clonagem e purificação de fragmento da proteína capsidial de banana streak ol virus cloning and purification of banana streak ol virus coat protein fragment

clonagem e purificação de fragmento da proteína capsidial de banana streak ol virus cloning and purification of banana streak ol virus coat protein fragment

;Ricardo Lombardi;Ricardo Harakava;Addolorata Colariccio
proc - the fourth ieee workshop on software technol for future embedded and ubiquitous syst, seus 2006 andthe second int workshop on collaborative comput, integr, and assur, wccia 2006 2010 Vol. 45 pp. 811-817
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lombardi2010pesquisaclonagem

Abstract

O objetivo deste trabalho foi clonar e induzir a expressão de fragmento da proteína capsidial de Banana streak OL virus (BSOLV-CP) em Escherichia coli, bem como purificar a proteína recombinante obtida. Empregou-se um par de iniciadores específicos para amplificar, em PCR, um fragmento de aproximadamente 390 pb, da região codificadora da porção central da BSOLV-CP. O fragmento obtido foi clonado em vetor pGEM-T Easy, subclonado em vetor pQE-30 e transformado em células de E. coli M15 (pREP4) por choque térmico. A expressão da proteína foi induzida por tiogalactopiranosídeo de isopropila (IPTG), e a proteína recombinante BSOLV-rcCP de 14 kDa foi detectada em Western blot e Dot blot. A expressão da proteína BSOLV-rcCP abre novas possibilidades para a obtenção de antígenos para a produção de antissoros contra o BSOLV.
The objective of this work was to clone and to induce the expression of a fragment of Banana streak OL virus coat protein (BSOLV-CP) in Escherichia coli, as well as to purify the obtained recombinant protein. Two specific primers were used for the PCR-amplification of approximately 390-bp fragment of the codifying region of the BSOLV-CP central portion. The obtained fragment was cloned in pGEM-T Easy vector, subcloned in pQE-30 expression vector and transformed into competent E. coli M15 (pREP4) cells by heat shock. The protein expression was induced by isopropyl thiogalactopyranoside (IPTG) and the 14-kDa BSOLV-rcCP recombinant protein was detected in Western and Dot blotting. The expression of the BSOLV-rcCP protein enables new approaches to the obtention of antigens for the antisera production against BSOLV.

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