Cloning and surface expression in Escherichia coli of a structural gene encoding a surface protein of Haemophilus influenzae type b.
Recombinant DNA technology was used to clone a gene coding for a surface protein of Haemophilus influenzae type b (Hib) into Escherichia coli. Chromosomal DNA from a clinical isolate of Hib was cleaved with EcoRI and ligated into plasmid vectors containing three different translational reading frames. E. coli carrying recombinant plasmids were screened in a colony blot-radioimmunoassay system by using murine monoclonal antibodies (mabs) directed against cell surface-exposed proteins of Hib. mab 7B2, which is specific for a Hib surface protein with an apparent molecular weight of 27,000 (27K), reacted with several recombinant strains of E. coli. Restriction analysis revealed the presence of a 9.1-kilobase DNA insert in each of these recombinant plasmids and also determined that both transcription and translation of the Hib gene(s) coding for the 7B2-reactive antigen were not dependent on the lac operator and promoter of the vectors. Radioimmunoprecipitation and Western blot analyses showed that the antigenic determinant recognized by mab 7B2 in these recombinant E. coli was present in a 27K protein. In addition, this 27K protein was shown to be both localized on the surface of these E. coli cells and accessible to antibody.
D000954:Antigens, Surface / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics* / Q000276:immunology; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins
D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins