K Balakrishna1, H S Murali, H V Batra. 1. Division of Microbiology, Defence Food Research Laboratory, Mysore, Karnataka 570011, India. kondurubk@gmail.com
Abstract
AIMS: Rapid detection of pathogenic Yersinia enterocolitica isolates by using antisera raised against recombinant attachment-invasion locus (Ail) protein. METHODS AND RESULTS: The complete gene (471 bp) encoding for the Ail protein was amplified by PCR and cloned in pQE 30 UA vector. The recombinant clones were selected by polymerase chain reaction (PCR). Recombinant protein was expressed using induction with 1 mmol l(-1) final concentration of isopropylthiogalactoside (IPTG). Polyclonal antibodies were raised in mice against this purified recombinant protein. An indirect plate ELISA was standardized based on rAil protein for the detection of Y. enterocolitica. Western blot analysis with the sera raised against recombinant Ail protein exhibited reaction at 17 kDa region of the native Ail protein present in pathogenic Y. enterocolitica standard strains and strains isolated from pork samples suggesting that the antigenicity of recombinant Ail protein was similar to that of native Ail protein. Nonpathogenic Y. enterocolitica and the other species of Yersinia, namely, Y. pseudotuberculosis, Y. intermedia, Y. kristenseni, Y. fredrickseni and also the Enterobacteriaceae organisms tested were not found reacting to polyclonal antisera against this recombinant Ail protein. CONCLUSION: The antibodies raised against recombinant Ail protein could specifically identify pathogenic Y. enterocolitica strains both by indirect plate ELISA and Western blot immunoassay. SIGNIFICANCE AND IMPACT OF THE STUDY: The method developed in this study may find application in the detection of pathogenic Y. enterocolitica not only from food and environmental samples but also from clinical samples.
AIMS: Rapid detection of pathogenic Yersinia enterocolitica isolates by using antisera raised against recombinant attachment-invasion locus (Ail) protein. METHODS AND RESULTS: The complete gene (471 bp) encoding for the Ail protein was amplified by PCR and cloned in pQE 30 UA vector. The recombinant clones were selected by polymerase chain reaction (PCR). Recombinant protein was expressed using induction with 1 mmol l(-1) final concentration of isopropylthiogalactoside (IPTG). Polyclonal antibodies were raised in mice against this purified recombinant protein. An indirect plate ELISA was standardized based on rAil protein for the detection of Y. enterocolitica. Western blot analysis with the sera raised against recombinant Ail protein exhibited reaction at 17 kDa region of the native Ail protein present in pathogenic Y. enterocolitica standard strains and strains isolated from pork samples suggesting that the antigenicity of recombinant Ail protein was similar to that of native Ail protein. Nonpathogenic Y. enterocolitica and the other species of Yersinia, namely, Y. pseudotuberculosis, Y. intermedia, Y. kristenseni, Y. fredrickseni and also the Enterobacteriaceae organisms tested were not found reacting to polyclonal antisera against this recombinant Ail protein. CONCLUSION: The antibodies raised against recombinant Ail protein could specifically identify pathogenic Y. enterocolitica strains both by indirect plate ELISA and Western blot immunoassay. SIGNIFICANCE AND IMPACT OF THE STUDY: The method developed in this study may find application in the detection of pathogenic Y. enterocolitica not only from food and environmental samples but also from clinical samples.