共表达PCV2 ORF2和猪IL-18基因的重组猪伪狂犬病毒对小鼠的免疫原性

Immunogenicity of a recombinant pseudorabies virus coexpressing ORF2 gene of PCV2 and porcine IL-18 gene in mice

摘要:

【目的】研制猪伪狂犬病毒(PRV)和猪圆环病毒2型(PCV2)的二联活疫苗,并用猪IL-18作为免疫佐剂。【方法】将猪IL-18基因插入到质粒pGO中,获得的重组转移质粒pGO18与猪PRV弱毒HB98株DNA共转染ST细胞,并进行空斑筛选和纯化; RT-PCR和Western blot分别从转录和蛋白水平鉴定其表达情况。将重组病毒PGO18和PGO、PRV弱毒株HB98、PCV2灭活商品苗和1640细胞培养基分别免疫6周龄雌性昆明小鼠, 4周后二次免疫,二免后4周用PCV2 DF强毒和PRV Min/A强毒接种小鼠。通过ELISA、血清中和试验和流式细胞术及攻毒保护试验评价重组病毒的免疫原性。【结果】获得了重组病毒PGO18,并且可在ST细胞内表达; PGO18可诱导小鼠机体产生PCV2的ELISA和PRV的中和抗体水平,刺激CD³⁺、CD⁴⁺、CD⁸⁺ T细胞亚群的增殖,且能有效抵抗PCV2和PRV强毒攻击。【结论】 IL-18基因可增强重组病毒的免疫效果,使重组病毒具有良好的免疫原性,有望成为防治PCV2和PRV的候选疫苗株。

Abstract:

[Objective] To develop a bivalent vaccine against pseudorabies virus(PRV) and porcine circovirus(PCV2), IL-18 was used as immunologic adjuvant.[Methods] Porcine IL-18 gene was inserted into vector pGO. The obtained recombinant transfer plasmid pGO18 was transfected into ST cells with PRV attenuated vaccine HB98 strain. Then plaque selection and purification were performed to obtain purified recombinant virus PGO18. RT-PCR and Western blot were used to demonstrate the expression of PGO18 from transcription and protein levels, respectively. Six-week-old female Kunming mice were immunized with recombinant virus PGO18 and PGO, commercial PCV2 inactivated vaccine, PRV attenuated vaccine HB98 strain, 1640 medium. Mice were vaccinated twice 4 weeks later and then challenged with the virulent PCV2 DF strain and PRV Min/A strain 4 weeks after the second immunization. ELISA, serum neutralization assay, flow cytometry and protect experiment were used to demonstrate the immunity of mice.[Results] The recombinant virus PGO18 was obtained, and it could express on ST cells. Mice vaccinated with PGO18 elicited high levels of humoral and cell immune response, and could also be protected against PCV2 and PRV challenge.[Conclusion] The recombinant virus possessed high safety and good immunogenicity. It may be a candidate vaccine strain against PCV2 and PRV infection.