Isolation of Brucella abortus ssb and uvrA genes from a genomic library by use of lymphocytes as probes

Abstract

BruceUa abortus proteins from virulent S2308 expressed from a pBluescript II SK-genomic library stimulated peripheral blood mononuclear (PBM) cell proliferation from cattle vaccinated with B. abortus S19. The method described here permits a rapid and directed approach to isolate genes encoding antigens of B. abortus that interact with lymphocytes primed to the living bacterium. The supernatants from the bacterial host JM109 (DE3) were cultured with freshly isolated bovine PBM cells. A total of 300 clones were evaluated. Ten clones were identified that stimulated T-lymphocyte proliferation. Among them, one clone with a 2.5-kb insert stimulated T-lymphocyte proliferation in all three animals, suggesting that the proteins encoded by genes within this fragment may represent immunodominant antigens. DNA sequencing of this clone reveals two large open reading frames (ORFs). ORF II has a high degree of similarity to the Escherichia coli ssb gene, which codes for the single-stranded DNA binding protein. ORF I, in the opposite direction to ORF II, shows similarity to the N terminus of the E. coli uvrA gene, which codes for one of the three subunits of the E. coli ABC excision nuclease. The observation that the PBM cells recognized and proliferated in response to proteins expressed from single clones provides a novel strategy to select bacterial antigens that may prove useful in designing alternative vaccines against brucellosis.B. abortus genomic library. The library of B. abortus S2308, a virulent strain of B. abortus, was constructed in the HindIII restriction site of the pBluescript II SK-phagemid. 5339 on July 16, 2020 by guest http://iai.asm.org/ Downloaded from

Lymphocyte proliferation in response to immunodominant antigens of Brucella abortus 2308 and RB51 in strain 2308-infected cattle

“…proteins rather than by the LPS 0 antigens. A previous study has also shown that blood lymphocytes from S19-vaccinated cattle exhibit proliferative responses to several recombinant S2308 fusion proteins which contain no S2308 LPS 0 antigens (17). In our study, each of the 22 fractions of S2308 and SRB51 did not contain a single protein but instead contained groups of proteins with similar molecular masses, and certainly only some proteins in each fraction stimulated lymphocyte proliferation.…”

Molecular characterization, occurrence, and immunogenicity in infected sheep and cattle of two minor outer membrane proteins of Brucella abortus

“…Three ATG codons are present in frame at nucleotide positions 94, 103, and 109. A putative Shine-Dalgarno sequence (GGAG) occurs 9 bp upstream from the third ATG; an identical ribosome-binding site has been reported to be present 10 bp 5Ј to the B. abortus ssb gene starting codon (46). If one considers the third ATG as the start codon, since its location downstream of the putative Shine-Dalgarno site is adequate, the omp10 ORF codes for a 126-amino-acid polypeptide with a calculated MM of 13,251 Da.…”

Characterization, occurrence, and molecular cloning of a 39-kilodalton Brucella abortus cytoplasmic protein immunodominant in cattle

“…In order to identify Brucella components that can stimulate lymphocytes from infected animals, one-and two-dimensional SDS-PAGE followed by cellular immunoblotting has been used (8-10); however, this strategy does not allow one to unambiguously exclude the interference of Brucella LPS chains present in the protein preparations. Another study used lymphocytes as probes to identify recombinant stimulatory Brucella proteins expressed by a genomic library (53). In this study, anion-exchange chromatography was used to purify a 39-kDa protein from brucellergen.…”