Acta parasitologica · 2025
Sequence Localization of SeGPx in S. digitata Genome Contigs and Determination of its Presence in the Whole Worm Extract.
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Jebaseelan Jefrillah, Natesan Sankar, Balakrishnan Anand Setty
Аннотация
PURPOSE: Lymphatic filariasis, caused by three major filarial species, is marked by immune evasion strategies involving antioxidant enzymes. The role of selenium-dependent glutathione peroxidase (SeGPx) in this process remains underexplored. This study aimed to identify and characterise SeGPx in Setaria digitata, a genomic analogue of Wuchereria bancrofti, and evaluate its potential as a diagnostic antigen.
METHODS: segpx sequences were identified through bioinformatics tools, including BLAST and UniProt databases. Due to limited nematode entries, a validated SeGPx sequence from Lymnaea stagnalis was used as a proxy in PSI-BLAST to identify homologues. Enzymatic activity was confirmed through spectrophotometric assay and activity staining on SDS-PAGE to confirm its enzymatic activity and molecular mass confirmation.
RESULTS: The segpx gene was localised within the S. digitata genome data (Contig 127, nucleotides 56,000-58,000). The enzyme assay showed a time-dependent decline in absorbance at 340 nm due to NADPH oxidation, plateauing after 13 min. Enzyme activity was calculated as 0.139 U, with a specific activity of 0.198 U/mg protein. A clear band at ~ 20 kDa was visualised via activity staining, confirming SeGPx presence.
CONCLUSION: Combined sequence-based identification and enzymatic validation confirm the functional presence of SeGPx in S. digitata. These findings support its role in oxidative stress mitigation and potential as a diagnostic antigen. The precise gene localisation offers a foundation for recombinant cloning, providing a streamlined alternative to conventional purification approaches for diagnostic development in filariasis.
METHODS: segpx sequences were identified through bioinformatics tools, including BLAST and UniProt databases. Due to limited nematode entries, a validated SeGPx sequence from Lymnaea stagnalis was used as a proxy in PSI-BLAST to identify homologues. Enzymatic activity was confirmed through spectrophotometric assay and activity staining on SDS-PAGE to confirm its enzymatic activity and molecular mass confirmation.
RESULTS: The segpx gene was localised within the S. digitata genome data (Contig 127, nucleotides 56,000-58,000). The enzyme assay showed a time-dependent decline in absorbance at 340 nm due to NADPH oxidation, plateauing after 13 min. Enzyme activity was calculated as 0.139 U, with a specific activity of 0.198 U/mg protein. A clear band at ~ 20 kDa was visualised via activity staining, confirming SeGPx presence.
CONCLUSION: Combined sequence-based identification and enzymatic validation confirm the functional presence of SeGPx in S. digitata. These findings support its role in oxidative stress mitigation and potential as a diagnostic antigen. The precise gene localisation offers a foundation for recombinant cloning, providing a streamlined alternative to conventional purification approaches for diagnostic development in filariasis.