Cloning,expression,structure and function prediction and protein localization of MDH gene of Echinococcus granulosus
In order to explore the biological function of malate dehydrogenase(MDH)gene in the growth and development of Echinococcus granulosus and preliminarily evaluate the potential of MDH as a vaccine candidate antigen,the EgMDH gene sequence was obtained from the NCBI GenBank database,and the specific primers were designed.The full-length sequence of the MDH gene was amplified by PCR using the cDNA of the protoscoleces of E.granulosus as a template.The physicochemical properties and structural characteristics of MDH protein were analyzed by bioinformatics software.The recombinant plasmid pET28a-MDH was constructed and transformed into E.coli BL21(DE3)competent cells.The expression of the protein was detected by SDS-PAGE,then the protein was purified and refolded,and the antigenicity of the protein was analyzed by Western-blot.The localization of the target protein in the protoscoleces was detected by indirect immunofluorescence assay.The relative transcription level of EgMDHgene mRNA in protoscoleces and adults was analyzed by real-time fluorescence quantitative PCR.The results showed that the EgMDH gene was 999 bp in length,encoding 332 amino acids.The protein molecular formula was C1639H2599N4450475S16,the relative molecular weight was 36 651.32,and the theoretical isoelectric point was 8.11.The encoded protein had no signal peptide and transmembrane region,containing 40 phosphory-lation sites,3 N-linked glycosylation sites,and 9 dominant B cell epitopes.The secondary structure wascomposedof α-helix(46.69％),randomcoil(31.93％),extendedchainstructure(16.87％)andβ-turn(4.52％).The cloned gene was 975 bp in length with a predicted molecular weight of 35.7 ku.Western-blotting showed that the recombinant protein could be recognized by rabbit polyclonal antibody against His tag and mouse polyclonal antibody against soluble whole protein of protoscoleces.Indirect immunofluores-cence assay showed that EgMDH protein was localized in the capsule wall of protoscoleces.Real-time fluorescence quantitative PCR showed that EgMDH gene was expressed in both adults and protoscoleces,and the transcription level of protoscoleces was significantly higher than that of adults(P＜0.05).The results of this study preliminarily indicated that EgMDH has the potential to be a vaccine candidate antigen,in order to provide candidate antigens for the subsequent development of E.granulosus vac-cine;and also provided a basis for the further study of the MDHgene of E.granulosus.
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