Banca de QUALIFICAÇÃO: SIBELE ANDRADE ROBERTO

Uma banca de QUALIFICAÇÃO de MESTRADO foi cadastrada pelo programa.
STUDENT : SIBELE ANDRADE ROBERTO
DATE: 22/04/2022
TIME: 08:30
LOCAL: Sala Virtual Remota
TITLE:

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KEY WORDS:

Nanobodies. HTLV-1. Nanoparticles. Biosensors. Diagnosis.

PAGES: 75
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

Human T-lymphotropic Virus type 1 (HTLV-1) is one of the etiologic agents of adult Tcell leukemia/lymphoma and HTLV-1-associated myelopathy, called tropical spastic paraparesis (HAM/TSP). About 10-20 million individuals are infected with the virus worldwide. The absence of symptoms and of screening methods for HTLV-1 makes diagnosis difficult and favors the transmission of the disease, which occurs mainly through contact with contaminated fluids. In Brazil, the tests applied for the diagnosis of the disease are restricted to the screening stages in blood donor samples. Thus, the availability of inputs to assist in the detection of infection, as well as its implementation at the public health level directed to the Brazil's Unified Health System (SUS) would be an alternative to facilitate the diagnosis of HTLV-1 infection. During the acute phase of infection, anti-p24 antibodies are produced, which makes this protein and the antibodies generated against it potential markers for diagnosis. The use of molecules with high stability and the ability to recognize molecular targets in a specific and sensitive way is a criterion for the effectiveness of diagnostic tests. In this perspective, the VHHs or camelid nanobodies stand out for presenting such characteristics. Therefore, this work proposes the use of anti-p24 HRVs for the construction of biosensors applicable to the development of assays for the diagnosis of infection caused by the HTLV-1 virus. From a previous characterization, it was possible to select 3 VHHs clones (anti-p24/C11, anti-p24/C52 and anti-p24/C86) after sequencing. However, only clones VHH-11 and VHH-52 were effectively recombined in vector pET22b(+) and then subjected to expression in E.coli strain BL21(DE3) and SHuffle® T7 Express. Of the expression conditions evaluated to obtain the HHV C52 in soluble form, the one that proved to be most effective was in E.coli strain SHuffle® T7 Express with induction at 30ºC and 0.3 mM of IPTG. After purification by Immobilized Metal Ion Affinity Chromatography (IMAC), Western blot and ELISA results confirmed, respectively, the presence of VHH and the permanence of its reactivity against p24. Given the standardization of the synthesis of gold nanoparticles (AuNPs) by the Turkevich method, the conditions for bioconjugation of VHH-52 at 8 µg concentration and pH 9, to AuNPs in the presence of 75 mM of the stabilizing agent acrylic acid Nhydroxysuccinimide-ester (NHS) were established. Finally, the immunoreactivity of the biosensors by DOT BLOT assay demonstrated the ability of the bioconjugates to recognize different concentrations of HTLV-1 p24. The results achieved demonstrate the potentiality of HHV-52 as an input for the constitution of immunosensors. However, additional assays are necessary to determine the application of the biosensors based on VHH antibodies for the detection of HTLV-1 p24.

BANKING MEMBERS:
Externo à Instituição - NILSON IVO TONIN ZANCHIN
Externo à Instituição - WALTER RICARDO BRITO - FIOCRUZ-MG
Presidente - 582.482.422-34 - NAJLA BENEVIDES MATOS - UNIR