| Abstract: | Chromosomal DNA from Streptococcus sanguis FW213 was partially digested with EcoRI and ligated into the positive-selection cloning vector pOP203(A2+). The ligation mixture was used to transform Escherichia coli K-12, and 4,500 transformants were examined. The tetracycline-resistant colonies had inserts averaging 3.2 kilobases. The entire colony bank was screened by colony immunoassay with polyclonal rabbit serum raised against S. sanguis FW213 whole cells. Thirty recombinant colonies produced stable positive reactions of various intensities, indicating that S. sanguis antigens could be expressed in E. coli. Restriction endonuclease digestion of these clones suggested that 26 of the clones were unique. Only two clones, VT616 and VT618, gave positive reactions with fimbria-specific antisera. That the gene coding for the antigen was located on the plasmid was confirmed by demonstrating that the presence of the plasmid was linked to antigen production. Western immunoblot analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that both clones produced a fimbrial peptide of Mr 30,000. The two recombinant plasmids were shown by Southern analysis and restriction mapping to contain the same 6-kilobase EcoRI fragment inserted in opposite orientations. Southern hybridization confirmed that this fragment is present in S. sanguis genomic DNA. The Mr 30,000 protein gene was expressed in both orientations, suggesting that the fimbrial promoter is located on the 6-kilobase fragment. These results show that at least one streptococcal fimbrial gene can be cloned and expressed in E. coli. |
