Vector development for the expression of foreign proteins in the vaccine strain Brucella abortus S19

Abstract

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae.

FIG. 1

Diagrammatic representation of plasmid pBEV-REP. The thin line represents pBBR4MCS sequences. The unshaded box represents the cloned B. abortus S19 fragment containing the promoter (Prom), regulatory sequences, and signal peptide (SP) of the bcsp31 gene. The nucleotide and peptide sequences of the first 31 amino acids and the linker sequences are indicated. The shaded box represents the repetitive T. cruzi reporter protein. The consensus sequence of the repeat is indicated.

FIG. 2

Expression of the reporter protein in B. abortus S19. Western blot analysis was done with a rabbit serum antirepeat protein. Lanes: 1, B. abortus S19(pBEV-REP) periplasmic content; 2, B. abortus S19(pBEV-REP) protoplastic fraction; 3, B. abortus S19(pBEV-REP) whole-cell extract; 4, B. abortus S19(pBEV) whole-cell extract; 5, E. coli S17.1(pBEV-REP) whole-cell extract. Standard molecular mass (MW) marker positions are indicated.

FIG. 3

Western blot of purified GST-REPEATS with pooled sera from two mice. (A) Sera from mice inoculated with B. abortus S19 (control) at 10 and 30 days postinfection. (B) Sera from mice inoculated with recombinant B. abortus S19(pBEV-REP) at 10, 18, 23, and 30 days postinfection. Prestained molecular mass (MW) marker positions are indicated.

FIG. 4

KELA results for pooled sera from mice inoculated with either B. abortus S19(pBEV-REP) or B. abortus S-19 (control) collected at 10, 18, 23, and 30 days postinfection. In all cases, the sera were tested against B. abortus LPS and the T. cruzi repetitive protein. The mean slope values (10³) are shown; error bars indicate standard deviations.