CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES >

2020 , Vol. 38 >Issue 2: 194 - 201

DOI: https://doi.org/10.12140/j.issn.1000-7423.2020.02.010

ORIGNAL ARTICLES

Expression, localization and preliminary evaluation of diagnostic value of Echinococcus granulosus phosphoglycerate mutase

  • Hong-yu SONG ,
  • Yu-qing LIANG ,
  • Rui-qi HUA ,
  • Yuan SHI ,
  • Ai-guo YANG ,
  • Li GUO ,
  • Dong-bo YUAN ,
  • Yue XIE ,
  • Guang-you YANG
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  • 1 Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
    2 Sichuan Center for Animal Disease Control and Prevention, Chengdu 610041, China

Received date: 2019-09-19

  Online published: 2020-05-11

Supported by

Supported by Sichuan Science and Technology Program(2015NZ0041)

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Abstract

Objective To clone and express Echinococcus granulosus phosphoglycerate mutase (EgPGAM), analyze its immunoreactivity and distribution in protoscoleces, hydatid cysts and adult worms, and preliminarily evaluate the diagnostic value.Methods Protoscolex RNA was extracted and reverse transcribed to cDNA, from which EgPGAM gene was amplified by PCR and sequenced. The physicochemical properties, conserved domains, antigen epitopes and homology of EgPGAM were analyzed by bioinformatics softwares. Mega 5.0 software was used to analyze the characteristics of the sequence, and the phylogenetic tree was constructed with the neighbor joining method. The EgPGAM gene was inserted into the pET32a plasmid, and transferred into Escherichia coli BL21(DE3) for induction of expression. The expressed proteins were purified by nickle column, and further analyzed by SDS-PAGE for verifying the expression products, and Western blotting for immunoreactivity. The distribution of EgPGAM in protoscoleces, hydatid cysts and adults was analyzed by immunofluorescence assay. The diagnostic value of EgPGAM was evaluated by indirect enzyme-linked immunosorbent assay(ELISA).Results The EgPGAM gene is 756 bp in length, encoding 251 amino acids. The encoded protein has a relative molecular weight Mr about 28 000, an isoelectric point of 8.29, containing no signal peptide or transmembrane region. EgPGAM protein contained 12 highly-conserved amino acid sites, 3 conserved catalytic sites, one substrate-binding site and 7 B epitopes. The EgPGAM had 99%, 94%, 78%, 57%, 45% and 4% amino-acid sequence homology with PGAMs of E. multilocularis, Taenia solium, Clonorchis sinensis, Caenorhabditis elegans, Homo sapiens and Ovis aries, respectively. SDS-PAGE showed that EgPGAM was highly expressed in E. coli BL21(DE3), mainly in soluble form. Western blotting showed reactions of EgPGAM with serum samples from E. granulosus-infected sheep. Immunofluorescence assay revealed that the EgPGAM was localized mainly on the tegument and hooks of protoscoleces, the germinal layer of the hydatid cyst and the cyst wall parenchyma of adults. The sensitivity and specificity of EgPGAM-based indirect ELISA were 55.6%(15/27) and 86.4% (89/103), respectively.Conclusion EgPGAM was widely distributed in the larvae and adult worms of E. granulosus. The recombinant EgPGAM had apparent immunoreactivity, but with low sensitivity in the indirect ELISA test. Therefore, EgPGAM is not a suitable candidate of diagnostic antigen for echinococcosis.

Key words： Echinococcus granulosus; Phosphoglycerate mutase; Prokaryotic expression; Immunofluorescence localization; Enzyme-linked immunosorbent assay

Cite this article

Hong-yu SONG , Yu-qing LIANG , Rui-qi HUA , Yuan SHI , Ai-guo YANG , Li GUO , Dong-bo YUAN , Yue XIE , Guang-you YANG . Expression, localization and preliminary evaluation of diagnostic value of Echinococcus granulosus phosphoglycerate mutase[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020 , 38(2) : 194 -201 . DOI: 10.12140/j.issn.1000-7423.2020.02.010

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