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Microb Pathog. 2011 Oct;51(4):285-90. doi: 10.1016/j.micpath.2011.03.012. Epub 2011 Jun 1.

The Mycoplasma gallisepticum α-enolase is cell surface-exposed and mediates adherence by binding to chicken plasminogen.

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Shanghai Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, 518 Ziyue Road, Shanghai 200241, PR China.


The α-enolase protein is reported to be an adhesin in several pathogenic bacterial species, but its role in Mycoplasma gallisepticum is unknown. In this study, the M. gallisepticum α-enolase gene was adapted to heterologous expression in Escherichia coli by performing overlapping polymerase chain reaction with site-directed mutagenesis to introduce A960G and A1158G mutations in the nucleotide sequence. The full-length mutated gene was cloned into a pGEM-T Easy vector and subcloned into the expression vector pET32a(+) to construct the pET-rMGEno plasmid. The expression of rMGEno in E. coli strain DE3 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGEno exhibited α-enolase catalytic activity that it could reflect the conversion of NADH to NAD(+). Mouse antiserum to α-enolase was generated by immunization with rMGEno. Immunoblotting and immunofluorescence assay with the antiserum identified α-enolase on the surface of M. gallisepticum cells. Enzyme-linked immunosorbent assay characterized rMGEno as a chicken plasminogen binding protein. An adherence inhibition assay on immortalized chicken fibroblasts (DF-1) demonstrated more than 77% inhibition of adhesion in the presence of mouse antiserum, suggesting that α-enolase of M. gallisepticum participates in bacterial adhesion to DF-1 cells.

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