Construction And Immunogenicity Of PCV2 Vaccine Using Baculovirus System

Porcine circovirus type 2 (PCV2) can cause an infection both separately and secondarily. In recent years, the infection by PCV2, including post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis nephropathy syndrome (PDNS), reproductive failure, porcine respiratory syndrome complex (PRDC), piglet congenital tremor, proliferative necrotizing pneumonia (PNP), "unknown hyperthermia ", is prevalent throughout the world and greatly harmful to the pig industry. Clinically, PCV2 extensively leads to immune suppression and immune activation, reducing immunity of the pigs, slowing down the growth of pigs and causing a large outbreak of other diseases.At present, there is no effective treatment for the disease, and vaccines are required for prevention. In Western Europe and the United States, there have been commercial vaccines in the market, mainly consisting of inactivated vaccine and subunit vaccine. In domestic market, inactivated vaccines are also available. However, due to PCV2's low titer in cell proliferation, difficulty of in vitro culture, the high cost of inactivated vaccine, the induction of body stress due to injection of multiple times, the development of cheap, safe and effective new vaccine is the focus of the study.This study explored the new PCV2 vaccine by constructing the recombinant baculovirus vaccines and ORF2 subunit vaccine, and investigated the immunogenicity of the two vaccines by mice experiments. The work in detail is as following:1. The construction of ORF2 recombinant baculovirus and the verification of its immunogenicitypcDNA3.1-iel gene was synthesized, PFastBac-VSV/G-SV40 vector (BamHI) pcDNA3.1--iel (Bg1II, BamHI) were digested and connected. The CMV promoter on the vector is replaced by iel promoter. Gene encoding EGFP was inserted into the baculovirus genome under the control of the iel promoter, to acquire the recombinant baculovirus AC-V-EGFP. Similarly, the complete PCV2 ORF2 reading frame was inserted under the iel promoter to obtain the recombinant baculovirus AC-V-ORF2. These two recombinant baculovirus were transducted to mammalian cells and transfected into Sf9 cells in vitro, respectively and high expression of target genes was detected. The recombinant baculoviruses were injected intramuscularly to mice, with a dose of 10 pfu/mL 100ul. Three weeks after the first immunization, ELISA antibody and neutralizing antibody were tested. Three weeks after the strengthening immunization, the mean ELISA antibody titer of Ac-V-ORF2 group reached 1:1152, and the neutralizing antibody titer reached 1:6. Therefore, Ac-V-ORF2 has shown a nice immune effect, suggesting that it could serve as a new PCV2 vaccine candidate.2. The construction of Baculovirus-mediated ORF2 subunit vaccine and the study on its immunogenicityPCV2 ORF2 gene was synthesized and transfer vector pFastBacDual-2ORF2 was constructed. This vector was transformed into DH10Bac E.coli competent cells, and the shuttle plasmid Bacmid-2ORF2 was obtained by blue-white screening. The vector was transfected into Sf9 cells to obtain baculovirus Ac-2ORF2. When this baculovirus was transfected into Sf9 insect cells, ORF2 gene was abundantly expressed in the cells. The expressed ORF2 protein was prepared as subunit vaccine to immunize 4-6 weeks old BALB/c mice in three doses:106cells/mL,5 x 106cells/mL and 107cells/mL. The results showed that three weeks after the first immunization, ELISA antibody and neutralizing antibody were detected in mice vaccinated with 106cells/mL,5×10 cells/mL and 107 cells/mL. After the booster immunization, antibody levels in these three groups continued to increase. The highest level was in the group of 5×106 cells/mL, in which ELISA antibody titer was up to 1:2133, and neutralizing antibody titer was up to 1:8.33. The results indicated that the ORF2 protein expressed by baculovirus system has nice immunogenicity, and the vaccine can be considered as a new PCV2 subunit vaccine candidate.