Construction and functional test of a chicken MHC-I (BF2*15)/peptide tetramer

文献类型： 外文期刊

第一作者： Liu, Guangliang

作者： Liu, Guangliang;Wang, Qun;Tong, Tiegang;Xiao, Yihong;Bai, Yu

作者机构：

关键词： chicken MHC class I;BF2*15-BSP and beta 2 microglobulin genes;BF2*15/peptide tetramer

期刊名称：VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY （ 影响因子：2.046； 五年影响因子：2.217 ）

ISSN： 0165-2427

年卷期： 2008 年 122 卷 1-2 期

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收录情况： SCI

摘要： The major histocompatibility complex class I (MHC class I) peptide tetramer is a sensitive and valuable tool to evaluate antigen-specific cytotoxic T lymphocytes (CTLs) of many animal species. To date, no chicken MHC class I peptide tetramer has been reported. In this report, we describe construction and functional evaluation of a chicken MHC-I (BF2*15)/peptide tetramer. To construct the chicken MHC class I peptide tetramer, genes of the chicken MHC-I alpha chain (BF2*15) and beta 2 microglobulin (Ch beta 2m) were synthesized by RT-PCR from the total RNA of PBMCs and the signal sequences were deleted. The BF2*15 was then fused with the BirA substrate peptide (BSP) sequence at the C terminus. Next, the synthesized PCR products of BF2*15 and Ch beta 2m were cloned into the expression vector pET-28a (+) and expressed in Escherichia coli strain BL21 (DE3). Highly purified BF2*15-BSP heavy chain and Ch beta 2m were obtained by a Ni2(+) NTA column affinity purification, yielding approximately 1.6 mg of BF2*15-BSP and 2.4 mg of Ch beta 2m per 1 g of the pelleted bacteria. The purified BF2*15-BSP heavy chain and Ch beta 2m were refolded with synthetic peptide originated from infectious bronchitis virus nucleoprotein (IBV N71-78) in refolding buffer to generate the monomer of BF2*15/peptide complex. The monomer was then biotinylated and tetramerized using PE-labeled streptavidin. Upon functional evaluation of the construct by using flowcytometry, we observed that 3.65% of CTLs were specific to IBV nucleoprotein. This demonstrates that the CTL response of IBV-infected chicks could effectively be evaluated using the prepared MHC-I BF2*15/ peptide tetramer. (C) 2007 Elsevier B.V. All rights reserved.

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