Análise do uso de Bacillus subtilis como hospedeiro para expressão de VHH anti-fosfolipase de Bothrops jararacussu

Abstract

The Variables Heavy-chain Homodimers (VHHs), also called camelid nanobodies, are unusual and unique IgG antibody fragments from animals of the camelid family, which achieve high antigen-binding affinity and specificity. Currently, they are potential agents for applications in the treatment or diagnosis of snakebites. Different hosts have already produced recombinant nanobodies, but there are still challenges in this production that need to be overcome. In order to overcome these problems, we explored the use of Bacillus subtilis for the expression of VHHs. In chapter I of this dissertation we present a review article that initially raised the main studies that justify the choice of B. subtilis in research and industries. We correlated the characteristics of this host linked to the various genetic engineering strategies to produce biomolecules efficiently and economically. Chapter II discusses the results resulting from the use of B. subtilis strain KO7 as a host to produce an anti-phospholipase VHH. The B. subtilis KO7 was transformed with plasmid pHEN1 and subsequent induction of protein expression was carried out, confirmation of expression was performed by the western-blot technique and protein functionality was evaluated via flow cytometry assay. The results showed that the transformation using the pHEN1 vector was efficient and the inductions using the IPTG inducer or lactose were satisfactory. The VHH protein was expressed in the soluble portion of the bacterial lysate with a molecular mass of approximately 11 kDa. There was a high recognition of anti-phospholipase VHH against the myotoxic proteins Bothropstoxin I (BthTX-I) and Bothropstoxin II (BthTX-I) from Bothrops jararacussu, confirming that B. subtilis KO7 is a potential host to produce soluble VHHs and with its functionality preserved for later clinical use.