immunology of infectious and parasitic diseases - XXXVII Congress ...

Title: IDENTIFICATION BY PHAGE DISPLAY OF THE MYCOBACTERIUM
TUBERCULOSIS ANTIGEN
SEBASTIÃO MARCOS TAFURI 1,2 ; FABIANA DE ALMEIDA ARAÚJO
SANTOS 2 ; MAYARA INGRID SOUSA LIMA 2 ; LÉA DUARTE DA SILVA
MORAIS 2 ; LUIZ FERNANDO ALMEIDA MACHADO 2 ; ISABELA MARIA
BERNARDES GOULART 3 ; MARCELO SIMÃO FERREIRA 4 ; AÉRCIO
SEBASTIÃO BORGES 4 ; ANDRÉ LUIS DE MORAES CARVALHO 5 ; LUIZ
RICARDO GOULART 6 ; CARLOS UEIRA-VIEIRA 6 .
1
Mestrando do Programa de Ciências da Saúde da Faculdade de
Medicina/UFU;
2
Laboratório de Nanobiotecnologia da Universidade Federal de Uberlândia;
3
Centro de Referência Nacional em Hanseníase e Dermatologia Sanitária –
Hospital de Clínicas/UFU;
4
Setor de Moléstias Infecciosas do Hospital de Clínicas/UFU;
5
Programa Nacional de Controle da Tuberculose de Uberlândia-MG;
6
Instituto de Genética e Bioquímica/UFU.
Introduction: Tuberculosis is an infectious disease very important to public
health worldwide, becoming the second most lethal infectious process,
approximately 1,7 million deaths per year, second only HIV / AIDS. The big
challenge for tuberculosis control is the identification of individuals with the
bacillus, but that are showing the disease from those who have TB infection,
because the diagnostic methods to identify the pathogen still have a low
sensitivity, between 65% and 85%, and 10% of those individuals can develop
active process at some stage of life.
Methods and results: The methodology of phage display of biomolecules,
developed by Smith (1985), was used in this work. The coupling for purification
of IgG from a pool of sera from TB+ (N = 10) and contacts with PPD+ (N = 10),
PPD- (N = 10) as positive and negative controls were performed by using
magnetic beads conjugated to protein G. In the selection of ligands IgG TB+
individuals, PPD+,PPD-, was used a library of random amino acids in the
protein expressed from the bacteriophage pIII. The biopanning was composed
by three cycles of selection. The DNA and amino acid sequences of peptides
were analyzed and aligned using programs of bioinformatics. Individual phage
clones were amplified, purified and validated by ELISA in duplicates. For the
alignment of the peptides to each other and with specific proteins of MTB were
used a CLUSTALW2 and PEPSURF programs, respectively. The reactivity of
26 selected phage was measured by ELISA, and 11 (24.44%) showed higher
reactivity with sera from individuals with active tuberculosis and PPD+,
compared with PPD- individuals. The in silico analyzes showed that the
sequence of the peptides has a similarity with the MPT64 protein
immunodominant Mycobacterium tuberculosis complex, and the alignment
occurred in a possible antigenic region.