Identification Of The Structure Protein Of The Grass Carp Reovirus Type Ⅱ And Immunogenicity Analysis Of Partial Structure Protein

Grass carp hemorrhage disease（GCHD）,caused by grass carp reovirus（GCRV）,is one of the severe infectious diseases which seriously endangers the healthy development of grass carp breeding industry in China.It causes a large number of grass carps death every year,resulting in huge economic losses.The disease has been classified as a second kind of animal epidemic by the Ministry of Agriculture.The genome of GCRV is composed of 11segmented dsRNA.Based on the difference of viral genome sequence,GCRV can be divided into 3 genotypes in general.In recent years,the main prevalent genotype is genotypeⅡ,which is the most important pathogen of grass carp hemorrhagic disease.The 11 gene segments of GCRV encode corresponding functional proteins,but the function of each gene segment of GCRV genotype Ⅱ（GCRV Ⅱ）is still lack of systematic research.Previously,the research mainly focused on genome structure and sequence.Through the analysis and prediction of bioinformatics database,different researchers have different ideas about the proteins function of the GCRV -Ⅱ virus,which causes inconvenience in the basic virological research of GCRV -Ⅱ and the development of genetic engineering vaccine.In this research,the viral purification,LC-MS/MS,Western blot,IFA and animal experiments were used to identify the coding proteins of each gene segment of GCRV -Ⅱ and the immunogenicity of the target proteins was also analyzed,so as to provide theoretical basis for the basic research on the etiology of GCRV -Ⅱ virus and the development of prevention and control products such as genetic engineering vaccine.The research contents and results were as follows:1.Virus purification and LC-MS/MS analysis:The virus was massively amplified by GSB cells,and then purified by sucrose density gradient centrifugation.The purified GCRV -Ⅱ virions were analyzed by LC-MS/MS.The results showed that the signals of nine segments of GCRV -Ⅱ genome encoding proteins were detected,and proteins in abundance from high to low respectively were S6,S4,S3,S9,S1,S10,S11,S2,S5 gene encoding proteins,suggesting that these nine segments may encode structural proteins of the virus,and S6 and S4 genes may encode the main structural proteins of the virus.However,no signal was detected from the peptide segment of the protein encoded by S7 and S8 genes,which may encode the non-structural protein of the virus.2.Antibody preparation and structural proteins identification:Antibodies against S1 to S4,S6 to S8,S10 and S11 gene coding proteins have been prepared and preserved in the laboratory.In this study,the recombinant prokaryotic expression vectors of S5 and S9 gene were constructed,recombinant S5 and S9 gene coding proteins were induced and purified,and polyclonal antibodies against S5 and S9 gene coding proteins were prepared.Results of Western blot analyzing the 11 antibodies and purified GCRV -Ⅱ virions showed that the antibodies against S1,S3,S4,S6,S9,S10 and S11 encoded proteins reacted positively with virus particles,while the antibodies against S2,S5,S7 and S8 gene encoded proteins reacted negatively with virus particles.After GSB cells were infected with GCRV -Ⅱ,11 antibodies were used for IFA detection,and the results were consistent with those of Western blot.Neutralization test was used to analyze the neutralization activity of the 7 antibodies.GSB cells were infected by diluted antibodies mixed with GCRV -Ⅱ solution in equal volume.RT-qPCR was used to detect the virus content after infection.The results showed that the virus content of antibodies against S6,S9,S10 and S11 genes coding proteins significantly decreased,and the antibody against S9 coding protein had the lowest virus content.All the four antibodies showed a certain neutralization activity,among which the antibody against S9 gene coding protein showed the strongest neutralization activity.3.Immunogenicity analysis of S4 and S9 gene coding proteins:Grass carps were intraperitoneally injected with the purified S4 and S9 gene coding proteins after emulsifying alone or with adjuvant.The serum,spleen and cephalic kidney tissues of grass carps were collected after grass carps were immuned 21 d later respectively.The specific IgM antibody levels of serum were detected by indirect ELISA method and the mRNA relative expression of IFN,TLR-3,TLR-7,Mx and IgM genes in spleen and head kidney were detected by relative quantitative PCR method.The results showed that the OD_450nm values of grass carp serum were 0.6⁰.8 in the S4 and S9 gene coding proteins with adjuvant immunization group,0.3⁰.5 in the S4 and S9 gene coding proteins immunization group,and less than 0.2in the adjuvant immunization group and the control negative group.The mRNA expression levels of 5 immune factor genes in the S4 and S9 gene encoding protein with adjuvant immunization groups were about 7⁴5 times of those in the control group.The levels of specific antibodies in serum and mRNA expression of immune factors in tissues of the protein with adjuvant immunization groups were significantly higher than those of the control group（P<0.05）,while the mRNA expression levels of immune factors in tissues of the protein immunization groups were not significantly changed compare to those of the adjuvant immunization group and negative control group（P>0.05）.The results of pathogenicity protection test showed that after artificial infection of GCRV -Ⅱ isolate,the relative protection rate of S4 gene encoded protein with adjuvant immunization group was37.9%,and that of S9 gene encoded protein with adjuvant immunization group was 34.9%,both of which had certain immune protection effect on grass carp.In summary,the proteins encoded by the S1,S3,S4,S6,S9,S10,S11 gene segments of GCRV -Ⅱ isolates may be the structural protein of the virus,while the proteins encoded by S2,S5,S7 and S8 genes may be the non-structural proteins of the virus,including the S4 and S6 genes coding proteins may be the main structural proteins of the virus,and the S6,S9,S10,S11 gene coding proteins have neutralization activity.The proteins encoded by S4 and S9 have good immunogenicity and certain immune protection against the artificial infection with GCRV -Ⅱ isolate,which can be used as candidate antigens for the development of genetic engineering vaccines.