Molecular cloning and characterization of transgelin-like proteins mainly transcribed in newborn larvae of Trichinella spp.
Molecular cloning and characterization of transgelin-like proteins mainly transcribed in newborn larvae of Trichinella spp.
复制标题
DOI：
10.1016/j.vetpar.2010.12.031
复制
发表时间：
2011-05-31
影响因子：
2.6
通讯作者：
Takahashi, Yuzo
中科院分区：
文献类型：
--
作者：
Nagano, Isao;Wu, Zhiliang;Takahashi, Yuzo
以假旋毛虫肌肉幼虫为材料，构建了旋毛虫肌幼虫基因文库。其中一个克隆命名为Tp4，包含一个783bp的转录本，具有一个编码153个氨基酸(估计分子质量为16793Da)的单一开放阅读框。预测的Tp4的氨基酸序列表明，该克隆具有钙蛋白同源结构域，与家蚕或赤霉菌中存在的转胶蛋白样蛋白(钙蛋白家族成员)有大约50%的同源性。旋毛虫Tp4克隆的同源物也存在于旋毛虫的cDNA中，命名为TS4。旋毛虫转胶样蛋白(TS4蛋白)与Tp4蛋白的氨基酸序列比较表明，两者非常相似(同源性约94%)。实时荧光定量聚合酶链式反应结果显示，Tp4和Ts4基因在新生幼虫中的转录水平最高。Western印迹显示，重组Tp4和TS4蛋白分别与抗Tp4和TS4蛋白的抗体反应，迁移量为20 kDa。抗重组Tp4和Ts4蛋白的抗体在成虫和新生幼虫粗提物中强染色约9 kDa和8 kDa的两条带，而在肌幼虫中仅弱染色的蛋白质。然而，一项免疫细胞化学研究表明，Tp4蛋白存在于假旋毛虫肌肉幼虫的肌肉中。重组Tp4抗体水平从感染后第8天开始升高，感染后第13天最高，随后缓慢下降。(C)2010爱思唯尔B.V.保留所有权利。
A cDNA library was constructed from Trichinella pseudospiralis muscle larvae. One cDNA clone, designated Tp4, contained a cDNA transcript of 783 bp in length, with a single open reading frame that encoded 153 amino acids (16,793 Da as the estimated molecular mass). The predicted amino acid sequence of Tp4 showed that the clone had a calponin homology domain and was approximately 50% identical to the transgelin-like proteins (calponin-family members) present in Bombyx mori or Tribolium castaneum. A homologue of the Tp4 clone was also present in cDNA from Trichinella spiralis, and this clone was designated Ts4. A comparison of the amino acid sequence of the transgelin-like proteins from T. spiralis (Ts4 protein) with the Tp4 protein indicated that the two proteins are very similar (about 94% homology). Real time quantitative polymerase chain reaction results showed that the transcription level of the Tp4 and Ts4 genes was highest in newborn larvae. On Western blot, the recombinant Tp4 and Ts4 proteins migrated at 20 kDa when reacted to an antibody against the recombinant Tp4 and Ts4 proteins, respectively. An antibody against the recombinant Tp4 and Ts4 proteins strongly stained two bands migrating at approximately 9 and 8 kDa in the crude extracts from adult worms and newborn larvae, but only weakly stained proteins in muscle larvae. However, an immunocytochemical study showed that the Tp4 protein was present within the muscle of the muscle larvae of T. pseudospiralis. The antibody level against the recombinant Tp4 antigens in infected mice began to increase from 8 days post-infection, was highest in 13 days post-infection, and then slowly decreased. (c) 2010 Elsevier B.V. All rights reserved.