title: [Preparation and characterization of monoclonal antibodies against FlhF protein of Campylobacter jejuni].
abstract: OBJECTIVE
  To develop a prokaryotic expression system for Campylobacrer jejuni FlhF protein and prepare monoclonal antibodies(mAb) against this protein.
  
  
  METHODS
  The C. jejuni flhF gene was amplified and inserted into the expression plasmids, pET-30a(+) and pGEX-6p-1. Then the rHis-FlhF protein and rGST-FlhF protein were expressed and purified by affinity column chromatography. They were subsequently used as immunogen and detecting antigen to screen antibodies produced by hybridoma cells against these proteins. The titers of the mAbs were measured by indirect ELISA, and the specificities of the mAbs was evaluated by Dot-ELISA and Western blot analysis.
  
  
  RESULTS
  The recombinant expression plasmids pET-30a(+)-flhF and pGEX-6p-1-flhF were successfully constructed, and the fusion proteins rHis-FlhF and rGST-FlhF were produced at sufficient quantities. Two hybridoma cell lines were screened, designated 2C12 and 7A9, which secrete mAbs against FlhF stably. Their immunoglobulin subclasses were both IgG1. The titers of the ascites fluid were 1×10(9); and 2×10(7); respectively. Western blot analyses confirmed that the two mAbs both reacted with the rHis-FlhF fusion protein with good sensitivity. The Dot-ELISA results demonstrated that the two mAbs reacted specifically with Campylobacrer jejuni.
  
  
  CONCLUSIONS
  The mAbs against Campylobacrer jejuni FlhF protein with high specificity were successfully prepared.
authors: Jiangsu Key Laboratory of Zoonoses, Yangzhou University, Yangzhou 225009, China.
authors: Fangzhe
authors: F
authors: Ren
authors: Nan
authors: N
authors: Wang
authors: Yuwei
authors: Y
authors: Shang
authors: Xinan
authors: X
authors: Jiao
authors: Jinlin
authors: J
authors: Huang
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000276:immunology*; D016123:Campylobacter jejuni; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D020559:Monomeric GTP-Binding Proteins / Q000276:immunology*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D011994:Recombinant Proteins; C078317:flhF protein, Bacteria; D020559:Monomeric GTP-Binding Proteins