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Prostaspanin: A novel member of tetraspanin superfamily specifically expressed in the prostate

前列腺跨膜蛋白：四跨膜蛋白超家族的新成员，在前列腺中特异性表达

基本信息

批准号：
12671538
负责人：
NISHI Nozomu
金额：
$ 1.92万
依托单位：
Kagawa Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12671538/
关键词：
prostate androgen tetraspanin subtraction growth

项目摘要

We have isolated cDNA clones which are specifically expressed during androgen-dependent regrowth of rat ventral prostate by using PCR-based subtractive hybridization technique. One of the clones, AIEG322 (tentatively designated as prostaspanin), showed significant sequence homology with those of the members of tetraspanin superfamily. Among rat tissues examined, highlevels of prostaspanin mRNA expression was detected in the prostate and seminal vesicle. To investigate physiological function of prostaspanin in the prostate and to evaluate the possibility of utilization of the serum prostaspanin concentration as a marker of prostatic cancer, we set the highest priority for preparing anti-prostaspanin antibodies. Prostaspanin cDNAs were amplified from cDNAs derived from rat and human prostates, and the expression vectors for rat and human prostaspanin with a GST- and a hexahistidine-tag were prepared. The recombinant proteins were expressed in E. coli. However, the expressed proteins could not be solubilized, because of the formation of inclusion bodies. Due to the difficulties in using the recombinant proteins as antigens, we started to produce anti-oligopeptide antibodies. The use of an synthetic oligopeptide, FTQVWNTTM, corresponding to one of the extracellular domains of prostapanin (EC2), resulted in the production of high titer antiserum in rabbits. The antiserum recognized recombinant rat and human prostaspanin in Western blot analysis. Western blot analysis using rat tissue extracts and membrane fractions, however, showed that the antiserum could not react with native prostaspnin. Glycosylation at the aspargine residue in the nativ prostaspanin may explain the observation. Currently, we are trying to prepare anti-prostaspanin antibodies using a synthetic oligopeptide corresponding to different regions of the protein.

我们利用基于PCR的消减杂交技术分离了雄激素依赖性前列腺再生过程中特异表达的cDNA克隆。其中一个克隆AIEG 322（暂命名为prostaspanin）与四跨膜蛋白超家族成员的序列具有显著的同源性。在检测的大鼠组织中，在前列腺和精囊中检测到高水平的prostaspanin mRNA表达。为了研究前列腺组织中前列腺Aspanin的生理功能，并评估血清中前列腺Aspanin浓度作为前列腺癌标志物的可能性，我们将制备抗前列腺Aspanin抗体列为最优先事项。从来自大鼠和人前列腺的cDNA扩增前列腺aspanin cDNA，并制备具有GST-和六组氨酸-标签的大鼠和人前列腺aspanin的表达载体。重组蛋白在E.杆菌但表达产物不能溶解，形成包涵体。由于利用重组蛋白作为抗原的困难，我们开始生产抗寡肽抗体。使用合成的寡肽，FTQVWNTTM，对应于前列腺素（EC 2）的胞外结构域之一，导致在兔中产生高滴度的抗血清。抗血清识别重组大鼠和人前列腺aspanin在Western blot分析。然而，使用大鼠组织提取物和膜组分的Western印迹分析表明，抗血清不能与天然前列腺素反应。天然prostaspanin中天冬酰胺残基的糖基化可以解释这一观察结果。目前，我们正在尝试使用对应于蛋白质不同区域的合成寡肽来制备抗前列腺Aspanin抗体。