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

RET转化基因是通过将酪氨酸激酶序列连接到含有“Finger”的基因的一部分(命名为RFP)来激活的。氨基酸序列分析表明，RFP的前315个氨基酸与5‘端截短的RET酪氨酸激酶的氨基末端融合。为了进一步分析ret和FRP蛋白的结构和功能，我们开发了一种针对合成肽的单抗，该合成肽对应于蛋白质的148到163个氨基酸。分离的单抗命名为RFP-1(IgM)，可与免疫多肽反应，但不能与体外翻译的95kD的ret蛋白反应。相反，RFP-1在高表达RFP的HL-60早幼粒白血病细胞株提取液中识别60kD的体外翻译RFP产物和85kD的天然蛋白。这可能是由于ret和RFP蛋白的构象不同所致。通过亲和素-生物素复合体免疫过氧化物法，RFP-1对人生精细胞(成熟精子除外)和人睾丸肿瘤细胞90%以上的细胞核进行了强染色。此外，当检测其与其他正常成人组织的反应性时，20%-40%的细胞被染色于胃肠粘膜、肾小管和甲状腺滤泡的上皮细胞、胸腺、脾和淋巴结的淋巴细胞以及肝细胞。RFP-1阳性细胞均为核染色。由于免疫学研究的结果与前人对RFP mRNA表达的研究结果一致，RFP-1单抗很可能识别天然的RFP蛋白。为了进一步阐明其性质和功能，需要足够数量的天然蛋白质。我们相信，抗RFP单抗的获得有助于从细胞和组织中纯化RFP蛋白。

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.