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Abstract For the construction of BF2/peptide tetramer, large amount of recombinant chicken major histocompatibility complex (MHC) Ⅰ heavy chain (BF2-BSP) and light chain Chβ2m were expressed and purified. And a peptide originated from avian Infectious bronchitis virus (IBV) nucleoprotein (N71~78) was synthesized. Afterwards, BF2-BSP and Chβ2m were refolded with IBV N71~78 in refolding buffer to generate the monomer of BF2/peptide complex. Then the folded product was treated with enzymatic biotinylation by BirA enzyme and followed by a gel filtration column to purify the BF2/peptide fraction. Finally, the BF2/peptide tetramer was formed by mixing purified, biotinylated monomer with phycoerythrin(PE)-labeled streptavidin at a molar ratio of 5∶1. In order to test if the prepared BF2/peptide tetramer could be employed to detect the efficiency of specific T cell, the peripheral blood mononuclear cells (PBMCs) were isolated from the SPF chickens infected with IBV H52 at 10 days post infection and subjected to flow cytometry staining. The cytotoxitic T lymphocytes (CTLs) response of IBV-infected chicks was evaluated with the prepared BF2/peptide tetramer and 3.65% of CTLs frequency specific to IBV N was detected. All these results indicated that the prepared BF2/peptide tetramer can be used to evaluate the specific T cell immune responses of IBV.
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Received: 07 April 2008
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