Expression Of Reconstructed PCV2b Virus-like Particles In Eukaryotic System And Their Immunogenecity

Currently,the virus-like particles(VLPs)vaccine is one of the main direction in the development of the vaccine.The structural protein of the virus spontaneously forms VLPs,and it could highly imitate the structure of natural virus.So it could stimulate the body to produce last and efficient immune response.The VLPs vaccine that used for the prevention and control of the HBV and HPV are already commercialized.The VLPs vaccine used for influenza virus and HIV are also in the stage of research.PCV2 b capside protein(CP)could also assemble into VLPs.PCV2 b mainly infect the pigs,but the recent research showed that PCV2 b also have certain infectious on human.Currently there is no approved VLPs vaccine on the market.So we mainly engaged in the construction of reconstructed PCV2 b VLPs,and try to provide the experimental basis for other human or animal viruses on the research of VLPs vaccine by using the mature animal model experiment platform.The CP is the only structure protein and primary immunogenicity protein of PCV2 b,it could spontaneously forms VLPs with the structure of 20 surfaces.But the CP may be not completely assembled into VLPs,lead to the capsid protein exists in monomer form.However,the CP(169~180)of the capside protein is considered a decoy epitope,an immunodominant epitope,is not involved in protection,and diverts the immune responses away from protective epitopes.And it is a strategy to evade host immune responses,promoting disease or chronic infections.So,in order to prevent the decoy epitope exposed on the exterior surface to influence the activity of PCV2 VLPs vaccine,we reconstructed PCV2 b VLPs by replacing the decoy epitope CP(169~180)with a protective epitope CP(113~131)in Hansenula polymorpha expression system and Baculovirus Expression Vector System.And study the structure and immunogenecity of the reconstructed VLPs,analysis of the influence ofreconstruction.First,we expressed a PCV2 b capside protein by replacing the decoy epitope(named ?CP-H)and another protein by replacing the decoy epitope and deleting the NLS(named ??CP-H)in Hansenula polymorpha,and studied the structure and immunogenecity of the reconstructed VLPs.The results are as follows:(1)The CP could expressed a higher level without NLS;(2)We selected the highest expression level strain by q PCR;(3)We got the protein purified by Nickel affinity chromatography;(4)The reconstructed PCV2 capsid protein could assembled into VLPs under the transmission electron microscopy,but the structure of VLPs is not as compact as the capsid protein;(5)The CP(169~180)-directed antibody could induced by PCV2 b VLPs,but could not induced by the reconstructed VLPs.(6)The serum of mice immunized with reconstructed PCV2 b VLPs could induce higher PCV2 b antibody than PCV2 b VLPs.Then,we expressed PCV2 b capside protein by replacing the decoy epitope CP(169~180)with a protective epitope CP(113~131)in Baculovirus Expression Vector System,named ?CP-B.We also found that:(1)The reconstruction of PCV2 capsid protein affected VLPs formation;(2)PCV2b VLPs could induce CP(169~180)-directed antibody,but the reconstructed VLPs could not.(3)Also the serum of mice immunized with reconstructed PCV2 b VLPs could induce higher PCV2 b antibody than PCV2 b VLPs,In accordance with the reconstructed PCV2 b capsid protein expressed in Hansenula polymorpha.Through reconstructing the PCV2 CP in eukaryotic expression system,we solved the problem of the decoy epitope exposed on the exterior surface and improved the activity of VLPs vaccine.The significance of this study are as follows:(1)Laid a solid foundation on the development of veterinary and people used VLPs vaccine of PCV2b;(2)Provide a platform for the research on other VLPs vaccines used for prevention and control human viruses such as HIV and HCV;(3)The reconstruction strategy may also be adopted for others viruses like FMDV and HIV that manifest a decoy mechanism.