Expressão heteróloga e purificação da tityustoxina: obtenção da proteína recombinante Ts3 do escorpião Tityus serrulatus

• Ano de defesa: 2011
• Autor(a) principal: Anderson Oliveira do Carmo
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Genética; Escorpião Veneno; Tityus serrulatus; Toxinas; Proteínas recombinantes; Sorologia
• Link de acesso: http://hdl.handle.net/1843/BUOS-8FNGWJ

The scorpion Tityus serrulatus is a Brazilian species that has the most toxic venom and causes high number of accidents, especially in urban areas, and may lead to deaths in children and the elderly. The treatment of severe cases consists of serum therapy, but this method has some problems, such as obtaining the crude venom, maintenance of horses immunized and poor survival of these animals after such immunization. An alternative to solve these problems is the heterologous expression of the main toxins in Escherichia coli. Thus, the goal of this work was to obtain the heterologous expression of Ts3 toxin of T. serrulatus without toxicity for serum production. The nucleotide sequence of the mature Ts3 was amplified and cloned in bacterial expression vectors pET26b and pET32c. After cloning, the vectors pET26/Ts3 and pET32/Ts3 were expressed in E. coli Origami (DE3), C41 (DE3) and C43 (DE3). Recombinant Ts3 produced by pET26b/Ts3 vector was purified using a nickel resin. After purification, rabbits were immunized with recombinant Ts3. After six boosters of recombinant Ts3, T. serrulatus crude venom was applied in the last booster. The anti-recombinant Ts3 serum was characterized by ELISA and SPOT. Recombinant Ts3 showed no pharmacological activity when injected subcutaneously in mice. The anti-recombinant Ts3 serum showed a reasonable recognition of the crude venom by ELISA and, with only one booster of crude venom, this recognition increased. SPOT revealed increased presence of antibodies that recognize the C-terminal portion of this toxin. Anti-recombinant Ts3 serum was unable to neutralize the venom, but the serum with one booster of crude venom neutralized 2DL50. The low performance in the neutralization may be related to an inadequate conformation of recombinant Ts3 generating poor neutralizing antibodies. The use of recombinant toxins in the serum production needs improvement, but the results show that this is a promising method for the production of anti-venom serum.