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

（目的）我们试图在肾细胞癌（RCC）中分离特异性和/或高表达的基因。利用该基因产物的抗体，研究了rcc抗原的检测。（材料和方法）用来自肾细胞癌和正常肾组织的总rna进行差异显示。对RCC中表达的独特基因进行克隆、测序、查找同源基因并分析RCC和正常肾脏的mRNA表达。利用分离基因编码的胸腺素β -10（17-43aa）肽段，制备了日本白兔胸腺素β -10抗体。抗体通过与胸腺素β -10肽（17-43aa和36-43aa）结合的亲和柱特异性纯化，抗胸腺素β -10抗体（17-43aa）与生物素结合。采用酶联免疫吸附法（ELISA）检测胸腺肽beat-10和胸腺肽β -4抗体的反应性。采用夹心ELISA法，以抗胸腺素-10抗体（17-43aa）为捕获抗体，以抗胸腺素-10抗体（36-43aa）为示踪抗体，检测RCC组织提取液和培养上清液中的胸腺素-10抗原。（结果）通过差分显示分离到4个cDNA克隆。Northern blot结果显示，这些基因在肾细胞癌中高度表达。2个基因与胸腺肽β -10和HLA-DR α链同源，其余为未知基因。我们利用胸腺素-10肽（17-43aa）制备抗胸腺素-10抗体，并利用胸腺素-10（17-43aa和36-43aa）偶联亲和柱进行纯化。抗胸腺蛋白酶β -10抗体（36-43aa）与胸腺蛋白酶β -10特异性反应，而与胸腺蛋白酶β -4无特异性反应。利用抗胸腺肽β -10抗体，夹心ELISA法可在RCC组织提取液和培养的RCC上清液中检测到胸腺肽β -10抗原。（讨论）胸腺蛋白酶β -10可能是几种癌症的标记物，因为我们可以用抗体检测抗原。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