Bioinformatics characteristics and immunoreactivity of protein kinase A of Echinococcus granulosus

doi:10.12140/j.issn.1000-7423.2020.06.002

Abstract

Abstract:

Objective To investigate the bioinformatics characteristics of protein kinase A gene in Echinococcus granulosus (EgPKA), and analyze the immunoreactivity of its recombinant protein. Methods The coding region of EgPKA gene was amplified using primers designed based on the whole-genome sequencing data of Echinococcus granulosus, and the physicochemical properties, subcellular localization, and tertiary structure of EgPKA were comprehensively analyzed using bioinformatics methods. The recombinant plasmid of GS115/EgPKA-pPIC9K was constructed and induced by methanol in Pichia pastoris eukaryotic system. The recombinant EgPKA protein was purified by nickel-affinity chromatography with anti-His label, and the immunoreactivity of purified EgPKA was detected by Western blotting with serum of a patient with cystic echinococcosis and sera from mice 4, 8, 12 and 16 months after infected with E. granulosus protoscolex. Results The EgPKA gene had a full length of 1 053 bp and encoded 350 amino acid residues. A 48-bp deletion mutation (417-464 bp) occurred in the coding region. After mutation, the bases A⁴¹⁵, G⁴¹⁶ and T⁴⁶⁵ in the EgPKA coding region formed a new amino acid Ser¹⁵⁴. Bioinformatics analysis showed that EgPKA had theoretical isoelectric point of 8.63, instability coefficient of 29.04, fat coefficient of 82.49, and a total average hydrophilicity of -0.374, with no signal peptide and no transmembrane region. Cell-ploc 2.0 analysis showed that the EgPKA protein may be localized to the cell membrane, cytoplasm, mitochondria and nucleus. Western blotting analysis showed that the yield of recombinant protein EgPKA was highest after induction for 72 h in 1% methanol. The purified recombinant protein EgPKA could react specifically with sera of infected mice at 4, 8, 12 and 16 months post-infection, resulting in two protein bands at M_r 40 000 and 35 000, while no specific reaction occurred in sera from healthy mice and healthy humans. Conclusion The coding region of EgPKA has a gene deletion mutation, and the recombinant EgPKA protein has good immunoreactivity, which could be a potential diagnostic antigen for early diagnosis of echinococcosis.

Key words: Echinococcus granulosus, Protein kinase A, Bioinformatic analysis, Eukaryotic expression

CLC Number:

R383.33

FAN Jun-jie, HAN Xiu-min, Nur Fazleen Binti Idris, LI Kai, TAN Qing-qing, CAO Wen-qiao, LI Xiang, LIAO Peng, YE Bin. Bioinformatics characteristics and immunoreactivity of protein kinase A of Echinococcus granulosus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(6): 682-687.

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