Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

Radaelli, Paula; Fajardo, T. V. M.; Nickel, Osmar; Eiras, Marcelo; Pio-Ribeiro, Gilvan

doi:10.1590/s0100-204x2008001000020

Cited by 8 publications

(5 citation statements)

References 18 publications

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“…A purificação da fração IgG foi realizada por cromatografia de troca iônica em coluna de DEAE-SEPHACEL e quantificada por espectrofotometria (FAJARDO et al, 2007). A sensibilidade e a especificidade do antissoro produzido foram avaliadas em ELISA indireto (RADAELLI et al, 2008), com amostras de videiras sadias ou infectadas por 12 diferentes isolados de RSPaV dos quais oito foram previamente caracterizados molecularmente (RADAELLI et al, 2009;BASSO, 2010).…”

Section: O Rupestris Stem Pitting-associated Virus (Rspav) é O Agenteunclassified

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Produção de antissoro policlonal utilizando a proteína capsidial recombinante do Rupestris stem pitting-associated virus

et al. 2010

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Section: O Rupestris Stem Pitting-associated Virus (Rspav) é O Agenteunclassified

“…Esses resultados são semelhantes aos rendimentos médios de CP recombinantes expressadas para outros vírus de videira (FAJARDO et al, 2007;RADAELLI et al, 2008).…”

Section: O Rupestris Stem Pitting-associated Virus (Rspav) é O Agenteunclassified

Produção de antissoro policlonal utilizando a proteína capsidial recombinante do Rupestris stem pitting-associated virus

et al. 2010

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“…GVB displays a complex infection pattern; it is usually accompanied by other viruses of the Vitivirus genus, such as GVA and the yields in grapevine are low (Komínek et al, 2009). In addition the presence of PCR-inhibitory compounds in the host, such as polyphenols, tannins, and polysaccharides, may also inhibit virus detection (Radaelli et al, 2008). GVB is poorly immunogenic ; therefore, the production of high-quality virus-specific antibodies suitable for the large-scale detection of GVB is difficult.…”

Section: Resultsmentioning

confidence: 99%

“…Despite the development of nucleotide sequence-based detection methods, ELISA remains highly relevant for large-scale identification of grapevine virus. Currently, expressing recombinant proteins from cloned virus genes in Escherichia coli is used to produce virus-specific antisera against GVB Saldarelli et al, 2005;Radaelli et al, 2008). Thus, to produce antibodies with adequate sensitivity and specificity, better quality antisera obtained against recombinant proteins is necessary.…”

Section: Resultsmentioning

confidence: 99%

Detection and sequence analysis of grapevine virus B isolates from China

Hu¹,

Dong²,

Zhang³

et al. 2014

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Summary. -The presence of grapevine virus B (GVB) was detected in 188 grapevine samples from China by double antibody sandwich ELISA (DAS-ELISA) and reverse transcription-PCR (RT-PCR). The accuracy of detection by RT-PCR was confirmed by sequencing amplified PCR fragments. Seventeen samples were GVBpositive by DAS-ELISA and five by RT-PCR. The isolate QMW proved to be positive by RT-PCR only, and four isolates (DGWH, DGW, QM, and JFL) could be detected by both methods. Among the five GVB-positive samples detected by RT-PCR, two isolates were originally collected from Henan province and three from Liaoning province. The expected 722 bp DNA fragment, covering partial ORF3 through partial ORF5, was amplified from the five GVB infected samples. Sequence analysis revealed that the molecular variants΄ composition of GVB in the different isolates was complex. Clones of DGWH, DGW, QM, and JFL isolate shared high nucleotide identities, while the identities among the clones of isolate QMW varied. The variants of GVB isolates obtained in this study showed nucleotide identities from 81.1% to 97.9% among themselves, and 79.1% to 98.5% identity with five previously published GVB isolates in NCBI. The alignment of partial ORF3 and the phylogenetic relationships of ORF4 revealed that the molecular variants of Chinese GVB isolates could be clustered into three groups. Only isolate DGW was in the same group with the reported GVB isolates from other countries; the other four GVB isolates in this study were clustered into two groups.

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“…Atualmente, a expressão e purificação de proteínas virais recombinantes em Escherichia coli são uma alternativa para a obtenção de antígenos para a produção de antissoros (Radaelli et al, 2008). Para o BSOLV, ainda não foi realizada a produção e purificação da proteína capsidial recombinante.…”

Section: Introductionunclassified

Clonagem e purificação de fragmento da proteína capsidial de Banana streak OL virus

Lombardi

Harakava

Colariccio

2010

Pesq. agropec. bras.

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Resumo -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.Termos para indexação: Badnavirus, Escherichia coli, Musa, antissoro, bananeira, expressão de proteínas recombinantes. Cloning and purification of Banana streak OL virus coat protein fragmentAbstract -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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Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

Cited by 8 publications

References 18 publications

Produção de antissoro policlonal utilizando a proteína capsidial recombinante do Rupestris stem pitting-associated virus

Produção de antissoro policlonal utilizando a proteína capsidial recombinante do Rupestris stem pitting-associated virus

Detection and sequence analysis of grapevine virus B isolates from China

Clonagem e purificação de fragmento da proteína capsidial de Banana streak OL virus

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