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Aryl hydrocarbon receptor(Ah R), a ligand-dependent nuclear receptor, is involved in a diverse spectrum of biological and toxicological effects. Due to the lack of three dimensional(3D)crystal or nuclear magnetic resonance structure, the mechanisms of these complex effects of AhR remain to be unclear. Also, commercial monoclonal antibodies(mA bs) against human AhR protein(h Ah R), as alternative immunological tools, are very limited. Thus, in order to provide more tools for further studies on h Ah R, we prepared two m Abs(1D6 and 4A6) against h Ah R. The two newly generated m Abs specifically bound to amino acids 484–508(located in transcription activation domain) and amino acids 201–215(located in Per-ARNT-Sim domain)of h Ah R, respectively. These epitopes were new as compared with those of commercial m Abs.The m Abs were also characterized by enzyme-linked immunosorbent assay, western blot,immunoprecipitation and indirect immunofluorescence assay in different cell lines. The results showed that the two m Abs could recognize the linearized AhR s in six different human cell lines and a rat hepatoma cell line, as well as the h Ah R with native conformations. We concluded that the newly generated m Abs could be employed in AhR-based bioassays for analysis of environmental contaminants, and held great potential for further revealing the spatial structure of AhR and its biological functions in future studies.
Aryl hydrocarbon receptor (Ah R), a ligand-dependent nuclear receptor, is involved in a diverse spectrum of biological and toxicological effects. Due to the lack of three dimensional (3D) crystal or nuclear magnetic resonance structure, the mechanisms of these complex effects of AhR remain to be unclear. Also, monoclonal antibodies (mA bs) against human AhR protein (h Ah R), as alternative immunological tools, are very limited. , we prepared two m Abs (1D6 and 4A6) against h Ah R. The two newly generated m Abs specifically bound to amino acids 484-508 (located in transcription activation domain) and amino acids 201-215 (located in Per-ARNT- Sim domain) of h Ah R, respectively. These epitopes were new as compared with those of commercial m Abs. The m Abs were also characterized by enzyme-linked immunosorbent assay, western blot, immunoprecipitation and indirect immunofluorescence assay in different cell lines. resu lts showed that the two m Abs could recognize the linearized AhR s in six different human cell lines and a rat hepatoma cell line, as well as the h Ah R with native conformations. We concluded that the newly generated m Abs could be employed in AhR -based bioassays for analysis of environmental contaminants, and held great potential for further revealing the spatial structure of AhR and its biological functions in future studies.