Clonagem, sequenciamento, expressão e caracterização do gene tsh de uma cepa de Escherichia coli patogênica de aves / Cloning, sequencing, expression, and characterization of the tsh gene from an avian pathogenic Escherichia coli strain
Semina ciênc. agrar
; 27(2): 253-260, abr.-jun. 2006. ilus
Article
in Portuguese
| LILACS
| ID: lil-453094
Responsible library:
BR512.1
RESUMO
A hemaglutinina temperatura sensível (Tsh) pertence à família das serino-proteases autotransporte de Enterobacteriacea (SPATE), as quais são capazes de clivar diferentes substratos. Nós isolamos e caracterizamos o gene de Escherichia coli patogênica aviária (APEC) amostra APEC 13, sorotipo O2H9, clonado em pET 101. A região de 4.2 kb do DNA clonado codificou uma proteína de aproximadamente 140 kDa (r-Tsh). O plasmídio recombinante pET 101-tsh conferiu um fenótipo de hemaglutinação positivo para a linhagem BL21 (tsh) para eritrócitos de galinha. A proteína r-Tsh foi purificada em coluna de níquel e utilizada na produção de anticorpos anti-Tsh. Um fragmento de 1.6 kb foi amplificado e subclonado em pCR4, e a seqüência parcial mostrou alta homologia com outras seqüências analisadas. O anti-Tsh reagiu com as proteínas r-Tsh e Tsh nativa da amostra APEC13, como demonstrado pela técnica de Western blot, mostrando que a r-Tsh tem epitopos conservados e que sua antigenicidade foi preservada. O anti-Tsh também inibiu a atividade hemaglutinante das amostras APEC 13 e BL12/pET 101-tsh
ABSTRACT
The temperature-sensitive hemagglutinin (Tsh) belongs to a family of high-molecular-weight serineprotease autotransporters of Enterobacteriaceae (SPATEs), which can cleave different substrates. Weisolated and characterised the tsh gene from an avian pathogenic Escherichia coli (APEC) strain, APEC13serotype O2H9, which was cloned in pET101. The 4.2 kb region of cloned DNA coded one protein ofapproximately 140 kDa (r-Tsh). The recombinant plasmid pET101-tsh conferred to E. coli BL21 strain(tsh) the hemagglutination-positive phenotype against chicken erythrocytes. The r-Tsh was purified byNi-NTA column and used to produce antibody anti-Tsh. A 1.6 kb fragment of the tsh sequence was alsoamplified and cloned in pCR4, and a partial sequence showed high homology with other sequenceanalysed. The anti-Tsh reacted with the protein r-Tsh and native Tsh of APEC13, as demonstrated byWestern blot, showing that r-Tsh has conserved epitopes and that its antigenicity was preserved. Theanti-Tsh also inhibited the hemagglutinating activity of strains APEC13 and BL21/pET101-tsh
Full text:
Available
Collection:
International databases
Health context:
Neglected Diseases
Health problem:
Neglected Diseases
/
Zoonoses
Database:
LILACS
Main subject:
Virulence Factors
/
Escherichia coli
/
Hemagglutinins
Language:
Portuguese
Journal:
Semina ciênc. agrar
Journal subject:
Science
/
Environmental Health
Year:
2006
Document type:
Article
Affiliation country:
Brazil
Institution/Affiliation country:
Universidade Estadual de Londrina/BR