| The innate and acquired immunity of infected pigs induced by PRRSV infection are poor and delayed. The immune supression caused by PRRSV should be reponsible for the persistent viral infection. At present, studies on the mechanism of immune supression have been carried out from different aspects including the virus and the host. But little research was conducted on the effector cells. The PD-1/PD-L pathway mainly regulates the level of acquired immunity, and was demonstrated to be closedly related to immune escape and persistent infection in some human diseases. The aim of this study is to clone and identify the porcine PD-1 and to detect the effect of PRRSV infection on PD-1/PD-L pathway.An sequence was found by blast search in the EST database with a human PD-1 sequence. According to the obtained sequence, a specific primer pair was designed and RT-PCR was conducted using the cDNA of PBMC as the template. The ORF of porcine PD-1 is 867 bp in length and encodes a protein of 288 amino acids. Protein sequence comparison showed that the porcine PD-1 shares 63% and 54% identity with human and murine PD-1, respectively, and has a similar structure with two highly hydrophobic amino acid fragments, an extracellular domain and a cytoplasmic domain. Some functional amino acid residues are conserved among these three species. To address the interaction between porcine PD-1 and PD-L1, pcDNA-PD-1 was transfected into 293T cells. The transfected 293T cells were stained with porcine PD-L1-Fc protein.The FACS analysis showed that the cloned porcine PD-1 was bound successfully by the reported porcine PD-L1. Then the porcine PD-1 gene was transducted into porcine PBMC by recombinant pseudovirus and in vitro T cell proliferation assay was conducted using anti-CD3 antibody as the stimulator in the presence of soluble PD-L1-Fc protein. These results indicate that the porcine PD-1/PD-L1 interaction exhibits a pattern of negative regulation in the pig immune response, similar to that seen in its human and mice counterparts.Specific primers and fluorescent probes for porcine PD-1, PD-L1 and PD-L2 genes were designed respectively. The reaction conditions for real-time fluorescent quantitative RT-PCR were optimized and the standard curves were constructed based on the serially diluted recombinant plasmids pMD-PD-1,pMD-PD-L1 and pMD-PD-L2. The results indicated that the assays worked well with a good coefficient correlation (r2>0.99) and amplification efficiency (>96%) while the template concentration was from 102 to 108 copies/μL. The sensitivity was sufficient to detect the minimum template of 100 copies. The established assays were used to detect the expression of these three genes on porcine PBMC and the results indicated that the assays were highly sensitive, specific and well reproducible,and suitable to detect clinical specimens.The extracellular domains of PD-1 and PD-L1 were cloned into the prokaryotic expression vectors pGEX-6p-1 and pET32a respectively. The extracellular domain of PD-1 was expressed as a form of inclusion body and the extracellular domain of PD-L1 was expressed as a form of solube protein. These proteins were purified and immunized into the BALB/c mice. One mAb specific for porcine PD-1 and two mAbs specific for porcine PD-L1 were obtained by hybridizing the spleen cells of immunized mice with sp2/0 cells. Western blot and FACS analysis demonstrated that these mAbs can not only recognize the denatured proteins but also the proteins expressed on the surface of cells.The expression of the PD-1 and its ligands in effector cells and target cells after PRRSV infection was detected using the methods established above. At first, primary PAM cells infected with PRRSV were havested at different time points post infection. The results from real-time RT-PCR assay showed that the expression of PD-L1 was upregulated 2.2~6 times relative to the uninfected cells. But PD-L2 didn't change. A positive correlation between the virus load and the PD-L1 expression level was ascertained suggesting that PRRSV infection can upregulate the expression of PD-L1. Then PBMCs from piglets infected with PRRSV were detected the expression changes of PD-1. The expression of PD-1 at the mRNA level was upregulated by 3 times just 3 days after infection and reached the peak at 10-14dpi with a fold change of about 5. The results from FACS were in line with those from the real-time fluorescent quantitative RT-PCR assay. The expression levels of PD-1 on CD4+ or CD8+T cells of the infected pigs were 2-3 times higher than those of the uninfected pigs between 7-14dpi. Part of the survival pigs also expressed the PD-1 at a high level at 35dpi. The virus load in the serum was detected using a real-time fluorescent quantitative RT-PCR assay. The level of PD-1 expression was closely related to the virus load which suggested that PRRSV infection has an effect on PD-1/PD-L pathway.
|
PDF Full Text Request