Preparation and identification of mouse polyclonal antibody against human Nanog / 解放军医学杂志

ABSTRACT

Objective To prepare mouse polyclonal antibody against human Nanog by genetic immunization and to identify this antibody by Western blot and immunofluorescence. Method The antigenicity fragment (A16-V101) of human Nanog (hNanog) was chosen by analysis of Accelrys software, and its cDNA (258bp) was amplified from plasmid containing full-length cDNA of hNanog, then it was cloned into pBQAP-TT to construct recombinant plasmid pBQAP-TT-hNanog for genetic immunization. Mice were immunized with this recombinant plasmid and two other adjuvant plasmids-pCMVi-GMCSF and pCMVi-FIT3L, which help to enhance the antibody's generation. After 12 weeks, we obtained mouse anti-hNanog antibody from mice blood serum. The antibody titer was determined by enzyme-linked immunoadsordent assay (ELISA), and its specificity was identified by Western blot in human renal protein. Using this antibody, we detected hNanog expression in HKC cells of hNanog-AAV2 transfection. Results Recombinant plasmid pBQAP-TT-hNanog for genetic immunization was confirmed to be correct by restriction digestion and sequencing. The result of ELISA showed that the antibody titer was 1∶3 200. This antibody recognized a band of 34kD hNanog protein in human renal protein by Western blot. Immunofluorescence showed that Nanog protein was mainly located in the nuclei in hNanog transgene HKC cells. Conclusion Genetic immunization can offer mouse anti-hNanog polyclonal antibody of high titer and high specificity.