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

我们以前确定了一个肿瘤相关的靶抗原，ATM-1，为人类杀伤T细胞克隆5 B5，并发现ATM-1存在于肝细胞癌患者的血清中。为了克隆编码ATM-1的基因，我们使用针对部分纯化的ATM-1的单克隆抗体免疫学地筛选从人肝细胞癌细胞系SK-HEP-1获得的cDNA文库。然而，我们未能成功克隆ATM-1基因，可能是由于所用的单克隆抗体的特异性和敏感性较低。因此，我们改变了策略，并试图纯化ATM-1和确定的N-末端氨基酸序列的ATM-1。我们从肝癌患者血清中纯化ATM-1，通过SepharoseCL-6 B凝胶过滤，然后在抗ATM-1结合的Sepharose珠上进行亲和层析。作为对照，以相同的方式处理来自健康人的血清。纯化的样品进行SDS-聚丙烯酰胺凝胶电泳，分离的蛋白质转移到硝酸纤维素膜上进行电泳和染色。来自肝细胞癌患者血清的样品给出分子量约为55，000的蛋白质条带，这在来自健康人血清的样品中未观察到。经N-末端氨基酸序列分析，发现该蛋白条带含有两种蛋白质。在同源性搜索中，发现一种蛋白质具有免疫球蛋白重链高变区III的序列，而另一种蛋白质不具有任何已知的氨基酸序列。我们认为免疫球蛋白是作为与ATM-1的免疫复合物纯化的。我们将针对未知蛋白质的N-末端氨基酸序列的合成肽产生抗体，并合成氨基酸序列的寡聚体。我们将使用这些抗体和寡聚体克隆ATM-1基因。然后我们将研究ATM-1的生物学和临床意义。

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.