| Infectious bursal disease is an acute and highly pathogenic infectious disease caused by the Infectious Bursal Disease Virus,a double virus family,which is primarily characterized by immunosuppression.VP2 is the main structural protein constituting the outer layer of the IBDV capsid,which can induce neutralizing antibodies and have serotype specificity.The amino acid residue mutation in VP2 hypervariable region is an essential factor that causes its virulence mutation.Therefore,VP2 gene has become an crucial gene for IBDV immunoregulation,genetic variation and reproductive mechanism.IgY is the main antibody present in the serum of birds and reptiles.It has been found that Fc of IgG in mammal increases half-life period of the antigen in the vaccine and enhances the rate and efficiency of antigen capture.However,there is little research on whether IgY could show the similar functions in poultry.Adjuvant is believed to establish high and long-lasting immune responses by activating the innate immune system,thereby enhancing the adaptive immune response to an administered antigen.Therefore the application of adjuvant in vaccination is indispensable because adjuvant enhances the effects of vaccines.In our previous studies,we found that Taishan Pinus massoniana pollen polysaccharides(TPPPS)is an effective adjuvant for the inactivated and subunit vaccines.However,little is known about the effects of TPPPS on the recombinant VP2 and IgY based on eukaryotic expression system.In order to investigate whether the fusion expression of protein expressed by VP2 and IgY could indicate a higher immunogenicity and the Taishan pine pollen polysaccharides are able to enhance immune effects of this subunit vaccine.VP2-Fc recombinant plasmid construct,protein express and analysis.According to the VP2 gene sequence of IBDV and Fc gene sequence of IgY in birds published in Genbank,two pairs of primers were designed and synthesized to amplify the VP2 gene of IBDV and Fc gene by RT-PCR.The two genes were fused and recombined into PMD18-T to construct clone plasmid which had been identified by double digestion.After then,the resultant plasmid was digested and the product was recombined into pPIC9 eukaryotic expression vector and transferred to Pichia pastoris eukaryotic expression system.The transformant was selected,cultured and induced by methanol.At 24 h,48h,72 h and 96 h,the expression of recombinantplasmid and the immunoreactivity of the expressed product were detected by SDS-PAGE and Westem blotting respectively.The results showed that the recombinant VP2-Fc protein had immunoreactivity and the expression level was the highest at 72 h.Immune effects of TPPPS responses to fusion protein.In the animal experiment,a total of240 SPF chickens were randomly divided into six sterilized groups,with 40 chickens in each.Chickens were allowed to immunize for the first time at 3 days of age.0.2 mL VP2-Fc-TPPPS(40 mg/ml),VP2-Fc-TPPPS(25 mg/ml)VP2-Fc-TPPPS(10 mg/ml),VP2-Fc,VP2 subunit vaccine and PBS were boosted at 7 and 14 days after the first immunization to strengthen immunity.At the same time,three chickens from each group were selected randomly to determine titers of antibody,interleukin-2(IL-2),interleukin-4(IL-4)?-interferon(IFN-?)in serum,peripheral blood CD4+ and CD8+ T lymphocyte percentage and T lymphocyte transformation rate at 3,7,14,21,28,35,42 and 49 days respectively.One week after three vaccination,20 chickens were randomly selected from each group for animal protection test and the clinical symptoms and protection rate were recorded for 7 days.The results showed that VP2-Fc subunit vaccine could effectively induce anti-VP2 antibody,secrete IL-2,IL-4and IFN-?,increase CD4+,CD8+T lymphocyte percentage and T lymphocyte transformation rate,and provided 92% protection rate.In addition,TPPPS adjuvant can significantly improve the level of immune response.This results provides a basis for the development of this subunit vaccine. |
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