TGEV N Protein In Insect Cells Regulates Swine FcRn Expression And Transcytosis Of IgG

Porcine infectious gastroenteritis(TGE)is a highly contagious infectious disease in swine caused by TGEV infection.TGEV infection causes intestinal barrier dysfunction and disrupts intestinal homeostasis.The typical symptomatic pathway of TGEV infection is villous atrophy within 48?h,followed by crypt hyperplasia,concomitant with lethal watery diarrhea,and ultimately severe dehydration in piglets until death.It is the most important disease that threatens the global pig industry.Fc Rn is a heterodimer composed of two subunits of the alpha chain and the beta chain in the form of non-covalent bonds which plays an important role in immunoglobulin Ig G transport and metabolism.At present,there are few reports about the regulation of Fc Rn expression after pathogens infect human or animal body.Previous studies have shown that TGEV-infected IPEC-J2 cells can upregulate Fc Rn.As a structural protein of the virus,TGEV N protein can induces cellular immunity and plays an important role in the replication and transcription of viral genomic RNA.In this study,we utilized N protein to stimulate IPEC-J2 cells to explore the mechanism of up-regulation of Fc Rn expression and the functional domains.Additionally,we found that N protein mediated Fc Rn up-regulation promotes Ig G transcytosis.The main results of this study are as follows:1.Expression of TGEV N protein and preparation of rabbit anti-TGEV N protein polyclonal antibodyThe prokaryotic expression system was successfully utilized to induce the expression of TGEV N protein.The purified GST-N protein with an adjuvant was immunized the rabbits,succeed to prepare the rabbit anti-TGEV N.The polyclonal antibody had good specificity by Western blot.2.Expression of TGEV-N protein using the baculovirus expression systemThe baculovirus expression systems belong to eukaryotic expression systems.The expressed proteins can be processed and modified more complexly which makes the expressed proteins closer to the natural state.The baculovirus expression system was used to successfully express the TGEV-N protein.We confirmed the expression of TGEV N protein which was consistent with the predicted protein size by SDS-PAGE and Western blot.3.TGEV N Protein enhances Fc Rn expression via NF-κB Signaling PathwaysWe utilized N protein to stimulate IPEC-J2 cells and found that the protein can up-regulate Fc Rn expression in a dose-dependent manner by q PCR and Western blot.Phosphorylation and nuclear translocation of p65 is the typical marker of NF-κB activation.By Western blot and indirect immunofluorescence assay,we found that TGEV N increased phosphorylation of p65 and promoted nuclear translocation of p65 to activate NF-κB.Pre-treatment of cells with si RIG-I impaired Fc Rn expression by TGEV N protein.This result showed that RIG-I may be responsible for TGEV N-protein-induced Fc Rn expression.4.TGEV-N functional domains can regulate Fc Rn expressionIn order to further explore N functional domain,N protein truncations were expressed in this study.We utilized the TGEV N deletion-mutant expression plasmids to transfect IPEC-J2 cells.The result showed that the central region(128–252)of N protein is critical for Fc Rn activation.5.TGEV-N protein up-regulates Fc Rn-mediated Ig G transportWe used the IPEC-J2 cells to construct the transwell system,porcine biotin-Ig G was added into the transwell system and then detected the level of biotin-Ig G in opposite chamber by Western blot.We found that the transcytosis of Ig G was enhanced by Western blot.The result showed that recombinant TGEV N Protein mediated induced Fc Rn-Mediated Ig G.In this study,the N protein was expressed by eukaryotic expression system and the mechanism of N protein up-regulation of Fc Rn was explored.The biological significance of N protein up-regulation of Fc Rn was verified by the transwell system.