Expression of Sarcoptes scabiei Exon 5 Tropomyosin Gene as an Attempt to Develop Recombinant Antigen for Scabies Diagnosis
Background: Scabies diagnosis is greatly influenced by the experience of the examiner, since the diagnosis is rely on a complex diagnostic criteria. Antigen detection test is an alternative technique for diagnosing scabies in the future. Tropomyosin is a highly conserved protein which expressed by S...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Faculty of Medicine, Universitas Diponegoro
2025-01-01
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| Series: | Jurnal Kedokteran Diponegoro |
| Subjects: | |
| Online Access: | https://ejournal3.undip.ac.id/index.php/medico/article/view/47102 |
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| Summary: | Background: Scabies diagnosis is greatly influenced by the experience of the examiner, since the diagnosis is rely on a complex diagnostic criteria. Antigen detection test is an alternative technique for diagnosing scabies in the future. Tropomyosin is a highly conserved protein which expressed by Sarcoptes scabiei mites.
Objective: This research aimed to develop recombinant protein from exon 5 of the S. scabiei tropomyosin gene for further scabies diagnostic development.
Methods: An exon from tropomyosin gene was cloned into pLATE-51 plasmid using ligation independent cloning methods. Protein overexpression was done using Eschericia coli strain BL21. Insoluble protein was solubilized using 5 mM dithiothreitol (DTT). Protein purification was done with Ni-NTA affinity chromatography principle. Analysis of the resulting recombinant protein was done using SDS-PAGE, immunodot blotting, and western blotting.
Results: A 550-bp exon was successfully cloned into pLATE-51 plasmid. Overexpression result showed protein was mostly expressed in pellet (insoluble form). Confirmation using immunodot blotting showed the presence of recombinant protein from the overexpression. However, further confirmation using western blotting was unsuccessful to detect the intended recombinant protein.
Conclusion: The exon of S. scabiei tropomyosin gene could be cloned in pLATE-51 plasmid and produced recombinant protein. However, the protein yield was low and the protein is mostly insoluble, preventing the success of purification of the intended recombinant protein. |
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| ISSN: | 2540-8844 |