Identification and characterization of the immunogenic cytotoxic TvCP39 proteinase gene of Trichomonas vaginalis
Identification and characterization of the immunogenic cytotoxic TvCP39 proteinase gene of Trichomonas vaginalis
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DOI：
10.1016/j.biocel.2011.07.001
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发表时间：
2011-10-01
影响因子：
4
通讯作者：
Arroyo, Rossana
中科院分区：
文献类型：
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作者：
de los Angeles Ramon-Luing, Lucero;Javier Rendon-Gandarilla, Francisco;Arroyo, Rossana
TvCP39是一种39 kDa的半胱氨酸蛋白酶（CP），参与阴道毛滴虫的细胞毒性，已在阴道分泌物中发现，并在毛滴虫病患者中具有免疫原性。这项工作的目的是鉴定、克隆、表达和表征tvcp39基因。利用蛋白质组学方法鉴定了tvcp39基因，并利用PCR扩增、克隆和测序了该基因。TvCP39由915 bp的组织蛋白酶l样CP基因编码。表达成熟区对应的片段（TvCP39r），纯化，并用于兔多克隆抗体和功能检测。在一维和二维western blot检测中，抗tvcp39r抗体与富含毛滴虫蛋白酶的提取物中的两个类似28和27 kDa的蛋白带和三个类似28、27和24 kDa的蛋白点反应，这些蛋白带可能对应于成熟的和加工过的TvCP39肽酶片段。抗tvcp39r抗体与滴虫病患者阴道洗液中的寄生表面和天然TvCP39发生反应。此外，重组的TvCP39蛋白结合到HeLa细胞表面，并以浓度依赖的方式保护HeLa细胞单层免受滴虫的破坏。总之，我们的数据支持TvCP39是糖基化的表面蛋白酶之一，参与滴虫细胞毒性。因此，TvCP39是第一个在阴道绦虫中检测到的糖基化半胱氨酸蛋白酶。(C) 2011 Elsevier Ltd.版权所有。
TvCP39 is a 39 kDa cysteine proteinase (CP) involved in Trichomonas vaginalis cytotoxicity that has been found in vaginal secretions and is immunogenic in patients with trichomonosis. The goal of this work was to identify, clone, express, and characterize the tvcp39 gene. The tvcp39 gene was identified using a proteomic approach, and the complete gene was amplified using PCR, cloned, and sequenced. TvCP39 is encoded by a 915-bp cathepsin L-like CP gene. A fragment corresponding to the mature region (TvCP39r) was expressed, purified, and used to produce rabbit polyclonal antibodies and in functional assays. In one- and two-dimensional western blot assays, the anti-TvCP39r antibody reacted with two protein bands of similar to 28 and 27 kDa and three spots of similar to 28, 27, and 24 kDa in trichomonad proteinase-rich extracts that could correspond to the mature and processed fragments of the TvCP39 peptidase. The anti-TvCP39r antibody reacted with the parasitic surface and the native TvCP39 present in vaginal washes from patients with trichomonosis. Moreover, the recombinant TvCP39 protein bound to the surface of HeLa cells and protected HeLa cell monolayers from trichomonal destruction in a concentration-dependent manner. In conclusion, our data support TvCP39 as one of the surface proteinases that is glycosylated and is involved in trichomonal cytotoxicity. Thus, TvCP39 is the first glycosylated cysteine proteinase detected in T. vaginalis. (C) 2011 Elsevier Ltd. All rights reserved.