Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis.
Background Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are mor...
Published in: | PLOS Neglected Tropical Diseases |
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ftdoajarticles:oai:doaj.org/article:23e52a803e854beaadd9cc3ce31f0c96 2023-05-15T15:16:25+02:00 Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis. Wagner J T Santos Diego H C Tavares Artur L Castro Neto Marília B Nascimento Rafael Dhalia Alessandra L Albuquerque Carlos H N Costa Franklin B Magalhães Antônio M Rezende Osvaldo P de Melo Neto 2020-07-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0008488 https://doaj.org/article/23e52a803e854beaadd9cc3ce31f0c96 EN eng Public Library of Science (PLoS) https://doi.org/10.1371/journal.pntd.0008488 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0008488 https://doaj.org/article/23e52a803e854beaadd9cc3ce31f0c96 PLoS Neglected Tropical Diseases, Vol 14, Iss 7, p e0008488 (2020) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2020 ftdoajarticles https://doi.org/10.1371/journal.pntd.0008488 2022-12-31T10:07:33Z Background Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL. Methodology/principal findings Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis. Conclusion The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLOS Neglected Tropical Diseases 14 7 e0008488 |
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Open Polar |
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Directory of Open Access Journals: DOAJ Articles |
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English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
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Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Wagner J T Santos Diego H C Tavares Artur L Castro Neto Marília B Nascimento Rafael Dhalia Alessandra L Albuquerque Carlos H N Costa Franklin B Magalhães Antônio M Rezende Osvaldo P de Melo Neto Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Background Visceral leishmaniasis (VL) is a major neglected disease, potentially fatal, whose control is still impaired by inefficient and/or expensive treatment and diagnostic methods. The most promising approach for VL diagnosis uses serological assays with recombinant proteins, since they are more efficient and easier to perform. Tests developed for the human form of the disease, however, have not been shown to be efficient for its diagnosis in the canine host, the major reservoir for the American VL. Methodology/principal findings Here, we describe a systematic approach aimed at the production of a new chimeric protein potentially able to be used for both human and canine VL diagnosis and based both on in silico gene design and experimental data. Starting from the previous identification of Leishmania infantum recombinant antigens efficient for the diagnosis of either human or canine VL, three of the best performing antigens were selected (Lci2, Lci3 and Lci12). After a preliminary evaluation validating the chimeric approach, DNA fragments encoding predicted antigenic regions from each protein, enriched with repeats, were joined in various combinations to generate a total of seventeen chimeric genes optimized for prokaryotic expression. These were assessed for optimal expression and purification yield, with four chimeric proteins being efficiently produced. Their diagnostic potential was then evaluated through ELISA assays with sera from VL afflicted humans and dogs. After two rounds of gene design, the results showed high levels of sensitivity for the best chimeric protein, named Q5, in humans (82%) and dogs (100%) with 100% specificity in comparison with healthy controls. A single non-specific reaction was seen with serum from individuals with tegumentary leishmaniasis. Conclusion The newly described chimeric protein is potentially useful for the detection of both humans and dogs afflicted with VL, with its use in rapid tests necessary for validation as a new diagnostic tool. |
format |
Article in Journal/Newspaper |
author |
Wagner J T Santos Diego H C Tavares Artur L Castro Neto Marília B Nascimento Rafael Dhalia Alessandra L Albuquerque Carlos H N Costa Franklin B Magalhães Antônio M Rezende Osvaldo P de Melo Neto |
author_facet |
Wagner J T Santos Diego H C Tavares Artur L Castro Neto Marília B Nascimento Rafael Dhalia Alessandra L Albuquerque Carlos H N Costa Franklin B Magalhães Antônio M Rezende Osvaldo P de Melo Neto |
author_sort |
Wagner J T Santos |
title |
Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis. |
title_short |
Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis. |
title_full |
Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis. |
title_fullStr |
Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis. |
title_full_unstemmed |
Gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis. |
title_sort |
gene design, optimization of protein expression and preliminary evaluation of a new chimeric protein for the serological diagnosis of both human and canine visceral leishmaniasis. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2020 |
url |
https://doi.org/10.1371/journal.pntd.0008488 https://doaj.org/article/23e52a803e854beaadd9cc3ce31f0c96 |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 14, Iss 7, p e0008488 (2020) |
op_relation |
https://doi.org/10.1371/journal.pntd.0008488 https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0008488 https://doaj.org/article/23e52a803e854beaadd9cc3ce31f0c96 |
op_doi |
https://doi.org/10.1371/journal.pntd.0008488 |
container_title |
PLOS Neglected Tropical Diseases |
container_volume |
14 |
container_issue |
7 |
container_start_page |
e0008488 |
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