The Preparation And Immunogencity Study For PCV2Virus Like Particles Vaccine

Some kinds of diseases are related with porcine circovirus type2in porcine groups including postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephrophathy syndrome (PDNS), congenital tremor (CT) etc, which have caused significant wasting losses to the swine industry in many parts of the world. At present, the immune effect of domestic traditional inactivated vaccine is not very good, and the price of overseas PCV2subunit vaccines is too expensive. So it is important for us to develop effective, low-cost and safe PCV2subunit vaccine. The ORF2of PCV2encodes the major structural capsid (Cap) protein, which is also the major immunogenic protein, has the ability to independently self-assemble and form virus-like particles (VLPs) in vitro. We successfully make the Cap protein highly soluble in Escherichia coli, which provide an good way for developing the PCV2VLP vaccine.A pairs of primers was designed and the ORF2gene was amplified from PCV2NJ strain genome DNA as template. The ORF2gene of PCV2NJ Strains was optimized and synthesized according to the E.coli codon usage preference, which were inserted into a prokaryotic expression vector pQZ1, and transformed into DH5a competent calls; the positive plasmid was designated as pQZ-Cap, which was transformed and expressed in BL21competent cells; The protein was identified by SDS-PAGE and Western-blot with monoclonal antibody; Results showed that there was a more significant band at about27kDa conforming to the expected size, which indicated that the gene could be expressed in E.coli. Western-blot analysis showed that the products has highly solubility and good reacting ability.Simultaneously, the recombinant Cap protein self-assembled into VLPs in vitro when observed through electron microscope. The Cap-like particles had a diameter of17nm, a shape that resembled PCV2Cap, and reacted with the PCV2serum, forming the antigen-antibody complex. The Cap protein as an antigen, different dose after the quantity by UV spectrophotometer emulsified with SPPEIC206adjuvant to prepare experimental vaccines, which was used to immunize two-three weeks piglets, and the commercial vaccine was used as control antigen. ELISA antibody titer for each group was detected at14days and21days following vaccination to evaluate the humoral immune response. Results indicated that swine immunized with this VLP vaccine generated perfect antibody response with100%percent qualified at21days post one sole vaccination and could be completely protected from pathogenic virus attack. In addition, we found that the immunopotentiator CVC1302could greatly boost the immune response against the vaccine when compared with groups without CVC1302. In summary, the VLP particles pose perfect immunity and could be used as subunit vaccines to prevent PCV2infection.Relations between expression yield and growth conditions was studied in this paper, and it is found that the optimal expression condition for the recombinant protein were:2%transfer,37℃,220rpm, when LB medium with OD600being5.0, the recombinant strains was placed in15℃for half past hour. Then the solution was add to IPTG which is0.2mmol/L and incubated24h in15℃. After induction under optimal conditions and ultrasonic disruption, we can obtain the more ORF2recombinant protein.