稳定表达猪CD163受体的MARC-145细胞系的构建

Construction of MARC-145 cell line stably expressing porcine CD163 receptor

为了构建稳定表达猪CD163(pCD163)受体的MARC-145细胞系,从猪肺泡巨噬细胞(PAMs)中提取总RNA,通过RT-PCR方法扩增pCD163基因,将pCD163基因克隆到慢病毒载体pLVX-IRES-mCherry中,获得重组质粒pLVX-pCD163-IRES-mCherry。将重组质粒pLVX-pCD163-IRES-mCherry与辅助质粒psPAX2、VSVG共转染293T细胞,获得具有感染能力的慢病毒粒子,使用慢病毒感染MARC-145细胞,用嘌呤霉素初步筛选阳性细胞,采用终点稀释法筛选出稳定表达pCD163受体的MARC-145细胞。通过RT-PCR扩增pCD163基因及测序分析表明细胞系基因组中存在pCD163受体编码序列,IFA和Western blot试验验证了pCD163受体蛋白在细胞系中稳定表达。用不同谱系的猪繁殖与呼吸综合征病毒(PRRSV)GXNN1396(lineage 8)、GXNN202004a(lineage 1)、GXGG202007(lineage 3)分别感染稳定表达pCD163的MARC-145细胞系与MARC-145细胞,PRRSV毒株在表达pCD163的MARC-145细胞系中能够更快达到最高病毒滴度。结果表明,成功构建了稳定表达pCD163的MARC-145细胞系,为快速分离各种谱系PRRSV毒株和相关研究奠定了基础。

In order to construct MARC-145 cell line stably expressing porcine CD163(pCD163)receptor,total RNA was extracted from porcine alveolar macrophages(PAMs).pCD163 gene was amplified by RT-PCR and cloned into the lentiviral vector pLVX-IRES-mCherry,yielding the recombinant plasmid pLVX-pCD163-IRES-mCherry.The pLVX-pCD163-IRES-mCherry together with the helper plasmids psPAX2 and VSVG were co-transfected into 293 T cells.The supernatants of co-transfected 293 T cells containing lentiviral particles were used to inoculate MARC-145 cells,then MARC-145 cells were screened with puromycin.MARC-145 cell lines stably expressing pCD163 receptor(MARC-145-pCD163)were screened by the end-point dilution method.The results of RT-PCR amplification of the pCD163 gene and sequencing analysis showed that the pCD163 receptor gene was integrated in the genome of MARC-145 cells.IFA and Western blot verified that the pCD163 receptor protein was stably expressed in the MARC-145 cell line.MARC-145-pCD163 and MARC-145 cells were infected with porcine reproductive and respiratory syndrome virus(PRRSV)isolates GXNN1396(lineage 8),GXNN202004 a(lineage 1),GXGG202007(lineage 3),respectively.The results showed that compared with MARC-145 cells,PRRSV isolates could reach rapidly to the highest virus titer in MARC-145-pCD163.These results showed that the MARC-145 cell line stably expressing pCD163 was successfully constructed,which laid the foundation for the rapid isolation of various lineages of PRRSV strains and related studies.