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Gene cloning and characterization of new HLA class II antigens

新 HLA II 类抗原的基因克隆和表征

基本信息

批准号：
60490021
负责人：
NAKATSUJI Takako
金额：
$ 3.07万
依托单位：
Tokai University Shcool of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1987
项目状态：
已结题

项目摘要

1. A cDNA clone encoding a new HLA class II antigen <alpha> chain, named DO<alpha>, was isolated from a human CDNA library using the DR<alpha>, DO<alpha> and DP<alpha> cDNA probes.The nucleotide sequence of the DO<alpha> cDNA was distinct from those of the DR<alpha>, the DO<alpha>, and the DP<alpha> cDNA, but showed some characteristic features of the class II antigen <alpha>-chain. We also isolated and identified genomic clones specifying the DO<alpha> gene. Genomic Southern hybridization analyses of cell lines with different HLA-DR serotypes indicated the existence of a single DO<alpha> gene that exhibited little restriction enzyme polymorphism.2. To describe the function of gene products from this new DO locus, DO gene clone was introduced. The resultant transfectant cells were analyzed their expression and function using biochemical and cellular-immunological procedure.3. A genomic clone specifying a new HLA class II antigen <beta> chain pseudogene, named DV<beta>, was isolated from a human genomic phage library using a DO<beta> cDNA probe.Southern hybridization and nucleotide sequence analyses identified the <beta>2 domain exon (exon 3) with several deleterious mutations and the CP-TM-CY exon (connecting peptide, transmembrane, and cytoplasmic exon, exon 4), but the first, second and fifth exons of normal <beta> chains were entirely missing. The nucleotide sequence of these two exons were distinct from those of other class II <beta>-chain genes, but slightly more related to the DQ<beta> and the DX<beta> genes than other class II genes. Cosmid clone analysis around the DV<beta> gene placed DV<beta> between DX<alpha> and DQ<beta>, 15 kb upstream from DX<alpha>, which was consistence with that around DX<alpha> which was hown by RFLP(restriction fragment length polymorphism studies).

1. 使用 DR<α>、DO<α> 和 DP<α> cDNA 探针从人 cDNA 文库中分离出编码新的 HLA II 类抗原 <α> 链的 cDNA 克隆，命名为 DO<α>。DO<α> cDNA 的核苷酸序列与 DR<α>、DO<α> 和 DP<α> cDNA 的核苷酸序列不同。 DP<α> cDNA，但显示出II类抗原<α>链的一些特征。我们还分离并鉴定了特异DO α 基因的基因组克隆。具有不同HLA-DR血清型的细胞系的基因组Southern杂交分析表明存在单个DOα基因，该基因表现出很少的限制性内切酶多态性。2．为了描述这个新 DO 基因座的基因产物的功能，引入了 DO 基因克隆。采用生化和细胞免疫学方法分析所得转染细胞的表达和功能。 3．使用 DO<β> cDNA 探针从人类基因组噬菌体文库中分离出一个指定新的 HLA II 类抗原 <β> 链假基因的基因组克隆，命名为 DV<β>。Southern 杂交和核苷酸序列分析鉴定出具有多个有害突变的 <β>2 结构域外显子（外显子 3）和 CP-TM-CY 外显子（连接肽、跨膜和细胞质外显子（外显子 4），但正常 β 链的第一、第二和第五外显子完全缺失。这两个外显子的核苷酸序列与其他II类β链基因的核苷酸序列不同，但与DQβ和DXβ基因的相关性比其他II类基因稍高。 DVβ基因周围的粘粒克隆分析将DVβ置于DXα和DQβ之间，距离DXα上游15kb，这与RFLP(限制性片段长度多态性研究)显示的DXα周围的粘粒克隆一致。