Identification and characterization of a hemagglutinin gene from Prevotella intermedia
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In this study, we describe the cloning of a gene encoding hemagglutinin from Prevotella intermedia to characterize hemagglutinin function and elucidate its possible role in the infectious process mediated by the organism. We screened genomic libraries created in Escherichia coli JM109, using antibodies against P . intermedia , and the positive clones were subsequently tested for their hemagglutinating activity, using rabbit red blood cells. We isolated a clone of strain 0543 in which, a 3.6-kb P. intermedia chromosomal DNA fragment was inserted into the pUC18 plasmid. Our deletion analysis-based subcloning procedures identified a hemagglutinin gene—designated phg. The open-reading frame of phg encoded a 35-kDa protein, and the expression of a recombinant protein of the predicted size was observed in a fusion protein expression system. While the DNA analysis of phg revealed no homology to known DNA sequences, the putative promoter, ribosome binding, and transcriptional termination sequence were found in the flanking DNA regions. In the N-terminus of the deduced amino acid sequence, we found a consensus leader peptide sequence for secretory proteins. These findings indicate that hemagglutinin might be expressed on the surface of P. intermedia to mediate its function.
Abstract
In this study, we describe the cloning of a gene encoding hemagglutinin from Prevotella intermedia to characterize hemagglutinin function and elucidate its possible role in the infectious process mediated by the organism. We screened genomic libraries created in Escherichia coli JM109, using antibodies against P . intermedia , and the positive clones were subsequently tested for their hemagglutinating activity, using rabbit red blood cells. We isolated a clone of strain 0543 in which, a 3.6-kb P. intermedia chromosomal DNA fragment was inserted into the pUC18 plasmid. Our deletion analysis-based subcloning procedures identified a hemagglutinin gene—designated phg. The open-reading frame of phg encoded a 35-kDa protein, and the expression of a recombinant protein of the predicted size was observed in a fusion protein expression system. While the DNA analysis of phg revealed no homology to known DNA sequences, the putative promoter, ribosome binding, and transcriptional termination sequence were found in the flanking DNA regions. In the N-terminus of the deduced amino acid sequence, we found a consensus leader peptide sequence for secretory proteins. These findings indicate that hemagglutinin might be expressed on the surface of P. intermedia to mediate its function.
참고문헌 (28)
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