The Role Of PNCR Ligands On The Process Of Pig Lymphocytopenia Induced By PCV2

Porcine circovirus type 2?PCV2?causes porcine circovirus associated disease?PCVAD?,which can lead to immunosuppression of pigs,making it more susceptible to infection by other pathogens and causing the pig industry huge loss.Lymphocytopenia is a characteristic symptom of PCVAD,but the mechanism by which PCV2 infection causes a decrease in the number of lymphocytes in piglets has not been clear.As a representative CD4⁺T cell in lymphocytopenia,it is an important type of immune cell in the body's immune system.It can mediate the body's resistance to pathogenic microorganism infection.Natural killer?NK?cells have lymphocyte killing activity.It plays an important role in the regulation of the number of lymphocytes in the body.NK cells recognize and kill target cells by combining natural cytotoxicity receptors?NCRs?on the surface with corresponding ligands?NCRLs?expressed on the surface of target cells.Studies have shown that PCV2 infection can significantly reduce the number of CD4⁺T cells in piglets,while the number of NK cells increases.However,whether the increase in the number of NK cells in PCV2 infected pigs is related to the decrease in the number of CD4⁺T cells,and whether the combination of NCRs and NCRLs mediates this process has not been reported so far.In this study,three kinds of porcine NCRs?p NCRs?extracellular domain and human Ig G Fc fusion protein eukaryotic expression vectors were constructed,and it was transfected into SP2/0 cells to obtain p NCRs-h Fc fusion protein used for flow cytometry to detect the expression level of p NCRs ligand on the cell surface.Then,PCV2copy number in the peripheral blood of pigs at different time after PCV2 infection was detected by real time PCR.Flow cytometry was used to detect the number of CD4⁺T cells and apoptosis rate,the number of NK cells and the expression level of p NCRs ligand on the surface of CD4⁺T cells at different time after PCV2 infection.Finally,the correlations between PCV2 copy number in porcine peripheral blood,the number of CD4⁺T cells and apoptosis rate,the number of NK cells,and the expression level of p NCRs ligand on the surface of CD4⁺T cells were analyzed.The research obtained the following experimental results:1. According to the TMHMM Server v.2.0 forecast result and the known extracellular domains of human NCRs,the gene sequences of the encoding p NCR1?NM?001123143.1?,p NCR2?XM?021098670.1?and p NCR3?EU282355.1?extracellular domains were identified as:1 aa?255 aa,1 aa?192 aa,and 1 aa?95 aa.The gene sequences are 1 bp?765 bp,1 bp?576 bp and 1 bp?285 bp.According to the forecast extracellular domains,genes encoding p NCR1,p NCR2,and p NCR3 extracellular domains and human Ig G Fc?MS820429?encoding genes were synthesized.The human Ig G Fc gene was cloned into the eukaryotic expression vector pc DNA3.1 to successfully construct pc DNA3.1-h Fc.Then,the three genes encoding the extracellular domains of p NCRs were respectively cloned into the eukaryotic expression vector pc DNA3.1-h Fc,and vectors pc DNA3.1-p NCR1-h Fc,pc DNA3.1-p NCR2-h Fc and pc DNA3.1-p NCR3-h Fc were successfully constructed.2. The constructed eukaryotic expression vectors were transfected into SP2/0 cells,and the supernatant was collected 24 hours later.After purification,SDS PAGE and western blotting tests showed that the three fusion proteins were successfully expressed with contents of 1.6103?g/?L,1.9697?g/?L,2.3540?g/?L,and purity of 94%,93%,and91%.Flow cytometry results showed that the expressed fusion proteins could be used to detect the p NCRLs on the CD4⁺T cell surface by flow cytometry.3.5-week-old weaned piglet was infected with PCV2 at the doses of 4×10⁵TCID₅₀.Blood was collected from the anterior vena cava at 0 d,7 d,14 d and 28 d post infection,and lymphocytes were isolated.The copy numbers of PCV2 in peripheral blood was detected by quantitative PCR,and the results showed that the number of PCV2 copies in peripheral blood was respectively 3.15×10⁵,3.76×10⁵,4.65×10⁵at 7 d,14 d,and 28 d post infection.Flow cytometry was used to detect the number of CD4⁺T cells in the blood of PCV2 infected piglets.The results showed that compared with the control group,the number of CD4⁺T cells decreased significantly at 14 d and 28 d post infection?p<0.05,p<0.01?,and was time-dependent.The results of flow cytometry detection of CD4⁺T cell apoptosis in the blood of PCV2-infected piglets showed that compared with the control group,the apoptosis rate of CD4⁺T cells increased significantly at 14 d and 28 d post infection?p<0.05,p<0.01?,and was time-dependent.The results of flow cytometry detection of NK cells in porcine peripheral blood showed that NK cells increased significantly compared with the control group at 14 d and 28 d post infection?p<0.05,p<0.05?.The results of flow cytometry detection of p NCRs ligand expression on the surface of CD4⁺T cells showed that compared with the control group,there was no significant difference in the expression levels of p NCR1L and p NCR3L on the surface of CD4⁺T cells of piglets infected with PCV2?p>0.05?,while the expression level of p NCR2L was significantly higher than that of the control group at 7 d,14 d and 28 d post infection.In this study,we obtained three fusion proteins p NCR1-h Fc,p NCR2-h Fc and p NCR3-h Fc that can be used for cell surface p NCR1L,p NCR2L and p NCR3L flow cytometry.Analysis of the correlation between PCV2 copy number in pig peripheral blood,CD4⁺T cell number and apoptosis rate,and NK cell number showed that PCV2 copy number was negatively correlated with CD4⁺T cell number,positively correlated with CD4⁺T cell apoptosis rate,positively correlated with the number of NK cells.Analysis of the correlation between p NCR2L expression level on the surface of porcine peripheral blood CD4⁺T cells and PCV2 copy number,CD4⁺T cell number and apoptosis rate showed that p NCR2L expression level was positively correlated with PCV2 copy number,negatively correlated with CD4⁺T cell number,positively correlated CD4⁺T cell apoptosis rate.The research results provide a basis for further exploring the mechanism of PCV2infection leading to piglet lymphocytopenia.