Cloning, expression, characterization, and immunological properties of citrate synthase from Echinococcus granulosus
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The larval stages of the tapeworm Echinococcus granulosus (Cestoda: Taeniidae) are the causative agent of cystic echinococcosis, one of the most important parasitic zoonoses worldwide. E. granulosus has a complete pathway for the tricarboxylic acid cycle (TCA), in which citrate synthase (CS) is the key enzyme. Here, we cloned and expressed CS from E. granulosus (Eg-CS) and report its molecular characterization. The localization of this protein during different developmental stages and mRNA expression patterns during H2O2 treatment were determined. We found that Eg-CS is a highly conserved protein, consisting of 466 amino acids. In western blotting assays, recombinant Eg-CS (rEg-CS) reacted with E. granulosus-positive sheep sera and anti-rEg-CS rabbit sera, indicating that Eg-CS has good antigenicity and immunoreactivity. Localization studies, performed using immunohistochemistry, showed that Eg-CS is ubiquitously expressed in the larva, germinal layer, and adult worm sections of E. granulosus. Eg-CS mRNA expression levels increased following H2O2 exposure. In conclusion, citrate synthase might be involved in the metabolic process in E. granulosus. An assessment of the serodiagnostic potential of rEg-CS based on indirect ELISA showed that, although sensitivity (93.55%) and specificity (80.49%) are high, cross-reactivity with other parasites precludes its use as a diagnostic antigen.
KeywordsEchinococcus granulosus Citrate synthase Tricarboxylic acid cycle Immunohistochemical localization mRNA expression
We are grateful to Chunyan Li, Hongyu Song, and Ruiqi Hua for their help and suggestions. We would like to thank the native English speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript.
NW and GYY conceived and designed the study and critically revised the manuscript. JFZ, CG, NXS, XBG, and WML performed the experiments, analyzed the data, and drafted the manuscript. XRP and YX conducted the data analysis. HZ and GYY revised the manuscript. All authors have approved the final manuscript.
This research was funded by the National Natural Science Foundation of China [grant number 31672547].
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
All animals included in this study were purchased from the Laboratory Animal Center of Sichuan Agricultural University. All procedures were carried out in strict accordance with the Guide for the Care and Use of Laboratory Animals and with approval from the Animal Ethics Committee of Sichuan Agricultural University (Ya’an, China; Approval No. 2013–028).
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