RESUMO
RT-PCR evaluation of the activity of eight Ixodes persulcatus salivary gland genes shows clear distinctions in their expression depending of the stage of tick feeding. Out of them, only Salp 10 and Salp 15 proteins may be regarded as candidates for protective antigens to develop anti-tick and anti-Borrelia vaccines. Firstly they play an important role in feeding a tick and modifying a host's immune response. Secondly, the increasing expression of the salp 10 and salp 10 genes begins at early tick feeding stages. Thirdly, the activity of these genes increases with the beginning of feeding by tens and hundreds times and keeps at this level until the third tick feeding stage is over.
Assuntos
Epitopos Imunodominantes/genética , Ixodes/genética , Vacinas contra Doença de Lyme/genética , Glândulas Salivares/metabolismo , Proteínas e Peptídeos Salivares/genética , Animais , Feminino , Regulação da Expressão Gênica , Ixodes/fisiologia , Doença de Lyme/prevenção & controle , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinas Combinadas/genéticaRESUMO
By using the guanidine-isothiocyanate test, the authors isolated a summary RNA preparation from Ixodes persulcatus salivary gland extracts. Activity products of the genes responsible for the expression of some salivary proteins were first identified using the RT-PCR. It has been shown that, firstly, I. persulcatus synthesizes at least 3 transcripts homologous to the respective salivary components of the related species I. scapularis, the translation product of which is likely to be immunodominant antigens; secondly, the number of each of these transcripts, as in I. scapularis, depends on the stage of tick feeding. The changes in the expression of each transcript are specific: monotonously increasing changes in Salp 17 and cyclic ones in Salp 16, and synthesis, only when the ticks are fully ingested, in Salp 25.
Assuntos
Comportamento Alimentar , Ixodes/genética , Glândulas Salivares/metabolismo , Proteínas e Peptídeos Salivares/genética , Animais , Feminino , Regulação da Expressão Gênica , Ixodes/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The recombinant plasmids have been constructed encoding the synthesis of a full-sized diphtheria toxin from its own or PR, PL-promoters of bacteriophage lambda in Escherichia coli cells. The high level constitutive synthesis of toxin results in slow cell growth and plasmid elimination. The toxin was mainly detected in the periplasm, partially in the membrane and to a less extent in the cytoplasm and culturing medium. The dimeric form of toxin was found in the cytoplasm. Participation of toxin B-subunit in secreting of the toxin into culturing medium is discussed. Proteolytic degradation of the synthesized toxin in different Escherichia coli strains was demonstrated. The process takes place in cytoplasm and periplasm mainly. The main enzyme participating in the process is a La-protease. The data on proteolysis obtained by immunoprecipitation immunoblotting, affinity chromatography and in mini-cells of Escherichia coli are summarized.