Construção de uma biblioteca de anticorpos (scFv) apresentada em fagos para seleção, análise e caracterização de antígenos do carrapato bovino (Boophilus microplus)

Abstract

CAPÍTULO I:

The amplification of fragmentos scFv (single chain fragments variable) of immunized animals is an important method to generation of monoclonal antibodies. In this work chickens had been immunized with proteins of the larval and adult phases of the Boophilus microplus for the generation of an antibodies combinatorial library. Total RNA of spleen had been extracted and amplification reactions (PCR) had been made using specific primers of the antibodies variable regions with the purpose to get all the variability of the immune repertoire in the construction of the library. Five cycles of selection (biopanning) had been made for capture of clones scFv reactive to total proteins of larvae and adults ticks. The affinity of clones to proteins was confirmed by ELISA demonstrating an increase of the affinity of clones to the ligante throughout the five cycles of selection. The sequencing of clones demonstrated a predominance of identical clones for the library using both larvae and adults proteins. The recombinant antibodies had been used for selection and characterization of epitope to proteins of the catle tick for use as vaccine in the control of this parasite.

CAPÍTULO II:

With the objective of selecting and characterizing new ticks vaccine targets, we developed a combinatorial antibody fragment library (scFv), expressed in the capsid of bacteriophages, produced from tick larval and adult protein immunized chicken Ig repertoire These antibodies (scFv) recognize a protein of the total larval and adult s extract of the proximally 80 Kd, by western blotting tests, and the sequence of these reactive proteins was checked by N-terminal sequencing. The band corresponded to a GP80 protein with high similarity and is found in huge quantities in parasite eggs and larval stages. With the objective of selecting mimotopes of B. microplus by phage displayed epitope characterization, the recombinant antibodies with major frequency were submitted to a selection against a constricted peptide library displayed on phages. The cross reactivity was confirmed by the recognition of one of those recombinant antibodes (scFv) to a motif similar to GP80 sequence and others peptide (Serotonin receptor, Notch-like protein, Paramiosin and Antigen B membrane) obtained from peptide library selection. This work confirms the GP80 as a vaccine candidate and points out the usefulness of phage display methodology for selection of new represents the selection efficiency of new B. microplus.vaccine targets.