Effect Of Pp38 Gene Of Marek's Disease Virus On Toll-Like Receptor Pathway

Marek's disease(MD)is an infectious neoplastic disease with high cell-binding characteristics,which is caused by Marek's Disease Virus(MDV)infection in poultry.Marek's disease virus(MDV)is a herpes virus that causes T cell tumors,but the mechanism of tumorigenesis is still not very clear.All MDV-induced tumors belong to serotype I.The three genes associated with type 1 MDV-induced tumors were 132-bPr,pp38,meq genes.The expression of 3800 phosphoprotein(pp38)encoded by the pp38 gene in tumors and cell lines is different.Type I specific pp38 complex is the only MDV-specific antigen that can be detected in MDV-induced tumors and non-productive tumor cell lines so far.And pp38 has been shown to inhibit the immune response of the body.CVI988 belongs to MDV I vaccine strain and RBI B belongs to MDV type I virulent strain.In this study,the pp38 gene sequence was amplified from the total RNA of CEF cells infected with MDV virulent strain RBIB and vaccine strain CVI988 by PCR.The results showed that about 870bp of pp38 gene fragment could be amplified from the vaccine strain and the virulent strain.The results of the sequence analysis showed that the rest was identical except that the amino acids at positions 107 and 109 were mutated.In order to further understand the mechanism of action of pp38 in immune response after MDV infection,the amplified gene fragment was digested with restriction endonuclease Hind? and Xhol,and then inserted into the eukaryotic expression vector pCMV-C-Flag cleaved by Hind?and Xho? to construct pCMV-pp38-Flag plasmid.The constructed plasmids were transfected into CEF cells and the expression of pp38 phosphoprotein was verified by indirect immunofluorescence assay(IFA)and western-blot.The correct construction of eukaryotic expression vectors pCMV-CV1988-pp38 and pCMV-RB1B-pp38 provided a test material for further study of the differences in the mechanism of inhibition of the cellular immune response between the MDV type I and the pp38 gene.In order to study the effect of Marek's disease virus pp38 protein on the innate immunity of host cells,the eukaryotic expression vectors pCMV-CVI988-pp38 and pCMV-RB1B-pp38 were transfected into CEF cells,and stimulated by poly(I:C),RNA was collected at 6,12,24 hours to obtain cDNA.The TLR3,TLR5,TLR15,TLR21,TLR2,TLR4,and TLR3 signal transduction genes were detected by fluorescence quantitative method at each sampling time.The results showed that pp38 had a certain inhibitory effect on TLR3-TRIF pathway and affected the expression of inflammatory factors in downstream pathways,and the differences of pp38 at different molecular factors were obvious.The expression levels of IRAK4,TRAF6,IFNA,IFNB and other genes in MyD88 pathway and other cytokines were also detected.The experimental results show that the effect of pp38 on the expression of these cytokines is related to the virulence of MDV.This study found that the MDV vaccine strain and the virulent strain pp38 protein had an inhibitory effect on the TLR3-TIRF pathway,and this inhibition was present betweent different strains.The effect of pp38 on the expression of TLR2,7,15 and some downstream cytokines was related to virulence.These results provide a basis for studying the response of pp38 proteins of different MDV strains to the innate immunity of cells in vitro and in vivo,and also provide a basis for studying the differences between pp38 immunosuppressive strains in different virulence strains,which will be more beneficial to clarified the mechanism of pp38 in MDV-induced tumors.