Construção de uma biblioteca de anticorpos (scFv)apresentada em fagos para seleção, análise e caracterização de antígenos do carrapato bovino (Boophilusmicroplus)
CAPÍTULO I:The amplification of fragmentos scFv (single chain fragments variable) ofimmunized animals is an important method to generation of monoclonalantibodies. In this work chickens had been immunized with proteins of the larvaland adult phases of the Boophilus microplus for the generation of an antibodiescombinatorial library. Total RNA of spleen had been extracted and amplificationreactions (PCR) had been made using specific primers of the antibodies variableregions with the purpose to get all the variability of the immune repertoire in theconstruction of the library. Five cycles of selection (biopanning) had been made forcapture of clones scFv reactive to total proteins of larvae and adults ticks. Theaffinity of clones to proteins was confirmed by ELISA demonstrating an increase ofthe affinity of clones to the ligante throughout the five cycles of selection. Thesequencing of clones demonstrated a predominance of identical clones for thelibrary using both larvae and adults proteins. The recombinant antibodies hadbeen used for selection and characterization of epitope to proteins of the catle tickfor use as vaccine in the control of this parasite.CAPÍTULO II:With the objective of selecting and characterizing new ticks vaccine targets, wedeveloped a combinatorial antibody fragment library (scFv), expressed in thecapsid of bacteriophages, produced from tick larval and adult protein immunizedchicken Ig repertoire These antibodies (scFv) recognize a protein of the total larvaland adult?s extract of the proximally 80 Kd, by western blotting tests, and thesequence of these reactive proteins was checked by N-terminal sequencing. Theband corresponded to a GP80 protein with high similarity and is found in hugequantities in parasite eggs and larval stages. With the objective of selectingmimotopes of B. microplus by phage displayed epitope characterization, therecombinant antibodies with major frequency were submitted to a selection againsta constricted peptide library displayed on phages. The cross reactivity wasconfirmed by the recognition of one of those recombinant antibodes (scFv) to amotif similar to GP80 sequence and others peptide (Serotonin receptor, Notch-likeprotein, Paramiosin and Antigen B membrane) obtained from peptide libraryselection. This work confirms the GP80 as a vaccine candidate and points out theusefulness of phage display methodology for selection of new represents theselection efficiency of new B. microplus.vaccine targets.
Advisor:Andrea Queiroz Maranhao; Luiz Ricardo Goulart Filho; Foued Salmen Espindola; Matias Pablo Juan Szabó; José Miguel Ortega
School:Universidade Federal de Uberlândia
Source Type:Master's Thesis
Keywords:Boophilus microplus Fragmento scFv Antibodies ScFv fragments Phage display library Peptide
Date of Publication:04/30/2007