RESUMO
We have cloned and expressed the gp63 gene of Leishmania major in BCG to develop a recombinant vaccine against zoonotic cutaneous leishmaniasis. Two different expression systems were investigated. The first system consists of pAN, a Mycobacterium paratuberculosis promoter, which drives expression of ORF2, an open reading frame in IS900. This system allows the production of heterologous polypeptides as hybrids with the ORF2 gene product. The second expression system relies on the production of antigenic fragments as fusion proteins with the N-terminal region of Mycobacterium fortuitum beta-lactamase. Both constructs resulted in the production of Gp63 in BCG. The ability of the two recombinant BCG strains to induce protective immunity against a challenge with L. major amastigotes was evaluated after vaccination of susceptible (BALB/c), and resistant (C57BL/6) mice. Recombinant BCG producing Gp63 as a hybrid protein with the N-terminal region of the beta-lactamase elicited significant protection against a challenge with L. major in BALB/c-immunized mice.
Assuntos
Leishmania major/genética , Leishmaniose Cutânea/prevenção & controle , Metaloendopeptidases/imunologia , Mycobacterium bovis/imunologia , Vacinas Sintéticas/imunologia , Animais , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/imunologia , Sequência de Bases , Western Blotting , Expressão Gênica , Vetores Genéticos , Leishmania major/crescimento & desenvolvimento , Leishmaniose Cutânea/mortalidade , Ativação Linfocitária , Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mycobacterium/genética , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Fatores de Tempo , Vacinação , Vacinas Sintéticas/administração & dosagemRESUMO
A fibrinogenase from Vipera lebetina venom was isolated by gel filtration in a Superose 12 column prep grade HR 16/50 and by ion-exchange in a Mono Q HR 5/5 column. The purified enzyme, which was obtained with a yield of 8 mg from 60 mg of crude venom, is a glycoprotein having an isoelectric point of 5.9 +/- 0.1 and a mol. wt of 26,000 +/- 1000 as estimated by SDS-PAGE. The biochemical characterization of the enzyme revealed that it hydrolyzes readily the B beta chain of fibrinogen and the A alpha chain as well as fibrin and casein. Over a pH range from 4 to 11 the enzyme was not inactivated by a 20 min treatment at 90 degrees C. The isolated fibrinogenase is inhibited by ethylenediamine tetraacetic acid, dithiothreitol and L-cysteine but not by phenylmethylsulfonyl fluoride. On the other hand, it is activated by Ca2+ and Mg2+. Purified fibrinogenase up to a dose of 100 micrograms/mouse shows no toxicity and has no hemorrhagic activity.