ABSTRACT
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.
Subject(s)
Corynebacterium diphtheriae/genetics , Diphtheria Toxin/genetics , Gene Expression Regulation, Bacterial/genetics , Animals , Corynebacterium diphtheriae/classification , DNA, Bacterial , Male , Mice , Polymerase Chain Reaction , Rabbits , Sequence Analysis, DNAABSTRACT
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.
Subject(s)
Animals , Male , Mice , Rabbits , Corynebacterium diphtheriae/genetics , Diphtheria Toxin/genetics , Gene Expression Regulation, Bacterial/genetics , Corynebacterium diphtheriae/classification , DNA, Bacterial , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
The dengue virus (DV) causes one of the most important arthropod-borne human viral diseases throughout the tropical and subtropical countries. However, the morbidity and mortality of DV infections could be reduced with an early hospitalization care and a rapid risk identification of developing the dengue haemorrhagic fever (DHF). The nonstructural glycoprotein 1 (NS1) has been pointed as a reagent for immune-assay diagnostic test optimization. To evaluate this potential, recombinant DV2-NS1 proteins (rNS1) were produced from Escherichia coli (NS1EC) and insect cells (NS1IC) expression. The tests were performed by analysis of a human serum panel reacted against different rNS1 forms. The results demonstrated high correspondence between the DV positive sera and the assay results using native or refolded forms of either NS1IC or NS1EC. Also, the IgG and IgM anti-rNS1 level profiles showed distinct distribution, depending on protein form and disease status. However, the IgM anti-rNS1 reactions did not show sensibility to detect the DV in primary infections. The data obtained from the paired serum samples reactivity comparison suggested a heterogeneous human immune response and absence of correspondence between the IgG and IgM profile levels. Moreover, a patient with negative reference test could be detected by specific IgG anti-rNS1 assays presented here. Therefore, these results sustain the usefulness of dengue nonstructural proteins, in particular the NS1, in diagnostic tests as a complementary reagent.
