Molecular Cloning And Expression Regulation Of CD8α In Duck

With the farming output and consumption increasingly growing in duck industry, the viral disease of duck becomes serious and complicated year by year, which resulted in the huge losses to the duck industry. CD8molecule is a double strand glycoprotein existed on the surface of T cell, which composes of two dissimilar subunits CD8a and CD8β.It is also a major marker on the surface of the T lymphocyte surface, which plays an important role in the immune defense of mammals and birds. The CD8a gene takes part in thymus differentiation and signal transduction during T cell activated, while the CD8β only assisted with the CD8a to play biological functions.The study has been demonstrated that the CD8a gene expression was regulated by CD8α-specific enhancer and silencer. Another study has been demonstrated that DNA methylation of CD8a gene affected by some factors, such as the thymus high mobility box protein and IL4, which resulted in abnormal expression. However, duCD8a gene involved in transcriptional regulation and epigenetic regulation was not been descripted up to date.In this study, the sequence of cDNA and the promoter in duCD8a gene were cloned, and expression regulation was analyzed by disease model infected duckling virus hepatitis, its results are as follows:The coding sequence (CDS) of CD8a from Jinding duck, Cherry Valley Peking duck, Muscovy, mallard and spot-billed duck were obtained by RT-PCR from their spleen, whose length is about714bp. And the cDNA sequence was cloned by RACE, which including the5’UTR of61bp, the3’UTR of849bp, the open reading frame of714bp. Besides, The CDS of CD8a was compared among susceptible group, resistant group and control group treated with duckling virus hepatitis, the nucleotide differences existed in exon2.The CD8a gene of Jinding duck, Cherry Valley duck, muscovy, mallard and spot-billed duck was cloned and genomic organization was analyzed. The CD8a gene among5populations all composed of six exons and five introns, and all the boundary of exon/intron has GT/AG conserved sequences except the first intron. but their length was different, which spanned6568bp,6567bp,6488bp,6568bp and6577bp, respectively.The phylogeny of CD8a gene was analyzed in different poultry(Jinding duck, Cherry Valley Peking duck, muscovy, mallard, spot-billed duck, domestic chicken, red Jungle fowl, zebra finch and turkey). The results indicated that the amino acid sequence showed the highest degree of identity between Cherry Valley Peking duck and mallard, and the lowest degree of identity between muscovy and domestic chicken. In addition, three conserved amino acid residues(Cys49, Cysl19and Cysl82) were discovered. Finally, the a-helices and p-strands in the structure of protein were also similar, which showed that CD8a evolution was very conservative in poultry.The expression of CD8a mRNA in some organs and cells of duck (The muscle, lung, liver, brain, cerebellum, thymus, spleen, kidney, lymphocytes) was detected by RT-PCR and real-time RT-PCR, which revealed that the CD8mRNA was much higher in the thymus spleen, lung and lymphocytes than in other organs and the expression level was the lowest in muscle and brain. CD8a mRNA was identified on white pulp of spleen by in situ hybridization, and positive cells of the susceptible group were more obvious than the control group. CD8a molecular was detected identified on splenic corpuscle and periarterial lymphatic sheath of spleen by immunohistochemistry.The CD8a mRNA levels were examined in some organs (such as cerebellum, thymus, spleen and so on) treated with polyriboinosinic polyribocytidylic acid (PolyⅠC) and duckling virus hepatitis. The result showed that the CD8mRNA levels was significantly down-regulated at48h compared to their levels at12h post-infection or24h post-infection with Polyl:C and duckling virus hepatitis (P<0.05). In addition, levels of cytokine (such as IFN-α、IFN-γ) were quantified using the ELISA kit. The result showed that both IFN-aand IFN-yhad a similar changing trend to CD8a mRNA expression.The eukaryotic expression vector pEGFP-C1-CD8a was constructed and expressed in NIH-3T3cells successfully. EGFP-CD8a fusion protein was identified to locate at the membrane and cytoplasm of NIH-3T3cells.The CD8a gene promoter was cloned by genome walking, the sequence of the promoter was analyzed by online software of bioinformatics (http://www.cbrc.jp/research/db/TFSEARCH.html) The DNA sequence of2480bp was amplified including56bp in exon1. And some transcriptional factor binding sites including CdxA、Nkx-2、GATA-1、SRY and so on were detected, whose function were relevance with cell proliferation, apoptosis.The promoter missing mutants were constructed (-625/-1bp,-1110/-1bp,-1413/-1bp,-2151/-1bp) according to the sequence of CD8a gene promoter, and then subcloned into pGL3.2basic vectors to construct luciferase report gene vectors, respectively. The recombinant vectors were transfected into DT40cells with Lipofectamine2000, and the transcriptional activities were detected. The results indicated that CD8a gene promoter had obviously promoter activity. The sequence from-625to-1110of5’flanking region had the strongest promoter activities, including two positive (-625/-1and-625/-1110) regulatory domains. Then the sequence of regulatory domains in the susceptible group, the resistance group and control group was compared, three mutations (-985T>C,-552G>A,-484G>A)were found, which resulted in transcription factor binding sites increasing or CpG island reducing.The CD8a gene methylation status of duck blood was compared. The results showed that CD8a gene methylation in the susceptible group was significantly higher than the other groups, but that difference was not significant (P>0.05). Meanwhile, the second CpG Island was found that methylation status was higher in the susceptible group than the other groups.The global level of methylation in the susceptible ducks, the resistance ducks and control ducks were detected by MethyFlash Quantification Kit. The results showed that the global methylation level in the susceptible ducks was significantly higher than the resistance ducks and control ducks (P<0.05).