Cloning and expression of the porcine attaching and effacing-associated (paa) gene of enteropathogenic Escherichia coli.
Porcine enteropathogenic Escherichia coli (PEPEC) produce an outer membrane protein (intimin) called Paa (porcine attaching and effacing-associated), which is involved in the pathogenesis of E. coli in piglets with diarrhea. The paa gene of a PEPEC strain isolated in Paraná, Brazil, was amplified by polymerase chain reaction, sequenced, and cloned into the pTrcHisTOPO2 vector. The deduced amino acid sequence encoded by the paa gene of PEPEC from Paraná, Brazil, showed 99% homology to the sequences from other PEPEC strains. In this study, the overexpression of recombinant Paa (rPaa) using alternative induction strategies was attempted. The auto-induction protocol showed excellent results for rPaa protein production with 0.4% (w/v) lactose. The rPaa protein is insoluble and was purified with Triton X-100 wash as a total antigen. This method produced a relatively high yield of rPaa. rPaa was recognized by serum from pigs immunized with the PEPEC strain. These results suggest that rPaa could be included in the development of a vaccine against swine colibacillosis.
Programa de Pós Graduação em Ciência Animal, Universidade Estadual de Londrina, Londrina, PR, Brasil.
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DA
Pereira
Laboratório de Protozoologia Veterinária, Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina, Londrina, PR, Brasil.
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EC
Teixeira Florian
Laboratório de Imunologia Animal, Departamento de Ciências Patológicas, Universidade Estadual de Londrina, Londrina, PR, Brasil.
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MA
Ono
Laboratório de Protozoologia Veterinária, Departamento de Medicina Veterinária Preventiva, Universidade Estadual de Londrina, Londrina, PR, Brasil.
C Nachi
CN
Rossi
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Vidotto
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MC
Vidotto
D000818:Animals; D003001:Cloning, Molecular; D054308:Enteropathogenic Escherichia coli / Q000235:genetics* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000382:microbiology / Q000517:prevention & control / Q000662:veterinary*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D034421:Sus scrofa / Q000382:microbiology; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology / Q000517:prevention & control; D015533:Transcriptional Activation
D029968:Escherichia coli Proteins; C520614:Paa protein, E coli