Study On The Trace Dioxins Biossay Relate To The Aryl Hydrocarbon Receptor(ahr) System

Objective By the mechanism of the aryl hydrocarbon receptor (AhR) binding aryl hydrocarbon receptor translocator (ARNT) with the interaction of TCDD. This project is aiming at establishing one convenient, rapid and specially dioxins detection method.Methods (1) Amplified the genes encoding AhR and ARNT by RT-PCR using the total RNA purified from the liver of C57BL/6J mice as templates. The fidelity of PCR was determined by agarose electrophoresis and DNA sequence analysis. Ligated the DNA fragments purified from the agarose into expression vector pGEX-5X-1 to construct 5 recombinants. Then transfered the identified recombinant into the competent cells of E.coli BL21(DE3).To explore the condition of induction and the intracellular location of the prokaryotic expression system,and affinity purify the fusion proteins by Glutathione SepharoseTM 4B beads or blood coagulation factor Xa. (2) Prepared the polyclonal antiserum of AhR-PAS and AhR-C through immunizing the obtained fusion proteins to rabbits. (3) Prepared the hepatic cytosol of C57BL/6J mice, and then quantify the total proteins by Bradford to calculate the content of AhR. (4) Incubated AhR and GST-ARNT-PAS which molar ratio is 1:1 with different TCDD graded concentrations and precipitated the mixtures with Glutathione SepharoseTM 4B beads. Study the binding condition, binding degree and the dose response relation of the two proteins by Western Blot.Results (1) Acquire the aimed genes and express fusion proteins. (2) Acquire the polyclonal antiserum through immunizing the rabbits. (3) Purify the actived AhR from C57BL/6J mice hepatic cytosol. (4) The Incubation of two proteins with the min TCDD quantity is 0.25 pmol. (5) If the proteins for interaction is abundant, the Western Blot of TCDD shows positive correlation between the two proteins.