| Norovirus is an important zoonotic virus,which can infect people,pigs,dogs,cattle,cats and rodents.Norovirus belongs to the Caliciviridae family and is a non-enveloped virus composed of a single stranded plus RNA genome.Norovirus is currently divided into 10 gene groups(GI-GX).These genotypes were further subdivided into more than 49 genotypes.Genomes GI,G? and GIV can infect humans.Since 2002,G? genotype 4(G?.4)has been the most commonly detected genotype in outbreaks and is associated with a large number of AGE cases worldwide.However,since 2014,the G?.17 genotype has received more attention as the genotype has spread from Asia to other countries.In the winter of 2016,the G?.P16/G?.2 genotype was widely prevalent in Asian countries.In this study,the preparation of norovirus prokaryotic expression protein and ELISA method were used to establish the purification and immune evaluation of norovirus viruslike particles as well as the study of the cellular mechanism of antigen presentation,so as to determine the immune effect and antigen presentation mechanism of the candidate vaccine of G?.P16/G?.2 recombination norovirus.Firstly,the VP1 gene of norovirus was amplified and cloned into p ET32a(+)vector to construct the recombinant plasmid p ET32a-VP1.PET32a-VP1 was transformed into Escherichia coli BL21(DE3),and IPTG was added to induce the expression and optimize the induction conditions.The purified protein was purified by His labeled nickel ion protein purification column,and the purity of the protein was determined by SDS-PAGE.The purified protein was mixed with Freund's complete adjuvant,and BALB/C mice were immunized by intraperitoneal injection to prepare polyclonal antibodies and establish an ELISA method.Then,norovirus virus-like particles were prepared by SF9 suspension cells in large quantities,and purified by Q Focurose HPR purification column.Polyclonal antibodies were prepared by using the purified virus-like particles.Finally,dendritic cells and macrophages were isolated from mice,and virus-like particles were co-cultured with them to observe changes in the levels of related cytokines to explore the mechanism of virus-like particles in vivo.The results showed that the norovirus VP1 gene with a size of 1 659 bp was successfully amplified.The recombinant plasmid p ET32a-VP1 was successfully constructed by PCR detection.The recombinant plasmid was transformed into BL21(DE3),and the 70×103 VP1 recombinant protein was induced by IPTG.The optimal induction conditions were 37 ? and 0.6 mmo L/L IPTG for 5 h.The recombinant protein was expressed in the form of inclusion body,and the purified production was identified as the target protein of a single band by SDS-PAGE.The results of established ELISA method showed that the optimal encapsulation concentration of recombinant protein VP1 was 2.5?g/m L,the optimal dilution of serum was 1:100,the optimal incubation time was 1.5h,and the titer of polyclonal antibody was 1:1638400.SF9 suspension cells successfully expressed virus-like particles,and SDS-PAGE identified the anion chromatographic column as a single electrophoresis target band.The titer of mouse immunoassay polyclonal antibody was1:204800.In vitro experiments showed that norovirus VLP could be recognized by DC cells,but could not effectively activate DC cells and induce DC cell maturation.Norovirus VLP could be recognized by Macrophages and effectively activate them.Activated Macrophages overexpress their surface MHC-?,CD40,CD80,and CD86,promote Macrophages recognition and phagocytosis of VLP,and secrete cytokines such as IL-6,IL-12p70,and TNF-? to promote antigen presentation of Macrophages.Macrophages can present norovirus antigens to Naive CD4+T cells to induce differentiation of Th0 cells towards Th1(cellular immunity)and Th2(humoral immunity)cells,with a preference for Th1(cellular immunity).VLP can induce Macrophages from M2 type to M1 type.Norovirus VLP activates Macrophages through the NLRP3 pathway for T cell antigen presentation. |
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