Characterisation of a Plancitoxin-1-Like DNase II Gene in Trichinella spiralis
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| Authors: | Chengshui Liao Mingyuan Liu Xue Bai Pan Liu Xuelin Wang Tingting Li Bin Tang He Gao Qingsong Sun Xidong Liu Ying Zhao Feng Wang Xiuping Wu Pascal Boireau Xiaolei Liu |
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| Institution: | 1. Key Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Jilin University, Changchun, People''s Republic of China.; 2. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, People''s Republic of China.; 3. National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai, People''s Republic of China.; University of Melbourne, Australia, |
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| Abstract: | BackgroundDeoxyribonuclease 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. {"type":"entrez-nucleotide","attrs":{"text":"XM_003370715.1","term_id":"339256039","term_text":"XM_003370715.1"}}XM_003370715.1) contains the HKD motif in its C-terminus domain.Methodology/Principal FindingsIn 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 {"type":"entrez-nucleotide","attrs":{"text":"KF984291","term_id":"594551321","term_text":"KF984291"}}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.ConclusionsThis 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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