Tc323, una nueva proteína de T. cruzi con potencial diagnóstico

GOMEZ, KARINA A

Medellin

Simposio; IX SIMPOSIO INTERNACIONAL DE BIOLOGÍA CELULAR Y MOLECULAR DE LA ENFERMEDAD DE CHAGAS; 2022

Universidad de Antioquia, Medellin

Chagas illness, a potentially life-threatening disease, is an infection caused by the hemoflagellate parasite Trypanosoma cruzi. Since nowadays none of the available diagnostic methods disclose ~100% specificity and sensitivity during chronic phase of Chagas disease (CCD), the WHO advises the use of at least two of distinct serological tests for the reliable diagnosis of CCD. From this perspective, we evaluated the diagnostic utility of a hypothetical protein T. cruzi, called Tc323, which is only present in this parasite. This protein was identified as the target of a single-chain variable fragment recombinant antibody (scFv 6B6) isolated from a phage display library constructed from B cells of chronic Chagas heart disease patients. Phylogenetic analysis showed that Tc323 is highly conserved throughout evolution in all T. cruzi lineage but it lacks orthologous in other kinetoplastid parasites. Due to experimental limitations to produce the full-length protein as recombinant, structural predictions using RaptorX server allowed us to foretell six structural domains (D1-D6) which were cloned into pRSET-A vector and expressed in E. coli BL21 Rosetta (DE3) cells. In addition, B cell epitopes predictors mapped that almost all of the protein is immunogenic, while the majority of antigenic sequences are within D3 and D6 domains. After optimization of direct ELISA by checkerboard titration, preliminary results showed that recombinant His-tag D3 and D6 domains were recognized by antibodies present in plasma from individuals with CCD but not from those coming from patients with Leishmania and non-infected donors. Our finding allowed the identification of two recombinant domains containing Tc323 as a promising immunodiagnosis candidates for chronic Chagas disease in human.