Molecular cloning of highly expressed genes in renal cell carcinoma using cifferential display system

（目的）本研究试图分离肾细胞癌（RCC）中特异性和/或高表达的基因。利用基因产物的抗体对肾细胞癌抗原的检测进行了研究。（材料与方法）采用差异显示技术对肾癌组织和正常肾组织的总RNA进行分析。对肾细胞癌中特异表达的基因进行克隆、测序，寻找同源基因，并分析其mRNA在肾细胞癌和正常肾组织中的表达。利用分离的胸腺素β 10（Thymosin beta-10，17- 43 aa）基因编码的多肽，在日本白色家兔体内制备了抗Thymosin beta-10抗体。使用与胸腺素β-10肽（17- 43 aa和36- 43 aa）缀合的亲和柱特异性纯化抗体，并且将抗胸腺素β-10抗体（17- 43 aa）与生物素缀合。使用胸腺素β-10和胸腺素β-4肽通过ELISA分析这些抗体的反应性。用双抗体夹心ELISA法检测胸腺素β 10抗原， 关于我们 以抗胸腺素β 10抗体（17- 43 aa）为捕获抗体，抗胸腺素β 10抗体（36- 43 aa）为示踪抗体，从肾细胞癌组织和肾细胞癌培养上清中分离纯化。（结果）通过差异显示技术分离得到4个cDNA克隆。北方印迹分析表明，这些基因在肾癌中均呈高表达。其中2个基因与Thymosin beta-10和HLA-DR α链同源，其余为未知基因。我们用胸腺素β-10肽（17- 43 aa）制备了抗胸腺素β-10抗体，并用胸腺素β-10（17- 43 aa和36- 43 aa）缀合的亲和柱纯化。抗胸腺素β-10抗体（36- 43 aa）与胸腺素β-10特异性反应，但不与胸腺素β-4反应。用抗Thymosin beta-10抗体进行夹心ELISA，可在RCC组织提取物和RCC培养上清中检测到Thymosin beta-10抗原。（讨论）胸腺素β-10可能是几种癌症的标志物，因为我们可以使用抗体检测抗原。少

(Purposes) We had tried to isolate specifically and/or highly expressed genes in renal cell carcinoma (RCC). Using antibodies of the gene products, The detection of antigen from RCCs was investigated.(Materials and Methods) Differential display was performed with total RNAs from renal cell carcinoma and normal kidney tissues. Unique genes expressed in RCC were cloned, sequences, searched homologous genes and analyzed mRNA expression in RCCs and normal kidneys. Using the peptide of Thymosin beta-10(17-43aa), which was coded by an isolated gene, we developed anti-Thymosin beta-10 antibody in Japanese white rabbit. Antibodies were specifically purified using affinity columns conjugated with Thymosin beta-10 Peptides(17-43aa and 36-43aa) and anti Thymosin beta-10 antibody(17-43aa) was conjugated with biotin. The reactivity of these antibodies were analyzed by ELISA using Thymosin beat-10 and Thymosin beta-4 peptides. Sandwich ELISA was performed to detect Thymosin beta-10 antigen in extrac … More tions from RCC tissues and cultured supernatants of RCCs, using anti Thymosin beta-10 antibody(17-43aa) as capture antibody and anti Thymosin beta-10 antibody(36-43aa) as tracer antibody.(Results) Four cDNA clones were isolated by Differential display. These genes were highly expressed in renal cell carcinomas by Northern blot analysis. Two genes were homologous with Thymosin beta-10 and HLA-DR alpha chain, the others were unknown genes. We developed anti Thymosin beta-10 antibody using Thymosin beta-10 peptide(17-43aa) and purified using Thymosin beta-10(17-43aa and 36-43aa) conjugated affinity columns. Anti Thymosin beta-10 antibody(36-43aa) was specifically reacted with Thymosin beta-10,but not with Thymosin beta-4. Thymosin beta-10 antigen was able to detected in the extraction of RCC tissues and cultured supernatants of RCCs by sandwich ELISA using anti Thymosin beta-10 antibodies.(Discussion) Thymosin beta-10 might be a marker for several carcinomas, because we could detect the antigen using the antibodies. Less