| Porcine epidemic diarrhea virus(PEDV)mainly causes severe diarrhea,dehydration and vomiting in neonatal piglets,and it also causes diarrhea,agalactia,and abnormal reproductive cycles in pregnant sows.Since 2010 outbreaks of PED caused by PEDV variant have increased,resulting in huge economic losses in many Asian and European countries.Previous research suggested that PEDV could strongly inhibit host type I interferon response,but its regulatory mechanism has not been fully understood.Exploring new viral proteins which regulate host antiviral response would help to reveal the mechanism by which PEDV used to escape the host antiviral response.Considering many different PEDV strains in the field and PEDV mainly affects 2-weeks-old piglets,and the disease always showes acute onset and short duration,the development of a method that can be widely used for the detection of different strains of PEDV and early diagnosis of PEDV is necessary.In this study,the non-structural protein 7 gene of PEDV was identified for the first time,and its sequence characteristics,prokaryotic expression and immunogenicity were analyzed,then its eukaryotic expression and subcellular localization and the effect on type I interferon response were investigated.In addition,we established a ELISA by using purified purified Nsp7 as coating antigen,and the method was used for the detection of different strains of PEDV and early detection of PEDV.The thesis are composed of the following three parts:1.Bioinformatics analysis,prokaryotic expression and polyclonal antibody preparation of PEDV Nsp7In order to understand the structural characteristics and immunogenicity of PEDV Nsp7,the Nsp7 sequence,physicochemical properties and spatial structure were analyzed by bioinformatics.Based on this,prokaryotic expression and purification and preparation of anti-Nsp7 polyclonal antibody were performed.The results of bioinformatics showed that Nsp7 gene was 249 bp in length,it was highly conserved among different strains of PEDV.Nsp7 was stable protein and existed in monomer form and its tertiary structure was mainly composed of four alpha helix.SDS-PAGEshowed that Nsp7 was expressed efficiently in Escherichia coli and purified successfully.The purified Nsp7 was used to prepare polyclonal antibody.ELISA showed that the titer of the prepared antibody was 1:32 000.The results of Western blot and indirect immunofluorescence assay showed that the polyclonal antibody could specifically recognize the recombinant Nsp7 protein and PEDV infected cells,which laid a foundation for the subsequent study of subcellular localization and function of Nsp7 and the establishment of ELISA for detecting anti-Nsp7 antibody.2.Eukaryotic expression,subcellular localization and effect on type I IFN response of Nsp7To investigate eukaryotic expression,subcellular localization and effect on type I interferon(IFN)response of nonstructural protein 7(Nsp7)of porcine epidemic diarrhea virus(PEDV),the Nsp7 gene was cloned and inserted into the eukaryotic expression vector p CAGGS.The expression and subcellular localization of Nsp7 in transfected cells were determined by Western blot and indirect immunofluorescence assay respectively.The effect of Nsp7 on type I IFN response was evaluated by dual luciferase reporter gene assay,ELISA,real-time quantitative PCR and VSV-GFP bioassay respectively.Western blot and indirect immunofluorescence assay showed that Nsp7 was highly expressed and localized mainly in cytoplasm.Dual luciferase reporter gene assay indicated that Nsp7 strongly inhibited the IFN-? promoter activity.ELISA results showed that Nsp7 could significantly inhibit the expression of IFN-? in the protein level.Real-time quantitative PCR results showed that Nsp7 significantly down-regulated the transcription level of some ISGs(ISG15,ISG20 and ISG56)genes.VSV replication assay revealed that Nsp7 significantly inhibited type I IFN antiviral activity induced by poly(I:C).Our results implied that Nsp7 was a highly conserved protein of PEDV and exhibited antagonistic function on type I IFN response.The results have laid a foundation for exploring the innate immunity evasion mechanism of PEDV and guiding the clinical application of vaccines.3.Development and application of a PEDV Nsp7-based indirect ELISA for antibodyConsidering many PEDV strains in the field and PEDV mainly affects2-weeks-old piglets,and it also showed acute onset and short duration,the development of a method that could be widely used for the detection of different strains of PEDV and early diagnosis of the virus is imperative.The results of Experiment One showed that the expressed PEDV Nsp7 had good immunogenicityand the Nsp7 gene was highly conserved among different strains of PEDV.In view of this,an indirect ELISA was established by using the purified Nsp7 protein as the coating antigen.The ELISA could be used to detect antibodies from different strains of PEDV.The optimal reaction conditions were as follow: optimal amount of coating antigen was 0.20?g/well and coating condition was at 37? 1h then 4? overnight;serum sample working dilution was 1:300,and its incubation time was 2h;working dilution of HRP-labelled second antibody was 1:10000,and its incubation time was1.5h;TMB substrate incubation time was 15 min.Serum sample was determined as positive when its S/P?0.1694 and negative when its S/P<0.1398 respectively.The ELISA was specific,reproducible and sensitive.Forty samples of suspected PEDV serum samples were tested by the established ELISA,and the coincidence rate between the ELISA and the commercial kit was 95%.The ELISA established in this study can be used clinically to detect the antibody level of different strains of PEDV,and it also has the potential for early diagnosis of PEDV,providing a basis for the development of effective measures to control PEDV. |
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