Macromolecular Modification And Biological Effect Analysis Of Capsid Protein Of Porcine Circovirus

Porcine circoviruses belongs to genus Circovirus of the family Circoviridae,are icosahedral nonenveloped,with a size of about 17nm containing a circular single stranded DNA molecule of about 1.7 Kb.Porcine circovirus type 2(PCV2)is mainly associated with a disease was called postweaning multisystemic wasting syndrome(PMWS).PMWS is currently considered to be a vital infectious swine viral disease that has a serious economic impact on the cosmopolitan pig farming industry,so the vaccine of porcine circovirus is research and development will bring great economic benefits for the worldwide pig industry.Open reading frame 2(ORF2)of PCV2 encodes a main structural capsid(Cap)protein,which is also the major antigen of virus and inducing protection of pig against PMWS.The expression of PCV2-Cap protein in vitro can assemble spontaneously into virus like particles(VLPs).VLPs have been widely used as vaccine because of it have high safety and trigger a forceful immune response.The recombinant protein of PCV2?Cap42—233 was optimized by bioinformatics and is achieves successfully in classic Escheichia coli expression system.PCV2?Cap42—233 protein of inclusion body expression was dissolved by high concentration urea,then was isolated,purified by Ni2+ affinity chromatography.The urea of PCV2?Cap42—233 protein solution was removed by gradient dialysis,then we obtains the water soluble PCV2?Cap42—233 protein nanoparticles without urea.The components of buffer should be optimized by experiment and we obtains PCV2?Cap42—233 protein nanoparticles of analogous VLPs.Mannose receptor(MR)is a surface receptor and is mainly expressed on antigen presenting cells(APCs)in the immune system,it increased the capacity of uptake,processing and presentation of antigen presenting cells for mannosylated protein.Mannosylated protein can target mannose receptors to stimulate a more efficient immune response.Oxidized mannan was obtained by sodium periodate under certain conditions.Which need monosaccharide of mannan have a structure of containing two aldehyde groups.Mannosylated PCV2?Cap42-233 protein nanoparticles of analogous VLPs containing Schiff bases,was prepared by aldehyde groups of oxidized mannan convalent attachment to the amine groups of PCV2?Cap42—233 protein nanoparticles of analogous VLPs.Then we determine the Schiff base molecules of mannosylated PCV2?Cap42—233 protein nanoparticles of analogous VLPs was degraded at the Lysosomal environment of pH=5.0,the protein was released from mannosylated protein at the Lysosome.Therefore,mannosylated PCV2?Cap42—233 protein nanoparticles of analogous VLPs not only have capacity to recognize mannose receptors on antigen presenting cells,but also have the characteristics of the nano-sized delivery system is gated by pH-sensitive dynamic covalent bond.PCV2?Cap42—233 protein nanoparticles of analogous VLPs and mannosylated PCV2?Cap42—233 protein nanoparticles of analogous VLPs together with different adjuvants,the subunit vaccines were prepared.Then mice were immunized by intraperitoneal injection with the subunit vaccines we obtained.The concentration of PCV2 antibody,IL-4,IL-2,IFN-? in mice was detected and we find that mannosylated PCV2?Cap42—233 protein nanoparticles of analogous VLPs could target to APCs and mannosylated antigens can enhance significantly the humoral immune.