Expression Of Porcine Circovirus Type 2 CAP Gene In Pichia Pastoris And Development Of Water Solubility Adjuvants

Porcine circovirus type 2 (PCV2) can cause a series of diseases, collectively known as porcine circovirus associated disease (PCVAD), including postweaning multisystem wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). PCV2 can damage the immune system of pigs, causing mixed or secondary infection of porcine parvovirus, Haemophilus parasuis, pasteurellosis and other diseases, which causing huge economic losses to pig industry. Cap protein encoded by ORF2, is the main structural protein of PCV2, possessing PCV2 major epitopes, and becomes the main target for development of PCV2 vaccines and serodiagnostic methods. In this study, a new water solubility adjuvant CPH was explored and used in PCV2 vaccine. And PCV2 Cap protein was expressed in Pichia Pastoris expression system. The main contents were as following:1. Development of new PCV2 inactivated vaccine adjuvantAdjuvants of CP (aqueous high-molecular polymer)、CPH(CP as basic adjuvants added protein H) and CPT(CP as basic adjuvants added protein T) were evaluated by mice immunization experiment, the results showed that CPH could enhance the antibody levels induced by inactivated PCV2. Then optimum concentration of adding protein H was screened, and ultimately a comparison was analyzed with oil adjuvants (ISA206 and Gel) as control. The results showed that carbomer with 0.8mg/mL protein H provided the highest enhancement better than Gel oil adjuvants and similar with ISA206 both in cellular and humoral immune responses. The pig experiment results of PCV2-CPH、PCV2 Cap-CPH and PCV2-ISA206 vaccines indicated that all these vaccines can induce similar antibody levels, and provide similar protective immune against PCV2 challenge. CPH adjuvant don’t cause any serious daminge reactions upon intramuscular administration in animals and cost lowly. Hence, it may provide an usfull adjuvant for the future PCV2 vaccine applications.2. Expression of the Cap pritein of PCV2 in pichia pastorisThe recombinant plasmid pPIC9K-Capo (containing the complete PCV2-Cap gene sequence) and pPIC9K-Capo△NLS (containing the complete PCV2 Cap genes with deletion of NLS) were constructed according to the codon bias of the methylotrophic yeast P. pastoris. Then the recombinant vectors were transformed into P.pastoris GS115 by electroporation, the recombinant yeast strains, GS 115/pPIC9K-Capo and GS115/pPIC9K-Capo△NLS, were screened.The results of SDS-PAGE showed that the size of the recombinnnat secreting products Capo and CapoANLS was approximately 43kd and 27kd, respectively. The recombinant Capo protein was larger than the real Cap of PCV2 by about 10kd. It indicated that NLS may affect on the expression of the Cap protein in pichia pastoris. And CapANLS protein (without NLS) was consisting with the expected size and was well secreted. Western-blot results confirmed that both Capo and Cap△NLS proteins expressed by Pichia expression system had the antigenic characteristics of PCV2. However, electron microscopy observation results showed that only Capo proteins formed virus-like particles. It made an important foundation for PCV2 subunit vaccine based on the Cap expressed in pichia pastoris.3. Immunogenicity of recombinant Cap protein expressed in Pichia expression systemAntigenic characteristics of Cap protein expressed in Pichia system were analyzed through the methods of sandwich ELISA. The results showed that both the Capo and CapANLS protein expressed in yeast expression had antigenic properties, and the deletion of NLS would reduce the antigenic reaction. Different vaccines were prepared by mixing CPH with Cap expressed in Pichia pastoris、Cap expressed in baculovirus expression system and inactived PCV2. Immunization experiments showed that all the vaccines can induce high levels of PCV2 antibodies and strong lymphocyte proliferation responses, however the Capo△NLS +CPH group induced the lowest level of neutralizing antibodies. These illustrated that neutralizing antibodies may have relation with NLS. It made a foundation on the molecular designing for developing subunit vaccine against PCV2.