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

Brazilian Journal of Medical and Biological Research

D.v. Nascimento²  E.m.b. Lemes²  J.l.s. Queiroz²  J.g. Silva Jr.²  H.j. Nascimento²  E.d. Silva²  R. Hirata Jr.¹  A.a.s.o. Dias¹  C.s. Santos¹  G.m.b. Pereira¹  A.l. Mattos-guaraldi¹  G.r.g. Armoa¹ 

[1] ,Fundação Oswaldo Cruz Instituto de Tecnologia em Imunobiológicos

关键词: Fragment B;    Diphtheria toxin;    Diphtheria;    dtb gene;    E. coli gene expression;    Immobilized metal affinity;   

DOI  :  10.1590/S0100-879X2010007500032

来源: SciELO

【 摘 要 】

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.

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