Prokaryotic Expression Of Proteins Involved In The Dioxins Bioassay System

The objective of this project is to establish one rapid and specially dioxin detection method.This dissertation mainly contained two parts.The first part is the amplification of DNA sequence and the reconstruction of expression plasmid.The second part is the preparation of polyclonal antibody special to AhR. The content of this study provide the basis for establishment of dioxin bioassay system and related research on AhR.In this paper,the genes encoding AhR and ARNT were amplified by RT-PCR using the total RNA purified from the liver of C57BL/6J mice as templates and the oligonucleotide primers with restrictive sites at both ends for cloning purposes. The fidelity of PCR was determined by agarose electrophoresis and DNA sequence analysis. We ligated the DNA fragments purified from the agarose into expression vector pGEX-5X-1 by T₄ DNA ligase. The positive recombinant were validated by digestion of restrictive endonuclease. Then we transfered identified recombinant into competent cells of E.coli BL21(DE3). Screening the positive clones which could express fusion proteins with predicted molecular weight after adding IPTG through SDS-PAGE analysis. Preserved the positive clones for future using.In order to prepared polyclonal antiserum especially to AhR, we plan to express and purify the two fragments of it in the first place. We found that most of the expressed AhR-C located in cytoplasm in the form of soluble protein,whereas the induced protein AhR-PAS mainly existed in the inclusion body. So we purified AhR-C by affinity adsorption of Glutathione Sepharose? 4B,but acquired the protein of AhR-PAS through purification of inclusion bodies.Then we prepared the polyclonal antiserum to AhR through immunizing the obtained fusion proteins to rabbits.