遗传 ›› 2011, Vol. 33 ›› Issue (3): 246-250.doi: 10.3724/SP.J.1005.2011.00246

• 研究报告 • 上一篇    下一篇

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

张莹, 胡丽玲, 谢伟, 孙蓓, 左爱军, 张镜宇   

  1. 天津医科大学内分泌研究所/代谢病医院, 天津市激素与发育重点实验室, 天津 300070
  • 收稿日期:2010-07-06 修回日期:2010-10-22 出版日期:2011-03-20 发布日期:2011-02-25
  • 通讯作者: 张镜宇 E-mail:zhangjingyu2000@eyou.com
  • 基金资助:

    国家自然科学基金项目(编号:30800955)和天津市自然科学基金重点项目(编号:09JCZDJC21000)资助

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

ZHANG Ying, HU Li-Ling, XIE Wei, SUN Pei, ZUO Ai-Jun, ZHANG Jing-Yu   

  1. Key Laboratory of Hormone and Development of Tinajin City, Institute of Endocrinology / Metabolic Disease Hospital, Tianjin Medical University, Tianjin 300070, China
  • Received:2010-07-06 Revised:2010-10-22 Online:2011-03-20 Published:2011-02-25
  • Contact: ZHANG Jing-Yu E-mail:zhangjingyu2000@eyou.com

摘要: 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。

关键词: 维甲酸X受体α, 巢式PCR, 免疫共沉淀

Abstract: 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 hu-man retinoid X receptor-α was prepared successfully, which makes a basic for further study of its function.

Key words: retinoid X receptor-α, nest-PCR, co-immunoprecipitation