CAJ | 학술논문

目的研究人工克隆表达的芳香烃受体核转位蛋白(ARNT)与天然芳香烃受体(AhR)特异性结合及其对制备的多克隆抗体识别的情况,通过两者结合与二噁(口英)(TCDD)的剂量反应关系,探索TCDD生物检测方法.方法 (1)以小鼠肝组织总RNA为模板,通过RT-PCR扩增目的基因AhR-PAS[periodicity(Per)/Aryl hydrocarbon nuclear translocatar(ARNT)/single-minded(Sim)]端、AhR羧基末端(AhR-C端)、ARNT-PAS端,构建含有目的基因的pGEX-5X1重组表达质粒,并采用大肠杆菌原核系统进行克隆表达.(2)用上述克隆表达的AhR片段蛋白(AhR-PAS、AhR-C)作为抗原免疫家兔,制备多克隆抗体.(3)从C57BL/6J纯系小鼠肝组织中提取具有结合活性的天然AhR复合物,将天然AhR复合物与结合在谷胱甘肽硫转移酶(GST)上面的融合蛋白GST-ARNT-PAS,分别在0.25、0.50、1.00、2.00 pmol TCDD存在的条件下进行结合,通过亲和吸附和免疫印迹观察所形成的蛋白复合物的量.结果 (1)成功构建含有目的基因AhR-PAS、AhR-C和ARNT-PAS的重组质粒,并通过大肠杆菌原核系统表达出具有结合活性的目的蛋白;(2)免疫家兔制得的多克隆抗体AhR-PAS、AhR-C的检测下限分别为5、1 ng;(3)测得该法制备的小鼠肝脏匀浆中总蛋白质的浓度为60.5 mg/ml;在TCDD存在的条件下,克隆得到的融合蛋白GST-ARNT-PAS能够与天然AhR复合物特异性地结合,并测得检测下限为0.25 pmol,约80 Pg TCDD.结论成功地建立了基于克隆表达的芳香烃受体系统的TCDD生物检测方法,且检测下限达到pg级别.
목적 연구인공극륭표체적방향경수체핵전위단백(ARNT)여천연방향경수체(AhR)특이성결합급기대제비적다극륭항체식별적정황,통과량자결합여이오(구영)(TCDD)적제량반응관계,탐색TCDD생물검측방법.방법 (1)이소서간조직총RNA위모판,통과RT-PCR확증목적 기인AhR-PAS[periodicity(Per)/Aryl hydrocarbon nuclear translocatar(ARNT)/single-minded(Sim)]단、AhR최기말단(AhR-C단)、ARNT-PAS단,구건함유목적 기인적pGEX-5X1중조표체질립,병채용대장간균원핵계통진행극륭표체.(2)용상술극륭표체적AhR편단단백(AhR-PAS、AhR-C)작위항원면역가토,제비다극륭항체.(3)종C57BL/6J순계소서간조직중제취구유결합활성적천연AhR복합물,장천연AhR복합물여결합재곡광감태류전이매(GST)상면적융합단백GST-ARNT-PAS,분별재0.25、0.50、1.00、2.00 pmol TCDD존재적조건하진행결합,통과친화흡부화면역인적관찰소형성적단백복합물적량.결과 (1)성공구건함유목적 기인AhR-PAS、AhR-C화ARNT-PAS적중조질립,병통과대장간균원핵계통표체출구유결합활성적목적 단백;(2)면역가토제득적다극륭항체AhR-PAS、AhR-C적검측하한분별위5、1 ng;(3)측득해법제비적소서간장균장중총단백질적농도위60.5 mg/ml;재TCDD존재적조건하,극륭득도적융합단백GST-ARNT-PAS능구여천연AhR복합물특이성지결합,병측득검측하한위0.25 pmol,약80 Pg TCDD.결론 성공지건립료기우극륭표체적방향경수체계통적TCDD생물검측방법,차검측하한체도pg급별.
Objective To study the specific binding of the artificial clonal aryl hydrocarbon receptor translocator(ARNT) with the natural aryl hydrocarbon receptor(AhR) and the recognization by polyclonal antibody. The dose-response relationship with tetrachlo-redibenzo-dioxin (TCDD) was also studied to develop TCDD detection method and the binding degree related to dose response. Methods (Ⅰ) 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-5XI recombinants. The recombinant plasmids were expressed in E. coli. (2) The rabbits were immuned by the eional fusion proteins: AhR-PAS, AhR-C to prepare the polyclonal antibody. (3) The natural AhR from the hepatic eytosol 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. Conclusion A TCDD detection method based on the aryl hydrocarbon receptor system was established and the detection limit might reach pg grade.