Generation and characterization of a recombinant chimeric protein (rCpLi) consisting of B-cell epitopes of a dermonecrotic protein from Loxosceles intermedia spider venom

Abstract

A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

Identification and characterization of B-cell epitopes of 3FTx and PLA2 toxins from Micrurus corallinus snake venom

“…(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) protective antibodies (protective B-cell epitopes) is an interesting perspective in the Toxinology field (Mendes et al, 2013).…”

“…Our group has also recently engineered, by molecular biology techniques, a chimeric protein composed by validated epitopes from a dermonecrotic protein present in the spider Loxosceles intermedia venom. Using a rabbit model, preincubation of anti-chimeric protein serum was capable of neutralizing 95% of the dermonecrotic activity of the recombinant toxin (Mendes et al, 2013). This chimeric protein was further used, in combined protocol with crude venom (3 doses of venom and 9 doses of chimeric protein), to immunize horses (Figueiredo et al, 2014).…”

A multi-protease, multi-dissociation, bottom-up-to-top-down proteomic view of the Loxosceles intermedia venom

“…In particular, those from the Loxosceles genus are already being used in at least four general application fronts, viz. : as therapeutic anti-venom sera 8 ; as tools in molecular and cellular biology research; and as aids in drug development and production of selective and environmentally friendly bioinsecticides 5 . Peptides originating from the venom of Thrixopelma pruriens have been used in the treatment of pain and inflammation 9 ; the T×2–5 and T×2–6 neuropeptides from the Phoneutria nigriventer venom, for treating erectile dysfunctions 10 ; and distinct bioactive peptides from spider venoms, in the treatment of diverse diseases, such as cancer 11 .…”

Biotechnological Trends in Spider and Scorpion Antivenom Development

“…In 2013, Mendes et al constructed a chimeric protein containing three epitopes of LiD1, a dermonecrotic toxin present in the venom of L. intermedia . When rabbits were immunized with the recombinant chimeric protein (rCpLi), they were able to elicit a protective immune response against whole venom from both L. intermedia and L. gaucho , comparable to rabbits immunized with the natural toxin [189]. …”