Genetic and antigenic characterisation of elongation factor Tu from Mycoplasma mycoides subsp. mycoides SC.
Specific serodiagnosis of contagious bovine pleuropneumonia (CBPP) is hampered by the low antibody titers against Mycoplasma mycoides subsp. mycoides small-colony type (MmmSC) antigens in calf serum due to persistent infections and by the existence of cross-reactions among the members of the mycoides cluster. In order to identify potential diagnostic antigens, we have constructed a genomic library from MmmSC which was screened with antibodies from naturally-infected animals. Using this strategy, a genome fragment has been isolated and characterised. The complete nucleotide sequence of this fragment revealed the presence of several open reading frames, including that of translation elongation factor Tu (EF-Tu), whose product was responsible of the positive reaction observed when expressed in E. coli. The organisation of this MmmSC genome region differed from that of other Mycoplasma species whose complete genome sequences are known, but was similar, by PCR amplification analysis of genomic DNA, to other members of the mycoides cluster, such as Mycoplasma capricolum subsp. capricolum (Mcc). Nevertheless, the MmmSC and Mcc amplicons could be distinguished by digestion with restriction enzymes AseI or HindIII, strategy that could be used as a tool for differential diagnosis of infections caused by members of the mycoides cluster. The full recombinant EF-Tu was produced in E. coli, after correction of an unusual tryptophan codon by site-directed mutagenesis, and used to investigate anti-EF-Tu circulating antibodies in bovine sera.
Alonso
José M Martin
JM
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain.
Prieto
Miguel
M
Parra
Francisco
F
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis* / Q000382:microbiology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015723:Gene Library; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009175:Mycoplasma Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D009176:Mycoplasma mycoides / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D010444:Peptide Elongation Factor Tu / Q000235:genetics* / Q000276:immunology; D011002:Pleuropneumonia, Contagious / Q000175:diagnosis* / Q000382:microbiology; D016133:Polymerase Chain Reaction
D000942:Antigens, Bacterial; D004269:DNA, Bacterial; D010444:Peptide Elongation Factor Tu