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Clonagem e expressão génica de potenciais candidatos antigénicos do parasita Trypanosoma brucei brucei
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A doença do sono ou tripanossomose humana africana é uma patologia
parasitária exclusivamente africana, cujo agente etiológico do género Trypanosoma é transmitido pela picada da mosca tsé-tsé, pertencente ao género Glossina. Afeta milhões de pessoas e animais anualmente, representando um grande fator de atraso no desenvolvimento do continente africano. Caracteristicamente negligenciada, poucos
fundos são disponibilizadas para o controlo e investigação da Tripanosomose Africana.
Não existe vacina para a doença e os fármacos disponíveis para o tratamento são pouco eficientes e apresentam elevada toxicidade.
Amplificou-se a partir de cDNA extraído de formas sanguíneas de T. b. brucei a sequência génica da enzima TSA, sugerindo a expressão da enzima TSA em formas sanguíneas de T. b. brucei. Duas proteínas recombinantes de T. brucei – Glicoproteína Invariável de Superfície (ISG) e a região N-terminal da Trans-sialidase (nTSA) foram
subclonadas no vetor comercial pET28a. Através da subclonagem dos genes de interesse ISG e TSA neste plasmídeo de expressão procariota - pET28a - foi possível produzir as proteínas recombinantes ISG e nTSA em Escherichia coli. Expressaram-se as proteínas recombinantes em várias linhagens celulares de E. coli e imunoidentificaram-se ambas através de immunoblot com soro de animais positivos para T. b. brucei. Imunidentificou-se a proteína recombinante nTSA através de immunoblot frente a soro de animais imunizados com o plasmídeo capacitado de expressão em sistema eucariota nTSApVAX1. Avaliou-se o potencial imunogénico dos
plasmídeos ISGpVAX1, nTSApVAX1 e PLCpVAX1 através de imunização génica do modelo experimental da THA. Processou-se soro de animais imunizados com estes plasmídeos através de ELISA para deteção de anticorpos anti-T. b. brucei.
Os três protótipos vacinais utilizados neste estudo foram capazes de induzir anticorpos da classe IgG reativos ao extrato proteico de T. brucei brucei, sugerindo então a expressão em formas sanguíneas do parasita dos antigénios ISG, TSA e PLC.
Contrariamente ao que tem sido sugerido pela literatura até então, foi possível amplificar de cDNA de formas sanguíneas de T. b. brucei o gene TSA, sugerindo pela primeira vez a expressão deste gene nas formas sanguíneas de T. b. brucei.
Sleeping sickness or human African trypanosomiasis is an exclusively African parasitic disease, whose etiologic agent of the genus Trypanosoma is transmitted by the bite of the tsetse fly, which belongs to the genus Glossina . It affects millions of people and animals, representing a major factor in the delayed development of the African continent. Characteristically neglected, very few funds are available for the control and investigation of THA. There is no vaccine for the disease and the drugs available for treatment are inefficient and have high toxicity. The genomic sequence of the enzyime TSA was amplified from cDNA obtained of the blood forms of T. b. brucei, which is suggestive of the positive expression of the enzyme in these blood forms. Two recombinant proteins from T. brucei - Invariable Surface Glycoprotein (ISG) and the n-terminal portion of the trans-sialidase (nTSA) were subcloned in the commercial vector pET28a. Subcloning of the genes of interest ISG and TSA in this plasmid of procariote expression - pET28a - enabled production of the recombinant proteins ISG and nTSA in Escherichia coli. These recombinant proteins were expressed in several Escherichia coli cell lineages and were immunoidentified by means of immunoblotting using serum of animals positive for T. b. brucei. The recombinant protein nTSA was also immunoidentified using immunoblotting against sera of animals immunized with the plasmid expressing an eucariotic system nTSApVAX1. The immunogenic potential of the plasmids ISGpVAX1, nTSApVAX1 and PLCpVAX1 was evaluated through genetic immunization in the experimental model of THA. Sera of the animals immunized with these plasmids were processed using ELISA to detect anti-T. b. brucei antibodies. The three vaccine prototypes used in this study were able to induce the production of antibodies of the IgG class which reacted to the T. b. brucei extract, suggesting therefore the expression, in the blood forms of the parasite, of the antigens ISG, TSA and PLC. Contrary to what has been suggested in the literature so far, it was possible to amplify the TSA gene from cDNA of the bloodstreams forms of T. b. brucei, suggesting for the first time the expression of the TSA gene in the bloodstreams forms of T. b. brucei.
Sleeping sickness or human African trypanosomiasis is an exclusively African parasitic disease, whose etiologic agent of the genus Trypanosoma is transmitted by the bite of the tsetse fly, which belongs to the genus Glossina . It affects millions of people and animals, representing a major factor in the delayed development of the African continent. Characteristically neglected, very few funds are available for the control and investigation of THA. There is no vaccine for the disease and the drugs available for treatment are inefficient and have high toxicity. The genomic sequence of the enzyime TSA was amplified from cDNA obtained of the blood forms of T. b. brucei, which is suggestive of the positive expression of the enzyme in these blood forms. Two recombinant proteins from T. brucei - Invariable Surface Glycoprotein (ISG) and the n-terminal portion of the trans-sialidase (nTSA) were subcloned in the commercial vector pET28a. Subcloning of the genes of interest ISG and TSA in this plasmid of procariote expression - pET28a - enabled production of the recombinant proteins ISG and nTSA in Escherichia coli. These recombinant proteins were expressed in several Escherichia coli cell lineages and were immunoidentified by means of immunoblotting using serum of animals positive for T. b. brucei. The recombinant protein nTSA was also immunoidentified using immunoblotting against sera of animals immunized with the plasmid expressing an eucariotic system nTSApVAX1. The immunogenic potential of the plasmids ISGpVAX1, nTSApVAX1 and PLCpVAX1 was evaluated through genetic immunization in the experimental model of THA. Sera of the animals immunized with these plasmids were processed using ELISA to detect anti-T. b. brucei antibodies. The three vaccine prototypes used in this study were able to induce the production of antibodies of the IgG class which reacted to the T. b. brucei extract, suggesting therefore the expression, in the blood forms of the parasite, of the antigens ISG, TSA and PLC. Contrary to what has been suggested in the literature so far, it was possible to amplify the TSA gene from cDNA of the bloodstreams forms of T. b. brucei, suggesting for the first time the expression of the TSA gene in the bloodstreams forms of T. b. brucei.
Descrição
Palavras-chave
Parasitologia médica Doença do sono Clonagem genética
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical