Generation and characterization of novel monoclonal antibodies against murine and human TARSH proteins
Generation and characterization of novel monoclonal antibodies against murine and human TARSH proteins
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DOI：
10.1089/hyb.2007.0520
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发表时间：
2007-12-01
期刊：
影响因子：
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通讯作者：
Maruyama, Mitsuo
中科院分区：
文献类型：
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作者：
Uekawa, Natsuko;Nishioka, Tomohisa;Maruyama, Mitsuo
TARSH/Abi 3bp最初是通过双杂交酵母系统作为NESH-SH 3的新靶标分离的。我们已经确定了小鼠TARSH（mTARSH）作为细胞衰老相关基因，因为它在小鼠胚胎成纤维细胞衰老的早期阶段具有强大的诱导作用。我们还发现，该基因的表达在人肺癌细胞系和原发性肺肿瘤中显著降低，而在正常条件下主要表达。这一证据表明，TARSH参与了应激诱导的衰老和预防癌症的发展;然而，对其分子机制知之甚少。为了进一步揭示该分子的生理功能，我们建立了大鼠抗TARSH单克隆抗体（MAb）。在大肠杆菌中表达重组His标记的部分小鼠TARSH蛋白，亲和纯化并作为抗原免疫大鼠。通过酶联免疫吸附试验筛选杂交瘤，我们产生了6个稳定的杂交瘤细胞系，产生抗鼠TARSH蛋白的抗体，其中包括3个克隆，代表与人TARSH的交叉反应性。我们确定了它们的同种型，并进一步检查了免疫印迹、免疫沉淀和免疫荧光显微镜的能力或局限性，从而实现了最适合每种应用的抗体。因此，这些单克隆抗体应该是非常有用的工具，研究TARSH的表达和以下的生物功能，在细胞衰老和肿瘤抑制。
TARSH/Abi3bp was originally isolated as a novel target of NESH-SH3 by a two-hybrid yeast system. We have already identified murine TARSH (mTARSH) as a cellular senescence-related gene because of its robust induction in the early phase of mouse embryonic fibroblast cellular senescence. We have also revealed that the expression of this gene was dramatically reduced in human lung cancer cell lines and primary lung tumor, while it was predominantly expressed in normal conditions. This evidence suggests that TARSH is involved in both stress-induced senescence and prevention of cancer development; however, little is known about its molecular mechanisms. To reveal the further physiological function of this molecule, we established rat anti-TARSH monoclonal antibodies (MAb). Recombinant His-tagged partial mouse TARSH protein was expressed in Escherichia coli, affinity purified and used as an antigen to immunize rats. Hybridomas were screened by enzyme-linked immunosorbent assay, and we generated six stable hybridoma cell lines that produced antibody against murine TARSH protein, including three clones that represented cross-reactivity with human TARSH. We determined their isotypes and further examined capabilities or limitations in immunoblotting, immunoprecipitation, and immunofluorescence microscopy, realizing the most suitable antibody for each application. These MAbs should therefore be very useful tools for the study of TARSH expression and for following biological function in cellular senescence and tumor suppression.