BF2/肽四聚体的构建及其在特异性T细胞检测上的应用

为构建可溶性鸡 组织相容性复合体(MHC)Ⅰ 类分子（BF2）/肽复合物，大量表达并纯化了可生物素化的MHC Ⅰ类分子重链（BF2-BSP）和轻链（Chβ2m），并合成了鸡传染性支气管炎病毒（IBV）核蛋白N71~78 T细胞表位肽，将其与纯化后的重组蛋白BF2-BSP和Chβ2m在重折叠缓冲液中复性。复性后在快速蛋白液相色谱仪（FPLC）上分离出复性组装成功的BF2/肽单体复合物。将该单体复合物在体外进行生物素化，其产物再次采用FPLC通过分子筛分离出生物素化后的BF2/肽单体复合物；然后将生物素化后的BF2/肽单体复合物与藻红蛋白（PE）标记的链霉亲和素按一定比例反应生成BF2/肽四聚体。为了检测所制备BF2/肽四聚体是否能应用于检测特异性T细胞，从感染IBV H52株10 d后的SPF鸡分离外周血单核细胞（PBMC），染色后采用流式细胞术检测，结果表明，所制备的BF2/肽四聚体可用于检测IBV N蛋白特异性T淋巴细胞，检测1周龄SPF鸡接种H52后10 d时针对N蛋白的特异性T细胞比率为3.65%。

Construction of a BF2/Peptide Tetramer and Its Application on the Detection of Specific T Cell

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.