RESUMO
Two cDNAs coding homologous putative metalloproteases (Metis 1 and Metis 2, expected molecular weights of 55.6 and 56.0kDa, respectively) were identified from the hard tick Ixodes ricinus. The expression of Metis genes was induced in salivary glands during tick blood meal. RNA interference was used to assess the role of both Metis 1 and Metis 2 in tick feeding. It was found that salivary gland extracts lacking Metis 1-2 had a restricted ability to interfere with fibrinolysis. RNAi against Metis 1-2 also induced a high mortality rate. An immune reaction was raised in repeatedly bitten animals against Metis 1 and 2. Vaccination of hosts with the recombinant Metis 1 protein produced in a eukaryotic system partially interfered with completion of the blood meal. Although vaccination did not alter the survival rate or feeding time of ticks, their weight gain and oviposition rate were reduced. This will affect their reproductive fitness in the field. We believe this is the first report of an anti-tick vaccine trial using a metalloprotease derived from I. ricinus.
Assuntos
Ixodes/genética , Metaloproteases/genética , Glândulas Salivares/enzimologia , Infestações por Carrapato/prevenção & controle , Vacinação/métodos , Sequência de Aminoácidos , Animais , Comportamento Alimentar , Feminino , Fibrinólise , Biblioteca Gênica , Inativação Gênica , Ixodes/enzimologia , Ixodes/imunologia , Masculino , Metaloproteases/imunologia , Metaloproteases/metabolismo , Dados de Sequência Molecular , Família Multigênica , Oviposição , Interferência de RNA , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteínas e Peptídeos Salivares/genética , Proteínas e Peptídeos Salivares/imunologia , Alinhamento de Sequência , Infestações por Carrapato/imunologia , Aumento de PesoRESUMO
The effect of the 5' non-translated region (5'NTR) on hepatitis C virus (HCV) morphogenesis in insect cells is investigated in this study. Expression in baculovirus-infected cells of a sequence encoding the C and E1 structural proteins under the control of the very late promoter P10 (AcSLP10-C-E1) led to the synthesis of C and C-E1 complexes, essentially found in dense reticular material associated with the ER and sedimenting at a density of 1.24-1.26 g ml(-1). Addition of the 5'NTR upstream of the C-E1 sequence (AcSLP10-5'NTR-E1) prevents translation from the initiating codon, probably because of the presence of five AUG codons in this sequence. When cells were co-infected with these two viruses, virus-like particles (VLPs) were found in the cytoplasm. The size and shape of these VLPs were variable. Concomitantly, a shift in the sedimentation profile from 1.24-1.26 to 1.15-1.18 g ml(-1) was observed, suggesting an association of C/E1 with the ER membrane. A unique vector was then constructed bearing a mutated 5'NTR (mutation of the five AUGs) and the sequence encoding all of the structural proteins and part of NS2 (5'NTRm-C-E1-E2-p7-NS2Delta). Translation of structural proteins was restored and electron microscopic observation of a cytoplasmic extract showed the presence of icosahedral particles with a density of 1.15-1.18 g ml(-1).