Preparation Of Polyclonal Antibody And Preliminary Study On Function Of VP39 Protein Of Grass Carp Reovirus Genotype Ⅲ

Grass carp(Ctenopharyngodon Idella),as the common aquatic products in diet life,the annual production of grass carp has broken through 20% of the total output of freshwater breeding in the country,and there is still a rising trend every year.However,the Grass carp reovirus(GCRV)hemorrhagic disease caused by grass carp reovirus(GCRV)caused huge losses and threats to the healthy development of grass carp breeding industry.Due to the variety of GCRV,existing vaccines or antiviral drugs cannot fully prevent and control the harm caused by GCRVS.Therefore,it is necessary to study the gene coding and protein function of different genotypes of GCRV.At present,the study of GCRV mainly focuses on type Ⅰ and type Ⅱ,but the replication mechanism of type Ⅲ GCRV is relatively unknown,and the function of VP39 protein encoded by its S9 sequence is still unknown.In this study,the biological function of VP39 protein will be preliminarily explored by using a variety of biological technologies.It mainly includes the following three parts:1.Bioinformatics analysis of VP39 protein and preparation of polyclonal antibodyWe analyzed the basic physical and chemical properties of VP39 by bioinformatics method,and found that VP39 is a hydrophilic protein with the size of 39.19 KDa,without signal peptide and transmembrane structure.The fat index and instability index of VP39 protein were 91.19 and 39.69,respectively.Vp39 protein was identified as stable protein,and its half-life was predicted to be 30 hours under cell culture conditions.The secondary structure of VP39 protein consisted of 39.27% α helix,4.52% βcorner and 42.37% random curl,and the remaining 13.84% was predicted to be extended chains.In addition,VP39 was predicted to have 20 serine phosphorylation sites,15 threonine phosphorylation sites and 3 tyrosine kinase phosphorylation sites.We cloned VP39 gene by PCR and constructed recombinant plasmid p ET28a-VP39.The recombinant plasmid was transferred into BL21 receptor cells by prokaryotic expression,and a large number of target fusion proteins were induced and purified.Mice were immunized with the obtained protein solution to prepare VP39 polyclonal antibody,and the resulting polyclonal antibody was identified.Colony PCR results showed that the plasmid p ET28a-VP39 was constructed successfully and the sequencing results were correct.SDS-PAGE results showed that VP39 was about39 KDa in size and soluble in PBS,which was consistent with the predicted results.Western Blot analysis showed that the prepared VP39 polyclonal antibody could not only identify HIS-VP39 fusion protein,but also accurately identify VP39 protein in GCr V-infected cells,but could not identify uninfected CIK cells,showing good specificity.At the same time,VP39 polyclonal antibody still maintains effective detection ability under the condition of 10000 times dilution.In addition,the results of q PCR and Western Blot showed that the expression level of VP39 increased continuously during virus infection.The preparation of VP39 polyclonal antibody can be used for pathogen detection and diagnosis of GCRV-104,and also lay a foundation for further study of the biological function of VP39.2.Biological analysis of VP39 proteinGCRV-104 virus was concentrated and purified by sucrose density gradient centrifugation.After centrifugation,GCRV-104 virus was enriched between 40% and50% sucrose concentration.A large number of intact virions were observed by transmission electron microscopy.In order to identify the structural type of VP39 protein,Western Blot analysis showed that VP39 was a structural protein and formed a part of GCRVB-104 capsid,which also confirmed the prediction of VP38 as a structural protein and VP15 as a non-structural protein in the existing literature.Dot Blot analysis further identified VP39 as a coat protein,which was also confirmed by virus neutralization experiments with antibodies against VP39.After incubation with GCRV-104 virus,VP39 multiple antibodies effectively reduced the replication level of the virus in a dose-dependent way.By IFA assay and cell transfection identification,we confirmed that VP39 protein was localized in the cytoplasm.In addition,we conducted dynamic analysis of the expression of three GCRV proteins,and the results showed that both VP38 and VP39 were expressed in the middle stage of infection,and the expression levels increased continuously in the later stage.However,the difference is that the expression level of VP39 is much lower than that of VP38 at the same time,which may suggest that although they are both coat shell proteins,they play completely different functions in the process of virus infection.The analysis results also showed that non-structural protein VP15 was expressed in the early stage of virus infection,and the expression level increased first and then decreased.We upregulated the expression of VP39 in GCO cells and infected the virus,showing that the replication level of GCRV was increased compared to the control group.This is further evidence that VP39 plays an important role in viral replication.3.Screening and analysis of VP39 protein interaction polypeptidesThe binding peptide N’-APRLSHHIAHHH-C’ of VP39 protein was screened by phage display technology,and the interaction between protein and peptide was further verified by Dot Blot analysis.To explore the role of screening peptides,we treated cells with different concentrations of peptides and infected the virus to observe their effect on viral replication.q PCR results showed that the replication level of GCRV was significantly increased after polypeptide treatment.However,the influence of p H on the virus infection process was ignored in the experiment.With the increase of peptide concentration,the p H value of culture medium was also downregulated.Therefore,we used HCl to down-regulate p H as the control group.In the experimental group,the polypeptide-incubated cells were simultaneously neutralized with Na OH.The results showed that the p H down-regulation did promote the viral replication,while the p H neutralized peptides did not affect the viral replication.Nevertheless,we screened four polypeptide homologous genes with potential interaction with VP39 protein in the Grass carp gene library on NCBI,namely,retinoic acid inducible protein I.estrogen receptor-alpha,Toll-like receptor 18,and C-X-C motif chemokine receptor3.1 are presented.This will expand a new perspective for the in-depth exploration of the replication mechanism of GCRV-104 virus in the host.