Molecular cloning and expression of antigens from Actinobacillus actinomycetemcomitans in Escherichia coli

Abstract

In order to study the role of membrane proteins in mucosal colonization by Actinobacillus actinomycetemcomitans, a plasmid library was constructed by ligating EcoRI-digested genomic DNA from A. actinomycetemcomitans strain Y4 into EcoRI-digested and dephosphorylated pUC13 DNA. A second library was constructed by ligating Sau3A-digested and size-fractionated A. actinomycetemcomitans strain Y4 DNA into BamHI-cleaved and dephosphorylated pUC13 DNA. The DNA was transformed into Escherichia coli strain MC1022 and plated onto Luria-Bertani agar containing 100 μg/ml ampicillin and X-gal. Recombinant colonies were examined for the expression of A. actinomycetemcomitans antigens by colony immunoscreening using rabbit antiserum to strain Y4. Nine positive clones were isolated. Three of these clones produced proteins that reacted with rabbit antiserum to strain Y4 on a Western transfer, and seven of these clones reacted with rabbit antiserum to strain Y4 as measured by indirect immunofluorescence. One reactive clone contained an 8.4 kb plasmid, which directed the synthesis of a peptide with an M r of 20,000 as measured by SDS-PAGE. Partial DNA sequence analysis revealed the presence of a signal-peptide leader sequence at the amino terminus suggesting that the native protein is a membrane component of A. actinomycetemcomitans. Thus A. actinomycetemcomitans antigens can be expressed in E. coli, and some of these proteins may be expressed on the E. coli cell surface.