Effects of a Sjogren's syndrome-associated mutation of the TAP2 gene on the diversity of HLA-bound antigen peptides

干燥综合征相关的 TAP2 基因突变对 HLA 结合抗原肽多样性的影响

• 批准号: 11670446
• 负责人: KUMAGAI Shunichi
• 金额: $ 2.5万
• 依托单位: Kobe University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670446/
• 关键词: TAP2 Gene; antigen peptides; Sjogren's syndrome; peptide transporter; HLA

1) Preparation of the mutated TAP2 gene.The A to G mutation was introduced at the 1729^<th> base of TAP2 gene by the method of Shuldier. A.R.et al (Analyt. Biochem. 194, 9-15, 1991), and the mutated (TAP2^*Bky2) and wild (TAP2^*b) type cDNA were prepared.2) Esablishment of TAP1 and TAP2 genes transfected cell lines.TAP1 and TAP2 genes (TAP2^*B or TAP2^*Bky2) were transfected to TAP genes-deficient cell line, T2, by electroporation method. Two cell lines. T2/Bky2 and T2/B, which were different only at the codon 577, were established.3) Collection of HLA-bound peptides from the established cell linesAntigen peptides bound to HLA-B5 were collected by anti-HLA column using anti-HLA-B5 monoclonal antibody (4D12). After filtration with 5,000 dalton cut-off filter, peptide fraction was collected.4) Analysis of HLA-bound peptidesThe fraction was applied on a reversed-phase HPLC, and some peaks were collected. The repertory and amino acid sequence of HLA-bound peptides are now further analyzed by mass-spectrometry.

1)突变的TAP 2基因的制备：通过Shuldier的方法在TAP 2基因的1729个碱基处引入A至G突变<th>。A.R.et al（Analyt. 194，9-15，1991），并获得突变型（TAP 2 ^* Bky 2）和野生型（TAP 2 ^*B）cDNA。2）TAP B和TAP 2基因转染细胞系的建立。两种细胞系。3）从建立的细胞系中收集HLA结合肽用抗HLA-B5单克隆抗体（4D 12）通过抗HLA柱收集与HLA-B5结合的抗原肽。用5，000道尔顿截止过滤器过滤后，收集肽级分。4）HLA结合肽的分析将该级分应用于反相HPLC，收集一些峰。现在通过质谱法进一步分析HLA结合肽的库和氨基酸序列。

项目成果

Morinobu, A., et at: "Bucillamine suppresses human Th1 cell development by a hydrogen peroxide-independent mechanism."J.Rheumatol. 27. 851-858 (2000)

Morinobu, A. 等人：“布西拉明通过不依赖过氧化氢的机制抑制人类 Th1 细胞发育。”J.Rheumatol。

Akio Morinobu: "Bucillamine suppresses human Th1 cell development by a hydrogen peroxide-independent mechanism"J.Rheumatol.. In press.

Akio Morinobu：“布西拉明通过不依赖过氧化氢的机制抑制人类 Th1 细胞发育”J.Rheumatol.. 正在出版。

Akio Morinobu: "Association of glutathione S-transferase M1 homozygous null genotype with disease susceptibility to Sjogren's syndrome in Japanese."Arthritis Rheum.. 42・12. 2612-2615 (1999)

Akio Morinobu：“谷胱甘肽 S-转移酶 M1 纯合无效基因型与日本人干燥综合征疾病易感性的关联。”Arthritis Rheum.. 2612-2615 (1999)。

Shinichi Kondo: "Factor XII Tenri,a novel cross-reacting material negative factor XII deficiency,occurs through a proteasome-mediated degradation"Blood.. 93・12. 4300-4305 (1999)

Shinichi Kondo：“因子 XII Tenri，一种新型交叉反应物质阴性因子 XII 缺乏症，通过蛋白酶体介导的降解而发生”Blood.. 93・12 (1999)

Shinichi Kondo: "Factor XII Tenri, a novel cross-reacting material negative factor XII deficiency. occurs through a proteasome-mediated degradation."Blood.. 93・12. 4300-4305 (1999)

Shinichi Kondo：“因子 XII Tenri，一种新型交叉反应物质阴性因子 XII 缺乏症。通过蛋白酶体介导的降解而发生。”Blood.. 93・12 (1999)