细胞核受体LXRβ的体外酶标测活方法的建立与应用  

Establishment of ELISA Method for Screening Agonists of the Rattus Norvegicus LXRβ

作　　者：蔡怀涵[1] 王璐[1] 谢元翼[1] 刘旭东[1] 宋青[1] 

机构地区：[1]北京科技大学应用科学学院生物科学与技术系,北京100083

出　　处：《中国生物工程杂志》2010年第7期63-67,共5页China Biotechnology

基　　金：2009年北京市教委共建项目资助

摘　　要：目的:克隆并表达大鼠细胞核受体LXRβ配体结合区(LBD)序列,并进行配体依赖性受体与共激活因子结合区短肽相互作用的酶标法分析,建立简易经济可靠的体外高通量筛选LXR调节剂方法。方法:从含大鼠LXRβcDNA序列的pSG5/rLXRβ上扩增LBD区序列,测序核实序列后,将此序列克隆入表达载体,构建原核表达载体pET28a(+)-rLXRβ-LBD;将重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达重组蛋白,通过SDS-PAGE、Western blotting检测及Ni2+亲和层析柱分离纯化;建立ELISA方法检测配体依赖性受体-共激活短肽相互作用。结果:重组蛋白rLXRβ-LBD在大肠杆菌中高效表达(占菌体总蛋白30%),SDS-PAGE显示在36kDa处有免疫特异性蛋白,纯化后全长蛋白占90%以上。ELISA结合测试显示在激动剂猪脱氧胆酸二甲酰胺存在时,rLXRβ-LBD能与共激活短肽结合,亲和力与双荧光酶报告基因测活结果吻合。结论:成功克隆并表达rLXRβ-LBD序列,建立起ELISA筛选方法,为LXR-LBD配体功能活性的研究和配体的高通量筛选提供新方法。Objective：Nuclear receptor LXR ligand-binding domain was cloned and heterogeneously expressed in E.coli, followed by Ni-affinity chromatographic purification. Purified protein was used to establish an ELISA method for screening LXR activators. Method： A DNA fragment encoding LXR ligand-binding domain was amplified by PCR using pSG5/rLXRβ as template. The DNA fragment was sequenced and cloned into an E.coli expression vector pET28a（＋） which was transformed into E.coli strain BL21（DE3） for expression. Expressed protein was purified by Ni-affinity chromatography and analyzed using SDS-PAGE and Western blotting. A synthetic peptide FITCEAEEPSLLKKLLLAPANTQ was used along with HRP-conjugated anti-FITC polyclonal Fab antibody in an ELISA method to detect ligand-dependent receptor/co-activator interaction. Result： rLXRβ-LBD was successfully expressed in E.coli in high yield （30% of total protein）. After affinity purification, full-length target protein was more than 90%. Using ELISA-based co-activator recruiting assay, LXR activator hyodeoxycholic acid dimethyl amide had an apparent Kd of 2.5μmol/L, which was in good agreement of a cell-based dual reporter gene assay. Conclusion： An in vitro LXR ligand screening method was established which may be further improved for highthroughput drug screening.

关 键 词：细胞核受体 LXR 胆固醇 冠心病 高通量药物筛选 

分 类 号：Q78[生物学—分子生物学]