多聚精氨酸蛋白转导域-凋亡素融合蛋白的原核表达及其生物活性 |
| |
引用本文: | 赵健,肖伟,傅文彬,高鹏,袁勤生,刘建文. 多聚精氨酸蛋白转导域-凋亡素融合蛋白的原核表达及其生物活性[J]. 中国生物制品学杂志, 2009, 22(5) |
| |
作者姓名: | 赵健 肖伟 傅文彬 高鹏 袁勤生 刘建文 |
| |
作者单位: | 华东理工大学生物反应器工程国家重点实验室,上海,200237 |
| |
摘 要: | 目的原核表达多聚精氨酸蛋白转导域-凋亡素融合蛋白,并检测其生物活性。方法应用PCR法扩增Arg9-VP3序列,与载体pET-43.1a连接后,转化E.coliBL21(DE3),IPTG诱导表达。表达产物经Ni2+-NTA纯化后,进行肠激酶裂解、超滤浓缩,并检测其生物活性。结果重组表达质粒pET-43.1a-Arg9-VP3经酶切鉴定和序列分析,证明构建正确。转化E.coliBL21(DE3)后,重组蛋白获得可溶性表达。纯化的融合蛋白纯度达90%以上,可抑制HeLa细胞增殖。结论已成功地在大肠杆菌中表达了多聚精氨酸蛋白转导域-凋亡素融合蛋白,纯化的融合蛋白具有诱导HeLa细胞凋亡的能力。
|
关 键 词: | 多聚精氨酸 蛋白转导域 凋亡素 融合蛋白 原核表达 生物活性 |
Prokaryotic Expression and Biological Activity of Poly-Arg Protein Transduction Domain-Apoptin Fusion Protein |
| |
Abstract: | Objective To express poly-Arg protein transduction domain(PTD)-apoptin fusion protein in prokaryotic cells and determine its biological activity. Methods Arg9-VP3 sequence was amplified by PCR and inserted into vector pET-43.1a, and the constructed recombinant plasmid pET43.1a-Arg9-VP3 was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was purified by Ni2+-NTA chromatography, lysed with enterokinase, concentrated by ultrafiltration and determined for biological activity. Results Both restriction analysis and sequencing proved that recombinant plasmid pET43.1a-Arg9-VP3 was constructed correctly. Recombinant fusion protein was expressed in a soluble form, reached a purity of more than 90% after purification and showed inhibitory effect on the proliferation of HeLa cells. Conclusion Poly-Arg PTD-apoptin fusion protein was successfully expressed in E. coli and purified , which induced the apoptosis of HeLa cells. |
| |
Keywords: | Poly-Arg Protein transduction domain(PTD) Apoptin Fusion protein Prokaryotic expression Biological activity |
本文献已被 万方数据 等数据库收录! |
|