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首页> 外文期刊>Molecular reproduction and development >Novel human testis-specific cDNA: Molecular cloning, expression and immunobiological effects of the recombinant protein.
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Novel human testis-specific cDNA: Molecular cloning, expression and immunobiological effects of the recombinant protein.

机译：新型人类睾丸特异性cDNA：重组蛋白的分子克隆，表达和免疫生物学作用。

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A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-lambdagt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122-124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S-transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the approximately 17 kDa recombinant TSA-1, and a approximately 24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility. Mol. Reprod. Dev. 60: 1-12, 2001. Copyright 2001 Wiley-Liss, Inc.
机译：使用差异展示-聚合酶链反应获得睾丸特异性cDNA片段。通过用睾丸特异性cDNA片段筛选人睾丸-lambdagt10-cDNA文库，获得了编码精子抗原的新型cDNA，命名为TSA-1。它具有471个碱基对的新型开放阅读框（ORF），编码156个氨基酸。计算机生成的翻译蛋白质的计算分子量为17.4 kDa，在氨基酸122-124处含有潜在的N-糖基化位点。氨基酸序列的亲水性分析表明该蛋白是膜​​锚定肽。通过使用各种人体组织的Northern印迹和RT-PCR-Southern印迹方法对组织特异性进行的广泛分析表明，TSA-1仅在人的睾丸中特异性表达。根据体外转录和翻译实验的结果，将TSA-1（ORF）亚克隆到pGEX-6P-3载体中，并使用谷胱甘肽S-转移酶基因融合系统进行表达。针对纯化的重组蛋白的抗体（Ab）在Western印迹程序中特异性识别人精子提取物中约17 kDa的重组TSA-1和约24 kDa的条带。重组TSA-1 Ab在间接免疫荧光中识别人精子的顶体，赤道，中部和尾部区域，并通过免疫珠结合技术（IBT）与活人精子结合，对人体产生明显的浓度依赖性抑制作用精子顶体反应。这些发现表明，新颖的精子特异性重组TSA-1在精子功能中起作用，并且可能在避孕疫苗的开发以及男性不育的具体诊断和治疗中具有应用。大声笑责备。开发人员60：1-12，2001。版权所有2001 Wiley-Liss，Inc.。

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