Construction and functional test of a chicken MHC-I (BF2*15)/peptide tetramer.
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 beta2 microglobulin (Chbeta2m) 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 Chbeta2m were cloned into the expression vector pET-28a (+) and expressed in Escherichia coli strain BL21 (DE3). Highly purified BF2*15-BSP heavy chain and Chbeta2m were obtained by a Ni(2+) NTA column affinity purification, yielding approximately 1.6mg of BF2*15-BSP and 2.4mg of Chbeta2m per 1g of the pelleted bacteria. The purified BF2*15-BSP heavy chain and Chbeta2m were refolded with synthetic peptide originated from infectious bronchitis virus nucleoprotein (IBV N(71-78)) 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.
Liu
Guangliang
G
Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, People's Republic of China.
Wang
Qun
Q
Tong
Tiegang
T
Xiao
Yihong
Y
Bai
Yu
Y
Liu
Shengwang
S
Wu
Donglai
D
D000818:Animals; D002645:Chickens / Q000276:immunology*; D003001:Cloning, Molecular; D015395:Histocompatibility Antigens Class I / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D001613:beta 2-Microglobulin / Q000235:genetics
D015395:Histocompatibility Antigens Class I; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin