Preliminary Study On The Induction Of Regulatory T Cells By N,M And GP5 Proteins Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine Reproductive and Respiratory Syndrome(PRRS)is a porcine disease caused by the Porcine Reproductive and Respiratory Syndrome Virus(PRRSV),which is characterized by reproductive disorders in pregnant sows and respiratory distress in growing pigs.The disease first broke out in North America and Europe,and then spread to Asia and other regions,causing a great impact on the global pig industry.The annual economic loss to the global pig industry is about 560 million U.S.dollars.PRRSV is a single-stranded positive-stranded RNA virus with an envelope.It is divided into two genotypes,European type and North American type,namely genotype I and genotype II.Regulatory T cells(Tregs)have the function of regulating the body’s autoimmunity.A large number of studies have shown that Tregs also play a regulatory role in the process of virus infection of the body.Induction of Tregs is a very important pathogenic mechanism of PRRSV.Cytokines are critical to the development and function of Tregs.Many studies have shown that the induction of Tregs by PRRSV is closely related to the changes of cytokines.In this study,the PRRSV JXA1-R strain was multiplied by inoculating Marc-145 cells,the total RNA of the virus was extracted from the cell fluid containing PRRSV,reverse transcription was used to generate c DNA,and the N,M and GP5 genes of PRRSV were amplified by PCR,the sizes were 420 bp,525 bp and 603 bp,respectively.Construct the cloning vector p MD19-T-M,p MD19-T-N,p MD19-T-GP5,and then construct the lentiviral expression vector p LVX-IRES-Zs Green1-N,p LVX-IRES-Zs Green1-M,p LVX-IRES-Zs Green1-GP5,and pass the bacterial solution PCR was used for identification,the positive clones that were sent to the biological company for sequencing,the expression vector with the correct sequencing result was re-amplified and the plasmid was extracted with the kit.We used lentiviruses containing plasmids encoding PRRSV N,M and GP5 proteins to transduce monocyte-derived dendritic cells(Mo DCs)to study PRRSV N,M,and GP5 proteins,respectively its influence on the secretion of cytokines related to the induction of regulatory T cells(Tregs).Results compared with the no-load control group,the PRRSV N protein transduction group had significant up-regulation of IL-2,IL-4,IL-10,TNF-α and TGF-β m RNA,and no significant change in IL-6m RNA;the PRRSV M protein transduction group did not detect IL-2 m RNA,IL-6m RNA was significantly up-regulated,IL-4 m RNA was significantly down-regulated,and no significant changes in TNF-α,IL-10 and TGF-β m RNA;the PRRSV GP5 protein transduction group did not detect IL-2 m RNA,IL-4,IL-6,TNF-α and TGF-βm RNA were significantly up-regulated,but IL-10 m RNA was significantly down-regulated.Commercially available ELISA kits were used to evaluate the contents of TGF-β and IL-10 in the supernatant of DCs after 48 h of transduction with PRRSV N,M and GP5 proteins.Results compared with the no-load control group,in the supernatant of DCs transduced with the lentivirus encoding PRRSV N protein,the contents of TGF-β and IL-10 were significantly increased;in the supernatant of DCs transduced by the lentivirus encoding PRRSV M protein,the contents of TGF-β and IL-10 were no significant change;in the supernatant of DCs transduced by the lentivirus encoding PRRSV GP5 protein,the content of TGF-β was significantly increased,and the content of IL-10 was significantly reduced.This study shows that PRRSV N protein has the ability to induce Tregs,PRRSV M protein does not have the ability to induce Tregs,and PRRSV GP5 protein has the possibility of inducing Tregs.This study lays the foundation for further exploring the mechanism of PRRSV inducing Tregs.