Proteína Recombinante do vírus da Artrite Encefalite Caprina com potencial antigênico. / Recombinant protein of the caprine arthritis encephalitis virus antigenic potential.

AUTOR(ES)
FONTE

IBICT - Instituto Brasileiro de Informação em Ciência e Tecnologia

DATA DE PUBLICAÇÃO

11/12/2009

RESUMO

The present study had two objectives, first to survey the main studies on recombinant proteins and vaccine experiments against CAEV and other lentivirus and second, to produce a recombinant protein from the Caprine Arthritis Encelhalitis virus with antigenic potential for use as antigen in serological diagnoses in a bacterial and plasmid expression system. Regarding the first objective the research of the last ten years (1998-2008) was revised in the literature regarding vaccines against animal lentivirus. Regarding the production of CAEV recombinant protein, the CAEV gene was analyzed to determine which region of the gag gene codified for the virus p28. The region considered was of the 971-1606 nucleotide of the CAEV Cork deposited in the gene bank. The primers were designed from the start of the sequence (20bp) and the end of the sequence (21bp). The p 28 gene was linked to the gag in pUC19 plasmid and then subcloned in pET32b. Both the constructions were sequenced and purified by affinity chromatography. PCR was performed on the colony and the plasmids extracted by alkaline lysis. The best expression condition of the recombinant protein was tested at 37oC and room temperature, using isopropyl -D-1-thiogalactopyranoside (IPTG) as inductor at the concentrations of 0.5, 1 and 3 mM. To verify the result of the expression, the proteins were analyzed by electrophoresis in polyacrylamide gel. Western blot was performed to assess their antigenic potential using the recombinant protein as antigen and the serum from a proven positive animal as primary antibody. Sequencing confirmed the correct insertion of the p28 gene fragment in the two plasmids, which presented a band of 636 bp after PCR in electrphorosis in agarose gel. The E. coli bacteria expressed the protein under various conditions. At 37oC the bacteria expressed the protein at lower intensities and there was no expression at the 0.5 mM IPTG concentration. The protein was expressed at room temperature at all the concentrations and at 1 mM IPTG the protein presented greater intensity. Based on the results the treatment of 1 mM IPTG and room temperature was considered the best expression condition of the p28 recombinant protein in E. coli BL21. When analysing the antigenic potential of the recombinant protein in western blot it reacted with the positive goat serum and presented a strong band characteristic of 28 kDa molecular weight. It was concluded that the CAEV p28 recombinant protein was produced by the bacteria and it presented an antigenic potential that could probably be used as antigen in diagnostic tests.

ASSUNTO(S)

lentivírus expressão de p28 clonagem western blot antigenicidade reproducao animal lentivirus p28 expression cloning western blot antigenicity

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