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The immunohistological study of the ret oncogene product by a monoclonal antibody raised against a synthetic peptide

针对合成肽的单克隆抗体对 ret 癌基因产物的免疫组织学研究

基本信息

批准号：
62570168
负责人：
TAKAHASHI Masahide
金额：
$ 1.28万
依托单位：
Aichi Cancer Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570168/
关键词：
ret oncogene rfp gene oncogene monoclonal antibody 合成ペプチド

项目摘要

The ret transforming gene was activated by the joining of a tyrosine kinase sequence to part of a "finger"-containing gene (designated rfp). Analysis of the amino acid sequence indicated that the first 315 amino acids of rfp were fused to the amino terminus of the 5' truncated ret tyrosine kinase. To further analyze the structure and function of the ret and frp proteins, we have developed a monoclonal antibody against a synthetic peptide corresponding to amino acids 148 to 163 of the proteins. The isolated MoAb, designated RFP-1 (IgM), was reactive with the immunizing peptide but not with a 95 kD in vitro translated ret protein. In contrast, RFP-1 recognized a 60 kD in vitro translated rfp product and a 85 kD native protein in extracts made from HL-60 promyelocytic leukemia cell line in which rfp is highly expressed. This might be due to the difference in conformations between the ret and rfp proteins. By the avidin -biotin complex immunoperoxidase method, RFP-1 strongly stained over 90% of the neclei of human spermatogenic cells, except mature spermatozoon, and of human testicular tumor cells. In addition, when its reactivity with other normal adult tissues were examined, 20-40% of cells were stained in epithelial cells of the gastroineteinal tract mucosae, the kidney tubules, and the thyroid gland follicles; lymphocytes of the thymus, the spleen, and the lymph nodes; and hepatocytes. All of RFP-1 positive cells showed nuclear staining. Since the results of immunological study is consistent with the previous study on expression of rfp mRNA, it is likely that RFP-1 MoAb recognizes the native rfp protein. To further elucidate the nature and function, sufficient amounts of the native protein will be required. We believe that the availabilty of the anti-rfp monoclonal antibody could facilitate purification of the rfp protein from cells and tissues.

ret转化基因通过将酪氨酸激酶序列连接到含有“指”的基因（称为rfp）的一部分而被激活。氨基酸序列分析表明，rfp的前315个氨基酸与5'截短的ret酪氨酸激酶的氨基末端融合。为了进一步分析ret和frp蛋白质的结构和功能，我们开发了一种针对合成肽的单克隆抗体，该合成肽对应于蛋白质的氨基酸148至163。分离的单克隆抗体，命名为RFP-1（IgM），与免疫肽反应，但不与95 kD的在体外翻译的ret蛋白。相反，RFP-1识别60 kD的体外翻译的RFP产物和85 kD的天然蛋白质的提取物中制成的HL-60早幼粒细胞白血病细胞系，其中RFP是高度表达。这可能是由于ret和rfp蛋白质之间构象的差异。用亲和素-生物素复合物免疫过氧化物酶法，RFP-1对除成熟精子外的90%以上的人生精细胞和人睾丸肿瘤细胞的细胞核均有强染色。此外，当检查其与其他正常成人组织的反应性时，20-40%的细胞在胃肠道粘膜、肾小管和甲状腺滤泡的上皮细胞;胸腺、脾脏和淋巴结的淋巴细胞;以及肝细胞中被染色。RFP-1阳性细胞均为核染色。由于免疫学研究的结果与先前关于rfp mRNA表达的研究一致，因此RFP-1 MoAb可能识别天然rfp蛋白。为了进一步阐明性质和功能，需要足够量的天然蛋白质。我们相信，抗rfp单克隆抗体的获得将有助于从细胞和组织中纯化rfp蛋白。