文献汇报鸭坦布苏病毒多表位核酸疫苗.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,*,abstract,Tembusu virus(TMUV)is a newly emerging pathogenic flavivirus that is causing massive economic loss in Chinese poultry industry;,until now,there is no effective vaccine or drug for its prevention.,Epitope-based vaccination is a promising approach to achieve protective immunity and to avoid immunopathology.In present study,based on in,silico epitope selection,we optimized and proposed a polytope DNA vaccine(pVAX1-rTEM)consisting,B-cell and T cell epitopes,from the TMUV envelope(E)protein.The immunogenicity and protective efficacy of constructed polytope DNA vaccine was assessed by in vitro and in vivo experiments.In in vitro assays,the expressed pVAX1-rTEM showed reactivity with Tembusu positive serum.Its protective efficacy against TMUV infection was evaluated in ducks.,The results showed that pVAX1-rTEM was highly immunogenic and could elicit high titer neutralizing antibodies and cell-mediated immune responses.These results indicate that pVAX1-rTEM may be a promising candidate vaccine for prevention of TMUV infection.,引言（节选）,the envelope(E)protein of TMUV is the major surface protein of the virion that mediates binding to the cellular receptor and subsequent fusion event between viral and host.Meanwhile,the flavivirus E protein is the major antigenic target of neutralizing antibodies,which have many significant epitopes.,引言（节选）,DNA vaccines,such as epitope-based vaccines,are a promising area of research due to their remarkable safety,profile and ease of manufacture,relative to proteins and other biologics.,This vaccine is capable of stimulating effective B-cell,T-cell and cytotoxic immune responses while avoiding potentially hazardous and undesirable side effects.,Construction of polytope insert,Firstly,the B cell epitopes of E protein were predicted using program Predicting Antigenic Peptides(imed.med.ucm.es/Tools/antigenic.pl).,T cell epitopes were predicted by RANKpep online Software(bio.dfci.harvard.edu/RANKPEP).,To minimize interference between adjacent epitopes,each was separated from its neighboring epitope by a glycine and a serine codon.,Plasmid construct,A,510 bp synthetic gene,was constructed by assembling a tandem array of epitopes from the TMUV E protein.This consisted of six B cell amino acid(aa)sequences,two T cell sites,and a universal T cell help epitope,(PADRE motif).,The inframe fusion of the,Kozak motif,with the initiation codon of polytope gene was confirmed by DNA sequencing,in order to enhance the level of transcription.To increase the immunogenicity of the synthetic polytope,the Cytidine Phosphate Guanosine,(CpG)motif,was introduced downstream.Amino acid linkers,(glycineserine),were incorporated between the two neighbor epitopes to ensure,maintenance of natural conformation,.In end,the nucleotide was synthesized by Genscript Biotechnology Co.Lid(Nanjing,China)and was cloned into the pUC plasmid and named pUC-rTEM.,Plasmids for DNA immunization,The rTEM gene was subcloned from the synthesized pUC-rTEM.,Amplicon cloned in frame into the eukaryotic vector pVAX1(+)according to the standard method.,Transfection,Identification of rTEM by RT-PCR and Western blotting,Duck vaccination experiment,ELISA for serum antibody titers,Neutralizing activity of antibodies against DTMUV JS804,Cytokine assays,Statistical analysis,结果讨论致谢参考文献,