Produção dos fragmentos de anticorpos recombinantes scFv-N e scFv-S1 e suas aplicações na detecção e diferenciação do Vírus da Bronquite Infecciosa
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2009-06-22
Autores
Caetano, Aline Gonçalves [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O vírus da Bronquite infecciosa (VBI) é um Coronavírus aviário que infecta aves domésticas de corte e postura, ocasionando grandes perdas econômicas na indústria avícola. Dada a natureza altamente contagiosa e aguda da doença, há uma grande necessidade do desenvolvimento de métodos diagnósticos que possam ajudar na detecção e/ou caracterização de estirpes variantes do VBI. Sendo assim, para auxiliar no diagnostico laboratorial da infecção, foi construída uma biblioteca de fragmentos de anticorpos monoclonais pela técnica de Phage-display. Para tanto, após a imunização de galinhas com a estirpe vacinal H120, foi extraído o RNA total do baço das aves imunizadas e amplificadas as cadeias variáveis leve e pesada que foram unidas por linker, originando o fragmento gênico de cadeia única scFv. Após a realização de três ciclos de seleção foram obtidos 400 clones que foram avaliados em ensaios de ELISA e Western blotting para averiguação da especificidade dos mesmos frente às proteínas da estirpe H120. Após realização dos testes foram selecionados dois clones, um que apresentou grande reatividade para com a proteína de nucleocapsídeo (N) (scFv-N) e o outro com reatividade para com a subunidade 1 da glicoproteína de superfície (S) (scFv-S1). O anticorpo scFv-S1 quando utilizado em ensaio de vírus-neutralização em ovos embrionados mostrou titulo significativo de proteção. Já em testes de ELISA utilizando estirpes de referência e isolados brasileiros de campo do VBI, o anticorpo scFv-N foi capaz de detectar todas as estirpes (H120, M41, Arkansas, IBVPR05, IBVPR02, IBVPR01, IBVSC01), enquanto que o scFv-S1 pode discriminar as estirpes pertencentes ao sorotipo Massachusetts (H120, M41 e IBVSC01) das demais estirpes variantes avaliadas. Os fragmentos de anticorpos scFv-N e scFv-S1 também mostraram bons resultados quando utilizados na técnica...
Infectious bronchitis virus (IBV), the coronavirus of the chicken, is one of the main causes of economic loss within the poultry industry, affecting the performance of meattype and egg-laying domestic fowls. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect and/or characterize IBV strains. In order to improve the laboratory diagnosis of IBV infection, phage-displayed recombinant antibody library derived from splenic mRNA of chickens immunized with H120 vaccine strain of infectious bronchitis virus (IBV) was constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction (PCR) of the individual heavy (VH) and light (VL) chain variable gene segments. After three rounds of panning selection, ten scFv phage display antibodies of 400 randomly chosen clones were demonstrated to react with IBV antigens by ELISA. The western blot analysis selected two scFv antibodies reacting strongly with nucleocapsid (N) (scFv-N) protein or subunit 1 of spike glycoprotein (S1) (scFv-S1) of IBV. The anti-S1 scFv antibody showed a significant neutralization titre in embryonating chicken egg test. In ELISA analysis using reference IBV strains and Brazilian field isolates, the anti-N scFv antibody was able to detect all strains (H120, M41, ARKANSAS, IBVPR05, IBVPR02, IBVPR01, IBVSC01), while the anti-S1 could discriminate Massachusetts serotype (H120, M41 and IBVSC01) between variant strains. A scFv-based indirect immunoperoxidase (IP) procedure was also applied to detect infectious bronchitis virus (IBV) antigens in formalin-fixed tracheal tissue sections. Thus, the results showed that scFv-N and scFv-S1 antibodies can be used for the detection and differentiation of IBV strains.
Infectious bronchitis virus (IBV), the coronavirus of the chicken, is one of the main causes of economic loss within the poultry industry, affecting the performance of meattype and egg-laying domestic fowls. Given the highly contagious and acute nature of the disease, there is an urgent need for the development of diagnostic assays that can detect and/or characterize IBV strains. In order to improve the laboratory diagnosis of IBV infection, phage-displayed recombinant antibody library derived from splenic mRNA of chickens immunized with H120 vaccine strain of infectious bronchitis virus (IBV) was constructed as single chain variable fragments (scFv) by overlap extension polymerase chain reaction (PCR) of the individual heavy (VH) and light (VL) chain variable gene segments. After three rounds of panning selection, ten scFv phage display antibodies of 400 randomly chosen clones were demonstrated to react with IBV antigens by ELISA. The western blot analysis selected two scFv antibodies reacting strongly with nucleocapsid (N) (scFv-N) protein or subunit 1 of spike glycoprotein (S1) (scFv-S1) of IBV. The anti-S1 scFv antibody showed a significant neutralization titre in embryonating chicken egg test. In ELISA analysis using reference IBV strains and Brazilian field isolates, the anti-N scFv antibody was able to detect all strains (H120, M41, ARKANSAS, IBVPR05, IBVPR02, IBVPR01, IBVSC01), while the anti-S1 could discriminate Massachusetts serotype (H120, M41 and IBVSC01) between variant strains. A scFv-based indirect immunoperoxidase (IP) procedure was also applied to detect infectious bronchitis virus (IBV) antigens in formalin-fixed tracheal tissue sections. Thus, the results showed that scFv-N and scFv-S1 antibodies can be used for the detection and differentiation of IBV strains.
Descrição
Palavras-chave
Anticorpos monoclonais, Bronquite infecciosa em ave domestica, Vírus da bronquite infecciosa, Detecção viral, Infectious bronchitis virus, Recombinant monoclonal antibodies, Viral detection
Como citar
CAETANO, Aline Gonçalves. Produção dos fragmentos de anticorpos recombinantes scFv-N e scFv-S1 e suas aplicações na detecção e diferenciação do Vírus da Bronquite Infecciosa. 2009. xviii, 78 f. Tese (doutorado) - Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 2009.