Preparation And Immunogenicity Evaluation Of Porcine Circovirus Type 2 Virus-like Particles

Postweaning Multisystemic Wistar Syndrome(PMWS)is mainly caused by Porcine circovirus type 2(PCV2),and 6~16 weeks of age weaned pigle ts are prone to the disease.The mainly clinical symptoms is slow growth,difficulty breathing,severe diarrhea,jaundice,extremely swollen lymph nodes.Since the first report in China in 2000,PCV2 infection has spreaded more than 20 provinces,autonomous regions and municipalities,and the positive infection rate in the herds has range from 21% to 96% to date.What's more,PCV2 can destroy the host immune system,leading to immunosuppression and resulting in mixed infection with other diseases.Therefore,PCV2 infection has caused huge annual economic losses to swine industry.At present,there is no effective means for the disease treatment,and vaccination has been proved to be the most effective approach for the disease prevention and control.The traditional inactivated vaccine has the problems of low virus proliferation titer,high production cost,and the risk of inactivation,so it has been gradually replaced by subunit vaccine,especially virus-like particles.Cap protein can be assembled into VLPs because it is the only structural protein of the circovirus and can induce complete protective immunity against PC V2 infection.In this study,based on the the cap sequence of PCV2 laboratory stock,cap gene with yeast codon optimization was synthesized and secretory expression in Pichia pastoris expression system for VLPs assembly.Animal experiments were carried out for evaluation the immunogenicity of PCV2 VLPs in mouse model.This study has achieved the following results:1.The recombinant plasmids p PICZ?-A-AHCap,p PICZ?-A-AHCap-Fla,p PICZ?-A-SUMO-AHCap and corresponding yeast expression strains GS115-p P ICZ?-A-AHCap,GS115-p PICZ?-A-AHCap-Fla,GS115-p PICZ?-A-SUMO-AHCap were successfully constructed.2.The recombinant proteins AHCap,AHCap-Fla,SUMO-AHCap were expressed in secretory form by yeast expression strains GS115-p PICZ?-A-AHCap,GS115-p PICZ?-A-AHCap-Fla,GS115-p PICZ?-A-SUMO-AHCap,respectively.3.The results of transmission electron microscopy showed that the Cap protein(SUMO-AHCap)with S UMO tag was not formed VLPs,while AHCap at the N-terminal deletion of 32 amino acids and AHCap(AHCap-Fla)at the C-terminal fusion flagellin adjuvant can clearly see VLPs with a diameter of about 20 nm.The recombinant protein AHCap formed more VLPs than AHCap-Fla.4.The purified recombinant protein AHCap and AHCap-Fla was quantified,and the immunogenicity was detected by mouse immunization.The results of indirect ELISA showed that PCV2 specific antibody was raised in the blood of the experimental group(AHCap and AHCap-Fla vaccination group),the expression of IFN-? and IL-2 was increased,and the virus was not detected in the serum.Meanwhile,the antibody titer and cytokines exoression(IFN-??IL-2)in AHCap-Fla immunization group was higher than that of protein AHCap immunization group,indicating that the recombinant protein AHCap-Fla and AHCap had good immunogenicity,and the recombinant protein AHCap-Fla had better immunogenicity.In summary,the recombinant protein AHCap and AHCap-Fla expressed by Pichia pastoris expression system could self-assemble into VLPs and had good immunogenicity,which provides a new idea for the development of PCV subunit vaccine.