Research On Flagellin Used As Molecular Adjuvant In Epitopes Vaccine

As the ligand of TLR5, flagellin is a powerful immune agonist which couldactivate a broad range of cells including innate and adaptive immune cells.Researches indicated that flagellin can boost cytokines production by innate immunecells, induce T and B lymphocytes migration toward to secondary lymph nodes, andTLR5+CD11+and T lymphocytes activation. The N and C terminals of flagellin, witha composition of the highly conservedα-helix, are the recognition sites of TLR5.While the middle sequences, with hypervariable region which can represent theantigenicity of flagellin, could be replaced by foreign epitope fragments. Thestructural plasticity of flagellin promises constructing multiple flagellin-relatedfusion proteins, and their function have been observed in animal experiments.Moreover, the C terminal of flagellin is a stimulator for inflammasome Nlrc4, whichis a part of body defense by actives apoptosis signal pathway.Porcine circovirus type2is the main pathogen causing post weaningmultisystemic wasting syndrome(PMWS) in pig. In the past15years, PCV2infection and PMWS outbreaks around the whole world caused serve economic loss.As the main antigenicity of PCV2, capsid protein(CP) includes A,B,C,D,E immuneepitopes domain, which could be recognized by PCV2antibodies.The purpose of this study is constructing flagellin-virus epitope fusion proteins,used as epitope protein vaccine, observing the immune stimulated effect of flagellinpart used as a molecule adjuvant. The53-147amino acids from PCV2CP wereselected as virus epitope peptide, given a name as Pd, used to replace the middlesequences (190-405amino acids) of flagellin, and the given a named as NPC. At the same time, a fusion between virus epitope peptide Pd and flagellin C-terminalfragment was made with a named as CP, which aimed to explore the impact ofinflammasome activation on immune system.Virus epitope Pd was synthesized by forth-round overlapping-PCR and thencloned into pET28a vector. The N and C terminal fragments of flagellin wereacquired by PCR with a plasmid containing flagellin gene as template. By digestionand ligation, resultant expressed plasmids contain flagellin N and C fragment-virusepitope (pET28a-NPC) and flagellin C fragment-virus epitope (pET28a-CP) wereacquired. After expressed in BL21, the fusion proteins were purified by Ni2+affinitychromatography and molecular chromatography, and used to immunize mice forevaluating their immunal function.Mice were immunized with NPC and CP with or without adjuvant206on day1,21. On day-1,7,14,28,35, the mice were bled for collecting sera. By ELISA test,PCV2specific antibodies in mice sera were evaluated.The main results include:Flagellin NC fragments-virus epitope fusion proteins NPC has goodimmunologic cross-reactivity with anti-PCV2antibodies.Flagellin NC fragments-virus epitope fusion proteins NPC and flagellin Cfragment-virus epitope fusion protein CP both have good immune antigenecity,while virus epitope peptide Pd alone has poor immune antigenecity.The recognition of anti-serum derived from mice immuned with NPC and CPwith PCV2VLP is quite high, NPC has a potencial to be a epitope protein vaccinefor PCV2. Flagellin NC fragments could be an effective molecular adjuvant.