| As one of the biggest pig population countries in the world,the occurrence of Porcine circovirus virus(PCV2)and Pseudorabies(PR)infection were quite serious in China.Vaccination has become the most commonly used and effective means to prevent the occurrence of diseases.But the traditional immune prevention methods existed many problems,and virus live vector vaccines had many advantages,such as the effective immunity of live vaccines,low cost and high safety of inactivated vaccine,which made it become one of the main directions of vaccine research and development in the current and future.IL-18 expressed genes were strong adjuvant to induce T,B and NK cells reaction.IL-18 could be used in the treatment of bacteria,viruses,infectious diseases,allergic diseases and tumors.In addition,we can achieve standardization production and greatly improve the practical value by cloning the IL-18 gene to the vector.Therefore,IL-18 gene was inserted to the previously build recombinant plasmid p GO,and the new recombinant plasmid was called p GO18,then co-transfected into ST cells with PRV attenuated vaccine HB98 strain.We eventually obtained a recombinant pseudorabies virus coexpressing PCV2 Cap and porcine IL-18 gene,which had laid a solid foundation for the development of PCV2 live vector vaccine,the development and utilization of porcine IL-18 adjuvant effect and construction of a recombinant PRV vaccine coexpressing porcine IL-18 gene and protective antigen gene.In this study,the recombinant plasmid p GEMT-IL18 was digested by Bgl ?to acquire IL-18 gene,and then cloned to phosphorylated plasmid p GO which were processed same with p GEMT-IL18.The accuracy of recombinant plasmid p GO18 were determined by PCR,enzyme digestion and sequencing.Then the recombinant plasmid p GO18 were transferred into ST cells with PRV attenuated vaccine HB98 genomic DNA,and recombinant virus PGO18 were acquired after green fluorescent plaque screening and purification.The expression of PGO18 on ST cells were identified by RT-PCR and Western blot from transcription and protein level,respectively.In order to research the potential value of recombinant pseudorabies virus PGO18 as a candidate vaccine strain,the biological characteristics,including cultural characteristics on ST cells,proliferation characteristics on ST,VERO,IBRS-2,PK-15 and IPEC cells,growth characteristics,genetic stability,security to mice,physical and chemical properties were studied.The above experiments showed that the recombinant virus PGO18 strain shared a similar culture and growth characteristics with parental strain PRV HB98.TCID50 on ST,VERO and IBRS-2 were closed to parent strain 107.75/0.1(m L).The genetic stability were good,and safety test showed that the recombinant virus were safe to mice.Physical and chemical properties test showed that the virus has the common gender with pseudorabies virus.To evaluate the immune effects of recombinant pseudorabies virus PGO18,1006-weeks-old female Kunming mice were randomly divided into 5 groups,and then immunized with recombinant virus PGO18 and PGO,commercial PCV2 inactivated vaccine,PRV attenuated vaccine HB98 strain and 1640 cell culture medium,respectively.Mice were vaccinated twice 4 weeks later and then challenged with the virulent PCV2 DF strain and PRV NY strain 4 weeks after the second immunization.ELISA and serum neutralization assay showed that mice vaccinated with PGO18 elicited high level of humoral immune response.T lymphocyte proliferation response and the amount of CD3+,CD4+,CD8+ subgroups of peripheral blood T-lymphocytes demonstrated that mice vaccinated with PGO18 elicited high level of cell immune response.Protection test showed that group of recombinant virus PGO18 could protect mice against PCV2 challenge.The above results suggested that IL-18 as the immune adjuvant could enhance the immune effect of recombinant vaccines,and it had a good immunogenicity.Therefore,PGO18 is expected to become the candidate vaccine strain for the prevention and control of PCV2 and PRV. |
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