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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Faculty of Medicine, Universitas Diponegoro
2025-01-01
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| Series: | Jurnal Kedokteran Diponegoro |
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| Online Access: | https://ejournal3.undip.ac.id/index.php/medico/article/view/47102 |
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| author | Alfin Harjuno Dwiputro Taufik Mulya Perdana Tri Baskoro Tunggul Satoto Wayan Tunas Artama |
| author_facet | Alfin Harjuno Dwiputro Taufik Mulya Perdana Tri Baskoro Tunggul Satoto Wayan Tunas Artama |
| author_sort | Alfin Harjuno Dwiputro |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-cc2cbfe74b374bd5964d33da36b9c285 |
| institution | Kabale University |
| issn | 2540-8844 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Faculty of Medicine, Universitas Diponegoro |
| record_format | Article |
| series | Jurnal Kedokteran Diponegoro |
| spelling | doaj-art-cc2cbfe74b374bd5964d33da36b9c2852025-08-20T03:32:24ZengFaculty of Medicine, Universitas DiponegoroJurnal Kedokteran Diponegoro2540-88442025-01-01141354310.14710/dmj.v14i1.4710233015Expression of Sarcoptes scabiei Exon 5 Tropomyosin Gene as an Attempt to Develop Recombinant Antigen for Scabies DiagnosisAlfin Harjuno Dwiputro0https://orcid.org/0000-0002-9253-9796Taufik Mulya Perdana1https://orcid.org/0000-0002-1036-3839Tri Baskoro Tunggul Satoto2https://orcid.org/0000-0003-4443-4751Wayan Tunas Artama3https://orcid.org/0000-0002-8504-7558Department of Parasitology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia, IndonesiaDepartment of Parasitology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia, IndonesiaDepartment of Parasitology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia, IndonesiaBiotechnology Research Center, Graduate Program, Universitas Gadjah Mada, Yogyakarta, Indonesia, IndonesiaBackground: 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.https://ejournal3.undip.ac.id/index.php/medico/article/view/47102diagnosis of scabiesrecombinant proteinsarcoptes scabieitropomyosin |
| spellingShingle | Alfin Harjuno Dwiputro Taufik Mulya Perdana Tri Baskoro Tunggul Satoto Wayan Tunas Artama Expression of Sarcoptes scabiei Exon 5 Tropomyosin Gene as an Attempt to Develop Recombinant Antigen for Scabies Diagnosis Jurnal Kedokteran Diponegoro diagnosis of scabies recombinant protein sarcoptes scabiei tropomyosin |
| title | Expression of Sarcoptes scabiei Exon 5 Tropomyosin Gene as an Attempt to Develop Recombinant Antigen for Scabies Diagnosis |
| title_full | Expression of Sarcoptes scabiei Exon 5 Tropomyosin Gene as an Attempt to Develop Recombinant Antigen for Scabies Diagnosis |
| title_fullStr | Expression of Sarcoptes scabiei Exon 5 Tropomyosin Gene as an Attempt to Develop Recombinant Antigen for Scabies Diagnosis |
| title_full_unstemmed | Expression of Sarcoptes scabiei Exon 5 Tropomyosin Gene as an Attempt to Develop Recombinant Antigen for Scabies Diagnosis |
| title_short | Expression of Sarcoptes scabiei Exon 5 Tropomyosin Gene as an Attempt to Develop Recombinant Antigen for Scabies Diagnosis |
| title_sort | expression of sarcoptes scabiei exon 5 tropomyosin gene as an attempt to develop recombinant antigen for scabies diagnosis |
| topic | diagnosis of scabies recombinant protein sarcoptes scabiei tropomyosin |
| url | https://ejournal3.undip.ac.id/index.php/medico/article/view/47102 |
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