Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum
Autor(a) principal: | |
---|---|
Data de Publicação: | 2019 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações do UNIOESTE |
Texto Completo: | http://tede.unioeste.br/handle/tede/4603 |
Resumo: | Fungal infections have been increasing significantly throughout the world due to the incidence of serious infectious diseases that primarily weaken immunocompromised patients. Due to high resistance to antifungal drugs in use, new molecules are becoming the target of researches and in this class are so-called antifungal peptides. These studies are being carried out due to the need to develop new active molecules against pathogenic fungi, since these are becoming increasingly resistant to existing drugs and increasing the number of immunocompromised hosts. This study investigated the presence of the antifungal peptide in Penicillium crustosum using as reference the homologous sequence of the PgAFP gene of Penicillium chrysogenum. Applying molecular biology techniques, the homologous PgAFP gene of P. crustosum was obtained and sequenced, as well as the peptide was isolated from the fungus growth in stressful liquid culture medium for expression of the peptide. The gene was amplified by the conventional PCR technique, in which specific oligonucleotides (primers) were designed for the PgAFP gene. The complete PcAFP gene sequence of 405 bp genomic fragment was obtained with primers designed upstream and downstream of the PgAFP gene. The genomic sequence was compared to sequences deposited in the NCBI database with the Blastn tool and the gene showed 96.30% identity to the P. chrysogenum PgAFP gene. Comparison of the complete PcAFP gene sequence with the PgAFP gene revealed a 279 bp region encoding the antifungal peptide. This sequence has been translated into its amino acid sequence and was compared to the PgAFP sequence of P. chrysogenum peptide model demonstrating 98.21% homology with this peptide. The PcAFP modeling revealed the conserved signal peptide of the antifungal family of proteins, three-dimensional conserved structure with 3 disulfide bonds and -core conserved domain found in the PAFs. The peptide was analyzed with the pI / Mw prediction tool, in which it obtained 6.48 kDa and pI 8.83. The extract obtained from the AFPIM culture medium supernatant showed a 6.9 kDa peptide on Tricine SDS-PAGE 16% polyacrylamide gel, however, not yet conclusive for the P. crustosum peptide. The extracellular crude extract from the liquid culture was tested for antimicrobial activity against bacteria and yeast. In the broth microdilution assay the microorganisms were exposed to different dilutions of the extracellular extract containing the peptide, where activity against Staphylococcus aureus and Candida albicans was obtained. In conclusion, the isolation of this peptide in P. crustosum will contribute with the characterization of a new antifungal peptide with potential application, besides increasing the knowledge about the structure and function of these bioactive molecules. Key words: AFP gene, Antifungal Peptides, Penicillium crustosum |
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Silva , José Luis da Conceiçãohttp://lattes.cnpq.br/9251091711372741Silva , José Luis da Conceiçãohttp://lattes.cnpq.br/9251091711372741Kadowaki , Marina Kimikohttp://lattes.cnpq.br/1819723253019762Rosado , Adriana Fiorinihttp://lattes.cnpq.br/7752079608717875http://lattes.cnpq.br/6675358404782115Menegotto, Juliana Beal2019-12-12T18:20:01Z2019-09-11MENEGOTTO, Juliana Beal. Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum. 2019. 55 f. Dissertação( Mestrado em Ciências Farmacêuticas) - Universidade Estadual do Oeste do Paraná, Cascavel, 2019.http://tede.unioeste.br/handle/tede/4603Fungal infections have been increasing significantly throughout the world due to the incidence of serious infectious diseases that primarily weaken immunocompromised patients. Due to high resistance to antifungal drugs in use, new molecules are becoming the target of researches and in this class are so-called antifungal peptides. These studies are being carried out due to the need to develop new active molecules against pathogenic fungi, since these are becoming increasingly resistant to existing drugs and increasing the number of immunocompromised hosts. This study investigated the presence of the antifungal peptide in Penicillium crustosum using as reference the homologous sequence of the PgAFP gene of Penicillium chrysogenum. Applying molecular biology techniques, the homologous PgAFP gene of P. crustosum was obtained and sequenced, as well as the peptide was isolated from the fungus growth in stressful liquid culture medium for expression of the peptide. The gene was amplified by the conventional PCR technique, in which specific oligonucleotides (primers) were designed for the PgAFP gene. The complete PcAFP gene sequence of 405 bp genomic fragment was obtained with primers designed upstream and downstream of the PgAFP gene. The genomic sequence was compared to sequences deposited in the NCBI database with the Blastn tool and the gene showed 96.30% identity to the P. chrysogenum PgAFP gene. Comparison of the complete PcAFP gene sequence with the PgAFP gene revealed a 279 bp region encoding the antifungal peptide. This sequence has been translated into its amino acid sequence and was compared to the PgAFP sequence of P. chrysogenum peptide model demonstrating 98.21% homology with this peptide. The PcAFP modeling revealed the conserved signal peptide of the antifungal family of proteins, three-dimensional conserved structure with 3 disulfide bonds and -core conserved domain found in the PAFs. The peptide was analyzed with the pI / Mw prediction tool, in which it obtained 6.48 kDa and pI 8.83. The extract obtained from the AFPIM culture medium supernatant showed a 6.9 kDa peptide on Tricine SDS-PAGE 16% polyacrylamide gel, however, not yet conclusive for the P. crustosum peptide. The extracellular crude extract from the liquid culture was tested for antimicrobial activity against bacteria and yeast. In the broth microdilution assay the microorganisms were exposed to different dilutions of the extracellular extract containing the peptide, where activity against Staphylococcus aureus and Candida albicans was obtained. In conclusion, the isolation of this peptide in P. crustosum will contribute with the characterization of a new antifungal peptide with potential application, besides increasing the knowledge about the structure and function of these bioactive molecules. Key words: AFP gene, Antifungal Peptides, Penicillium crustosumInfecções fúngicas vêm aumentando significativamente em todo o mundo, devido à incidência de doenças infecciosas graves que debilitam principalmente pacientes imunocomprometidos. Em virtude da elevada resistência aos medicamentos antifúngicos em uso, novas moléculas estão tornando-se alvo de pesquisas. Enquadram-se nessas os chamados peptídeos antifúngicos. Esses são estudados por causa de sua capacidade de atingir as células alvo sem danificar o organismo hospedeiro. Assim, este estudo investigou a presença do peptídeo antifúngico no Penicillium crustosum utilizando como referência a sequência homóloga do gene PgAFP de Penicillium chrysogenum. Ao aplicar técnicas de biologia molecular, o gene homólogo PgAFP de P. crustosum foi obtido e sequenciado, bem como o peptídeo foi isolado, a partir do crescimento do fungo em meio de cultivo líquido estressante para expressão do peptídeo. O gene foi amplificado através da técnica de PCR convencional, na qual foram desenhados oligonucleotídeos (primers) específicos para o gene PgAFP. A sequência completa do gene PcAFP de 405 pb foi obtida com primers desenhados na região a montante e a jusante do gene PgAFP. A sequência genômica foi comparada com sequências depositadas no banco de dados do NCBI com a ferramenta Blastn. Após essa ação, o gene mostrou 96,30% de identidade ao gene PgAFP do P. chrysogenum. A comparação da sequência completa do gene PcAFP com o PgAFP permitiu revelar uma região de 279 pb codificante para o peptídeo antifúngico. Esta sequência foi traduzida em sua sequência de aminoácidos, comparada ao modelo do peptídeo antifúngico PgAFP do P. chrysogenum e demonstrou homologia de 98.28% com esse. A modelagem do PcAFP revelou o peptídeo sinal conservado da família das proteínas antifúngicas, estrutura tridimensional conservada com 3 pontes dissulfeto e domínio conservado -core encontrado nas PAFs. O peptídeo foi analisado com a ferramenta para previsão do pI/Mw, que indicou massa molecular de 6,48 kDa e pI 8,83. O extrato obtido a partir do sobrenadante do meio de cultura AFPIM mostrou um peptídeo de 6,9 kDa em gel de poliacrilamida Tricina SDS-PAGE 16%, contudo, ainda não conclusivo quanto ao peptídeo de P. crustosum. O extrato bruto extracelular do cultivo líquido foi testado quanto à atividade antimicrobiana contra bactérias e levedura. No ensaio de microdiluição em caldo, os microrganismos foram expostos a diferentes diluições do extrato extracelular contendo o peptídeo. Nesse, obteve-se atividade inibitória contra Staphylococcus aureus e Candida albicans. Em síntese, ao concluir o isolamento desse peptídeo em P. crustosum contribuiremos com a caracterização de um novo peptídeo antifúngico com potencial para aplicação na saúde, além de aumentar o conhecimento sobre a estrutura e função dessas moléculas bioativas.Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2019-12-12T18:20:01Z No. of bitstreams: 2 JULIANA_ MENEGOTTO_2019.pdf: 2265494 bytes, checksum: fcd846ebdf1f9ead87a1449a931e3e8c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)Made available in DSpace on 2019-12-12T18:20:01Z (GMT). No. of bitstreams: 2 JULIANA_ MENEGOTTO_2019.pdf: 2265494 bytes, checksum: fcd846ebdf1f9ead87a1449a931e3e8c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2019-09-11application/pdfpor6588633818200016417500Universidade Estadual do Oeste do ParanáCascavelPrograma de Pós-Graduação em Ciências FarmacêuticasUNIOESTEBrasilCentro de Ciências Médicas e Farmacêuticashttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessGene AFPPenicillium crustosumPeptídeos AntifúngicosSequenciamentoAFP geneAntifungal PeptidesPenicillium crustosumSequencingCIENCIAS DA SAUDE::FARMACIAIsolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosumIsolation and sequencing of PcAFP gene coding for antifungal peptide of Penicillium crustosuminfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis7878055067573953101600600600-89404397133878492676997636413449754996reponame:Biblioteca Digital de Teses e Dissertações do UNIOESTEinstname:Universidade Estadual do Oeste do Paraná (UNIOESTE)instacron:UNIOESTEORIGINALJULIANA_ MENEGOTTO_2019.pdfJULIANA_ MENEGOTTO_2019.pdfapplication/pdf2265494http://tede.unioeste.br:8080/tede/bitstream/tede/4603/5/JULIANA_+MENEGOTTO_2019.pdffcd846ebdf1f9ead87a1449a931e3e8cMD55CC-LICENSElicense_urllicense_urltext/plain; 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dc.title.por.fl_str_mv |
Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum |
dc.title.alternative.eng.fl_str_mv |
Isolation and sequencing of PcAFP gene coding for antifungal peptide of Penicillium crustosum |
title |
Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum |
spellingShingle |
Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum Menegotto, Juliana Beal Gene AFP Penicillium crustosum Peptídeos Antifúngicos Sequenciamento AFP gene Antifungal Peptides Penicillium crustosum Sequencing CIENCIAS DA SAUDE::FARMACIA |
title_short |
Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum |
title_full |
Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum |
title_fullStr |
Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum |
title_full_unstemmed |
Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum |
title_sort |
Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum |
author |
Menegotto, Juliana Beal |
author_facet |
Menegotto, Juliana Beal |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Silva , José Luis da Conceição |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/9251091711372741 |
dc.contributor.referee1.fl_str_mv |
Silva , José Luis da Conceição |
dc.contributor.referee1Lattes.fl_str_mv |
http://lattes.cnpq.br/9251091711372741 |
dc.contributor.referee2.fl_str_mv |
Kadowaki , Marina Kimiko |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/1819723253019762 |
dc.contributor.referee3.fl_str_mv |
Rosado , Adriana Fiorini |
dc.contributor.referee3Lattes.fl_str_mv |
http://lattes.cnpq.br/7752079608717875 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/6675358404782115 |
dc.contributor.author.fl_str_mv |
Menegotto, Juliana Beal |
contributor_str_mv |
Silva , José Luis da Conceição Silva , José Luis da Conceição Kadowaki , Marina Kimiko Rosado , Adriana Fiorini |
dc.subject.por.fl_str_mv |
Gene AFP Penicillium crustosum Peptídeos Antifúngicos Sequenciamento |
topic |
Gene AFP Penicillium crustosum Peptídeos Antifúngicos Sequenciamento AFP gene Antifungal Peptides Penicillium crustosum Sequencing CIENCIAS DA SAUDE::FARMACIA |
dc.subject.eng.fl_str_mv |
AFP gene Antifungal Peptides Penicillium crustosum Sequencing |
dc.subject.cnpq.fl_str_mv |
CIENCIAS DA SAUDE::FARMACIA |
description |
Fungal infections have been increasing significantly throughout the world due to the incidence of serious infectious diseases that primarily weaken immunocompromised patients. Due to high resistance to antifungal drugs in use, new molecules are becoming the target of researches and in this class are so-called antifungal peptides. These studies are being carried out due to the need to develop new active molecules against pathogenic fungi, since these are becoming increasingly resistant to existing drugs and increasing the number of immunocompromised hosts. This study investigated the presence of the antifungal peptide in Penicillium crustosum using as reference the homologous sequence of the PgAFP gene of Penicillium chrysogenum. Applying molecular biology techniques, the homologous PgAFP gene of P. crustosum was obtained and sequenced, as well as the peptide was isolated from the fungus growth in stressful liquid culture medium for expression of the peptide. The gene was amplified by the conventional PCR technique, in which specific oligonucleotides (primers) were designed for the PgAFP gene. The complete PcAFP gene sequence of 405 bp genomic fragment was obtained with primers designed upstream and downstream of the PgAFP gene. The genomic sequence was compared to sequences deposited in the NCBI database with the Blastn tool and the gene showed 96.30% identity to the P. chrysogenum PgAFP gene. Comparison of the complete PcAFP gene sequence with the PgAFP gene revealed a 279 bp region encoding the antifungal peptide. This sequence has been translated into its amino acid sequence and was compared to the PgAFP sequence of P. chrysogenum peptide model demonstrating 98.21% homology with this peptide. The PcAFP modeling revealed the conserved signal peptide of the antifungal family of proteins, three-dimensional conserved structure with 3 disulfide bonds and -core conserved domain found in the PAFs. The peptide was analyzed with the pI / Mw prediction tool, in which it obtained 6.48 kDa and pI 8.83. The extract obtained from the AFPIM culture medium supernatant showed a 6.9 kDa peptide on Tricine SDS-PAGE 16% polyacrylamide gel, however, not yet conclusive for the P. crustosum peptide. The extracellular crude extract from the liquid culture was tested for antimicrobial activity against bacteria and yeast. In the broth microdilution assay the microorganisms were exposed to different dilutions of the extracellular extract containing the peptide, where activity against Staphylococcus aureus and Candida albicans was obtained. In conclusion, the isolation of this peptide in P. crustosum will contribute with the characterization of a new antifungal peptide with potential application, besides increasing the knowledge about the structure and function of these bioactive molecules. Key words: AFP gene, Antifungal Peptides, Penicillium crustosum |
publishDate |
2019 |
dc.date.accessioned.fl_str_mv |
2019-12-12T18:20:01Z |
dc.date.issued.fl_str_mv |
2019-09-11 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
MENEGOTTO, Juliana Beal. Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum. 2019. 55 f. Dissertação( Mestrado em Ciências Farmacêuticas) - Universidade Estadual do Oeste do Paraná, Cascavel, 2019. |
dc.identifier.uri.fl_str_mv |
http://tede.unioeste.br/handle/tede/4603 |
identifier_str_mv |
MENEGOTTO, Juliana Beal. Isolamento e sequenciamento do gene PcAFP codificante para peptídeo antifúngico de Penicillium crustosum. 2019. 55 f. Dissertação( Mestrado em Ciências Farmacêuticas) - Universidade Estadual do Oeste do Paraná, Cascavel, 2019. |
url |
http://tede.unioeste.br/handle/tede/4603 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.relation.program.fl_str_mv |
7878055067573953101 |
dc.relation.confidence.fl_str_mv |
600 600 600 |
dc.relation.department.fl_str_mv |
-8940439713387849267 |
dc.relation.cnpq.fl_str_mv |
6997636413449754996 |
dc.rights.driver.fl_str_mv |
http://creativecommons.org/licenses/by/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
http://creativecommons.org/licenses/by/4.0/ |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Estadual do Oeste do Paraná Cascavel |
dc.publisher.program.fl_str_mv |
Programa de Pós-Graduação em Ciências Farmacêuticas |
dc.publisher.initials.fl_str_mv |
UNIOESTE |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Centro de Ciências Médicas e Farmacêuticas |
publisher.none.fl_str_mv |
Universidade Estadual do Oeste do Paraná Cascavel |
dc.source.none.fl_str_mv |
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Universidade Estadual do Oeste do Paraná (UNIOESTE) |
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UNIOESTE |
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Biblioteca Digital de Teses e Dissertações do UNIOESTE |
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Biblioteca Digital de Teses e Dissertações do UNIOESTE |
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Biblioteca Digital de Teses e Dissertações do UNIOESTE - Universidade Estadual do Oeste do Paraná (UNIOESTE) |
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biblioteca.repositorio@unioeste.br |
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