Study On The Establishment And Characteristics Of Porcine Alveolar Macrophage Cell Line Expressing CD163 Stably

The Porcine Reproductive and Respiratory Syndrome(PRRS)is extensively distributes worldwide.It is an acute highly contagious viral disease characterized by porcine reproductive and respiratory syndrome virus(PRRSV)characterized by sow reproductive failure,respiratory symptoms,growth retardation and increased mortality in weaned piglets.In vitro investigation of cytokine secretion induced by PRRSV requires porcine alveolar macrophages(PAM)and interactions with immunocytes.However,existing immortalized PAM cells are non-permissive for PRRSV infection.The porcine CD163 receptor isolated from primary PAM(pPAM)could confer susceptibility to PRRSV infection,thus establishing a novel cell line to more conveniently explore PRRSV infection kinetics and the molecular mechanism of inducing the secretion of related cytokine.In this study,the coding region of the CD163 gene was amplified by pPAM and inserted into the chromosome of immortalized PAM cells by lentivirus expression system to stably express it with cell proliferation.The results showed that the mPAM-CD163 cells infected with high pathogenicity HP-PRRSV strain JXA1 could produce offspring viruses with titers higher than 10~9 copies/mL,and the cell line could be stable for at least 100 generations.In addition,we also studied the expression of relevant cytokines and Toll-like receptors(TLR)in mPAM-CD163 and pPAM inoculated with PRRSV.Compared with pPAM cells,mPAM-CD163 cells showed extremely similar patterns of viral replication and cytokine secretion.Therefore,the stable PAM cell line expressing CD163 in this study can be used to replace the primary PAM cells for in vitro experiments to study the mechanism of cytokine secretion and the interaction between PAM cells infected by PRRSV and immune cells.Primary PAM cells were obtained from the lungs of PRRSV?PCV2 negative piglets aged 4-6 weeks.The primers were designed according to the gene sequence of porcine CD163,and the porcine CD163 gene was amplified by PCR.The amplified CD163 gene fragment was ligated into the cloning vector pEASY-T1 to construct a cloning plasmid pEASY-T1-CD163.pEASY-T1-CD163 and pLV-sf GFP(2A)Puro were digested by restriction endonucleases Xba I and Xho I,respectively.The CD163 gene was cloned from vector plasmid pEASY-T1-CD163 into lentivirus gene overexpression vector pLV-sf GFP(2A)Puro.The linked products were transformed into E.coli Competent Cell JM109,the positive recombinant plasmid was identified as pLV-EGFP-CD163 by sequencing.The endotoxin-free pLV-EGFP-CD163 plasmid was added to 293T cells for culture,and the culture solution was collected to obtain virus particles.After the optimal concentration for the growth of immortalized PAM cells was selected by puromycin,the lentiviral particles were added to the medium containing puromycin for infection,the cell population containing the gene of interest were obtained.The cell population cells were diluted,inoculated into96-well plates,and monoclonal cell lines were obtained by limiting dilution.The selected monoclonal cell strain stably expressing CD163 was named mPAM-CD163.Western blot results showed that the CD163 protein was correctly expressed in mPAM-CD163 cells,and the molecular weight was about 130 KDa.The antibody blocking assay showed that PRRSV titer was reduced in a dose-dependent manner compared to the control group after anti-CD163 antibody was combined with CD163.Confocal results showed that CD163 protein mainly localized in the membrane of cells.Furthermore,this cell line allows PRRSV infection and produces progeny virus with a high titer of more than 10~9copies/mL,and this cell line can be passaged for at least 100 generations.This cell line was infected with the HP-PRRSV JXA1 strain,and the number of virus produced was lower than that of pPAM,but the trend was similar to pPAM.The pPAM and mPAM-CD163 cells were infected with different virulence highly pathogenic PRRSV JXA1 strain and the classical PRRSV SD1 strain.The expression levels of related cytokines(IL-4,IL-10,IFN-?,IFN-?)and Toll receptors(TLR-3,TLR-7)were determined by real-time PCR.The results showed that there were almost the same changes in cytokines between the cell lines constructed in this experiment and pPAM cells.Among them,the levels of 24 hpi IL-4 and 6,12 hpi IL-10 were different from those of primary cells,and the mRNA pattern of change in expression of Toll-like receptors was consistent.