Expression and purification of the 26 kDa periplasmic protein of Brucella abortus: a reagent for the diagnosis of bovine brucellosis.
Development of a single diagnostic test for brucellosis in animals is the top priority of present-day research in the field. There is currently a battery of serological tests relying mainly on the use of LPS (lipopolysaccharide) as an antigen, culminating in false positives due to serological cross-reactivity. Other problems include difficulties in antigen production and the associated biohazard risk. This has prompted the need to develop an alternative antigen to replace LPS. In the present study, we cloned and expressed a BP26 (26 kDa periplasmic protein) antigen gene (bp26) of Brucella abortus. The recombinant periplasmic protein [rBP26 (recombinant BP26)] was expressed to high levels in Escherichia coli and purified in a single step. The purified rBP26 was examined for its binding activity with antibodies in a serum derived from a rabbit immunized intramuscularly with whole-cell lysate of B. abortus, as well as with commercial Brucella antibody (Difco). The purified rBP26 was used to develop an in-house plate ELISA and was further tested with a panel of 75 bovine brucellosis sera samples characterized previously by conventional serological tests. The results of both were in excellent agreement. The results show that rBP26 has potential use in the diagnosis of brucellosis, both in the laboratory and in field-based conditions with high levels of sensitivity and specificity.
Division of Microbiology, Defence Research and Development Establishment, Jhansi Road, Gwalior, Madhya Pradesh, India.
Sanjay
S
Kumar
Urmil
U
Tuteja
Ashok
A
Kumar
Harsh Vardhan
HV
Batra
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D002003:Brucella abortus; D002007:Brucellosis, Bovine / Q000097:blood / Q000175:diagnosis*; D002417:Cattle; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000662:veterinary; D004926:Escherichia coli / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D033903:Periplasmic Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification*
D000907:Antibodies, Bacterial; C106040:BP26 protein, Brucella; D008565:Membrane Proteins; D033903:Periplasmic Proteins; D011994:Recombinant Proteins