串联标记的HMGB1融合蛋白表达载体的构建及其诱导表达与纯化
onstruction of tandem-tagged HMGB1 fusion protein expression vector and its prokaryotic expression and protein expression
目的 构建带有两个亲和纯化标签的HMGB1融合蛋白原核表达载体，并在原核细胞中进行表达与纯化。方法 以pET-14b-HMGB1-EGFP载体为模板，使用PCR方法扩增HMGB1编码区，酶切后连接至pET-14b-SBP-EGFP载体；对阳性克隆进行酶切、PCR和测序鉴定。转化BL21(DE3)大肠杆菌株，用IPTG诱导融合蛋白表达，并利用Ni2+-NTA亲和层析纯化蛋白。结果 所构建的His-SBP-HMGB1融合蛋白表达载体是正确的，该载体可在大肠杆菌内高效表达，用Ni2+-NTA纯化获得了相对分子质量约33 kD的融合蛋白。结论 成功构建了His-SBP-HMGB1融合蛋白原核表达载体，并在大肠杆菌中高效表达。这为以后深入研究HMGB1的细胞信号转导通路提供实验材料。
Objective To construct tandem-tagged HMGB1 fusion protein prokaryotic expreesion vector for the protein induction and purification. Methods The coding sequence of human HMGB1 was amplified by PCR with plasmid pET-14b-HMGB1 as the template and subcloned into the vector pET-14b-SBP. The positive clones were identified with enzyme digestion, PCR and sequencing. The plasmid was transformed into E. coli BL21(DE3) and induced with IPTG. The His-SBP-HMGB1 fusion protein was purified with Ni2+-NTA resin. Results The recombinant plasmid pET-14b-SBP-HMGB1 was proved correct and with a high efficiency of expression on the induction with IPTG. The fusion protein was purified with a molecular weight of 33 kD. Conclusion We successfully constructed a prokaryotic expreesion vector for His-SBP-HMGB1 and got an efficient expression in E. coli. This work provides an useful tool for further study on HMGB1-related signal transduction.
刘芸、王娟、姜勇、冯国开、罗海华、邓鹏
分子生物学生物工程学生物化学
HMGB1串联亲和纯化系统(TAP)原核表达载体蛋白纯化
HMGB1tandem affinity purification (TAP)prokaryotic expression vectorprotein purification
刘芸,王娟,姜勇,冯国开,罗海华,邓鹏.串联标记的HMGB1融合蛋白表达载体的构建及其诱导表达与纯化[EB/OL].(2010-02-05)[2025-06-16].http://www.paper.edu.cn/releasepaper/content/201002-279.点此复制
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