title: Recombinant antigen production for assays of intradermoreaction for diagnosis and surveillance of tuberculosis.
abstract: The goal of the present work was to develop reagents with potential for tuberculosis diagnosis. Genetic sequences of Mycobacterium tuberculosis secretion antigens were amplified by PCR, cloned into the Gateway(®) system, and expressed in Escherichia coli. The recombinant M. tuberculosis proteins were purified by metal affinity chromatography and preparative gel SDS-PAGE electrophoresis followed by electroelution and removal of endotoxins using Triton X-114. In total, seven recombinant proteins were obtained (ESAT-6, CFP10, TB10.3, TB10.4, MTSP11, MPT70, and MPT83). Delayed hypersensitivity reactions (DHR) was evaluated in Cavia porcellus and compared to the response using a standard purified protein derivative (PPD). All seven recombinant proteins produced a positive induration reaction in an intradermal test in guinea pigs previously sensitized with M. tuberculosis. When applied together, at a concentration of each recombinant protein 0.04 mg/mL, the intradermoreaction in C. porcellus was significantly higher than that obtained by standard PPD (p-value=0.00386).
authors.affiliation: Laboratory of Forensic Molecular Genetics, Institute of Criminology, 80010-100 Curitiba, Brazil.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Malaghini
authors.forename: Vanete
authors.initials: V
authors.lastname: Thomaz-Soccol
authors.forename: Christian Macagnan
authors.initials: CM
authors.lastname: Probst
authors.forename: Marco Aurelio
authors.initials: MA
authors.lastname: Krieger
authors.forename: Henrique
authors.initials: H
authors.lastname: Preti
authors.forename: Afranio
authors.initials: A
authors.lastname: Kritski
authors.forename: Carlos Ricardo
authors.initials: CR
authors.lastname: Soccol
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006168:Guinea Pigs; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012882:Skin Tests / Q000379:methods*; D014373:Tuberculin; D014376:Tuberculosis / Q000175:diagnosis* / Q000276:immunology
chemical_list: D000942:Antigens, Bacterial; D011994:Recombinant Proteins; D014373:Tuberculin; C072681:Mycobacterium tuberculosis antigens