| Since the porcine circovirus type 2 was found and identified in Canada in 1991, porcine circovirus disease and its associated diseases including postweaning multisystemic wasting syndrome (PMWS), nephropathy syndrome (PNDS).congenital tremors of piglet (CT) have became epidemic worldwide and the pathogen——porcine circovirus type 2 (PCV2) has been reported one of the most economically damaging virus worldwide in swine industry. Now, commercial PCV2 vaccines have been verified the most effective strategy of preventing porcine circovirus disease and its associated diseases. Commercial vaccines including inactivated virus vaccines, genomic subunit vaccines, chimeric vaccines have many disadvantages such as complicated production process, inconvenient transport and storage, high immune response and so on, so it is the trend to research innovative vaccines which have high purity, low immune response, low inoculation dosage, and convenient transport.Based on the advanced bioengineering technologies, the aim of this paper focuses on the research of large-scale culture process and highly effective particles purification methods of PCV2 ZJ/C strain, and the development of new PCV2 inactivated vaccines, which would make contributions to designing novel inactivated vaccines for PCV2 and improving the future veterinary application of the vaccines.Firstly, the microcarriers suspension culture processes of PCV2 ZJ/C strain were studied.Limiting dilution assay was carried out to acclimatize PK5 cell line by culturing on microcarriers, and based on that, the best culturing parameters of PK15 cell line and the optimized culturing technique of PCV2-ZJ/C was acquired by culturing them using microcarriers suspension culture techniques on microcarrier bioreactor.The results suggested that the titer of PCV2-ZJ/C virus which was acquired by using microcarrier suspension culture techniques was 108.3-8.5TCID50/mL, ten times more than using traditional roller bottle cultures.Secondly, the virus protein purification techniques from PCV2 ZJ/C suspension culture were optimized.Highly purified virus protein was obtained by the combination of pretreatment, concentration, inactivation and re-purification by size-exclusion chromatography from the PVC2 ZJ/C suspension culture with the virus titer of 108.3-8.5TCID50/mL. Immune efficacy was compared with other same kind vaccines by mice challenge test according to the quality standard of the PCV2 inactivated vaccine from announcement no.1893 issued by the Ministry of Agriculture of the People’s Republic of China, and the immune efficacy of these purified virus protein was more than 80% with the immune dosage of 20μg/sample(PCV20.34μg/mL). All these results suggested that the new PCV2 vaccine was better than the vaccines which are commercially available.Thirdly, the new type of lyophilized inactivated vaccine of PCV2 was developed.Using the technology of freeze-drying and antigen purifcation, ten different thermo-stable stabilizers (named B1,B2,......B10) and three optimal freeze-drying curves procedures were explored by orthogonal experiments. Three batch vaccines with the antigen concentration of 40μg virus protein per sample were prepared to produce the vaccines and optimize lyophilization process, then tested the vaccines according to the quantity standard of lyophilized vaccines, and detected their thermal stability, immune effects and preservation period. The results indicated that the preservation period of the new PCV2 lyophilized inactivation vaccine was 14 days at 37℃ and more than 18 months at 2-8℃. The PCV2 vaccine has a bright future in veterinary clinical application. |
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