Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum

文献类型： 外文期刊

第一作者： He, Suibin

作者： He, Suibin;Qi, Jingjing;Yu, Shengqing;Tan, Lei;Bao, Shijun;Qiu, Xvsheng;Wang, Xiaolan;Ding, Chan;He, Suibin;Yin, Yuncong;Fei, Rongmei;Ding, Chan

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关键词： Mycoplasma gallisepticum;Pyruvate kinase;Adhesion;Immunological characteristics

期刊名称：MICROBIAL PATHOGENESIS （ 影响因子：3.738； 五年影响因子：3.663 ）

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收录情况： SCI

摘要： The widespread avian pathogen Mycoplasma gallisepticum is a causative agent of respiratory disease. The wall-less prokaryotes lack some tricarboxylic acid cycle enzymes, therefore, the glycolysis metabolic pathway is of great importance to these organisms. Pyruvate kinase (PK) is one of the key enzymes of the glycolytic pathway, and its immunological characteristics in Mycoplasma are not well known. In this study, the M. gallisepticum pyruvate kinase fusion protein (PykF) was expressed in a pET system. The full-length of the gene was subcloned into the expression vector pET28a(+) to construct the pET28a-rMGPykF plasmid, which was then transformed into Escherichia coli strain BL21 (DE3) cells. The expression of the 62 kDa recombinant protein of rMGPykF in E. coli strain BL21 (DE3) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGPykF exhibited PK catalytic activity, which could reflect the conversion of NADH to NAD(+). Mouse anti-PykF antibodies were generated by immunization of mice with rMGPykF. Immunoblot and immunoelectron microscopy assays identified PykF as an immunogenic protein expressed on the surface of M. gallisepticum cells. Bactericidal assay showed that anti-rMGPykF antiserum killed 70.55% of M. gallisepticum cells, suggesting the protective potential of PykF. Adherence inhibition assay on immortalized chicken fibroblasts (DF-1) cells revealed more than 3931% inhibition of adhesion in the presence of anti-rMGPykF antiserum, suggesting that PykF of M. gallisepticum participates in bacterial adhesion to DF-1 cells. (C) 2015 Elsevier Ltd. All rights reserved.

分类号： R3

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