Molecular characterization of a putative protein disulfide isomerase from Babesia caballi
Molecular characterization of a putative protein disulfide isomerase from Babesia caballi
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DOI：
10.1017/s0031182005008516
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发表时间：
2005-08
期刊：
影响因子：
2.4
通讯作者：
H. Ikadai;R. Tsukada;M. Sasaki;R. Takashiro;N. Yokoyama;N. Kudo;I. Igarashi;T. Oyamada
中科院分区：
文献类型：
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作者：
H. Ikadai;R. Tsukada;M. Sasaki;R. Takashiro;N. Yokoyama;N. Kudo;I. Igarashi;T. Oyamada
我们制备了一株抗卡氏巴贝斯虫裂殖子的mAb，命名为mAb2H2，并将其用于筛选卡氏巴贝斯虫裂殖子mRNA高表达蛋白的cDNA表达文库。该基因全长1547个核苷酸，包含一个36个核苷酸的内含子。1398核苷酸开放阅读框预测了一个51 kDa的蛋白质，与其他物种的蛋白质二硫键异构酶(PDI)相似。PDI基因由19个氨基酸组成的N端信号序列和C端的四肽序列(His-Thr-Glu-Leu；HTEL)用于内质网(ER)的腔内滞留。在杆状病毒中表达的重组蛋白的表观质量为51 kDa，与天然的B.Caballi蛋白相同。此外，重组蛋白的内质网保留信号位点(HTEL)保留了其在昆虫细胞内质网中的功能。在间接免疫荧光抗体试验中，该51 kDa蛋白在胞外裂殖子中有较强的表达，在滋养体发育的早期不表达。有趣的是，详细的观察表明，抗P51抗体和mAb2H2对梨形抗体的反应非常不稳定，有的表现出一种或两种明亮的荧光图案。
We produced a mAb against the Babesia caballi extracellular merozoite termed mAb 2H2 and used it to screen a cDNA expression library prepared from B. caballi merozoite mRNA for highly expressed proteins. The complete nucleotide sequence of the cloned gene had 1547 nucleotides and contained a 36-nucleotide intron. The 1398 nucleotide open reading frame predicts a 51 kDa protein showing similarity to protein disulfide isomerase (PDI) from other species. The PDI gene had a predicted N-terminal signal sequence of 19 amino acids and a C-terminal tetrapeptide sequence (His-Thr-Glu-Leu; HTEL) for retention in lumen of the endoplasmic reticulum (ER). The recombinant protein expressed in baculovirus showed an apparent mass of 51 kDa, identical to that the native B. caballi protein. Moreover, the ER retention signal site (HTEL) of the recombinant protein retained its function in ER of insect cells. This 51 kDa protein was strongly expressed by extracelluar B. caballi merozoites in indirect immunofluorescence antibody tests, and was not expressed in the early phase of trophozoite development. Interestingly, detailed observation showed that the reaction of anti-P51 antibody and mAb 2H2 against pear-shaped forms was very erratic, some displaying one or two brightly fluorescent patterns.