Molecular Cloning And Immunological Characterization Of CD8α In Chinese Goose

CD8 molecule is a cell membrane glycoprotein, which plays an important role in the cell-mediated immune response. Hereby, the sequence of Chinese goose CD8 a gene was identified for the first time. The full-length cDNA of goose CD8 a is 1459 bp in length and contains a 711 bp open reading frame. The multiple sequence alignment analysis showed that goose CD8 a shared about 90% homology with the CD8 a of Muscovy duck and mallard in the sequence of nucleotide, and 80% in the deduced amino acid sequence. Phylogenetic analysis indicated that the waterfowl CD8 a formed a monophyletic group.Semi-quantitative RT-PCR was used to detect the transcript of CD8 a mRNA in the tissues of gosling.β-actin was amplified as a control for cDNA quantity and quality. Results showed that transcripts of CD8 a mRNA were high in the immune-related organs and mucosal immune system, including thymus, spleen, bursa of fabricius (BF) and skin; and high aboundance was also found in brain. Subsequently, the transcripts of CD8 a were compared in the immune-related tissues of goose and gosling. The highest transcripts were detected in the thymus and spleen in both goose and gosling. What’s more, the transcript of CD8 a in BF was far higher in gosling than that of goose. While in the cecum and instin, the mRNA level of CD8 a was lower in gosling than that of goose.In addition, the anti-viral defense efforts of goose CD8 a were further explored after new type gosling viral enteritis virus (NGVEV) infection. The results of semi-quantitative RT-PCR showed that the increases of CD 8 a were observed in the thymus, bursa of fabricius, and cecum in the acute infection and the chronic infection. Intrestingly, the transcription pro fling of CD8 a decreased in the instanal in the chronic infection.To characterize immunobiological activity of goose CD8 a, the goose spleen mononuclear cells (MNCs) were chosen as the responder, phytohemagglutinin (PHA), concanavalin A (ConA), polyriboinosinic polyribocytidylic acid (Poly I:C), and lipopolysaccharide (LPS) were chosen as the agonist, the CD8a mRNA transcripts after stimulation were investigated by real-time quantitative PCR (real-time qPCR) assay. Results documented the mRNA levels of CD8 a were significantly up-regulated when stimulated by PHA, ConA, Poly I:C, but not by lipopolysaccharide in spleen MNCs in vitro.After that, the anti-viral defense efforts of goose CD8 a were further explored in GPV-and NGVEV-infected spleen MNCs. In the infected model, the indirect immunofluorescent assay was used to detect the appearance of CD8 a in goose spleen MNCs at the 48 h,72 h and 96 h post infection. PHA-treated spleen MNCs were as positive control, and the expression of CD8 a has been significantly upregulated. In addition, the PBS, of which was as the negative control, had no effect on the expression of CD8 a. Results also documented that the number of CD8 a significantly increased from 48 h post infection with NGVEV and 72 h after GPV infection.To better understanding of viral-host interactions, the transcription profiles of immuen-related molecules (CD8 a and CD4) and anti-virus cytokines (IFN-a, IFN-y and IL-18) in goose spleen MNCs after NGVEV and GPV infection were analyzed by real-time qPCR.The transcriptional levels of CD8 a, CD4, IFN-a and IFN-y increased significantly (P< 0.05) at 48 h,72 h and 96 h, and the increase of IL-18 at 48 h and 96 h post NGVEV-infection. After infection with GPV, the significant up-regulation (P<0.05) of goose IFN-a, IFN-y and IL-18 were detected form 24 h post infection. CD4 mRNA transcriptions were significantly up-regulated (P<0.05) at 48 h and 72 h post-infection, CD8 a transcriptions were elevated significantly (P< 0.05) at 48 h,72 h, and 96 h post-infection. In parallel, the proflication of GPV was also detected during the prcess of infection. GP viral loads increased at the beginning of infection (24 h post-infection), and then decreased sharply.While, the proflication of GPV was detected at the 96 h post infection. The cytotoxic activity was programmed at 24 h,48 h,72 h and 96 h during spleen MNCs infection with NGVEV and GPV. After measuring the cell viability through detecting the absorbance of spleen MNCs at 450 and 630 nm wavelengths, the results showed that there were no significantly viability changes between the viral-infected and the control cells during NGVEV and GPV infection. It indicated that there were no proflication of T cells, and the increases of CD8 a mRNA level were triggered by the infection during the process.Accordingly, these findings would shed lights on the role of CD8 against viral infection in cellular immune system of waterfowl and contribute to provide information for the development of novel immunological assay.