title: Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum.
abstract: 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 39.31% inhibition of adhesion in the presence of anti-rMGPykF antiserum, suggesting that PykF of M. gallisepticum participates in bacterial adhesion to DF-1 cells.
authors.affiliation: Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China; Key Laboratory of Animal Disease Diagnosis & Immunology, College of Veterinary Medicine, Nanjing Agricultural University, No. 1 Weigang, Nanjing 210095, PR China.
authors.forename: Suibin
authors.initials: S
authors.lastname: He
authors.affiliation: Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Qi
authors.affiliation: Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China.
authors.forename: Shengqing
authors.initials: S
authors.lastname: Yu
authors.affiliation: Key Laboratory of Animal Disease Diagnosis & Immunology, College of Veterinary Medicine, Nanjing Agricultural University, No. 1 Weigang, Nanjing 210095, PR China.
authors.forename: Yuncong
authors.initials: Y
authors.lastname: Yin
authors.affiliation: Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Tan
authors.affiliation: Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China.
authors.forename: Shijun
authors.initials: S
authors.lastname: Bao
authors.affiliation: Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China.
authors.forename: Xvsheng
authors.initials: X
authors.lastname: Qiu
authors.affiliation: Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Key Laboratory of Animal Disease Diagnosis & Immunology, College of Veterinary Medicine, Nanjing Agricultural University, No. 1 Weigang, Nanjing 210095, PR China. Electronic address: feirongmei@njau.edu.cn.
authors.forename: Rongmei
authors.initials: R
authors.lastname: Fei
authors.affiliation: Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, 88 South University Ave., Yangzhou 225009, PR China. Electronic address: shoveldeen@shvri.ac.cn.
authors.forename: Chan
authors.initials: C
authors.lastname: Ding
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001422:Bacterial Adhesion; D002460:Cell Line; D002645:Chickens; D004926:Escherichia coli / Q000235:genetics; D005347:Fibroblasts / Q000382:microbiology; D015870:Gene Expression; D005822:Genetic Vectors; D015151:Immunoblotting; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D051379:Mice; D050296:Microbial Viability; D016253:Microscopy, Immunoelectron; D045686:Mycoplasma gallisepticum / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D010957:Plasmids; D011770:Pyruvate Kinase / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
chemical_list: D000907:Antibodies, Bacterial; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011770:Pyruvate Kinase
keywords: Adhesion; Immunological characteristics; Mycoplasma gallisepticum; Pyruvate kinase