Expression Of Porcine Circovirus Type2Cap Protein In Silkworms And Immune Efficacy Of Its Subunit Vaccine In Mice And Piglets

Porcine circovirus type2(PCV2) is the primary pathogen of postweaning multisystemic wasting syndrome (PMWS) and other PCV2associated disease (PCVAD). Vaccination is one of major measures for preventing these diseases. In this study, silkworm system was constructed to express Cap protein of PCV2and the expressed proteins were used in order to prepare the inactived vaccine against PCV2. Meanwhile, the immunogenicity of the prepared vaccine was analyzed in pigs and mice.Lungs, spleeds, livers and lymph nodes were collected from the pigs with clinical symptoms of PMWS and PCR was performed for PCV2detection in these tissues. PCV1-free PK-15cells were inoculated with the PCV2-positive samples to isolate PCV2. Five isolates of PCV2, which were designated as PCV2-1, PCV2-2, PCV2-3, PCV2-4, PCV2-5respectively, were obtained and confirmed by IFA, IPMA, and immunoelectron microscopy.The cap gene without nucleus-located signs was cloned and expressed in E.coli fistly.Then the expressed protein was purified and used to prepare the antibody in rabbit.The antibodies with the titer more than1:3200were used to detection of expressed Cap protein in silkworm. The cap gene of PCV2was amplified by PCR and the cap gene mutant was reconstructed artificially. The mutant of cap gene was cloned into the baculovirus carrying vector of silkworm system. The constructured vectors were homologously recombinated with the parental viruses in order to acquire the recombinant baculovirus. Then the Cap protein was expressed in the silkworm which was infected by the recombinant baculovirus containing the cap gene of PCV2. The purified protein was confirmed to be a fusion protein with a titer of1:3200by ELISA. The expression were proved to be PCV2empty particles mainly. It was estimated that the expression concentration of the empty particle of PCV2in the skillworm could be1.2-1.6mg per silkworm. The complete empty particle with a diameter of10nm was also observed by electron microscopy and colliadal gold immuno electron microscopy.Five-day-old baby silkworms or the lymph-blood were inoculated with105pfu recombination baculovirus. The infected silkworms expressing the Cap protein were collected and mixed with PBS by1:10(g/v), and then broken, and further subjected to filtration, centrifugation and inactivation. Inactivated vaccine was prepared by means of emulsification with206adjuvant.Mice were vaccinated by the PCV2Cap protein sub-unit vaccine. Antibody in sera and PCV2antigen in tissues of the vaccinated mice were detected to evaluate the immunogenicity of the vaccine.Meanwhile, porcine circovirus type2baculovirus vector vaccine produced by Boehringer Ingelhein Vetmedica, Inc, the healthy silkworm and non-vaccinated mice were served as controls. The results showed that the inoculated mice could generate specific antibody against PCV2, with a positive rate of10/10and a ELISA titer ranging from1:800to1:3200, which was similar to those of baculovirus vector vaccine group. PCV2antigen in tissues of the vaccinated mice showed negative in all vaccinated groups, and10/10positve rate in control group.14to21-day-old pigs without PCV2antibody were also vaccinated. Antibody in sera and PCV2antigen in tissues of the vaccinated pigs were detected. Porcine circovirus type2baculovirus vector vaccine produced by Boehringer Ingelhein Vetmedica,Inc and the healthy silkworm were served as controls.The results showed that the vaccinated pigs could induce specific antibody against PCV2. The positive rate of PCV2antibody was5/5in group administered with the vaccine of batch201001, with a lightly lower titer ranging from1:320to1:640, than group given with baculovirus vector vaccine. PCV2antigen detection in tissues of the immunized pigs showed0/5positive in group given with the vaccine of batch201001and5/5positive in control group.As a whole, PCV2Cap protein sub-unit vaccine could induce specific antibodies against PCV2, which was able to inhibit the replication of PCV2in mice and pigs, indicating that the porcine circovirus type2empty capsid particle expressed by silkworm system presents better immunogenicity and potential as effective vaccine.