Dados do Trabalho

Título

A Novel Recombinant Single Chain Antibody Exhibiting Broad Neutralization Activity on Bothrops pauloensis Snake Venom: In-Planta Expression Approach

Introdução

Available data show 4.5–5.4 million people get bitten by snakes annually. Of this, 1.8–2.7 million develop clinical illness and 81 000 to 138 000 die from complications. In Brazil, about 79% of snakebites are caused by Bothrops snakes. Bothropic envenomation is characterized by significant systemic alterations, primarily involving hemostasis disorders.

Objetivo (s)

In this study, we highlight the advantages of using recombinant antibodies over animal immunization for the production of specific and consistent antivenoms.

Material e Métodos

Briefly, RNA from the spleen of chickens immunized with Bothrops crude venom was used for cDNA synthesis and construction of the scFv library through PCR amplification and isolation of the light and heavy regions of the antibody chains for subsequent binding reaction into the pComb3XSS display vector and pharmagidia formation. Phages from the single-chain variable fragment (scFv) library were screened against crude B. pauloensis venom. The recombinant strain of E. coli TOP 10 was induced to express the soluble form of scFvs. Clones expressing scFv in solution were tested for their reactivity against crude B. pauloensis venom and neuwiedase. Nicotiana tabacum plants were transformed using the Agrobacterium tumefaciens strain. Through leaf processing, the recombinant scFv was purified by ion affinity chromatography. scFvs in bacterial and plant systems was tested for recognition in different venoms of the genus Bothrops by ELISA and immunoproteomics assays. scFvs were also analyzed for their ability to inhibit phospholipasic, proteolytic, fibrinogenolytic, coagulant and hemorrhagic activities.

Resultados e Conclusão

Notably, we have successfully expressed this antibody in plants, allowing us to evaluate its enzymatic and biological activities more comprehensively. Through proteolytic assays, we have demonstrated the remarkable ability of the antibody's scFv molecules to inhibit various actions of snake venom metalloproteinases present in the venom of B. pauloensis. These actions include fibrinogenolysis, azocaseinolysis, coagulation, and hemorrhaging. By specifically targeting one of the major classes of metalloproteinases, our antibody effectively neutralizes the toxic effects associated with envenomation, particularly those impacting systemic processes. Its successful expression in plants showcases its biotechnological applications, providing a promising alternative for developing a more effective antidote against snake venom.

Palavras-chave

Bothrops, envenoming, antivenom, scFv, antibodies