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Master's Dissertation
DOI
10.11606/D.60.2015.tde-01072015-103417
Document
Author
Full name
Eliza Vieira Davi
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2015
Supervisor
Committee
Russo, Elisa Maria de Sousa (President)
Cunha, Anderson Ferreira da
Dinamarco, Taísa Magnani
Title in Portuguese
Clonagem de fragmentos dos genes gag e env do HIV-1 e HTLV-1, expressão em Escherichia coli das proteínas gp21, p24 e gp46 do HTLV-1 e imunodetecção
Keywords in Portuguese
Diagnóstico
Escherichia coli
HIV-1
HTLV-1
Proteína recombinante
Abstract in Portuguese
O HIV-1 é o agente etiológico da síndrome da imunodeficiência adquirida (AIDS) e o HTLV-I da leucemia/linfoma de célula T no adulto (ATL) e da paraparesia espástica tropical ou mielopatia associada ao HTLV (HAM/TSP), principalmente. Ambos são retrovírus com genoma RNA e possuem o gene gag que codifica as proteínas p24 (HIV-1 e HTLV-1) e p19 (HTLV-1) que formam o capsídeo e a matriz do vírus, respectivamente, e o gene env que codifica as proteínas gp41 e gp120 (HIV-1) e gp21 e gp46 (HTLV-1) que compõem o envelope viral. Os primeiros anticorpos produzidos nas infecções por ambos os vírus são destinados a essas proteínas e os diferentes testes diagnósticos disponíveis no mercado usam uma combinação dessas proteínas virais. O diagnóstico precoce é de extrema importância para o controle da epidemia, tratamento dos indivíduos e planejamento dos gastos com saúde pública. Os kits diagnósticos usados em laboratórios clínicos, bancos de sangue e hospitais brasileiros para o diagnóstico destas viroses são na sua maioria de empresas estrangeiras e o Brasil despende milhares de reais importando esses materiais. No Brasil, há a necessidade e incentivo para a produção de sistemas de diagnóstico com tecnologia nacional. Neste trabalho, os genes das proteínas p24, gp41 e gp120 do HIV-1 e p19 do HTLV-1 foram clonados com sucesso em diferentes vetores e em diferentes linhagens de E. coli, porém essas proteínas não foram expressas. As proteínas gp21, p24 e gp46 do HTLV-1 foram produzidas em bactérias BL21(DE3) com vetor pET28a(+). Essas três proteínas foram solubilizadas dos corpos de inclusão, purificadas por IMAC e identificadas pelas técnicas de Western Blotting e por espectrometria de massas. As proteínas recombinantes gp21, p24 e gp46 foram reconhecidas pelos soros de indivíduos com HTLV-1 e não foram reconhecidas por soros de indivíduos com HIV-1 e saudáveis, o que confere a elas especificidade e grande potencial diagnóstico. Os resultados deste trabalho são os primeiros passos para atingir o objetivo maior de produzir todas as sete proteínas em maior escala e, por fim, chegar a produção de um kit diagnóstico sensível, específico e barato com tecnologia nacional, diminuindo os gastos com a importação destes produtos e fomentando a indústria biotecnológica nacional.
Title in English
Cloning of fragments of gag and env genes of HIV-1 and HTLV-1, expression of the proteins gp21, gp46 and p24 of HTLV-1 in Escherichia coli system and immunodetection
Keywords in English
Diagnosis
Escherichia coli
HIV-1
HTLV-1
Recombinant protein
Abstract in English
HIV-1 is the etiologic agent of acquired immunodeficiency syndrome (AIDS) and HTLV-I is the cause of adult T-cell leukemia/lymphoma (ATL) and HTLV-I-associated myelopathy or tropical spastic paraparesis (HAM/TSP). Both are retroviruses with RNA genome and possess the gene gag and env. The gag gene encodes for p24 protein (HTLV-1 and HIV-1) and p19 (HTLV-1) forming the viral capsid and matrix, respectively, and the env gene encodes for proteins gp120 and gp41 (HIV-1) and gp21 and gp46 (HTLV-1) making the viral envelope. The first antibodies produced in infections by both viruses are against these proteins and the various diagnostic tests on the market use a combination of those viral proteins. Early diagnosis is extremely important to control the epidemia, treatment of individuals and planning of public health expenditures. The diagnostic kits used in clinical laboratories, blood banks and in Brazilian hospitals for the diagnosis of these viruses are mostly from foreign companies. Brazil spends thousands of reais importing these materials. In Brazil, there is a need and incentive for the production of diagnostic systems with national technology. In this study, the genes of p24, gp41 and gp120 of HIV-1 and p19 of HTLV-1 have been successfully cloned in different vectors and different strains of E. coli, but these proteins were not expressed. The proteins gp21, gp46 and p24 of HTLV-1 were produced in bacteria BL21 (DE3) with vector pET28a (+). These three proteins were solubilized from inclusion bodies, purified by IMAC and identified by Western blotting techniques and mass spectrometry. The recombinant proteins gp21, p24 and gp46 were recognized by sera from patients with HTLV-1 and were not recognized by sera from individuals with HIV-1 and healthy people, which gives them great specificity and diagnostic potential. These results are the first steps to achieve the ultimate goal of producing all seven proteins on a larger scale and finally get the production of a diagnostic kit sensitive, specific and cheap with national technology, reducing spending on imports of these products and fostering the national biotechnology industry.
 
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Release Date
2017-06-30
Publishing Date
2015-07-06
 
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