旋毛虫弹性蛋白酶-1 的分子特征及其作为旋毛虫病诊断抗原的潜力。
Molecular characterization of a Trichinella spiralis elastase-1 and its potential as a diagnostic antigen for trichinellosis.
机构信息
Department of Parasitology, Medical College, Zhengzhou University, Zhengzhou, 450052, China.
出版信息
Parasit Vectors. 2020 Feb 24;13(1):97. doi: 10.1186/s13071-020-3981-y.
BACKGROUND
Trichinella spiralis muscle larval (ML) excretion/secretion (ES) antigen is the most widely used diagnostic antigen of trichinellosis, but preparation of ES antigen requires collecting worms from infected animals, and detection of specific IgG against ML ES antigen may result in a false negative at the early stage of infection. The aim of the study was to characterize T. spiralis elastase-1 (TsEla) and to evaluate its potential as diagnostic antigen for trichinellosis.
METHODS
The complete cDNA sequences of the TsEla gene were cloned and expressed, and recombinant (rTsEla) was purified. TsEla transcription and expression in different T. spiralis life-cycle stages was investigated by qPCR and western blotting, and its location in the nematodes was evaluated using an immunofluorescence assay (IFA). The antigenicity of rTsEla was investigated by western blotting analysis and ELISA. Anti-Trichinella IgG, IgM and IgE of experimentally infected mice and specific IgG antibodies of trichinellosis patients were assayed by rTsEla-ELISA and ES-ELISA.
RESULTS
The results of the qPCR and western blotting showed that TsEla was expressed in various T. spiralis life stages. Natural TsEla was detected in the soluble proteins and ES proteins of different life stages. IFA revealed that TsEla was identified in the whole nematodes of various stages, especially in the cuticle, stichosome and genital primordium of the parasite. Serum anti-Trichinella IgM, IgG and IgE in infected mice was first detected by rTsEla-ELISA at 6, 10 and 12 days post-infection (dpi), and reached 100% at 8, 14 and 14 dpi, respectively. When rTsEla-ELISA and ES-ELISA were used to detect anti-Trichinella IgG in sera of trichinellosis patients, the sensitivity was 97.37% (37/38) and 89.74% (34/38) (P > 0.05), and the specificity was 99.10% (220/222) and 98.20% (218/222), respectively (P > 0.05). The rTsEla cross-reacted with only one serum sample out of 20 samples from paragonimiasis patients and 7 samples from clonorchiasis patients.
CONCLUSIONS
rTsEla is valuable to early diagnosis of trichinellosis and could be an alternative diagnostic antigen to the ML ES antigens.
背景
旋毛虫肌肉幼虫（ML）排泄/分泌（ES）抗原是旋毛虫病最广泛使用的诊断抗原，但 ES 抗原的制备需要从感染动物中收集蠕虫，并且针对 ML ES 抗原的特异性 IgG 的检测可能导致在感染的早期出现假阴性。本研究的目的是表征旋毛虫弹性蛋白酶-1（TsEla），并评估其作为旋毛虫病诊断抗原的潜力。
方法
克隆并表达了 TsEla 基因的完整 cDNA 序列，并对重组（rTsEla）进行了纯化。通过 qPCR 和 Western blotting 研究了 TsEla 在不同旋毛虫生命周期阶段的转录和表达，并通过免疫荧光分析（IFA）评估了其在线虫中的定位。通过 Western blotting 分析和 ELISA 研究了 rTsEla 的抗原性。通过 rTsEla-ELISA 和 ES-ELISA 检测了实验感染小鼠的抗旋毛虫 IgG、IgM 和 IgE 以及旋毛虫病患者的特异性 IgG 抗体。
结果
qPCR 和 Western blotting 的结果表明，TsEla 在各种旋毛虫生活阶段均有表达。天然 TsEla 存在于不同生活阶段的可溶性蛋白和 ES 蛋白中。IFA 显示 TsEla 存在于各个阶段的整个线虫中，尤其是在寄生虫的角质层、刺体和生殖原基中。rTsEla-ELISA 在感染后 6、10 和 12 天首次检测到感染小鼠的血清抗旋毛虫 IgM、IgG 和 IgE，分别在 8、14 和 14 天达到 100%。当使用 rTsEla-ELISA 和 ES-ELISA 检测旋毛虫病患者血清中的抗旋毛虫 IgG 时，灵敏度分别为 97.37%（37/38）和 89.74%（34/38）（P>0.05），特异性分别为 99.10%（220/222）和 98.20%（218/222）（P>0.05）。rTsEla 仅与 20 例肺吸虫病患者血清样本中的 1 例和 7 例华支睾吸虫病患者血清样本中的 1 例发生交叉反应。
结论
rTsEla 对旋毛虫病的早期诊断具有重要价值，可作为 ML ES 抗原的替代诊断抗原。
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