Kakar S S, Chen L, Puri R, Flynn S E, Jennes L
James Graham Brown Cancer Center, University of Louisville, Kentucky 40202, USA.
J Histochem Cytochem. 2001 Dec;49(12):1537-46. doi: 10.1177/002215540104901207.
Pituitary tumor transforming gene 1 (PTTG1), recently cloned from human testis, is a potent oncogene that is expressed in most tumors. However, assessment of its potential value as a prognostic marker is dependent on the development of a suitable antibody. We have developed a rabbit polyclonal antibody, SK601, that is highly specific for the PTTG1 gene product using recombinant PTTG1 protein (24 kD) containing an N-terminal His(6) tag as the immunogen. The antiserum is capable of detecting recombinant PTTG1 protein in ELISA assays at a titer of 1:100,000. Use of the antibody as the probe in Western blotting analyses revealed a single band with the anticipated relative molecular weights of 52 kD from E. coli expressing the GST-PTTG1 recombinant protein, and 56 kD from COS-7 cells transfected with the PTTG1-GFP chimeric construct. A single band with a relative molecular weight of 28 kD was observed in extract of COS-7 cells transfected with PTTG1 cDNA. The antiserum immunoprecipitated a protein of relative molecular weight of 56 kD from the extracts of COS-7 cells transfected with the PTTG1-GFP chimeric construct. Immunohistochemical analysis of COS-7 cells transfected with this construct confirmed that the antibody detected and was specific for expressing the PTTG1-GFP recombinant protein. Screening of various normal human tissues (testis, ovary, and breast) by immunohistochemistry indicated that these tissues did not exhibit staining with the exception of testis, a tissue that had previously been shown to express PTTG1 mRNA. In contrast all of the tumor tissues (testicular tumor, ovarian tumor, and breast tumor) that were assessed exhibited intense staining. The results suggest that antiserum SK601 is highly specific for the PTTG1 protein and therefore should prove useful in further analysis of the expression and interactions of this protein, including its potential application as an immunohistochemical marker of human tumors.
垂体肿瘤转化基因1（PTTG1），最近从人睾丸中克隆得到，是一种在大多数肿瘤中都有表达的强效癌基因。然而，评估其作为预后标志物的潜在价值取决于合适抗体的开发。我们使用含有N端His(6)标签的重组PTTG1蛋白（24 kD）作为免疫原，开发了一种对PTTG1基因产物具有高度特异性的兔多克隆抗体SK601。该抗血清在ELISA检测中能够以1:100,000的效价检测重组PTTG1蛋白。在蛋白质印迹分析中使用该抗体作为探针，从表达GST-PTTG1重组蛋白的大肠杆菌中检测到一条预期相对分子质量为52 kD的单带，从转染了PTTG1-GFP嵌合构建体的COS-7细胞中检测到一条相对分子质量为56 kD的单带。在用PTTG1 cDNA转染的COS-7细胞提取物中观察到一条相对分子质量为28 kD的单带。该抗血清从转染了PTTG1-GFP嵌合构建体的COS-7细胞提取物中免疫沉淀出一种相对分子质量为56 kD的蛋白。对转染了该构建体的COS-7细胞进行免疫组织化学分析证实，该抗体能够检测并特异性识别表达PTTG1-GFP重组蛋白。通过免疫组织化学对各种正常人体组织（睾丸、卵巢和乳腺）进行筛查表明，除了先前已显示表达PTTG1 mRNA的睾丸组织外，这些组织均未出现染色。相反，所有评估的肿瘤组织（睾丸肿瘤、卵巢肿瘤和乳腺肿瘤）均呈现强烈染色。结果表明，抗血清SK601对PTTG1蛋白具有高度特异性，因此在进一步分析该蛋白的表达和相互作用，包括其作为人类肿瘤免疫组织化学标志物的潜在应用中应会证明是有用的。
