Study On Antiviral Immune Regulation Mechanisms Of EcTRIM44L And EcTRIM21 In Grouper,Epinephelus Sp.

As an important economic marine fish in China,grouper（Epinephelus spp.）has encountered serious disease threat when they are in intensive large-scale cultivation.Among them,Singapore grouper iridovirus（SGIV）and red spotted grouper nervous necrosis virus（RGNNV）were identified as two important viral pathogens of grouper diseases.In recent years,they have frequently broken out in grouper cultures,and caused significant economic losses.However,the research of molecular mechanism against viral infection is not deep enough in grouper.As a member of E3 ubiquitin ligase family,TRIM（tripartite motif）proteins play important roles in cell cycle regulation,cell apoptosis,signal transduction,protein processing and transportation,proteasome mediated protein degradation and the response of viral infection.In order to understand the effect of grouper’s immune related genes in viral infection and replication,the mechanisms of TRIM44L and TRIM21 in responding to SGIV and RGNNV infection were studied.In addition,the oral vaccine against RGNNV which applied biological vector was developed.The innovative results of research were summarized as follows.1.The immune regulation mechanism of Ec TRIM44L in RGNNV infection in grouper,Epinephelus coioides.According to the grouper spleen transcriptome EST sequence,grouper TRIM44L（Ec TRIM44L）was identified and cloned.The results of sequence analysis showed that a 393amino acid polypeptide encoded by Ec TRIM44L had 81.44%and 51.02%homology with the sequences of large yellow croaker and zebrafish,respectively.Ec TRIM44L contained two conserved domains:B-box domain and coiled coil domain.Fluorescent quantitative PCR（q PCR）analysis showed that after RGNNV infecting grouper spleen cells（GS）,the expression of Ec TRIM44L was significantly up-regulated,which suggested that Ec TRIM44L might be involved in RGNNV infection.Further studies showed that the overexpression of Ec TRIM44L in vitro could significantly enhance the CPE of viral lesions caused by RGNNV,and significantly up regulate the expression of RGNNV coat protein（CP）and RNA dependent RNA polymerase（Rd Rp）.At the same time,the overexpression of Ec TRIM44L significantly reduced the expression level of IFN signal key molecules and proinflammatory cytokines.In addition,overexpression of Ec TRIM44L negatively regulated the activity of ISRE,an interferon promoter induced by melanoma differentiation associated protein 5（MDA5）and mitochondrial antiviral signaling protein（MAVS）.In conclusion,the results of this study revealed for the first time that Ec TRIM44L can participate in RGNNV infection by negatively regulating the immune response of interferon induced by MDA5 and MAVS.2.The immune regulation mechanism of Ec TRIM21 in SGIV and RGNNV infection in grouper,Epinephelus coioides.According to the EST sequence,Ec TRIM21 was identified and cloned.The results of sequence analysis showed that Ec TRIM21 encodes a 557 amino acid polypeptide,which has38.92%and 31.13%homology with zebrafish and human,including RING,B-Box,PRY and SPRY domain.Subcellular localization analysis showed that Ec TRIM21 was proposed to encode a cytoplasmic protein.The q PCR analysis showed that the expression of Ec TRIM21was significantly up-regulated after the infection of grouper spleen cells by SGIV and RGNNV.In the spleen cells of grouper,the overexpression of Ec TRIM21 significantly inhibited the transcription of SGIV and RGNNV genes,while the down-regulation of Ec TRIM21 significantly promoted the transcription of SGIV and RGNNV genes.At the same time,the overexpression of Ec TRIM21 significantly increased the expression level of IFN related signal molecules and inflammatory cytokines.In conclusion,our results for the first time revealed that Ec TRIM21 positively regulated innate antiviral immune response to grouper virus infection.3.Development and evaluation of RGNNV oral vaccine which applied biological vector.The reults of viral infection showed that the median lethal dose of RGNNV to the Leopard coral grouper（Plectropomus leopardus）was 8.5×10⁴ TCID₅₀/0.05m L.A flexible linker was used to connect the sequence of RGNNV CP and grouper defensin protein（DP）,and a recombinant plasmid p ET28a-CP-DP was constructed.After IPTG induced expression in E.coli,bacteria containing CP-DP fusion protein were collected and fed to artemia as food.After a certain amount of fusion protein was enriched in artemia,then grouper fries were fed with the artemia.The results showed that CP-DP fusion protein,as an oral vaccine,significantly inhibited the replication of RGNNV in the tissues of Leopard coral grouper,and the inhibition effect was significantly stronger than single CP protein oral vaccine.The preliminary results showed that the immune protection rate was 100%.The results of this study provide important information for the development of the oral vaccine against RGNNV.In conclusion,this study revealed that Ec TRIM44L and Ec TRIM21 genes of grouper can regulate IFN and inflammatory response through different mechanisms,thus affecting virus infection and replication,which can provide a theoretical foundation for better understanding of the host defense mechanism of grouper against viral infection.The development of artemia biovector oral vaccine against RGNNV provides basis for exploring effective strategies for the prevention and control of neural necrosis virus.