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Basic sciences oral session 1—Genomics, replication and pathogenesis
O4-S1.02 Molecular cloning and expression of hydrogenosomal malate dehydrogenase of Trichomonas vaginalis
  1. S Ardalan1,
  2. C Lee2,
  3. G Garber2
  1. 1Faculty of Medicine, University of Ottawa, The Ottawa Hospital Research Institute Ottawa, Canada
  2. 2Faculty of Medicine, University of Ottawa, Division of Infectious Diseases, The Ottawa Hospital Ottawa, Canada

Abstract

Background Trichomoniasis, a sexually transmitted disease caused byTrichomonas vaginalis, is associated with adverse pregnancy outcomes, and increased risk of HIV acquisition. Malate dehydrogenase (MDH), which catalyses the interconversion of malate to oxaloacetate, has a pivotal role in the survival and pathogenicity of this amitochondrial protozoan. The objective of this study was to clone and express Malate dehydrogenase gene ofT vaginalis, and analyse the biological function of this hydrogenosomal enzyme.

Methods The MDH gene from a clinical isolate ofT vaginalis was amplified by PCR, and cloned into pET101/D-TOPO vector with a C-terminal 6XHis tag. Positive clones were screened and identified by restriction endonuclease digestion and sequence analysis. The plasmid pET101/D-MDH was then transformed into E.coli BL21(DE3) to express after IPTG induction. The expression product further analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The recombinant protein was purified with Ni-NTA agarose under native conditions. Western blot, using antibody raised against whole cellT vanginalis, was performed to determine the immunogenicity of purified recombinant protein.

Results The recombinant plasmid pET101/D-MDH was constructed successfully. High homology (98%) of nucleotide sequence was revealed between the cloned MDH and the corresponding gene. The recombinant protein showed a high expression level when induced with 1 mM IPTG at 37° C for 4 h. SDS-PAGE analysis showed that the recombinant MDH protein with the correct molecular weight (about 60 kDa) was expressed in E.coli BL21 (DE3). Western blotting revealed that the purified recombinant protein was specifically recognised by sera from mice infected with whole cellT vaginalis.

Conclusions A prokaryotic expression system ofT vaginalis Malate dehydrogenase gene has been established successfully. The immunogenicity of the recombinant protein has been tested. The present study shows that the recombinant MDH is specific and suitable for use as an antigen for detecting anti-Trichomonas vaginalis IgG antibodies. Our work has established a good foundation for future studies onT vaginalis vaccine construction.

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