新型单克隆抗体检测人睾丸中POTE蛋白的表达
Expression of POTE protein in human testis detected by novel monoclonal antibodies.
作者信息
Ise Tomoko, Das Sudipto, Nagata Satoshi, Maeda Hiroshi, Lee Yoomi, Onda Masanori, Anver Miriam R, Bera Tapan K, Pastan Ira
机构信息
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892-4264, USA.
出版信息
Biochem Biophys Res Commun. 2008 Jan 25;365(4):603-8. doi: 10.1016/j.bbrc.2007.10.195. Epub 2007 Nov 9.
The POTE gene family is composed of 13 highly homologous paralogs preferentially expressed in prostate, ovary, testis, and placenta. We produced 10 monoclonal antibodies (MAbs) against three representative POTE paralogs: POTE-21, POTE-2gammaC, and POTE-22. One reacted with all three paralogs, six MAbs reacted with POTE-2gammaC and POTE-22, and three MAbs were specific to POTE-21. Epitopes of all 10 MAbs were located in the cysteine-rich repeats (CRRs) motifs located at the N-terminus of each POTE paralog. Testing the reactivity of each MAb with 12 different CRRs revealed slight differences among the antigenic determinants, which accounts for differences in cross-reactivity. Using MAbs HP8 and PG5 we were able to detect a POTE-actin fusion protein in human testis by immunoprecipitation followed by Western blotting. By immunohistochemistry we demonstrated that the POTE protein is expressed in primary spermatocytes, implying a role in spermatogenesis.
POTE基因家族由13个高度同源的旁系同源物组成，这些旁系同源物在前列腺、卵巢、睾丸和胎盘中优先表达。我们制备了针对三种代表性POTE旁系同源物（POTE-21、POTE-2γC和POTE-22）的10种单克隆抗体（MAb）。其中一种与所有三种旁系同源物反应，六种MAb与POTE-2γC和POTE-22反应，三种MAb对POTE-21具有特异性。所有10种MAb的表位都位于每个POTE旁系同源物N端的富含半胱氨酸的重复序列（CRR）基序中。测试每种MAb与12种不同CRR的反应性发现，抗原决定簇之间存在细微差异，这解释了交叉反应性的差异。使用MAb HP8和PG5，我们能够通过免疫沉淀后进行蛋白质印迹法在人睾丸中检测到一种POTE-肌动蛋白融合蛋白。通过免疫组织化学，我们证明POTE蛋白在初级精母细胞中表达，这意味着其在精子发生中起作用。
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