中国医科大学学报

中国医科大学学报

中国医科大学学报 ›› 2012, Vol. 41 ›› Issue (1): 24–27.

• 基础医学 • 上一篇    下一篇

h-arrestin1重组蛋白不同亚型筛选及互作鉴定

刘彩红,王利利,冯艳玲,朱亚勤   

  • 收稿日期:2012-09-19 修回日期:2012-09-19 出版日期:2012-01-01 发布日期:2012-09-21
  • 通讯作者: 朱亚勤, E-mail: yzhu@mail.cmu.edu.cn
  • 作者简介: 刘彩红(1986-),女,硕士研究生
  • 基金资助:
    辽宁省自然科学基金项目(20092123);省重点实验室项目(2009S108)

Identification of hβ-arrestin1 Isoform A,B and Recombinant Protein Interaction with p80

Liu Cai-hong, Wang Li-li,Feng Yan-ling, Zhu Ya-qin   

  1. (Division of Cell Pathobiology ,The Key Laboratory of Medical Cell Biology,Ministry of Education, Department of Cell Biology ,College of Basic Medical Science, China Medical University, Shenyang 110001,China)
  • Received:2012-09-19 Revised:2012-09-19 Online:2012-01-01 Published:2012-09-21

摘要: 目的 构建GST/-arrestin1融合蛋白表达载体,筛选其不同亚型重组子,并诱导表达、纯化蛋白,初步进行蛋白亚型互作比较,为进一步功能研究提供实验基础。方法 提取人结肠癌细胞SW480的mRNA,反转录为cDNA。用PCR法扩增h?-arrestin1全长编码基因,通过SalI和NotI酶切位点将h?-arrestin1全长定向插入pGEX-5X-1中,构建原核表达载体pGEX-5X-1-?-arrestin1,并通过BglⅡ单酶切筛选A、B亚型,然后将重组质粒转入E.coli BL21 中,异丙基硫代β-D 半乳糖苷诱导表达,用谷胱甘肽-琼脂糖珠亲和纯化表达的GST/?-arrestin1A、B, SDS-PAGE鉴定,互作实验比较亚型的结合能力。结果 酶切及测序结果证明,成功构建了?-arrestin1A、B原核表达载体,并通过SDS-PAGE证实:经IPTG诱导表达及亲和纯化,从转化重组质粒的BL21菌株中获得了融合蛋白GST/?-arrestin1A、B亚型,通过GST Pulldown初步比较了重组?-arrestin1A、B蛋白亚型的结合能力结论 成功构建pGEX-5X-1-?-arrestin1A、B原核表达载体并确定了融合蛋白的诱导表达及纯化方法,且互作实验显示?-arrestin1A有较强的结合能力,为进一步研究?-arrestin1的生物学功能奠定了基础。

关键词: h-arrestin1, 克隆, 重组亚型表达, 蛋白互作

Abstract: Objective To construct GST/?-arrestin1 expression vectors, select different recombinant isoforms,and harvest purified fusion protein from E.coli. Methods Total RNA was extracted from human colon cancer cells SW480. The hβ-arrestin1 coding sequence was amplified by polymerase chain reaction(PCR). The full length of hβ-arrestin1 was directed into the pGEX-5X-1, to construct prokaryotic expression vector. Further, to enzymatically select recombinant subtype A, B, which were transformed into E.coli BL21, induced by IPTG and purified with glutathione beads. The purified products were analyzed by SDS-PAGE. Results Recombinant of pGEX-5X-1-β-arrestin1A、B were analyzed by restriction enzyme digestion and sequenced. GST/β-arrestin1 A、B induced by IPTG were purified from strain BL21. GST/β-arrestin1 A、B were confirmed by SDS-PAGE. GST Pulldown was employed to comfirm binding capacity of GST/β-arrestin1 A、B. Conclusion Recombinant GST/β-arrestin1 A、B expression vectors were successfully constructed, the specific recombinant isoforms induced were detected, and harvested with high purity for the further study of the biological function of β-arrestin1.

Key words: humanβ-arrestin1, cloning, recombinant isoforms expression , interaction of protein

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