Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently availab...

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Published in:	Brazilian journal of medical and biological research Vol. 43; no. 5; pp. 460 - 466
Main Authors:	Nascimento, D V, Lemes, E M B, Queiroz, J L S, Silva, Jr, J G, Nascimento, H J, Silva, E D, Hirata, Jr, R, Dias, A A S O, Santos, C S, Pereira, G M B, Mattos-Guaraldi, A L, Armoa, G R G
Format:	Journal Article
Language:	English
Published:	Brazil Associação Brasileira de Divulgação Científica 01-05-2010
Subjects:	Animals BIOLOGY Corynebacterium diphtheriae - classification Corynebacterium diphtheriae - genetics Diphtheria Diphtheria toxin Diphtheria Toxin - genetics DNA, Bacterial dtb gene E. coli gene expression Fragment B Gene Expression Regulation, Bacterial - genetics Immobilized metal affinity Male MEDICINE, RESEARCH & EXPERIMENTAL Mice Polymerase Chain Reaction Rabbits Sequence Analysis, DNA E. coli gene expression Diphtheria toxin Diphtheria dtb gene Fragment B Immobilized metal affinity
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Summary:	The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15 cells using the expression vector pQE-30. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0100-879X 1414-431X 1414-431X 0100-879X
DOI:	10.1590/S0100-879X2010007500032