硫氧还蛋白促进人胰岛素样生长因子-1在大肠杆菌中高效可溶表达

Thioredoxin Increases the Efficient and Soluble Expression of Insulin-Like Growth Factor-1 in E.coli

DOI：10.3969/j.issn.1673-1689.2019.04.008

中文关键词: 胰岛素样生长因子-1 硫氧还蛋白 大杨杆菌C43（DE3） 可溶 生物活性

英文关键词: Insulin-like growth factor-1,thioredoxin,E.coliC43（DE3）,soluble,bioactivity

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中文摘要:

为实现人胰岛素样生长因子-1（Insulin-like growth factor-1，IGF-1）在大肠杆菌中的高效可溶表达，获得大量具生物活性的IGF-1。应用融合PCR技术构建trx-igf1融合基因，克隆至pET30a载体。重组载体pET30a-Trx-IGF-1转化大肠杆菌C43（DE3），并进行条件优化大量表达可溶性蛋白Trx-IGF-1。利用His标签纯化表达产物，对纯化蛋白进行Western blot与生物活性分析。构建的pET30a-Trx-IGF-1重组载体转化大肠杆菌C43（DE3）后，在30 ℃培养条件下1 mmol/L IPTG诱导表达5 h，获得大量以可溶形式表达的融合蛋白Trx-IGF-1；采用Ni离子亲和层析，获得纯度达90%以上的融合蛋白，经Western blot检测具有IGF-1抗原活性。生物学活性检测显示，融合蛋白Trx-IGF-1能明显促进NIH3T3细胞增殖及细胞周期进展。本研究应用的载体蛋白Trx，能实现在大肠杆菌中高效可溶表达具生物活性的IGF-1，为包涵体蛋白在大肠杆菌中的可溶性表达提供借鉴。

英文摘要:

In order to overexpress soluble human insulin-like growth factor-1（IGF-1） inEscherichia coliand obtain a large number of bioactive proteins.A fusion gene containing trx and igf-1 was constructed via fusion of PCR technique and inserted into pET30a plasmid. Then the recombinant plasmid was transformed toE.colistrain C43（DE3） and a series of conditions optimization was carried out. After purification，target protein was identified by Western blot and determined for bioactivity. Trx-IGF-1 fusion protein was high expressed in soluble form in C43（DE3） after induction with 1mmol/L IPTG at 30 ℃ for 5 h. The recombinant protein reached a purity above 90% by using Ni2+ affinity chromatography and showed a strong antigenic ability by Western blot. The biological activity assay revealed that the fusion protein Trx-IGF-1 could significantly promote the proliferation and cell cycle progression of NIH3T3 cells. The carrier protein Trx can be used to realize the high-level soluble expression of bioactive IGF-1 in E.coli，which provide an evidence for the soluble expression of foreign proteins inE. coli.

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