Studies On The Mechanism Of The Coxsackie Virus B5 On The NF-?B Pathway

Coxsackie virus(Coxsackievirus,CVB),which belongs to the enterovirus genus,can cause colds,aseptic meningitis,myocarditis,epidemic pleurodynia.Coxsackie virus is divided into A,B two groups,A group including 1-22 and 24 type;B group including 1-6 type.Among them,coxsackievirus group B type 5 has become an important pathogen of hand foot mouth disease in China.Coxsackievirus B group 5 is divided into 5 'untranslated region(untranslated,region,UTR),3'-UTR and P1,P2,P3 encoding.P1 protein encoding region of capsid protein VP1,VP2,VP3 and VP4,P2 and P3 encoding region of non structural protein 2A,2B,2C,3A 3B,3C and 3D.Natural immunity is the body's first line of defense against invading pathogens.The host through the recognition of pathogen associated molecular patterns trigger a signaling cascade induced by type I interferon gene expression and inflammatory reaction,start the antiviral innate immune response and activate the acquired immune response,get rid of the infection.At the same time,the non structural protein encoding some viruses may protein factor and pathway of interaction,blocking the expression and inflammatory reaction type I gene interference occurred.At present,the research on CVB5 less,virus infection and replication mechanism is not clear,usually virus non structural protein in virus,play an important role in the replication process.Therefore,research on the influence of CVB5 on the innate immune response,and CVB5 which non structure protein of the innate immune response caused by the impact,provide theory based on the occurrence and propagation will prevent and control CVB5.This study includes three aspects:1)identification,culture and purification of coxsackievirus group B type 5.The experiment of extracting preserved in our laboratory CVB5 virus strain RNA,and reverse transcriptase.Using cDNA as a template,sequenced.The sequencing result in NCBI blast comparison,determine the laboratory preserved strain is coxsackievirus group B type 5.CVB5 in HEK293T extended culture and virus concentration respectively by three methods of purification,were polyethylene glycol 6000 concentrated isopropanol precipitation,PEG 6000 concentration of sucrose density gradient centrifugation and 50kD dialysis purification,preliminary verification and the infection of the virus.Methods find a relatively low loss,determination,and virus titer of the purified virus,OMOI,0.01MOI,0.1 MOI and 1MOI virus respectively in Oh,6h,12h,24h,The validation of 6h and 48h in the pathogenesis of the disease has laid a foundation for the further study on the effect of CVB5 on the NF-?B pathway in the innate immune response2)the effect of coxsackievirus B group on the NF-?B pathway in innate immune response.The first pre detected by luciferase reporter gene CVB5 of NF-?B promoter level of innate immune pathway.By CVB5 MOI and CVB5 were inoculated with different inoculation at different time to detect NF-?B promoter 0.01 MOI CVB5 levels found in the inoculated 12h when compared with the control group significantly decreased the NF-?B promoter.Through inoculation of CVB5 detection of p65 and TNF-? level of mRNA found that 0.01MOI CVB5 can significantly reduce the p65 and TNF-?mRNA levels in 12h after inoculation,and as the incubation time increased p65 and TNF-? mRNA levels also increased.The protein levels were inoculated with CVB5 p65 detection found that 0.01MOI CVB5 can significantly reduce the protein levels of p65 in 12h after inoculation.Combined with the experimental results of promoter level,mRNA level and protein level,it can be concluded that coxsackievirus group B type 5 is an inflammatory response that can reduce the NF-?B pathway in the innate immune response.3)the effects of the non structural proteins of coxsackievirus B group on the NF-kappa B pathway in innate immune response.Six kinds of non structural protein preserved in our laboratory CVB5 eukaryotic expression vector pcDNA3.1(+)-2A-2Flag?pcDNA3.1(+)-2B-2Flag?pcDNA3.1(+)-2C-2Flag?pcDNA3.1(+)-3AB-2Flag?pcDNA3.1(+)-3C-2FlagandpcDNA3.1(+)-3D-2Flag were transfected into HEK293T cells,detect the effeect of p65 and TNF-alpha mRNA levels.The results showed that 3D protein can significantly reduce the p65 level of mRNA,3AB protein can significantly reduce TNF-alpha mRNA levels.You can determine the coxsackievirus B 5 can reduce the influence of natural immune response in NF-kappa B pathway through this research,and identified some non viral structural protein play an important role in the process,lay the foundation for further study on Mechanism of the process of virus infection.