染后的不同时间点收集病毒，并在MARC - 145细胞上进行滴定。
扩增出预期大小的猪CD151 cDNA，其序列与已发表序列的同一性为99.7%。从转染了pcDNA - CD151的细胞培养物中获得了转基因细胞系PK15 - CD151，并证实了猪CD151的正确表达。PRRSV感染后，在该细胞系中检测到病毒RNA基因组和抗原。虽然在病毒感染的细胞系中未观察到明显的细胞病变效应，但检测到具有高病毒滴度的感染性病毒。该细胞系已传代30代以上，PRRSV感染后第10、20和30代之间的病毒滴度无显著差异。
用猪CD151 cDNA转染非允许性PK - 15细胞可使其获得对PRRSV感染的敏感性，表明猪CD151在PRRSV感染猪细胞中起重要作用。
