The Association Study Of Differential Expression Of MHC Class Ⅰ And Ⅱ Molecules With Pathogenicity Of MD In Spf Chickens Carrying Different MHC Haplotypes

There exists a strong and reproducible association between chicken majorhistocompatablity complex (MHC) haplotypes and the resistance to all kinds of diseases. Themost accepted example is related to lymphoproliferative tumor Marek's disease (MD). Inearlier stage, we had successfully established six SPF BW/G(n) lines chickens carryingdifferent MHC-B haplotypes based on genotypes at four MHC microsatellite DNA markers.In this study, the sequences of the polymorphic exons of duplicated BF and BG genes insubsequent4generations (5chickens of each haplotype) were determined by direct sequence.We prepared three kinds of anti-BF and anti-BG allo-antisera, and serologically confirmed theMHC-B haplotypes using serological typing method simultaneously. It was shown that therewas a highly genetic stability with no individual difference for the six haplotype lines.The expression of duplicated MHC-B BF and BLB genes were studied based onestablished MHC-B haplotypes. BF loci contain BF1and BF2genes, and BLB loci containBLB1and BLB2genes. In this study, we developed specific TaqMan probed real-timequantitative reverse transcription PCR (TaqMan qRT-PCR) methods based on the diagnosticnucleotide polymorphisms present in duplicated BF or BLB genes of B2and B19haplotypes.Spleen mRNA samples of MD-infected and control chickens of B2and B19haplotypes wereused to validate these TaqMan qRT-PCR methods. We observed that there was differenttranscribed change for duplicated BF and BLB genes of spleens in both lines during the latestage of the MDV infection (77dpi); furthermore, BF2and BLB2gene was dominantlytranscribed. Our findings verified the impact of diversified promoter sequences on thefunction of duplicated BF or BLB genes.To semi-quantitatively analyze differential expressions of MHC class I proteins ofMDV-infected and control chickens of MD-resistent BW/G3and MD-susceptible BW/G7lines, we cloned and expressed class I α chain protein coded by conserved BF sequences usingprokaryotic expression system and prepared the polyclonal antibody of the rabbit serum todetect MHC class I protein of different MHC-B haplotypes. The differential expression ofMHC class I protein of MDV-infected (5chickens of each line) and control chickens (3chickens of each line) of BW/G3and BW/G7lines were semi-quantitatively analyzed using the ImageQuant5.2software. As showed by Western blotting, the expression of MHC class Igenes of the resistant and susceptible chickens were up-regulated during the late stage of theinfection, and the expression of MHC class I genes of susceptible chickens was higher thanresistant chickens.In order to further study the association of the expression of duplicated BF or BLB genesin different kinds of splenocytes and bursa of Fabricius with the pathogenicity of MD, oneday old BW/G3(B2haplotype) and BW/G7(B19haplotype) chickens were divided intocontrol and infection group (18chickens of each group), The infection groups were infectedwith MDV Md5strain. After infection, the ration of CD4/CD8lymphocytes of PBL andspleens and B cells composition of spleens were detected using flow cytometry. In addition,three kinds of spleen lymphocytes were sorted using flow cytometry. MDV meq gene mRNAlevels of sorting cells and bursa of Fabricius were assessed by real-time fluorescencequantitative PCR. The disease resistance/susceptibility of BW/G3and BW/G7lines wereanalysed by clinical symptom, relative weight gain rate, the gross lesion, histological and pathologicalchanges (HE staining) of spleens and bursa of Fabricius and immunohistochemistry of viralantigens in spleens. The differential expression of duplicated BF or BLB genes of splenocytesB cell, CD4+and CD8+T cell and bursa of Fabricius were deceted using TaqMan qRT-PCRmethods per sampling time point and data were analysed using the SAS System. All of theresults above indicated that there was no significant difference of virus copy numbers betweenBW/G3line (B2haplotype) and BW/G7line (B19haplotype) after MDV infection, but theantigen amounts in BW/G7line spleens were dramatically higher than BW/G3line. The kindsof cells of spleens infected MDV were different between the lines, and the change of amountof different kinds of cells was induced correspondingly. The significantly up-regulatedtranscriptions of BF1gene in three kinds of spleen lymphocytes were detected in both lines ofchickens after MDV infection. The mRNA level of BF2gene was reduced in the MD mediumresistant chickens (BW/G3line), but increased in the susceptible chickens (BW/G7line). Inaddition, BLB genes mainly expressed in B cells, and the expression can be inhibited due toMDV infection. For bursa of Fabricius, the expression of BF1was up-regulated in thesusceptible chickens (BW/G7lines); meanwhile, the expression of BF2gene was increased inboth lines. So there was the close relationship between MHC I molecular and MDV, while the expression of BLB loci as the candidate gene for MD resistance was less affected by MDV.In conclusion, this study provides the basic information to explore the possibility ofMHC haplotypes to be used for the challenge experiments to investigate particularly thepathogenic mechanisms of and control measures to disease. In addition, it also helpsunderstanding the theoretical basis to study the host immune response mechanism after virusinfection.