猪CD151转基因PK-15细胞系构建及其对猪繁殖与呼吸综合征病毒的易感性

Establishment of a porcine CD151 transgenic PK-15 cell line susceptible to porcine reproductive and respiratory syndrome virus

【目的】建立猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)易感的猪CD151转基因PK-15细胞系,研究CD151分子在PRRSV感染猪源细胞中的作用。【方法】用RT-PCR从猪肺泡巨噬细胞中扩增CD151全长cDNA,测序正确后克隆入真核表达载体pcDNA3;用重组载体pcDNA-CD151转染PK-15细胞,经G418抗性筛选获得转基因细胞系PK15-CD151,用RT-PCR和免疫荧光试验检测CD151表达;用VR-2332株PRRSV分别感染PK-15细胞、PK15-CD151细胞、MARC-145细胞和3D4-CD163细胞,定期观察细胞病变,用RT-PCR和免疫荧光试验检测病毒RNA基因组和病毒抗原,用半数组织细胞感染剂量测定病毒滴度。【结果】从猪巨噬细胞中克隆得序列正确的猪CD151 cDNA;从重组载体转染的PK-15细胞培养中筛选得G418抗性细胞克隆,并能正确表达猪CD151分子;在PRRSV感染后,PK15-CD151细胞虽然不表现明显的细胞病变,但能检测到病毒RNA基因组和病毒抗原,并能产生较高滴度的感染性病毒;该细胞系已在体外传30代以上,第10、20、30代细胞的PRRSV滴度无明显变化。【结论】猪CD151基因转染能使非易感PK-15细胞获得对PRRSV的易感性,提示猪CD151参与PRRSV感染猪源细胞。

[ Objective] 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. [Methods] The full-length complementary DNA （eDNA） for porcine CD151 was amplified from porcine alveolar macrophages by reverse transcription-polymerase chain reaction （RT-PCR）, and subcloned into eukaryotie expression vector peDNA3. 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 eDNA 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 immunofluoreseence. At different time points post-infection, the virus was harvested and titrated on MARC-145 cells. [ Results] 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 eytopathic 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. [ Conclusion] Transfection of non-permissive PK-15 cells with porcine CD151 eDNA conferred the susceptibility to PRRSV infection, indicating an important role of the porcine CD151 in PRRSV infection of porcine cells.