Department of Parasitology, Gifu University Graduate School of Medicine, Yanagido 1-1, Gifu 501-1194, Japan.
Vet Parasitol. 2011 May 31;178(1-2):134-42. doi: 10.1016/j.vetpar.2010.12.031. Epub 2010 Dec 25.
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.
旋毛虫肌肉幼虫 cDNA 文库构建。一个 cDNA 克隆，命名为 Tp4，包含 783bp 的 cDNA 转录本，具有一个单一的开放阅读框，编码 153 个氨基酸（估计分子量为 16793Da）。Tp4 的预测氨基酸序列表明，该克隆具有钙调蛋白同源结构域，与存在于家蚕或赤拟谷盗中的迁移蛋白样蛋白（钙调蛋白家族成员）约有 50%的同一性。Tp4 克隆的同源物也存在于旋毛虫 cDNA 中，该克隆被命名为 Ts4。Ts4 蛋白的迁移蛋白样蛋白的氨基酸序列与 Tp4 蛋白的比较表明，两种蛋白非常相似（约 94%的同源性）。实时定量聚合酶链反应结果显示，Tp4 和 Ts4 基因的转录水平在新生幼虫中最高。在 Western blot 中，当与针对重组 Tp4 和 Ts4 蛋白的抗体反应时，重组 Tp4 和 Ts4 蛋白分别迁移到 20kDa。针对重组 Tp4 和 Ts4 蛋白的抗体在成虫和新生幼虫的粗提物中强烈染色两条约 9 和 8kDa 的带，但在肌肉幼虫中仅微弱染色蛋白。然而，免疫细胞化学研究表明，Tp4 蛋白存在于旋毛虫肌肉幼虫的肌肉中。感染小鼠对重组 Tp4 抗原的抗体水平从感染后 8 天开始增加，在感染后 13 天达到最高，然后缓慢下降。
