Characterisation of a plancitoxin-1-like DNase II gene in Trichinella spiralis.
Characterisation of a plancitoxin-1-like DNase II gene in Trichinella spiralis.
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旋毛虫中 Plancitoxin-1 样 DNase II 基因的表征
DOI：
10.1371/journal.pntd.0003097
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发表时间：
2014-08
影响因子：
3.8
通讯作者：
Liu X
中科院分区：
文献类型：
--
作者：
Liao C;Liu M;Bai X;Liu P;Wang X;Li T;Tang B;Gao H;Sun Q;Liu X;Zhao Y;Wang F;Wu X;Boireau P;Liu X
背景脱氧核糖核酸酶II（DNase II）是一种已知的酸性核酸内切酶，其催化DNA降解成寡核苷酸。在许多物种的基因组中只观察到一个或几个编码DNase II的基因。在旋毛虫（Trichinellaspiralis，T. spiralis）基因组;然而，没有一个得到证实。DNase II是一种单体核酸酶，在N-和C-末端含有两个拷贝的变体HKD基序。在这125个基因中，只有plancitoxin-1（1095 bp，GenBank登录号XM_003370715.1）在其C-末端结构域中含有HKD基序。方法/主要发现在这项研究中，我们克隆和特点的plancitoxin-1基因。然而，从T. spiralis序列均短于GenBank预测序列。有趣的是，在克隆序列的N-和C-末端有两个HKD基序。因此，具有较短序列的基因被命名为plancitoxin-1-like（Ts-Pt，885 bp），并已在GenBank中以登录号KF 984291保藏。在原核表达系统中表达重组蛋白（rTs-Pt），镍亲和层析纯化。Western blot分析表明，rTs-Pt能被T.感染旋毛虫的小鼠;抗rTs-Pt血清识别粗抗原，但不识别ES抗原。在所有T.实时定量PCR检测旋毛虫的发育阶段。免疫定位分析表明，Ts-Pt分布在整个新生幼虫（NBL），皮层的成人（Ad）和肌肉幼虫（ML）。DNase酶谱分析表明，表达蛋白具有阳离子非依赖性DNase活性。rTs-Pt在微酸性条件下（pH 4和pH 5）具有窄的最适pH范围，其最适温度为25°C、30°C和37°C。结论Ts-Pt在不同的T.首次证明了表达的DNA酶II蛋白在旋毛虫发育过程中具有特异性。spiralis具有核酸酶活性。
Background Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain. Methodology/Principal Findings In this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C. Conclusions This study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity.
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Wang L;Wang ZQ;Hu DD;Cui J
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