title: Construction of a cell-surface display system based on the N-terminal domain of ice nucleation protein and its application in identification of mycoplasma adhesion proteins.
abstract: OBJECTIVE
  To construct and demonstrate a surface display system that could be used to identify mycoplasma adhesion proteins.
  
  
  RESULTS
  Using the N-terminal domain of InaZ (InaZN) as the anchoring motif and the enhanced green fluorescent protein (EGFP) as the reporter, the surface display system pET-InaZN-EGFP was constructed. Then, the mgc2 gene which encodes an adhesin and the holB gene which encodes DNA polymerase III subunit delta' (nonadhesin, negative control) of Mycoplasma gallisepticum were cloned into the pET-InaZN-EGFP respectively. The fusion proteins were expressed in Escherichia coli BL21 (DE3). The distribution of the fusion proteins in E. coli cells was determined using SDS-PAGE followed by Western blotting, based on cell fractionation. Escherichia coli cell surface display of the fusion protein was confirmed by immunofluorescence microscopy. The results indicated that the fusion proteins were not only anchored to the outer membrane fraction but also were successfully displayed on the surface of E. coli cells. Adhesion analysis of E. coli harbouring InaZN-EGFP-mgc2 to host cells showed that the MGC2-positive E. coli cells can effectively adhere to the surfaces of DF-1 cells.
  
  
  CONCLUSIONS
  A surface display system using the InaZN as the anchoring motif and EGFP as the reporter was developed to identify putative adhesins of mycoplasma. Results indicated that adhesion by the cytadhesin-like protein MGC2 of mycoplasma can be reproduced using this surface display system.
  
  
  CONCLUSIONS
  This is the first construction of surface display system which could be used to identify the adhesion proteins of mycoplasma. The method developed in this study can even be used to select and identify the adhesion proteins of other pathogens.
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.|College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China.
authors: S
authors: S
authors: Bao
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
authors: S
authors: S
authors: Yu
authors: College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China.
authors: X
authors: X
authors: Guo
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
authors: F
authors: F
authors: Zhang
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
authors: Y
authors: Y
authors: Sun
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
authors: L
authors: L
authors: Tan
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
authors: Y
authors: Y
authors: Duan
authors: College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China.
authors: F
authors: F
authors: Lu
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
authors: X
authors: X
authors: Qiu
authors: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
authors: C
authors: C
authors: Ding
mesh_terms: D018829:Adhesins, Bacterial / Q000032:analysis* / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis* / Q000235:genetics; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D008856:Microscopy, Fluorescence; D009174:Mycoplasma / Q000235:genetics / Q000302:isolation & purification*; D017434:Protein Structure, Tertiary
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C063113:ice nucleation protein
keywords: Mycoplasma gallisepticum; adhesion protein; ice nucleation protein; identification; surface display