Study On The Effect Of Bovine IRF7 Protein Expression On Bovine Viral Diarrhea Virus Proliferation

Bovine viral diarrhea virus(BVDV)can cause a very complex bovine viral diarrhea-mucosal disease(BVD-MD)with multiple clinical symptoms,which seriously harms global animal husbandry The healthy development of the industry.Interferon regulatory factor 7(IRF7)is an important member of the IRF transcription factor family,and it is also a part of the positive feedback regulatory loop that is essential for sustained IFN response.Early q PCR and transcriptome data found that IRF7 was down-regulated in NCP BVDV-infected bovine peripheral blood mononuclear cells.However,the relationship between IRF7 and BVDV infection and how to regulate the type I IFN signaling pathway is still unclear.Accordingly,this study aims to determine the relationship between IRF7 and BVDV replication,and to provide a basis for elucidating the molecular mechanism of IRF7 in the innate immune and antiviral response.Purpose:(1)The structure and function of IRF7 gene were analyzed by bioinformatics method,the Bo IRF7 gene was cloned,the IRF7 protein was expressed by prokaryotic expression system,and the polyclonal antibody was prepared,which provided material for the subsequent experimental research.(2)The overexpression and interference cell model of Bo IRF7 was constructed to lay the foundation for the functional verification of Bo IRF7 gene.(3)To determine the relationship between Bo IRF7 and BVDV proliferation,and to provide a basis for clarifying the mechanism of Bo IRF7 in the innate immune response.Methods:(1)Bioinformatics software was used to predict and analyze the potential biological functions of Bo IRF7,and primers were designed with reference to the IRF7 gene sequence published in Gen Bank.Target genes of expected size were obtained by RT-RCR amplification,which were linked to p MD19-T vector and sequenced;In order to express highly efficient IRF7 protein,the cloned 1497 bp gene fragment was digested with the p ET-28 a vector plasmid with His label respectively,and then ligated into the recombinant plasmid,which was transformed into the expression strain of Escherichia coli,Positive clones were selected,identified by enzyme digestion and sequencing,and then expressed and purified.The purified protein was renaturated,and the renaturated protein was used as immunogens to immunize experimental rabbits,and the polyclonal antibody serum was obtained after three immunization,The antibody was purified,and the protein concentration was determined by BCA method.The antibody titer was determined by indirect ELISA.(2)Overexpression and interference lentiviral vectors were constructed,and lentivirions were packaged by HEK-293 T cells and infected host cells.Overexpression and interference effects of related genes were detected by RT-PCR and Western-blot,and fragments with better interference effects were screened out.(3)After infected with BVDV,the m RNA and protein expression of BOIRF7 gene were detected by real-time quantitative PCR and Western-blot.MDBK cells overexpressed and interfered with IRF7 were infected with NCP BVDV,and the same two sets of samples were prepared in parallel for RT-PCR and Western-blot detection,respectively,to verify the effect of NCP BVDV on virus proliferation.Results:(1)Through bioinformatics analysis of Bo IRF7,it was found that it had potential antigen,and the dominant epitopes of antigen were screened out.A 1497 bp gene fragment was successfully amplified from MDBK cells infected with BVDV.IRF7 protein was successfully expressed at 60 k Da,and the purified IRF7 protein concentration measured by BCA method was 2000?g/m L;The results of Western-blot showed that the prepared antibody had good specificity.The titer of IRF7 polyclonal antibody detected by indirect ELISA was 1? 128000,and the polyclonal antibody of IRF7 was successfully prepared.(2)Overexpression and interference of MDBK cell lines were successfully constructed,and interference fragments with better interference effect were screened out.(3)After BVDV virus infected MDBK cell lines with overexpression and interference,it was found that overexpression of IRF7 caused decreased BVDV replication,and interference with the expression of IRF7 resulted in enhanced BVDV replication.The results showed that the expression of IRF7 gene affected BVDV replication.Conclusion:(1)The bioinformatics method was used to predict that IRF7 protein had good antigenicity,and the polyclonal antibody prepared by prokaryotic expression system expressed IRF7 protein had good specificity.(2)Overexpression of IRF7 can significantly inhibit the proliferation of BVDV,while interference with the expression of IRF7 can promote the proliferation of BVDV.IRF7 plays an important role in inhibiting the proliferation of BVDV...