| The RING E3 s are multifunctional cytokines that belong to the E3 ubiquitin ligase,while herpes virus resists the host immune response by downregulating the expression of host RING E3 s or encoding RING E3 s analogues when infecting the host,thereby establishing persistent infection.Cyprinid herpesvirus 2(CyHV-2)is a highly pathogenic virus that seriously harms goldfish and Carassius carassius farming industries.Analysis of the genome of CyHV-2 revealed that ORF144 was a RING E3 s analogue.In this thesis,the CyHV-2 ORF144 gene was cloned and expressed,and the Carassius carassius gill cells(CCG)were constructed to explore the effect of ORF144 on the expression of immunerelated factors.1.Construction of Carassius carassius gill cell line(CCG)and sensitivity analysis of CyHV-2The gill tissue of Carassius carassius was cultured by trypsinization and successfully constructed a cell line by subculturing,which has been passaged to 90 times.The growth characteristics and sensitivity of CyHV-2 were analyzed for CCG,and the optimal culture temperature of CCG was 25°C,and the optimal serum concentration was 15-20% fetal bovine serum.The karyotype results showed that the number of chromosomes in the 50 th generation was 46.The efficiency of CCG transfection of GFP reporting genes exceeds 60%,indicating that CCG can be used for in vitro gene studies.CCGs infected with CyHV-2showed typical cytopathic effects(CPE),confirming viral proliferation by PCR,indirect immunofluorescence assay(IFA),and transmission electron microscopy(TEM).This cell line provides the basic material for virus isolation and gene function studies.2.CyHV-2 ORF144 prokaryotic expression and polyclonal antibody preparationThe full length of ORF144 gene was obtained by PCR amplification,and the recombinant vector p ET-ORF144 was constructed by linking it with p ET-32 a prokaryotic expression vector,which was transformed into E.coli cells,and the recombinant protein in the form of insoluble inclusion bodies with a size of about 46 k Da was obtained by IPTG-induced expression.The purified recombinant fusion protein immunized BALB/c mice obtained polyclonal antibodies,and ELISA detection showed antibody titer greater than 1:51200.Western Blot has validated that the antibody specifically recognizes fusion proteins and proteins in cells infected with CyHV-2.Indirect immunofluorescence showed that the protein was localized to the cytoplasm in a punctate manner.ORF144 localization provides clues to the biological function of proteins.3.Regulatory effect of CyHV-2 ORF144 on host immunity-related factorsOverexpression vectors(pc DNA3.1-ORF144,pc DNA3.1-ORF144-ΔRING and pc DNA3.1-STING)were constructed,and the effects on viral replication and immune gene expression were detected by real-time PCR after transfection of CCG.The results showed that overexpression of ORF144 significantly promoted the transcription of major capsid protein(MCP)and helicase ORF99 genes,and the deletion of the main RING domain significantly reduced the promotion effect on viral gene transcription.By quantifying the expression of immune genes,the results showed that ORF144 significantly inhibited the expression of STING,IRF3,IRF7,IRF9,MX1,INF-α,IL-1 and IL-17,while ORF144 inhibited the expression of STING-mediated interferon signaling molecules,such as IRF3,IRF7,IRF9,MXI,and this inhibition effect was weakened after the loss of the RING domain.The results showed that CyHV-2 ORF144 relies on the RING domain to inhibit the expression of immunocorrelated factors and STING-mediated interferon signaling molecules.In this study,CCG cell lines were successfully constructed,cloning CyHV-2 ORF144 gene,obtaining corresponding polyclonal antibodies,and confirming that ORF144 can inhibit the expression of related immune genes to promote viral replication,which provides a theoretical and material basis for studying the mechanism of CyHV-2 immune evasion. |
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