title: [Expression and purification of mucin 16 and preparation and  characterization of anti-mucin 16 monoclonal antibody].
abstract: OBJECTIVE
  To generate monoclonal antibodies (mAbs) against mucin 16 using purified recombinant protein of human mucin 16 N terminus with His tag (His-mucin 16N) as the antigen.
  
  
  METHODS
  Mucin 16 N terminus was cloned into a prokaryotic expression vector pET-32. His-mucin 16N was then expressed in E.coli and purified by the affinity chromotography. Cell fusion was performed after the BALB/c mice were immunized with the purified His-mucin 16N protein. We screened hybridoma cell strains producing mAbs against mucin 16. The specificity and titer of the antibodies were characterized with ELISA, Western blotting, immunofluorescent and immunohistochemical staining.
  
  
  RESULTS
  The recombinant protein of His-mucin 16N was expressed and purified. A few hybridoma cell strains which could secrete specific mAbs against mucin 16 were obtained, and one anti-mucin 16 mAb with good specificity and high titer was selected and purified. The isotype of this anti-mucin 16 mAb was determined as IgG1, which indicated that this anti-mucin 16 mAb could be used for ELISA, Western blotting, immunofluorescent and immunohistochemical staining. The endogenous expression of mucin 16 in various cancer cell lines or tissues was also examined with this anti-mucin 16 antibody by Western blotting and other immunoassays.
  
  
  CONCLUSIONS
  The recombinant protein of His-mucin 16N was expressed and purified successfully, with which we prepared anti-mucin 16 mAb with good specificity and high titer.
authors.affiliation: Hybridoma and Antibody Center, School of Life Sciences, Xiamen University, Xiamen, China. jamesyangyun1@126.com
authors.forename: Yun
authors.initials: Y
authors.lastname: Yang
authors.forename: Yu-ting
authors.initials: YT
authors.lastname: Ding
authors.forename: Hui-ting
authors.initials: HT
authors.lastname: Gong
authors.forename: Zhan-jiao
authors.initials: ZJ
authors.lastname: Yu
authors.forename: Xiao-tong
authors.initials: XT
authors.lastname: Li
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D018394:CA-125 Antigen / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D000911:Antibodies, Monoclonal; D018394:CA-125 Antigen; C451759:MUC16 protein, human; D008565:Membrane Proteins; D011994:Recombinant Proteins