RESUMO
We have constructed an aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica, harbouring mutations in aroA and trpE to investigate the use of such a strain as a live-attenuated vaccine. B. bronchiseptica aroA trpE was unable to grow in minimal medium without aromatic supplementation. Compared to the parental wild-type strain, the mutant displayed significantly reduced abilities to invade and survive within the mouse macrophage-like cell line J774A.1 in vitro and in the murine respiratory tract following experimental intranasal infection. Mice vaccinated with B. bronchiseptica aroA trpE displayed significant dose-dependent increases in B. bronchiseptica-specific antibody responses, and exhibited increases in the number of B. bronchiseptica-reactive spleen cells in lymphoproliferation assays. Immunised animals were protected against lung colonisation after challenge with the wild-type parental strain. With such a broad host range displayed by B. bronchiseptica, the attenuated strain constructed in this study may not only be used for the prevention of B. bronchiseptica-associated disease, but also for the potential delivery of heterologous antigen.
Assuntos
Aminoácidos Aromáticos/metabolismo , Vacinas Bacterianas/imunologia , Infecções por Bordetella/prevenção & controle , Bordetella bronchiseptica/imunologia , Mutação , Vacinas Atenuadas/imunologia , 3-Fosfoshikimato 1-Carboxiviniltransferase , Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/imunologia , Animais , Antranilato Sintase/química , Antranilato Sintase/genética , Antranilato Sintase/imunologia , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Infecções por Bordetella/microbiologia , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/crescimento & desenvolvimento , Bordetella bronchiseptica/patogenicidade , Linhagem Celular , Modelos Animais de Doenças , Feminino , Ativação Linfocitária , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Óperon , Análise de Sequência de DNA , Vacinação , Vacinas Atenuadas/administração & dosagemRESUMO
The trpE (or anthranilate synthetase) gene product has been used extensively as a fusion protein for the expression of a myriad of biologically active proteins. A trpE construct can be produced in high yield, is relatively resistant to proteolysis, and separates from the bulk of E. coli proteins because of its insolubility. We have isolated and characterized a monoclonal antibody against the TrpE protein for use as a detection and immunoaffinity reagent. The MAb, TRP 7.4, is highly specific for the TrpE protein and has a relative affinity of 1.0 ng. The antibody can also be used to detect TrpE constructs on Western blots. In addition, TRP 7.4 has been used to purify a TrpE-IL-6 fusion protein. These studies show the utility of this MAb as a tool for both research and protein purification.
Assuntos
Antranilato Sintase/isolamento & purificação , Anticorpos Antibacterianos/isolamento & purificação , Anticorpos Monoclonais/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Interleucina-6/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Antranilato Sintase/imunologia , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Proteínas de Bactérias/imunologia , Western Blotting , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/imunologia , Técnicas de Imunoadsorção , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Proteínas Recombinantes de Fusão/imunologia , SolubilidadeAssuntos
Antranilato Sintase/imunologia , Proteínas de Bactérias/imunologia , Escherichia coli/enzimologia , Glutamato Sintase/imunologia , Transaminases/imunologia , Antranilato Sintase/isolamento & purificação , Anticorpos , Proteínas de Bactérias/isolamento & purificação , Testes de Fixação de Complemento , Reações Cruzadas , Glutamato Sintase/isolamento & purificação , Testes de Precipitina , RadioimunoensaioAssuntos
Antranilato Fosforribosiltransferase/metabolismo , Antranilato Sintase/metabolismo , Escherichia coli/enzimologia , Pentosiltransferases/metabolismo , Antranilato Fosforribosiltransferase/imunologia , Antranilato Fosforribosiltransferase/isolamento & purificação , Antranilato Sintase/imunologia , Antranilato Sintase/isolamento & purificação , Especificidade de Anticorpos , Fenômenos Químicos , Química , Cloraminas , Testes de Fixação de Complemento , Glutamina , Imunodifusão , Lactoperoxidase , Testes de Precipitina , TransferasesRESUMO
An immunological study of anthranilate synthetase (ASase) has been initiated using quantitative precipitation, enzyme neutralization, and immunodiffusion methods. Cross-reactivity of anthranilate synthetase-anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase (ASase-PRTase) from Escherichia coli, Klebsiella aerogenes, and Salmonella typhimurium and ASase from Serratia marcescens and Pseudomonas putida was detected with antibodies to ?E. coli trypsin-treated ASase. Cross-reactivity of antigens was also obtained with S. marcescens anti-ASase. Indices of dissimilarity verified the overall structural similarity of ASase-PRTase from E. coli, K. aerogenes, and S. typhimurium and the divergence from S. marcescens ASase. Further divergence of these enzymes from ASase in B. subtilis and P. putida was apparent. Precipitation of ASase components I and II (ASase CoI and ASase CoII) was obtained using anti-ASase or antiserum fractionated to contain component-specific antibodies. Anti-ASase inhibited enzyme activity to binding to determinants on both subunits. Anti-ASase CoI inhibited the ammonia-dependent reaction and interfered with amide transfer from glutaminyl-ASase CoII. Anti-ASase CoII inhibited the glutamine reaction by blocking amide transfer. Enzyme neutralization experiments indicate more conservation of determinants at the active site region of ASase CoII compared to ASase CoI in the enterobacteria. A particulate form of ASase-PRTase in E. coli, K. aerogenes, and S. typhimurium could be distinguished by quantitative precipitation and immunodiffusion.