Molecular cloning and characterization of a 21 kDa protein secreted from Trichinella pseudospiralis
Molecular cloning and characterization of a 21 kDa protein secreted from Trichinella pseudospiralis
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DOI：
10.1079/joh200158
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发表时间：
2001-09
影响因子：
1.6
通讯作者：
I. Nagano;Zhiliang Wu;T. Nakada;A. Matsuo;Y. Takahashi
中科院分区：
文献类型：
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作者：
I. Nagano;Zhiliang Wu;T. Nakada;A. Matsuo;Y. Takahashi
从假螺旋旋毛虫cDNA文库中获得重组蛋白，该蛋白似乎是其排泄-分泌产物的一部分。利用感染后1个月的肌肉幼虫cDNA构建文库，并用假螺旋绦虫ES产物抗体进行免疫筛选。一个名为Tp21-3的克隆含有全长657 bp的cDNA转录物，其中包含一个开放阅读框，编码172个氨基酸（估计分子质量为19617 Da）。预测的Tp21-3与螺旋体克隆ORF 17.20 （GenBank，登录号U88239）的氨基酸序列相似性为76%。克隆Tp21-3编码的重组融合蛋白在大肠杆菌表达系统中得到并亲和纯化。Western blotting分析显示，Tp21-3重组蛋白在40 kDa处迁移，并与假螺旋体ES产物和假螺旋体感染血清的抗体发生反应。重组蛋白Tp 21-3在免疫组化染色上与假螺旋绦虫粗提物和ES产物在21 kDa的单带迁移反应，与假螺旋绦虫的棘细胞反应。
Recombinant protein was produced from the cDNA library of Trichinella pseudospiralis, which seemed to form part of the excretory–secretory (ES) products. The library was constructed from cDNA of muscle larvae at 1 month post-infection, and immunoscreened with antibody against T. pseudospiralis ES products. A clone, designated Tp21-3, contained a cDNA transcript of 657 bp in length with a single open reading frame, which encoded 172 amino acids (19617 Da in the estimated molecular mass). The predicted amino acid sequence of clone Tp21-3 had a similarity of 76% to that of clone ORF 17.20 (GenBank under accession number U88239) from T. spiralis. The recombinant fusion proteins encoded by clone Tp21-3 were produced in an Escherichia coli expression system and affinity purified. On Western blotting analysis, Tp21-3 recombinant proteins migrated at 40 kDa and reacted to antibody against T. pseudospiralis ES products and T. pseudospiralis-infected sera. Sera were developed against Tp 21-3 recombinant proteins, which reacted to a single band migrating at 21 kDa in crude worm extract and ES products from T. pseudospiralis on Western blotting analysis, and reacted with stichocytes of T. pseudospiralis on immunohistochemical staining.