[Construction of the dioxin bioassay method based on the clonal expressed aryl hydrocarbon receptor system].
OBJECTIVE
To study the specific binding of the artificial clonal aryl hydrocarbon receptor translocator (ARNT) with the natural aryl hydrocarbon receptor (AhR) and the recolonization by polyclonal antibody. The dose-response relationship with tetrachlo-rodibenzo-dioxin (TCDD) was also studied to develop TCDD detection method and the binding degree related to dose response.


METHODS
(1) The target genes including AhR-PAS, AhR-C and ARNT-PAS were amplified by RT-PCR by using the total RNA purified from the liver cells of C57BL/6J mice as templates to construct pGEX-5X1 recombinants. The recombinant plasmids were expressed in E. coli. (2) The rabbits were immuned by the clonal fusion proteins: AhR-PAS, AhR-C to prepare the polyclonal antibody. (3) The natural AhR from the hepatic cytosol of C57BL/6J mice was extracted. The artificial cloning expressed fusion protein:GST-ARNT-PAS and the natural AhR were incubated in different dose of TCDD. The quantity of the heterodimer through affinity adsorption and Western blots were measured.


RESULTS
(1) The target proteins including AhR-PAS, AhR-C and ARNT-PAS were successfully cloned and expressed in E. coli. (2) The detection limit of polyclonal antibody AhR-PAS and AhR-C were 5 ng and 1 ng, respectively. (3) The total protein concentration prepared from the liver cells was 60.5 mg/ml. The artificial clonal protein ARNT-PAS could specifically bind to the natural AhR complex with the existence of TCDD. The detection limit of TCDD was 0.25 pmol which was 80 pg approximately.


CONCLUSIONS
A TCDD detection method based on the aryl hydrocarbon receptor system was established and the detection limit might reach pg grade.
Wang
Zhuo
Z
Department of Non-Communicable Disease Control and Prevention, Tianjin Center for Disease Control and Prevention, Tianjin 300011, China.
Zhao
Na
N
Shen
Jun
J
Wang
Ying
Y
Tang
Nai-jun
NJ
Wu
Yun-tang
YT
Zhang
Wan-qi
WQ
Mi
Huai-feng
HF
D000818:Animals; D002478:Cells, Cultured; D057230:Limit of Detection; D008110:Liver Extracts / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D000072317:Polychlorinated Dibenzodioxins / Q000032:analysis*; D011817:Rabbits; D018336:Receptors, Aryl Hydrocarbon / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D008110:Liver Extracts; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon