[Expression and purification of mucin 16 and preparation and characterization of anti-mucin 16 monoclonal antibody].

ABSTRACT

AIM: 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. CONCLUSION: 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.