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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Section: Original Articles
Language : EN
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  1. Andriantsoanirina V, Ariey F, Izri A, Bernigaud C, Fang F, Charrel R, et al. Sarcoptes scabiei mites in humans are distributed into three genetically distinct clades. Clin Microbiol Infect 2015;21:1107–14. https://doi.org/10.1016/j.cmi.2015.08.002
  2. Karimkhani C, Colombara D V., Drucker AM, Norton SA, Hay R, Engelman D, et al. The global burden of scabies: a cross-sectional analysis from the Global Burden of Disease Study 2015. Lancet Infect Dis 2017;17:1247–54. https://doi.org/10.1016/S1473-3099(17)30483-8
  3. Engelman D, Yoshizumi J, Hay RJ, Osti M, Micali G, Norton S, et al. The 2020 International Alliance for the Control of Scabies Consensus Criteria for the Diagnosis of Scabies. Br J Dermatol 2020;183:808–20. https://doi.org/10.1111/bjd.18943
  4. Cox V, Fuller LC, Engelman D, Steer A, Hay RJ. Estimating the global burden of scabies: what else do we need?*. Br J Dermatol 2021;184:237–42. https://doi.org/10.1111/bjd.19170
  5. Osti MH, Sokana O, Gorae C, Whitfeld MJ, Steer AC, Engelman D. The diagnosis of scabies by non-expert examiners: A study of diagnostic accuracy. PLoS Negl Trop Dis 2019;13:1–13. https://doi.org/10.1371/journal.pntd.0007635
  6. Siddig EE, Hay R. Laboratory-based diagnosis of scabies: a review of the current status. Trans R Soc Trop Med Hyg 2022;116:4–9. https://doi.org/10.1093/trstmh/trab049
  7. Arlian LG, Morgan MS. A review of Sarcoptes scabiei: Past, present and future. Parasites and Vectors 2017;10:1–22. https://doi.org/10.1186/s13071-017-2234-1
  8. Naz S, Desclozeaux M, Mounsey KE, Chaudhry FR, Walton SF. Characterization of Sarcoptes scabiei tropomyosin and paramyosin: Immunoreactive allergens in scabies. Am J Trop Med Hyg 2017;97:851–60. https://doi.org/10.4269/ajtmh.16-0976
  9. Zhang R, Jise Q, Zheng W, Ren Y, Nong X, Wu X, et al. Characterization and evaluation of a Sarcoptes scabiei allergen as a candidate vaccine. Parasites and Vectors 2012;5:1–9. https://doi.org/10.1186/1756-3305-5-176
  10. Thieme F, Engler C, Kandzia R, Marillonnet S. Quick and clean cloning: A ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes. PLoS One 2011;6. https://doi.org/10.1371/journal.pone.0020556
  11. Obradovic J, Jurisic V, Tosic N, Mrdjanovic J, Perin B, Pavlovic S, et al. Optimization of PCR conditions for amplification of GC-rich EGFR promoter sequence. J Clin Lab Anal 2013;27:487–93. https://doi.org/10.1002/jcla.21632
  12. Li MZ, Elledge SJ. SLIC: A method for sequence- and ligation-independent cloning. Methods Mol Biol 2012;852:51–9. https://doi.org/10.1007/978-1-61779-564-0_5
  13. Upadhyay AK, Murmu A, Singh A, Panda AK. Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in escherichia coli. PLoS One 2012;7. https://doi.org/10.1371/journal.pone.0033951
  14. Bhatwa A, Wang W, Hassan YI, Abraham N, Li XZ, Zhou T. Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications. Front Bioeng Biotechnol 2021;9:1–18. https://doi.org/10.3389/fbioe.2021.630551
  15. Salazar-Anton F, Tellez A, Lindh J. Evaluation of an immunodot blot technique for the detection of antibodies against Taenia solium larval antigens. Parasitol Res 2012;110:2187–91. https://doi.org/10.1007/s00436-011-2747-z
  16. Malafaia CB, Guerra ML, Silva TD, Paiva PMG, Souza EB, Correia MTS, et al. Selection of a protein solubilization method suitable for phytopathogenic bacteria: A proteomics approach. Proteome Sci 2015;13:1–7. https://doi.org/10.1186/s12953-015-0062-9
  17. Mónico A, Martínez-Senra E, Cañada FJ, Zorrilla S, Pérez-Sala D. Drawbacks of dialysis procedures for removal of EDTA. PLoS One 2017;12:1–9. https://doi.org/10.1371/journal.pone.0169843
  18. Nurjayadi M, Afrizal R, Hardianto D, Agustini K. Variations of binding, washing, and concentration of imidazole on purification of recombinant Fim-C Protein Salmonella typhi with Ni-NTA Resin. J Phys Conf Ser 2019;1402. https://doi.org/10.1088/1742-6596/1402/5/055055
  19. Stave JW, Lindpaintner K. Antibody and Antigen Contact Residues Define Epitope and Paratope Size and Structure. J Immunol 2013;191:1428–35. https://doi.org/10.4049/jimmunol.1203198
  20. Bornhorst, Falke. Purification of Proteins Using Polyhistidine Affinity Tags. Methods Enzym 2000:77–91. https://doi.org/10.1007/978-1-4612-4278-9_4

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