Kurita A, Takizawa T, Takayama T, Totsukawa K, Matsubara S, Shibahara H, Orgebin-Crist M C, Sendo F, Shinkai Y, Araki Y
Department of Immunology & Parasitology, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
Biol Reprod. 2001 Mar;64(3):935-45. doi: 10.1095/biolreprod64.3.935.
A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.
一种名为TES101的单克隆抗体，是通过用同种异体小鼠睾丸匀浆免疫BALB/c小鼠，随后以免疫组织化学选择作为初始筛选方法制备的。通过用TES101反应蛋白的N端氨基酸序列搜索表达序列标签（EST）数据库，我们发现一个小鼠睾丸EST克隆编码的预测氨基酸序列与TES101蛋白序列匹配。对该克隆的序列分析表明，在DNA/蛋白质数据库中没有同源分子。根据从TES101蛋白的N端氨基酸分析获得的数据，推导的氨基酸序列包含一个25个氨基酸的信号肽区域和一个225个氨基酸的成熟蛋白区域，其翻译后的蛋白分子量为24093。Northern印迹分析显示，TES101蛋白的mRNA在睾丸中发现，但在所检测的任何其他小鼠组织中均未发现。Western印迹分析表明，在非还原条件下，TES101与SDS-PAGE上的一条38-kDa条带发生反应，而在还原条件下这种反应性消失。免疫电子显微镜研究表明，该分子主要位于精母细胞和精子细胞的质膜上，而不在支持细胞或包括睾丸间质细胞在内的间质细胞中。因此，TES101蛋白是一种主要存在于发育中的雄性生殖细胞表面的新分子。TES101蛋白可能在雄性生殖细胞形成的潜在过程中发挥作用。
