The Studies On The Effects Of TGEV Proteins On FcRn Expression In PK-15 Cells After The Activation Of TLRs

The host innate immune system is formed in the course of evolution which can recognize "self" and "non self".Mucosal immunity acts as the first defense against external pathogens.After the invasion,the "non self" can be recognized by PRRs.After the recognition,PRRs leads to a series of signaling transduction which activates some nuclear transcription factors transported into the nucleus.The activation promotes the expression of some genes in cells.The first confirmed PRRs are TLRs.Structural proteins and non-stuctual proteins’ recombinant eukaryotic expression plasmids of TGEV were constructed to study the effects on FcRn expression as well as the functional domains of the related proteins.On the other hand,the connection between the promotion of FcRn expression and TLRs signaling pathway after stimulated by TGEV,UV-TGEV and TGEV proteins was investigated.The researches were as follows: 1.The effection on FcRn expression after infected with TGEVThe mRNA and protein expression of FcRn in IPEC-J2 cells and PK-15 cells were detected by Real-Time Fluorescent Quantitative PCR,dual luciferase reporter system and Western Blot after infected with TGEV.The results indicated that TGEV could significantly promote the mRNA and protein expression of FcRn.2.The effections on FcRn expression when stimulated with TGEV proteinsAfter PK-15 cells were stimulated with different dose of UV-TGEV,the expression of FcRn was detected by Real-Time Fluorescent Quantitative PCR,dual luciferase reporter system and Western Blot.The results indicated that the expression of FcRn at both mRNA and protein levels could be upregulated by UV-TGEV which could be concluded that the expression of FcRn could be promoted by the structural proteins of TGEV.Compared with TGEV,a lower expression level of FcRn was found after PK-15 cells were stimulated with UV-TGEV.Except for the nucleic acid,the nonstructural proteins of TGEV may be also involved in the regulation of FcRn expression.3.Construction of TGEV proteins’ recombinant eukaryotic expression plasmidsThe RNA of TGEV WH-1 strain was extracted which was then synthesized into cDNA using reverse transcription polymerase chain reaction(RT-PCR).The specific primers of TGEV proteins were designed to amplify the protein genes which were then connected to pCAGGS-HA.Then the recombinant plasmids were tested to be correct by PCR,restriction enzyme digestion and sequencing.Real-Time Fluorescent Quantitative PCR and Western Blot were performed to detect the expression of FcRn after the recombinant plasmids were transfected into PK-15 cells.The result indicated that the expression of FcRn could be significantly promoted by M protein,N protein,E protein,nsp1,nsp5,nsp7,nsp8,nsp9 and nsp15.4.Construction of TGEV proteins’ truncated expression plasmidsIn order to investigate the structural domains of M protein and N protein,different truncated protein expression plasmids were constructed and transfected into PK-15 cells.Real-Time Fluorescent Quantitative PCR and Western Blot was then used to detect the mRNA level and protein level of FcRn.The functional domain of N protein was confirmed between residues 156 and 198,M protein was between residues 219 and 262.5.Explore the TLRs signaling pathway that related to the up-regulation of FcRn after stimulated with TGEV,UV-TGEV and proteins of TGEVThis study further explored whether the stimulation of TGEV,UV-TGEV and proteins of TGEV which could up-regulate the expression of FcRn could activate the TLRs signaling pathway or not.PK-15 cells were stimulated with TGEV and UV-TGEV after transfected with negative control plasmid of MyD88 and TRIF,the protein of FcRn was then tested with Western Blot.The result indicated that both TGEV and UV-TGEV could promote the expression of FcRn through TLRs signaling pathway.It was further confirmed that TGEV could activate TLR1,TLR2,TLR3,TLR4,TLR6 while UV-TGEV could activate TLR1 and TLR2.Nsp1,nsp5,nsp7,nsp8,nsp9 and nsp15 could activate TLR1,TLR2,TLR4 and TLR6,while protein E could activate TLR2,TLR4 and TLR6,N1 protein could activate TLR1,TLR4 and TLR6,M2 protein could activate TLR2 and TLR4.