title: [Preparation of monoclonal antibody against bovine phosphoenolpyruvate carboxykinase].
abstract: OBJECTIVE
  To prepare the monoclonal antibody against bovine phosphoenolpyruvate carboxykinase (PEPCK) and characterize its biological functions.
  
  
  METHODS
  The recombinant plasmid containing PEPCK gene was constructed and used to transfect Escherichia coli. After expression induction in E.coli, the recombinant protein PEPCK was purified and used to immunize BALB/c mice. After the spleen B cells in the immunized mice were fused with murine myeloma cells, the positive clones were identified and selected by indirect ELISA for titer determination. PEPCK mAb produced by the positive hybridoma cells was enriched and its biological functions were examined by Western blotting, immunohistochemistry and immunoprecipitation.
  
  
  RESULTS
  One hybridoma cell line steadily secreting PEPCK mAb was successfully generated, namely 3D36H. Western blotting, immunohistochemistry and immunoprecipitation showed that the PEPCK mAb was able to specifically bind to bovine PEPCK protein.
  
  
  CONCLUSIONS
  The bovine PEPCK mAb was prepared successfully and had a good ability and specificity.
authors.affiliation: Jiangsu Provincial Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
authors.forename: Meilei
authors.initials: M
authors.lastname: Sheng
authors.affiliation: Jiangsu Provincial Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Ministry of Health Key Laboratory of Antibody Technique, Nanjing Medical University, Nanjing 210029, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Ministry of Health Key Laboratory of Antibody Technique, Nanjing Medical University, Nanjing 210029, China.
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Ministry of Health Key Laboratory of Antibody Technique, Nanjing Medical University, Nanjing 210029, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Jiangsu Provincial Key Laboratory of Xenotransplantation, Nanjing Medical University, Nanjing 210029, China.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Dai
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood / Q000276:immunology*; D000918:Antibody Specificity; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D051379:Mice; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; C069767:phosphoenolpyruvate carboxylase kinase; D017346:Protein Serine-Threonine Kinases