慢病毒载体介导稳定表达CD163的PAM细胞系的建立及对PRRSV感染的研究

Generation of a Porcine Alveolar Macrophage Cell Line Stably Expressing CD163by Lentiviral Vector for the Production of Porcine Reproductive and Respiratory Syndrome Virus

本研究旨在通过慢病毒载体系统将猪源CD163转染入永生化的猪肺泡巨噬细胞(PAM)系(CRL-2843),建立稳定表达CD163的PAM细胞系(PAM-CD163)及验证该细胞系对PRRSV的易感性。用PCR方法从pJET1.2-CD163载体上扩增CD163基因编码区,通过酶切连接构建慢病毒表达载体pTrip-CMV-CD163-IRESpur。将该质粒与慢病毒包装质粒psPAX2和pMD2.G共转染293-T细胞,包装表达CD163的慢病毒。将收获的慢病毒在Polybrene的介导下转导至永生化PAM细胞,采用嘌呤霉素筛选和细胞有限稀释法筛选出稳定表达CD163的PAM细胞系。应用得到的细胞系进行PRRSV感染试验。经过RT-PCR、间接免疫荧光试验、Western blot和流式细胞术试验证实,筛选出1株能稳定表达CD163的PAM细胞系,命名为PAM-CD163。该株细胞对PRRSV高度易感,PRRSV在PAM-CD163细胞上的毒价可以达到105.0 TCID50·mL-1以上。建立了稳定表达CD163的PAM细胞系,可以用于PRRSV的分离培养和细胞受体的研究。

The objectives of this study were to use the lentiviral vector pTrip-CMV-IRES-pur plasmid to deliver porcine CD163 into a porcine alveolar macrophage （PAM） cell line （CRL- 2843）, to generate a cell line stably expressing CD163 （designated PAM-CD163）, and to evaluate its permissibility for PRRSV infection. The porcine CD163 coding sequences were amplified by PCR from pJET1.2-CD163 plasmid and inserted into downstream of CMV promoter in the lentivi- ral vector pTrip-CMV-IRES-pur plasmid. Monolayer of 293-T cells were cotransfected with three plasmids psPAX2, pMD2. G and pTrip-CMV-CD163-IRES-pur. The recombinant lentivirus ex- pressing CD163 was harvested in the culture fluid. For transduction, the immortalized PAM cells （CRL-2843） were exposed to lentivirus in the presence of polybrene. The transduced cells were selected with puromycin and single cell colonies were isolated and expanded for PRRSV infection assay. The CD163 gene was transcribed by RT-PCR and the protein was expressed as identified by indirect immunofluorescence assay, Western blot and flow cytometry analyses. A PAM cell line （CRL-2843） stably expressing CD163 （designated PAM-CD163） was susceptible to PRRSV infection with the titer of 10 5.0 TCID50 mL-1. A PAM cell line stably expressing CD163 was constructed and permissive to PRRSV infection. This cell line could be a valuable tool for PRRSV propagation and PRRSV cellular receptors study.