Avaliação de epitopos na proteina do capsideo de isolados do virus da tristeza dos citros (CTV)

AUTOR(ES)
DATA DE PUBLICAÇÃO

1999

RESUMO

Preliminary studies about the epitopes location in the capside protein of the isolates of the citrus tristeza virus of three isolate (´Pêra IAC , Pêra CB-22 and Pêra CB-104 ) were accomplished. In the first phase, the amplification was accomplished with specific direct and reverse primers of eight different regions of the CP gene, coding for three N-terminal peptides (aa 1 to 70, aa 1 to 120 and aa 1 to 170), three C-terminal peptides (aa 70 to 223, aa 120 to 223 and aa 170 to 223) and two internal peptides of CP (aa 37 to 64 and aa 64 to 90). In the second phase they were appraised two strategies for expression of the peptides: cloning in expression vector through AT cloning site (vector Pinpoint TM Xa-1 T promega), and cloning of the coding sequences in vectors of the system pET. Vector Pinpoint TM Xa-1, containing as insert the whole gene of CP, obtained as PCR s product after amplification of the three isolates cDNA, transformed competent cells E. coli JM109, but after induction with IPTG there was not expression of the peptides of interest. The vectors of the pET system didn t transform competent cells E. coli BL21 (DE3)pLysS. In the third phase, three recombinant proteins (MBP-CPC1V, CB-22 and CB-104), produced from the expression of the CP-C1V gene of the three isolates, they were broken with cyanogen bromide. The break products were evaluated through "Western Blotting" against monoclonal antibodies specific for CTV. The monoclonal IC-04.6, developed against the recombinant protein CB-22 detected epitopes, in an intense reaction, in peptides with mass esteemed in 27 kDa and 18 kDa, original of CB-22; the monoclonal 39-08, developed against the recombinant protein CB-104 detected epitopes, in a moderate reaction, in peptides with esteemed mass of 28 kDa and 14 kDa, original of CB-104; the monoclonal MCA-13, developed against the T-36 isolate from Florida, detected epitopes in original peptides of the recombinants proteins MBP-CPCTV and CB-104; the monoclonal 3DF1, developed against Spanish isolates of CTV, detected epitopes in original peptides of CB-22 and CB-104 and the monoclonal 3CA5, developed against Spanish isolates of C1V, just detected epitopes in a peptide with esteemed mass of 18 kDa present in CB-22. These results suggest that the five monoclonal recognizes different epitopes and that will be necessary new strategies to evaluate them

ASSUNTO(S)

citros anticorpos monoclonais virus de planta

ACESSO AO ARTIGO

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