期刊
MICROBIAL PATHOGENESIS
卷 51, 期 4, 页码 285-290出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.micpath.2011.03.012
关键词
Mycoplasma gallisepticum; alpha-Enolase; Plasminogen; Adherence
资金
- National Natural Science Foundation of China [30871883, 31001077]
- Shanghai Agriculture & Technology Foundation [2007-11-2]
- National Non-profit Institute of China [2006JB01, 2008JB12]
The alpha-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 alpha-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 alpha-enolase catalytic activity that it could reflect the conversion of NADH to NAD(+). Mouse antiserum to alpha-enolase was generated by immunization with rMGEno. Immunoblotting and immunofluorescence assay with the antiserum identified alpha-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 alpha-enolase of M. gallisepticum participates in bacterial adhesion to DF-1 cells. (C) 2011 Elsevier Ltd. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据