| :Porcine Circovirus 2(PCV2) is the smallest animal DNA viruses with a single-stranded circular DNA genome, which is icosahedral and nonenveloped. Porcine circovirus-associated disease (PCVAD) was first identified in the early 1990s, and then the France,USA, Korea, Japan, and some other countries also made it as a detailed report.PCV2 is related to PCVAD closedly and plays an important role in the development of it.PCV2 is recognized as an essential agent for porcine dermatitis and nephropathy syndrome (PDNS),postweaning multisystemic wasting syndrome (PMWS), reproductive disorder,porcine respiratory disease complex(PRDC)and some other disease.in order to obtain the PCV2 recombinant vaccine and provide theoretical foundation for the treatment,we constructed the recombinant PRV virus to express the major capsid protein with the attenuated vaccine parental strain in this study.We digest vector PG and plasmid pGEMT-ORF2 which contains the 702 bp ORF2 fragment of PCV2 with BamHI and then recoveried them. After dephosphorylation of the digested vector PG,we linked the ORF2 gene into vector PG and got the recombinant plasmid named PGO,and we proved the success of the construction of the transfer plasmid with biological technology of PCR, restriction enzyme digestion and sequencing. Then the genome of PRV attenuated vaccine and the transfer plasmid were transfered into ST cells with transfection techology. The recombinant PRV was obtained with three times of plaque assay and PCR test.Research of partial biological characteristics on the recombinant virus showed TCID50of the recombination virus on different cell lines are PK 104.125/0.1ml,ST 106.125/0.1ml,VERO104.625/0.1 ml,IBRS-2 106.25/0.1ml,The study of physicochemical characters on the recombinant virus indicat-ed that it is sensitive to trichloromethane,trypsase(0.25%),ultraviolet rays irradiating(30min),phen-ol(5%),sodium hydroxide(1%),The reasearch on safety testing indicated that the immunization of the recommbinant virus was safe for mice.the study of genetic stability showed that the inserted ORF2 of PCV2 played an excellent character on it.the expressing of the inserted gene in the recommbinant virus was identified by RT-PCR、inverted fluorescence microscope and IPMA.such studies laid foundation for the research and development of recombinant pseudorabies virus va-ccine expressing ORF2 gene of PCV2.In order to evaluate the immunogenicity of the recombinant virus,we immunized intramuscularly into the quadriceps muscle of 6-week-old female Kunming mice with commercial inactivated vaccine of PCV2,recombinant virus,DMEM culture solution,and boosted them with an equivalent dose 4 weeks later. Serum were collected on the third week after the first immunization to examine the humoral immune condition in mice each week by ELISA. Five mice of each group were killed 8 weeks later after the first immunization to evaluate the PCV2-specific lymphocyte proliferation response.The rest in each group were challenged with the virulent PCV2 LY strain and they were killed for the PCV2 ORF2-specific PCR test.The result in PGO group showed that the level of PCV2-specific humoral immune response was low for the first immunization but significant for the second immunization.and the PCV2 ORF2-specific lymphocyte proliferation response showed that the recombinant virus group played an obvious effect on it.challenge test showed that groups of recombinant virus and inactivated vaccine of PCV2 could protect the mice against challenge by PCV2 with a protective rate 3/5.These results suggest that the recombinant virus can be an excellent potential vaccine. |
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