DOI: https://doi.org/10.14710/dmj.v14i1.47102

Expression of Sarcoptes scabiei Exon 5 Tropomyosin Gene as an Attempt to Develop Recombinant Antigen for Scabies Diagnosis

Alfin Harjuno Dwiputro orcid scopus  -  Department of Parasitology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia, Indonesia
Taufik Mulya Perdana orcid scopus  -  Department of Parasitology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia, Indonesia
*Tri Baskoro Tunggul Satoto orcid scopus  -  Department of Parasitology, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, Indonesia, Indonesia
Wayan Tunas Artama orcid scopus  -  Biotechnology Research Center, Graduate Program, Universitas Gadjah Mada, Yogyakarta, Indonesia, Indonesia

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Abstract

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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Keywords: Diagnosis of scabies; Recombinant protein; Sarcoptes scabiei; Tropomyosin
Funding: Universitas Gadjah Mada

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Language : EN
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