Banca de DEFESA: MARCELA CRISTINA DE SOUZA SILVA

Uma banca de DEFESA de DOUTORADO foi cadastrada pelo programa.
STUDENT : MARCELA CRISTINA DE SOUZA SILVA
DATE: 15/08/2022
TIME: 13:30
LOCAL: Sala Virtual Remota
TITLE:

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KEY WORDS:

serum therapy; botropic toxins; BjussuMP-II; nanobody.

PAGES: 110
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

Driven by many advances in immunology and synthetic biology, different approaches and antibody formats are investigated for their ability to neutralize animal toxins. The Camelid Antibody Heavy Chain Variable Domain (VHH) or nanobody presents itself as a promising tool for diagnosis and therapy. VHH has a small size (~15 kDa), ease of genetic manipulation, low immunogenicity, ability to penetrate dense tissues, recognition of normally inaccessible epitopes and stability to changes in temperature and pH. Its peculiar characteristics have leveraged studies for its application regarding the neutralization of animal toxins. The high number of cases and the sequelae caused by snakebites make this condition a serious public health problem. Although serum therapy is effective against systemic damage, it has limitations in neutralizing the venom at the bite site. In addition, serotherapy agents may develop hypersensitivity reactions due to their heterologous nature. Thus, the need for more efficient, homogeneous and safe preparations has instigated the search for alternative processes and products for the treatment of snake envenomation. Therefore, this work proposes the in silico and in vitro characterization of Lama glama nanobodies previously selected against the metalloproteinase P-I, BjussuMP-II, from the venom of B. jararacussu. For this, camelid nanobodies against BjussuMP-II were previously selected using Phage Display technology. After gene characterization by sequencing and in silico analysis, 7 distinct clonal profiles were identified. In order to obtain soluble nanobodies for further assays, the gene sequences of the 7 clones were recombined in pET-22b(+) vector and expressed in E.coli bacteria strain BL21(DE3). The immunoreactivity of clones against BjussuMP-II was evaluated by enzyme immunoassay, which was maintained after purification by cobalt affinity chromatography column. The specificity of the nanobodies was also determined by Western blot, with recognition against BjussuMP-II and B. jararacussu total venom. In the evaluation of the proteolytic activity of BjussuMP-II on casein, VHH OL960543 showed inhibitory potential on the proteolytic activity of BjussuMP-II in all tested ratios. Through in vitro tests, it was possible to observe a 25% reduction in the levels of LDH released by murine endothelial cells (t-END), demonstrating the ability of VHH OL960543 to neutralize part of the toxic effects triggered by BjussuMP-II. In silico analysis, by molecular docking, of VHHs with BjussuMP-II demonstrated the possible interaction with amino acids present in the catalytic site, as well as in regions of great importance for the conformation and stability of the toxin of interest. CD spectra for VHH OL960543 estimated a secondary structure of 5.3% α helix, 44.7% β strand (3.3% left twisted; 29.2% relaxed; 12.2% right twisted) and 11.2% turn, in agreement with the theoretical structural model generated for the sequence (4.5% of α helix, 52.2% of β strand and 16.4% of turn), with calculated melting temperature (TM ) of 56.4º C. The results generated will be able to direct specific studies in the perspective of producing useful tools for the development of immunobiologicals based on camelid nanobodies.

COMMITTEE MEMBERS:
Externo à Instituição - CARLOS DELFIN CHASVEZ OLORTEGUI - UFMG
Externa à Instituição - ANDREA QUEIROZ MARANHÃO
Externo à Instituição - GILVAN PESSOA FURTADO
Externo à Instituição - MARCOS ROBERTO DE MATTOS FONTES - UNESP
Presidente - ***.335.462-** - SORAYA DOS SANTOS PEREIRA - UNIR