Construstion Of BF2/peptide Tetramer And Assessment Of The Infectious Bronchitis Virus-sepecific T Cells By Using The Prepared BF2/peptide Tetramer

The major histocompatibility complex classâ… (MHC classâ… ) peptide tetramer is a sensitive tool to evaluate antigen-specific cytotoxic T lymphocytes(CTLs). However, no chicken MHC classâ… peptide tetramer has been reported by now.In order to construct the chicken MHC classâ… peptide tetramer, genes of the chicken MHCⅠαchain(BF2*15) andβ2 microglobulin(Chβ2m) were amplified from the total RNA of peripheral blood monouclear cells(PBMCs) by RT-PCR and cloned into pMD18-T. Then the PCR products deleted the signal sequence at the 5' end of both the BF2 and Chβ2m genes and included the BirA substrate peptide(BSP) sequence at the 3' end of the BF2 gene were sub-cloned into the expression vector pET-28a(+) and named as pET-BF2-BSP and pET-Chβ2m. High levels of BF2-BSP and Chβ2m proteins were successfully expressed in E. coli strain BL21(DE3) transformed with the recombinant vectors pET-BF2-BSP and pET-Chβ2m. The recombinant target proteins were purified with a Ni²⁺NTA column including His-Bind Resin, and then high-quality recombinant proteins were recovered.BF2-BSP and Chβ2m were refolded with synthetic peptide originated from infectious bronchitis virus nucleoprotein(IBV N_71-78) in refolding buffer to generate the monomer of BF2/peptide complex. The folded product was then subjected to enzymatic biotinylation by BirA enzyme. After biotinylation the sample was run over a gel filtration column to purify the BF2/peptide fraction. The tetrameric complexes of biotinylated BF2/peptide were produced by mixing purified, biotinylated monomer with PE-labeled streptavidin at a molar ratio of 4:1.In order to test the function of the prepared BF2/peptide tetramer, one-week SPF chicks were immunized with 10^5.5EID₅₀ IBV H52 strain through intranasal inoculation. The PBMCs were isolated at 10 days post infection(d.p.i.) and used for flow cytometry staining. The CTLs response of IBV-infected chicks was evaluated with the prepared BF2/peptide tetramer and 3.65% CTLs frequency specific to IBV N was detected.To discuss the kinetics of the antibody titer and the IBV N-specific T cell when the SPF chicks were infected with the IBV, 125 SPF chicks were divided into three groups at random, namely vaccinated group, challenge control group and negative control group. The SPF chicks from the vaccinated group were immunized with 10^5.5EID₅₀ IBV H52 strain through intranasal inoculation. Five chicks were sacrificed every three days for pathogenesis observation, assessment of antibody titer and IBV N-specific T cell measured by prepared BF2/peptide tetramer. The chicks from vaccinated group and challenge control group were challenged with IBV M41 at the day 15 after vaccination. Also, the same assessments were taken on as described above. All the results were pooled together to investigate the interaction between the antibody titer and nucleoprotein-specific T cell after vaccination and challenge. The results indicated that the CTL activity was shown to be effective during the initial elimination of virus in the chicks, later control of infection probably depends on antibody, especially the IgG response, which likely plays a important role in preventing recrudescence of infection.