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Preparation and Identification of HLA-A＊1101 Tetramer Loading with Human Cytomegalovirus pp65 Antigen Peptide

ISSN号：1672-7681
期刊名称：《中国免疫学杂志：英文版》
时间：0
分类：R392-33[医药卫生—免疫学;医药卫生—基础医学] R373[医药卫生—病原生物学;医药卫生—基础医学]
作者机构：[1]Institute of Tissue Transplantation and Immunology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China, [2]Institute of Bioengineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China, [3]Clinical Experimental Center of the First Affiliated Hospital, Jinan University, Guangzhou 510632, China
相关基金：Acknowledgements This work was supported by grants from the National Natural Science Foundation of China （No.30230350, No.30572199 and No.30371651）.

作者： Fengyao Li[1], Lihui Xu[1,2], Qingbing Zha[1,3], Xiaoyun Chi[1], Qiantao Jia[1], Kianhui He[1]

关键词： HLA-A＊1101四聚物, 人类巨细胞病毒, PP65, 抗原肽, 制备, 鉴定, HLA-A＊ 1101, tetramer, cytomegalovirus, CD8^＋T cell, epitope

中文摘要：

MHC/peptide tetramer 技术广泛地被用来学习抗原特定的 T 房间，特别为识别在人的病毒特定的 CD8+ T 房间。tetramer 分子由 HLA 重链， 2-microglobulin (2m ) ， antigenic 肽，和荧光灯标签的 streptavidin 组成。为了推进，调查 HLA --*1101 对人的 cytomegalovirus (HCMV ) 限制了 CD8+ T 房间回答，我们建立了一条途径准备 HLA -- 有从 HCMV 的肽的 *1101 tetramer complexed。编码 HLA 的 cDNA --*1101 重链被克隆并且为 HLA 的 ectodomain 的原核生物的表示向量 -- 与 BirA 底层肽熔化的 *1101 (HLA -- 在它的 carboxyl 终点的 *1101 -BSP) 被构造。熔化蛋白质高度在 Escherichia coli 在优化条件下面被表示为包括身体。而且， HLA --*1101 -BSP 蛋白质面对 2m 和 HCMV 肽 pp6516-24 是 refolded (GPISGHVLK， GPI ) 。可溶的 HLA --*1101 -GPI 单体是 biotinylated 并且净化了到 95% 的纯净，它随后与 streptavidin 被相结合在 80% 的收益形成 tetramers。HLA --*1101 -GPI tetramers 能绑在病毒特定的 CD8+ T 房间，建议可溶的 HLA --*1101 -GPI tetramers 是生物学上功能的。这研究为 HLA 的进一步的评估提供基础 --*1101 对 HCMV 感染限制了 CD8+ T 房间回答。

英文摘要：

MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8^＋ T cells in humans. The tetramer molecule is composed of HLA heavy chain, β2-microglobulin （β2m）, an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A＊1101-restricted CD8^＋ T cell responses against human cytomegalovirus （HCMV）, we established an approach to prepare HLA-A＊1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A＊1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A＊1101 fused with a BirA substrate peptide （HLA-A＊1101-BSP） at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichla coli. Moreover, HLA-A＊1101-BSP protein was refolded in the presence of β2m and an HCMV peptide pp6516.24 （GPISGHVLK, GPI）. Soluble HLA-A＊1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of 〉 80%. The HLA-A＊1101-GPI tetramers could bind to virus-specific CD8^＋ T cells, suggesting soluble HLA-A＊1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A＊1101-restricted CD8^＋ T cell responses against HCMV infection.

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