Amplificação, clonagem e expressão de proteína recombinante do vírus da doença de Aujeszky em sistema de baculovírus para utilização em programa de controle e erradicação / Amplification, cloning and expression of the recombinant protein of the Aujesskys disease vírus in baculovirus system for use in control and eradication program

AUTOR(ES)

Régia Maria Feltrin Dambros

DATA DE PUBLICAÇÃO

2006

RESUMO

Aujeszkys disease (AD) is an infect-contagious illness that causes serious economical damages to the producer and the swine industry. Aiming to develop mechanisms and to improve technologies that are faster, more sensitive and more specific for diagnosis and for use in free areas or in ADs eradication programs, the sequence codifier of the glycoprotein E (gE) of Aujeszkys disease virus (ADV) was amplified, cloned and expressed. Through genetic engineering the sequence of the gE gene was propagated in an host organism. The gE was amplified by the technique of polimerase chain reaction (PCR), cloned in the vector pGem-T Easy and transformed in competent cells of Escherichia coli, DH-5a. The clone obtained was sub-cloned in the expression plasmid pFastBac1, which contains the promoter gene of the polyhedrin. The obtained subclone was analyzed inside for certification of its correct plasmid orientation with the restriction endonucleases BamH I and EcoR I. The sub-clone with the correct orientation had its DNA extracted and used for transposition inside the bacmid (recombinant baculovirus and helper plasmid with competent DH10Bac cell). Colonies with inserted gE were selected by the phenotype of the colony, which expresses white color when cloned. White recombinant colonies had their DNA extracted and used for cotransfection in insect cells Trichoplusia ni (BTI-Tn5B1-4). The recombinant-gE baculovirus was inoculated in cultured cells and expressed the recombinant gE by PCR and Western blotting. The recombinant-gE baculovirus containing only the gE gene of the VDA will be used for antigen and monoclonal antibodies production, which will aid in the development of a more sensitive, specific and safer for the use in VDA free regions.

ASSUNTO(S)

swine - diseases engenharia genética recombinant dna dna recombinante genetic engineering suíno - doeças doencas infecciosas de animais

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