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中国农学通报

中国农学通报 ›› 2014, Vol. 30 ›› Issue (2): 45-49.doi: 10.11924/j.issn.1000-6850.2013-1855

所属专题： 生物技术

• 畜牧 动物医学 蚕 蜂 • 上一篇    下一篇

盐藻丝氨酸/苏氨酸蛋白激酶基因DsSTPK的克隆、原核表达及纯化

陶晓迎 柴晓杰 薛飞 李秀娟 丛玉婷   

  • 收稿日期:2013-07-05 修回日期:2013-08-08 出版日期:2014-01-15 发布日期:2014-01-15
  • 基金资助:
    国家自然科学基金项目“盐藻中磷酸化转录因子对盐胁迫的分子响应”(30972240)；辽宁省教育厅科技研究项目“胰蛋白酶抑制剂基因在盐藻中的高效表达”(2008T023)。

Prokaryotic Expression, Purification and cloning of a novel Gene DsSTPK from Dunaliella salina

  • Received:2013-07-05 Revised:2013-08-08 Online:2014-01-15 Published:2014-01-15

摘要： 前期研究表明，盐藻丝氨酸/苏氨酸蛋白激酶DsSTPK在转录水平上的表达受盐胁迫诱导。为了进一步研究该蛋白激酶的生理功能，通过RT-PCR扩增DsSTPK的开放阅读框，并将其克隆至pET32a(+)载体，得到重组表达载体pET32a(+)-DsSTPK。将重组表达载体转化E. coli. BL21 (DE3)，IPTG诱导表达，然后对融合蛋白进行表达形式分析，利用His60 Ni离子重力柱进行纯化，所得产物进行SDS-PAGE和Western-blot鉴定。本研究成功构建了重组表达载体pET32a(+)-DsSTPK，经IPTG诱导后表达出的融合蛋白的分子量与预期相符；表达形式分析显示该融合蛋白在上清和包涵体中均存在；将上清蛋白纯化后获得了纯度较高的融合蛋白；Western-blot检测显示该融合蛋白能被抗组氨酸抗体特异性识别，具有良好的免疫学活性。DsSTPK的成功表达、纯化，为下一步制备抗体，然后进一步在蛋白质水平上研究其在盐藻耐盐机制中的作用奠定了基础。

关键词: 接触角, 接触角

Abstract: Previous studies indicated that the expression of a serine/threonine protein kinase DsSTPK of Dunaliella salina could be induced by salinity stress. In order to study the functions of DsSTPK, the Open Reading Frame (ORF) of DsSTPK gene was obtained through RT-PCR. The target fragment was subcloned into pET32a(+ ), and the recombinant expression vector pET32a(+ )-DsSTPK was transformed into E. coli. BL21 (DE3), then followed by induction of IPTG. The products were purified by His60 Ni Gravity Columns, identified by SDS-PAGE and Western-blot. The results indicated that the recombinant expression vector pET32a(+)-DsSTPK was constructed successfully, the molecular weight of the recombinant protein induced by IPTG was in line with expectation; And the recombinant protein existed both in the supernatant and in the form of inclusion body. Western-blot analysis showed that the recombinant protein could be identified specifically by the anti-his antibody. The research has laid a foundation for further studies on how the DsSTPK works in Dunaliella salina salinity stress signal transduction.