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