The expression and function of a Bacteroides gingivalis hemagglutinin gene in Escherichia coli.
Eight Escherichia coli JM 109 transformants generated from a clone bank of Bacteroides gingivalis 381 genomic DNA, were found to express B. gingivalis antigens. Quantitation of antigen expression by ELISA indicated that isopropyl-beta-D-thiogalactopyranoside (IPTG) was not necessary for antigen expression for any of the clones but that expression in 2 of the clones, ST 2 and ST 3, was increased in cells grown in the presence of IPTG. Western blot analysis revealed that the expressed protein of clone ST 2 has a molecular weight of 125,000 Dal. and that clone ST 3 contains multiple bands of 30 to 50 kdal which react with the anti-B. gingivalis antiserum. Three of the transformants were found to agglutinate sheep erythrocytes. Polyclonal monospecific antiserum to one of the transformants, clone ST 2, was found to react to 2 major bands of MWs 43,000 and 38,000 and minor bands of 115,000, 105,000, 32,000, and 30,000 Dal. present in B. gingivalis cell lysate preparations. Adsorption of anti B. gingivalis antiserum with cells of clone ST 2 reduced the hemagglutination inhibition activity of the antiserum 4-fold whereas antiserum to the clone itself inhibited B. gingivalis hemagglutination at a titer of 8 times that of normal rabbit serum. Immunoelectronmicroscopic studies using the antiserum to clone ST 2 indicate that the product of the cloned gene (hemagglutinin) is located on the B. gingivalis cell surface. A restriction map generated of the cloned B. gingivalis DNA fragment confirms the insert to be 3.2 kbases and indicates the possibility of a repeated sequence in the fragment.
Progulske-Fox
A
A
Tumwasorn
S
S
Holt
S C
SC
D001439:Bacteroides / Q000235:genetics*; D002999:Clone Cells; D004926:Escherichia coli; D006388:Hemagglutinins / Q000235:genetics*
D006388:Hemagglutinins