ITRAQ-based Quantitative Proteomics Analysis Of Host Proteins Profile Induced By The Different Virulence Viruses Of Genetype Ⅱ GCRV

Grass carp reovirus(GCRV)belongs to the genus aquareoviridae.The first fish virus isolated in China.More than 30 isolates have been reported.GCRV is a double-stranded RNA(ds RNA)virus with 11 segments encoding VP1-VP7 structural proteins and 4/5 non-structural proteins,respectively.The isolated GCRV can be divided into three genotypes I,II,and III.The virulent strain of GCRV genotype II is highly pathogenic to grass carp and causes hemorrhagic disease in grass carp at the fingerling stage,and the mortality rate can be as high as 80%.The virulent strain of GCRV genotype II has no obvious bleeding symptoms and a mortality rate of less than 30%.The molecular mechanism that causes the difference in virulence of the same genotype is still unclear.Proteomics analyzes the dynamic changes of protein composition,modification status and expression level from the perspective of cell or tissue,and understands the interaction and relationship between proteins,protein function and cell activity.Using comparative proteomic to study the regulation mode of grass carp infected with different virulence GCRV will help to understand the molecular mechanism of GCRV damage to the blood circulation system and the pathogenesis of grass carp hemorrhagic disease,as well as the possible reasons why the blood circulation system of grass carp infected with avirulent strain is not affected.Therefore,the research contents of this paper mainly include two aspects: First,an iTRAQ-based comparative proteomic was performed to analysis the differential protein profile of spleen of grass carp infected with virulent and avirulent isolates of grass carp reovirus genotype II,and the protein profile was analyzed by bioinformatics software.The analysis reveals the pathogenesis and molecular mechanism of grass carp hemorrhagic disease;second,the gene which may cause differences in the virulence of genotype II GCRV by Proteomics analysising will be verificated.The research contents and results are as follows:1.iTRAQ-based quantitative analysisBy iTRAQ-based protein quantitative analysis of grass carp spleen infected with different virulence genetype II GCRV(Hu Nan1307、GD1108),the results showed that there were 222 differential proteins,40 of which were up-regulated and 182 down regulated,after infection with virulent strain(Hu Nan1307),and 34 differential proteins,3 of which were up-regulated and 31 down regulated,after infection with avirulent strain(GD1108).Gene ontology annotation enrichment(GO)shows that 222 differential proteins of virulent strains are enriched into 46 subcategories of three main categories ‘molecular＿function’,‘cellular＿component’,and‘biological＿process’,mainly in ‘binding’,‘catalytic activity’,‘cell part’,‘cell’,‘cellular process’,and ‘metabolic process’;34 differential proteins of avirulent strains are enriched into30 subcategories,among which ‘binding’,‘catalytic activity’,‘organelle’,‘cell part’,‘cell’,‘cellular process’,and ‘metabolic process’ are the main enrichment biological processes.KEGG pathway analysis showed that the differential protein of the virulent strains was enriched to 217 signaling pathways,and the differential protein of the avirulent strains was enriched to 89 signaling pathways,such as ‘Metabolic pathways’,‘Carbon metabolism’,‘Fatty acid degradation’,‘Metabolism of xenobiotics by cytochrome P450’,‘Pyruvate metabolism’,etc.All of them were significantly enriched in both virulent strain and the avirulent strain.STRING analysis showed that the infection of virulent strains induces interaction networks caused by‘GCRV induced protein from the gene 2i’(gig2i),‘eukaryotic translation initiation factor 2-alpha kinase 2’(eif2ak2),‘DEXH(Asp-Glu-X-His)box polypeptide 58’(dhx58,LGP2),‘signal transducer and activator of transcription 1b’(stat1b)and other immune and metabolism-related protein.The results of RT-q PCR showed that the m RNA levels of RIG-I、MDA5、LGP2、Gig1、STAT1、IFI56、ISG152、Mx3、PEPCK、PYGL、GAPDH、gstz1 were consistent with the trend of proteomics data.Western blot showed that catalase and GAPDH were consistent with proteomic data.In this study,iTRAQ labeling technology was used to quantitatively analyze the protein expression profiles of grass carp infected with GCRV-Hu Nan1307 and GCRVGD1108,and to screen the different proteins in different experimental groups,which provided a new theoretical basis for the difference of pathogenic mechanism between strong and weak strains of genetype II GCRV.2.Validation of interaction between grass carp laminin receptor and genetype 2GCRVProteomics and transcriptomics analysis indicated that the 37 k Da/67 k Da laminin receptor(Lam R)of grass carp may have an effect on the proliferation of genetype II GCRV.By constructing the eukaryotic expression plasmid of grass carp Lam R,transfecting the GCRV non-sensitive cell line EPC and the sensitive cell line CIK,the cells stably expressing grass carp Lam R were selected by G418,and the construction of the grass carp Lam R stable expression cell line was verified by Western blot.The genetype Ⅱ GCRV was used for infecting EPC,CIK and stably expressed Lamr EPC,CIK.Detection of copy number difference of genetype II GCRV after inoculation.The results showed that the 927 bp grass carp laminin receptor complete ORF was obtained by PCR amplification,and successfully cloned into the eukaryotic expression plasmid p EYFP-Mem to obtain the p EYFP-Mem-Lam R recombinant plasmid,which was transfected and screened by G418 to obtain about 80% positive fluorescent cells.Western blot showed that LAMR was stably expressed in EPC and CIK cell lines.EPC-p EYFPMem,EPC-p EYFP-Mem-Lam R,CIK-p EYFP-Mem and CIK-p EYFP-Mem-Lam R cells were inoculated genetype Ⅱ GCRV with different initial concentrations.EPC-p EYFP-Mem and EPC-p EYFP-Mem-Lam R cells could not detect the proliferation of genetype II GCRV.Virus proliferation can be detected in CIK-p EYFP-Mem and CIK-p EYFP-Mem-Lam R,but there is no significant difference.The results showed that grass carp Lam R protein had no significant effect on the proliferation of genotype II GCRV.This study further studied the content of proteomics analysis,verified the proteins that may be related to the pathogenicity of genotype II GCRV virus,and provided a new theoretical basis for the study of the pathogenic mechanism of genotype II GCRV virus.