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Molecular biological studies on a hepatoma-associated target antigen for a human killer T-cell clone

人类杀伤 T 细胞克隆的肝癌相关靶抗原的分子生物学研究

基本信息

批准号：
01480222
负责人：
IMAWARI Michio
金额：
$ 3.26万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01480222/
关键词：
hepatoma killer T-cell tumor-associated antigen 細胞傷害性T細胞

项目摘要

We previously identified a tumor-associated target antigen, ATM-1, for a human killer T-cell clone 5B5, and found the existence of ATM-1 in sera of patients with hepatocellular carcinoma. To clone the gene that coded ATM-1, we screened the CDNA library obtained from a human hepatocellular carcinoma cell line, SK-HEP-1, immunologically using monoclonal antibodies raised against partially purified ATM-1. However we could not succeed in the cloning of ATM-1 gene possibly due to the low specificity and low sensitivity of the monocional antibodies used. Thus we changed the strategy and tried to purify the ATM-1 and determine the N-terminal amino acid sequence of the ATM-1. We purified ATM-1 from sera of patients with hepatocellular carcinoma by gel filtration on Sepharose CL-6B followed by affinity chromatography on anti-ATM-1 -bound Sepharose beads. As a control, serum from a healthy person was processed in the same way. Purified samples were subjected to SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to a nitrocellulose membrane electrophoretically and stained. The sample from sera of patients with hepatocellular carcinoma gave a protein band with a molecular wright of approximately 55, 000 that was not observed in the sample from serum of a healthy person. On N-terminal amino acid sequence analysis, the protein band was found to contain two proteins. On homology search, one protein was found to have a sequence of immunoglobulin heavy chain hypervariable region III, and the other protein did not have any known amino acid sequence. We thought that the immunoglobulin was purified as an immune complex with ATM-1. We will raise antibodies against the synthetic peptide for the N-terminal amino acid sequence of the unknown protein, and also synthesize oligomers for the amino acid sequence. We will clone the ATM-1 gene using these antibodies and oligomers. Then we will study the biological and clinical significance of ATM-1.

我们之前发现了人类杀伤t细胞克隆5B5的肿瘤相关靶抗原ATM-1，并在肝细胞癌患者的血清中发现ATM-1的存在。为了克隆编码ATM-1的基因，我们从人肝癌细胞系SK-HEP-1中筛选CDNA文库，使用针对部分纯化的ATM-1的单克隆抗体进行免疫筛选。然而，由于所用的单克隆抗体特异性和敏感性较低，我们未能成功克隆ATM-1基因。因此，我们改变策略，尝试纯化ATM-1并测定其n端氨基酸序列。我们先用Sepharose CL-6B凝胶过滤，然后用抗ATM-1结合的Sepharose珠亲和层析法从肝癌患者血清中纯化ATM-1。作为对照，用同样的方法处理健康人的血清。纯化后的样品进行sds -聚丙烯酰胺凝胶电泳，将分离的蛋白电泳转移到硝化纤维素膜上染色。从肝细胞癌患者的血清中提取的样本显示出一个分子量约为55000的蛋白带，这在健康人的血清样本中是没有观察到的。在n端氨基酸序列分析中，发现该蛋白带含有两个蛋白。同源性分析发现，其中一种蛋白具有免疫球蛋白重链高变区III的序列，而另一种蛋白没有已知的氨基酸序列。我们认为免疫球蛋白被纯化为与ATM-1的免疫复合物。我们将针对未知蛋白的n端氨基酸序列，对合成的肽段提出抗体，并合成该氨基酸序列的低聚物。我们将利用这些抗体和低聚物克隆ATM-1基因。然后我们将研究ATM-1的生物学和临床意义。