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

(目的)我们曾试图分离肾细胞癌(RCC)中特异和/或高表达的基因。利用基因产物的抗体检测肾癌组织中的抗原。(材料和方法)对肾癌组织和正常肾组织的总RNA进行差异显示。对肾细胞癌中表达的独特基因进行克隆、测序、同源基因搜索，并分析其在肾细胞癌和正常肾脏中的表达。利用分离基因编码的胸腺素β-10(17-43aa)多肽，在日本大白兔体内制备了抗胸腺素β-10抗体。抗体用亲和层析柱与胸腺素β-10多肽(17-43aa和36-43aa)偶联，抗胸腺素β-10抗体(17-43aa)与生物素偶联。用胸腺肽BEAT-10和胸腺肽β-4对抗体的反应性进行了酶联免疫吸附试验。用双抗体夹心ELISA法检测Exc…中胸腺素β-10抗原以抗胸腺素β-10抗体(17-43aa)为捕获抗体，以抗胸腺素-β-10抗体(36-43aa)为示踪抗体，从肾癌组织和肾癌细胞培养上清液中分离出4个克隆。Northern印迹分析表明，这些基因在肾癌组织中高表达。2个基因与胸腺素β10和人类白细胞抗原DRα链同源，其余为未知基因。我们用胸腺素β-10肽(17-43aa)制备了抗胸腺素β-10抗体，并用胸腺素β-10(17-43aa和36-43aa)偶联亲和层析柱纯化。抗胸腺素β-10抗体(36-43aa)可与胸腺素β-10特异反应，但不与胸腺素β-4反应。用抗胸腺素β-10抗体夹心ELISA法在肾癌组织提取液和肾癌培养上清液中检测到胸腺素β-10抗原。(讨论)胸腺素β-10可作为多种肿瘤的标志物。较少

(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