重组人维甲酸X受体α的克隆与表达及其与甲状腺受体的结合

The cloning, expression and the binding ability with TRβ1 of retinoid X receptor-α gene

维甲酸X受体α(Retinoid X receptor-α,RXR-α)属于核受体家族,在调节基因转录及信号转导方面发挥着重要的作用。为了深入研究RXR-α的生物学作用,文章利用RT-PCR技术扩增了人RXR-α基因,并将其克隆入原核表达载体pQE-30Xa,转化大肠杆菌M15[PREP4],异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,SDS-PAGE可见与预期大小相符的蛋白条带,Western blotting证实该条带为重组人RXR-α蛋白,并用Ni2-NTA柱进行亲和层析纯化。免疫共沉淀结果显示其具有与TRβ1结合的能力。电泳迁移率改变实验(EMSA)显示RXR-α与TRβ1形成的杂二聚体具有与DNA结合的能力。结果显示,文章已成功建立了重组人RXR-α的大肠杆菌表达系统,表达产物具有很好的生物学活性。

Retinoid X receptor-α（RXR-α）,a member of nuclear receptor family,is capable of mediating retinoid signaling pathways and plays a critical role in regulating target gene transcription.To further study the function of RXR-α,abundant of recombinant RXR-α protein in hand is necessary.In this study an intact RXR-α coding sequence was amplified by RT-PCR and subsequently inserted into expression plasmid vector pQE-30Xa to form the recombinant construct of pQE-30Xa/RXR-α.Thereafter,competent bacteria Escherichia coli M15 [PREP4] was transformed and the expression of RXR-α was induced by adding IPTG to the medium.Bacterially expressed recombinant RXR-α was purified by Ni-NTA affinity chromatography and verified by SDS-PAGE and Western blotting analyses.The results showed that a protein,with the molecular mass around 50 kDa,could be selectively recognized by anti-RXR-α antibody.Co-immunoprecipitation assay indicated that this recombinant RXR-α could effectively bind TRβ1 to form a heterodimer,which could specifically bind the target DNA fragment.This was confirmed by EMSA.In conclusion,the recombinant human retinoid X receptor-α was pre-pared successfully,which makes a basic for further study of its function.