Expressão e purificação de construtos de nanocorpos aplicados ao envenenamento ofídico

Abstract

Camelids, in addition to conventional antibodies, produce functional immunoglobulins G (IgG) lacking the light chain and the CH1 domain. The antigen recognition region of these IgGs is constituted by a single domain, called VHH or nanobody. VHHs, one tenth the size of conventional antibodies, have high similarity to human VH, ability to cross dense tissues and low immunogenicity. They are capable of recognizing antigenic sites inaccessible to human antibodies, have high solubility, stability and can be produced in microorganisms. These characteristics have allowed applications in scientific research, diagnosis and development of biopharmaceuticals. Considering that in Brazil about 30,000 people are victims of snakebites annually; that the species of the Bothrops genus are the main responsible for the disease; that phospholipases are major proteins in the composition of the venom and play a role in the pathophysiology of bothropic envenomation and that serum therapy has limitations mainly related to the local damage of envenomation, our research group seeks to developalternative tools for the treatment of snakebite. As so, VHHs capable of recognizing and inhibiting phospholipases isolated (BthTX-I and II) from Bothrops jararacussu venom were selected. Dimeric VHH constructs were designed by our group with a view to increasing the affinity and neutralizing capacity of monomeric VHHs. The present work aimed to express and purify dimeric VHH constructs and evaluate the ability to recognize phospholipases BthTX-I and II by enzyme immunoassay. After the gene synthesis of the VHH dimers, the clones were expressed in a microbial system using the E. coli SHuffle plysY. Protein expression was induced with 0.3 mM IPTG. Purification was performed by affinity chromatography using cobalt resin. The purified material was analyzed by polyacrylamide gel electrophoresis. To evaluate the ability to recognize phospholipases, flat-bottomed polystyrene microtiter plate were adsorbed with 500 ng per well of BthTX-I, BthTX-II and Bothrops jararacussu venom. The dimeric constructs of VHHs were able to recognize bothropic phospholipases and the tested venom at different dilutions (1 µg, 500 ng, 250 ng, 125 ng) in ELISA. Preliminary data encourage the use of nanobodies and related constructs in the development of nextgeneration antivenoms.