Cloning, expression and purification of the Enterohemorrhagic Escherichia coli virulence factors Intimin, EspA and EspB for serodiagnosis and vaccine applications.

VILTE, DA; LARZÁBAL, M; ELIZONDO AM; CATALDI, AA; MERCADO, EC

Montevideo

Simposio; 7º Seminario de la OIE en Biotecnología, 12º Simposio Internacional de la Asociación Mundial de Laboratorios de Diagnóstico Veterinario.; 2005

Shiga toxin (Stx)-producing Escherichia coli which are capable of causing intestinal attaching and effacing (A/E) lesion are known as enterohemorrhagic E.coli (EHEC). This enteric pathogen is associated with hemorrhagic colitis, thrombotic thrombocytopenic purpura, and hemolytic-uremic syndrome, the leading cause of acute renal failure in children. Intimin, Esp (EPEC-secreted-protein) A and Esp B, some of the proteins responsible of the A/E lesion, are recognized by HUS patients antisera and have been proposed as candidate vaccines for humans and cattle. Taking into account that Argentina has a serious health hazard due to EHEC in its paediatric population, and being the cattle one of the recognized STEC reservoirs we now describe the production of C- terminal third intimin and Esp recombinant proteins using His6-tagged fusions. The pRSET-A expression vectors containing N-(His)6 tag was constructed using the PCR amplified sequences of EspA, EspB, and C-terminal third of gamma and theta variants of intimin from EHEC strains isolated from diarrhoeic calves. Plasmids were introduced in Escherichia coli BL21 (DE3) pLysS competent cells. His6-tagged proteins were induced by addition of 0.5mM IPTG and purified under denaturing conditions by Ni-NTA agarose affinity chromatography. Reference polyclonal antibodies raised against recombinant O157 EspA, EPEC EspB and O157 gamma intimin reacted with the respective purified products. The expression level achieved was between 5% and 34% of the total protein present in lysates. Recombinant protein yields varied from 0.7 to 1.7 mg per gram of wet weight cells. The purified proteins in combination with other immunodominant antigens may be used in detecting antibodies in sera of HUS patients and will be assayed as vaccines to prevent cattle intestinal colonization by EHEC.