| Coxsackieviruses B5 is a member of the genus Enterovirus,family Picomaviridae,characterized by a single-stranded positive RNA genome.The genome of CVB5 is divided into 5’untranslated region(UTR).3’-UTR and P1,P2,P3 protein coding region.P1 region encodes VP1,VP2,VP3 and VP4 capsid proteins,P2 and P3 encoding 2A,2B,2C,3A,3B,3C,3D nonstructural protein.CVB5 mainly through the respiratory tract and digestive tract infection host,can cause hand,foot and mouth disease(HFMD),aseptic meningitis,myocarditis,type I diabetes and other diseases.In recent years,CVB5 in Asia Pacific and Europe and the United States has repeatedly caused HFMD and aseptic meningitis outbreak in China also showed an increasingly active trend.At present,due to the less research of CVB5,the virus infection and replication mechanism is not clear,usually the virus nonstructural protein in the virus proliferation and host disease process plays an important role.The virus can escape the immune response of host cells by regulating the expression of its own proteins,which can promote the replication of virus and persistent infection.The aim of this study was to investigate the effect of CVB5 on the interferon beta(IFN-β)in host cells and to play a major role in the nonstructural proteinThere are three parts in this research:1、Construction of eukaryotic expression vector of CVB5 nonstructural protein and its expression in HEK293T cellsThe CVB5 virus stored in the laboratory was inoculated on HEK239T cells for proliferation.The results of VP1 sequencing were compared with those of CVB5 sequences in GenBank,and the results were the highest(99%)with sequence<JX843811.1>.Therefore,according to the sequence of nonstructural protein in sequence<JX843811.1>The lab’s CVB5 nonstructural protein amplification primers.Viral RNA was extracted from CVB5 and amplificate the nonstructural proteins after the reverse transcription.Since the base sequence of the nonstructural protein 3B is too short,it is co-amplified with the 3A protein(i.e.3AB).Insert the genes of the six nonstructural proteins(2A,2B,2C,3AB,3C,3D)into the tag vector pcDNA3.1-2Flag to construct the eukaryotic expression vector(pcDNA3.1-2A/2B/2C/3AB/3C/3D-2Flag).The eukaryotic expression vectors of six nonstructural proteins were identified by PCR,double digestion and sequencing analysis.Six eukaryotic expression vectors were transfected into HEK293T cells,and the eukaryotic expression vector constructed by Western blot could be stably expressed in eukaryotic cells.2、Effects of CVB5 on different levels of IFN-β in HEK293T cellsThe transcription factor expression plasmid(pIFN-β-Luc)and reporter gene plasmid(pRL-TK)were transfected into HEK293T cells.After 24h of transfection,the cells were inoculated with different infection complexes(OMOI,0.01MOI,0.1MOI)CVB5,after 12h,the samples were harvested for double luciferase assay to analyze the effect of CVB5 on promoter level of IFN-β.Harvest samples after 6h/12h infecting with different multiplicity of CVB5(OMOI,0.01MOI,0.1MOI)to HEK293T cells,RNA detected by real-time quantitative PCR detection and analysis the effects of CVB5 on the mRNA level of IFN-β.And the effects of CVB5 on the mRNA level of IFN-β upstream transcription factor IRF-3.Harvest samples after 12h infecting with different multiplicity of CVB5(OMOI,0.01MOI,0.1MOI)to HEK293T cells,the effects of CVB5 on the expression of EFN-βand IRF-3 protein were analyzed by WB.3、Exploration of specific CVB5 nonstructural proteins that play a role in IFN-βThe six kinds of non-structural protein eukaryotic expression vector was transfected into HEK293T cells after transfection of 24h samples were harvested,RNA detected by real-time quantitative PCR detection,non structural proteins play a major role of IFN-β mRNA level analysis in CVB5.Results:3AB,3C and 3D in six kinds of nonstructural proteins of CVB5 could inhibit the expression of IFN-P mRNA in cells,and the inhibitory effect of 3AB protein on IFN-β was the strongest.Analysis of non structural proteins that play a major role in EFN-β upstream transcription factor IRF-3 mRNA in CVB5.Results:six kinds of non structural proteins of CVB5 could inhibit the level of IRF-3 mRNA,and 3AB,3C and 3D protein had the most significant inhibitory effect on the level of IFN-β mRNA.In this paper,the eukaryotic expression vector of CVB5 was constructed successfully and expressed stably in HEK293T cells.It was confirmed that CVB5 had no effect on the level of IFN-β promoter,and had a significant inhibitory effect on the mRNA level of IFN-β.By further examination,it was confirmed that CVB5 had the same inhibitory effect on the mRNA level of IFN-β transcription factor IRF-3.The major nonstructural proteins of CVB5 which inhibit the level of IFN-β and IRF-3 were 3AB,3C and 3D in mRNA.These results lay the foundation for the further study of the function of CVB5 nonstructural proteins and the regulation of viral proteins on natural immune responses. |
