Cloning Of Ctenopharyngodon Idella Trap1 And Its Anti-apoptosis Function Involved In Grass Carp Reovirus Infection

Grass carp reovirus(GCRV) is the major pathogen which leads to serious grass carp hemorrhagic disease, and have a bad effect on the development of grass carp aquaculture. GCRV, as a double-strand RNA(ds RNA) virus containing 11 ds RNA segments, encodes 12 proteins and is classified as Reoviridae aquatic reovirus genus. During the GCRV infection, the gene transcriptional expression level and protein synthesis of host cell could be changed, the cell fusion, apoptosis and many other symptoms also are induced. Apoptosis, which is one of the programmed cell death, has been confirmed and regulated by genes. Virus-induced apoptosis have been proved having two-way interaction between host cell and virus. When the host cell be resistant to virus infection by apoptosis, apoptosis is also beneficial for virus particles releasing.Firstly, apoptosis is detected in CIK cells by various methods during GCRV infection in this study. The tumor necrosis factor receptor-associated protein 1(Ci Trap1) of Grass carp is identified and analyzed by two-dimensional electrophoresis(2-DE). The rapid amplification of c DNA ends(RACE) assays is succeeded in Ci Trap1 and the function of Ci Trap1 involved in GCRV induced apoptosis is investigated by RNA interfere experiment successfully. All the study are presented into three parts:1. Detection of apoptosis in CIK cells during GCRV infection. GCRV-induced apoptosis is detected in different ways. The apoptotic bodies are observed by fluorescence microscope with DAPI staining in CIK cells when GCRV infection at a multiple of infection(MOI) of 10. Genomic DNA are extracted from virus-infected CIK cells at 0, 3, 6, 12, 24, 36, 48 and 60 h(MOI=1), and analyzed by agarose gel electrophoresis. As a result. “DNA ladder” appears early from 24 h after GCRV infection obviously. Using TUNEL assays, signals of apoptosis are also found in GCRV infected CIK cells at 12 and 24 h with a MOI of 5 separately. At the same time, the transcriptional level of Caspase-3 is highly upregulated at 12 h after virus infection(MOI=1) as well as the ratio of Bax/Bcl-2 at 8 h. Furthermore, apoptosis is quantified by cell analyzer using annexin V labeling following virus infection at 12, 24, 36 and 48 h, and quantified that apoptotic rate in CIK cells during GCRV infection is gradually increasing and significantly higher than the controls.2. Proteomic identification and characterization of Ci Trap1. The different proteins between virus-infected and non-infected CIK cells are compared by 2-DE and mass spectrometry(MS) at 6 h p i, 12 h p i and 24 h p i. The protein spot 1061 is identified as the Trap1 and its translational level in infected cells is higher above three times at 24 h. The full length c DNA of Ci Trap1 is amplified by RACE experiment which including 2762 bp, contains an opening reading frame of 2157 bp encodeing a peptide of 718 amino acids. Phylogenetic analyses indicate that the Ci Trap1 shared 87% identity with its homologue from zebrafish(Danio rerio). The Ci Trap1 gene is inserted into p EGFP-N1 vector and the recombinant plasmid p EGFP-Ci Trap1 is transfected into CIK cells for subcellular localization. As a result, GFP-Ci Trap1 fusion protein is expressed and located mainly in the cytoplasm, and the expression of GFP-Ci Trap1 fusion protein is confirmed with western blotting., The expression of Ci Trap1 gene in CIK cells are validated upregulated significantly with poly(I: C) stimulation and GCRV infection3. Regulation of virus-induced apoptosis by Ci Trap1. Partial sequences of PTEN induced putative kinase 1(PINK1) and Sorcin are amplified from the c DNA of CIK cells to investigate the role of Ci Trap1 in the process of GCRV-induced apoptosis in CIK cells, and these two genes are affirmed to interacted with Trap1 and involved in regulation of apoptosis. With GCRV infection, the m RNA expression levels of PINK1 and Sorcin in CIK cells are found upregulated. Three si RNA oligos are designed for Ci Trap1 and the Ci Trap1-si RNA2 is choosed for its best silencing effect later. CIK cells treated with Ci Trap1-si RNA2 are infected with GCRV at a MOI of 1. Then, cells were collected at 36 h post-infection and analyzed with cell analyzer. The analysis of data indicates that the percentage of total apoptotic cells in CIK cells treated with si RNA is higher than treated with scrambled si RNA and the controls.In summary, apoptosis occurred in CIK cells during GCRV infection, which is demonstrated by a series of methods. Anti-apoptotic factor Trap1 protein is identified by 2-DE and MS, and the full length c DNA of Ci Trap1 is also successfully amplified. RNAi-mediated silencing of Ci Trap1 in CIK cells resulted in the increased rate of virus-induced apoptotic cells, which is also indicated that Ci Trap1 in CIK cells is involved in the GCRV induced apoptosis as an anti-apoptotic factor.