Molecular analysis of the M protein of Streptococcus equi and cloning and expression of the M protein gene in Escherichia coli.
A Streptococcus equi gene bank was constructed in the bacteriophage lambda gt11 cloning vector, and hybrid phage plaques were screened with S. equi M protein antiserum. A hybrid phage expressing the S. equi M protein (lambda gt11/SEM7) was identified and lysogenized into Escherichia coli Y1089. The cloned M protein appeared in immunoblots as three polypeptides with relative molecular weights of 58,000, 53,000, and 50,000. When reacted with S. equi M protein antiserum in an agar double-diffusion assay, the cloned M protein formed a line of identity with a protein in an acid extract of S. equi. Furthermore, lambda gt11/SEM7 protein inhibited opsonization of S. equi by antiserum to S. equi M protein. In addition, the recombinant protein expressed determinants of the antigen in the immune complexes of purpura hemorrhagica. Native M protein obtained from S. equi and recombinant M protein showed very similar molecular weight distributions on immunoblots, appearing as multiple closely spaced bands with molecular weights ranging from 52,000 to 60,000. Antisera prepared separately against each of the acid-extracted polypeptides shown to be important in serum bactericidal responses (molecular weight, 29,000) and nasopharyngeal local antibody responses (molecular weights, 41,000 and 46,000) of the horse each reacted with all three polypeptides in an acid extract. Moreover, antisera against protoplasts and against recombinant M protein of S. equi also reacted with these polypeptides. These results suggest that the entire M protein molecule of S. equi is present in these preparations and that the fragments in acid extracts carry overlapping segments.
Galán
J E
JE
Department of Veterinary Microbiology, New York State College of Veterinary Medicine, Cornell University, Ithaca 14853.
Timoney
J F
JF
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics*; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D002352:Carrier Proteins; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D006734:Horse Diseases / Q000276:immunology; D006736:Horses / Q000382:microbiology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D011693:Purpura / Q000276:immunology / Q000662:veterinary; D011994:Recombinant Proteins / Q000276:immunology; D013291:Streptococcus / Q000235:genetics* / Q000276:immunology
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D010446:Peptide Fragments; D011994:Recombinant Proteins; C014689:streptococcal M protein