Produção, caracterização e avaliação da imunogenicidade do complexo protéico MSP1 recombinante (rMSP1a erMSP1b) de isolado paranaense do anaplasma marginale
The Anaplasma marginale (Rickettsiales: Anaplasmataceae) is a pathogen that cause bovine anaplasmosis. This disease occurs in world wide, and it is more frequently in tropical and subtropical areas, causing important economic losses in cattle herds. Six Major Surface Protein (MSP1a, MSP1b, MSP2, MSP3, MSP4 and MSP5) have been identified in A. marginale. The function of MSP1a and MSP1b is adhesion in bovine erythrocytes and tick cells. The molecular mass of these proteins is 105kDa for MSP1a and 100kDa for MSP1b. The objectives of this work were clone, produce and characterizate recombinant proteins from MSP1a and MSP1 and to evaluate the humoral immune responses in mice immunized with Escherichia coli BL21 recombinant expressing rMSP1a and rMSP1 of A. marginale. The msp1? and msp1b gene obtained from a PCR assay of A. marginale from Parana isolated, were cloned into vectors pET102 and pET101/D-TOPO, respectively. After certification of the recombinant vector construction, these vectors were transformed into E. coli BL21 Star and the proteins were expressed on E. coli BL21Star after induction by IPTG and purified in resin charged with the nickel ion. Antibodies anti-MSP1a and anti-MSP1b reacted with rMSP1a and MSP1b showing a molecular mass of 70 kDa to 105 kDa and 100 kDa, respectively. rMSP1a and rMSP1b were inoculated in chickens to produce IgY anti-MSPs. Bovine erythrocytes were agglutinated with BL21/rMSP1a and BL21/rMSP1b and, this agglutination was inhabited by the presence of the IgY anti rMSP1a and rMSP1b, confirming the adhesion function of these proteins. Indirect immunofluorescence assay (IFA) was performed using IgY anti-rMSP1a and rMSP1b, and we observed reaction in the external membrane of the recombinant E. coli BL21. Additionally, Balb/c were immunized with BL21/rMSP1a and rMSP1b, and the production of whole IgG and IgG2a were determined by indirect ELISA. The animals immunized with BL21/rMSP1a had a strong humoral response (whole IgG and IgG2a), while the mice immunized with BL21/rMSP1b presented a weak response after three immunizations. Western blotting of whole IgG was detected against the proteins rMSP1a and rMSP1b. Sera of mice immunized with BL21/rMSP1a reacted with BL21 and rMSP1a with molecular masses varying from 70 to 105 kDa and a band of 20 kDa suggesting the break of the protein. Sera from mice immunized with BL21/rMSP1b reacted with BL21 and rMSP1b in a molecular mass of 100 kDa. In conclusion, the results confirmed the adhesion function of rMSP1a and rMSP1b in the bovine erythrocytes invasion mechanism and showed the importance of these recombinant proteins in the development of a new vaccine for the bovine anaplasmosis control. In addition these results also demonstrated that BL21 containing the rMSP1a and arMSP1b in the outer membrane are able to produce immune response in mice, and could be supposed that will also be able to induce protection against the bovine anaplasmosis.
Advisor:Odilon Vidotto; Odilon Vidotto [Orientador].; João Luis Garcia; Amauri Alcindo Alfieri
Source Type:Master's Thesis
Cattle - Parasites
Date of Publication:02/16/2006