title: [Preparation and characterization of monoclonal antibodies specific for CjaA protein of Campylobacter jejuni].
abstract: OBJECTIVE
  Expression,  purification of Campylobacter jejuni CjaA protein and development of monoclonal antibodies (mAbs) against this protein.
  
  
  METHODS
  The C. jejuni cjaA gene was amplified and inserted into the expression plasmids,  pGEX-6p-1 and pET30a (+). The purified rGST-CjaA protein was used as an immunogen in 8-week-old BALB/c mice,  and injected subcutaneously. The purified rHis-CjaA protein used as a detecting antigen for screening mAbs against CjaA was prepared. The specificity of mAbs was characterized by Dot-ELISA and Western blot assays.
  
  
  RESULTS
  The recombinant expression plasmids,  pGEX-6p-1-cjaA and pET30a(+)-cjaA were obtained. The sizes of the recombinant proteins,  rGST-CjaA and rHis-CjaA,  were consistent with their predicted size. Specific reaction was found between CjaA positive serum and expressed protein by Western blot assay,  confirming its identification as a Campylobacter jejuni immunogen. Three hybridoma cell lines,  designated 2B6,  3C2 and 4F11,  secreting mAbs against CjaA were obtained. Their immunoglobulin subclasses were all IgG1. The ELISA titers of the ascites fluid were 1:1×10(5);,  1:2×10(5); and 1:4×10(5);,  respectively. Western blot analysis confirmed that the three mAbs reacted with the rHis-CjaA fusion protein but not the His tag. The Dot-ELISA results demonstrated that the three mAbs only with CjaA and not the tags for the expression vectors.
  
  
  CONCLUSIONS
  The successful preparation of three mAbs specific for the CjaA protein lays the foundation for further study regarding the biological characteristics of CjaA and the pathogenesis of C. jejuni.
authors.affiliation: Jiangsu Key Lab. of Zoonosis,  Yangzhou University,  Yangzhou 225009,  China.
authors.forename: Jin-lin
authors.initials: JL
authors.lastname: Huang
authors.forename: Yan-xin
authors.initials: YX
authors.lastname: Yin
authors.forename: Yuan-qing
authors.initials: YQ
authors.lastname: Hu
authors.forename: Gong
authors.initials: G
authors.lastname: Zhang
authors.forename: Xiu-fan
authors.initials: XF
authors.lastname: Liu
authors.forename: Xin-an
authors.initials: XA
authors.lastname: Jiao
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology*; D016123:Campylobacter jejuni / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology* / Q000302:isolation & purification*
chemical_list: D018528:ATP-Binding Cassette Transporters; D026921:Amino Acid Transport Systems, Neutral; D000911:Antibodies, Monoclonal; C503090:CjaA protein, Campylobacter jejuni; D011994:Recombinant Proteins