基于蛋白质结构预测的胰岛素样生长因子-1的制备及活性分析

Preparation and activity evaluation of insulin-like growth factor 1 based on protein structure prediction

摘要:

为了更经济高效地制备胰岛素样生长因子-1（insulin-like growth factor-1，IGF-1），本研究采用计算机模拟技术对3种IGF-1融合蛋白进行蛋白质结构预测与分子对接，筛选出酶切最适配的IGF-1融合蛋白表达形式。利用基因工程技术构建并鉴定了IGF-1融合蛋白原核表达载体，并转化大肠杆菌Origami B （DE3）菌株，获得重组子；经异丙基-β-D-硫代吡喃半乳糖苷（isopropyl-β-D-1-thiogalactopyranoside，IPTG）诱导表达后，对菌体的破菌上清进行亲和层析、脱盐、凝血酶酶切及酶切产物亲和层析纯化得到目的蛋白；通过3T3细胞增殖法建立活性评价体系并对获得的IGF-1进行活性测定。结果显示，构建的IGF-1融合蛋白原核表达载体序列正确，获得的重组子在25℃、0.05 mmol/L IPTG诱导16 h时融合蛋白为可溶性表达，经初步纯化、凝血酶酶切、再次纯化后可获得纯度大于90%的IGF-1目的蛋白，在建立的活性评价体系下测得制备的IGF-1比活为2.47×10⁵ U/mg，与市售标准品接近。本研究建立了一条用于制备IGF-1的完整工艺路线，为IGF-1药物的研制及工业化生产奠定基础。

关键词:胰岛素样生长因子-1;虚拟预测;基因重组;活性分析

Abstract:

In order to prepare insulin-like growth factor 1 (IGF-1) more economically and efficiently, the structure prediction and molecular docking of three IGF-1 fusion proteins were performed by computer simulation. The most suitable expression form of IGF-1 fusion protein was screened out. A prokaryotic expression vector of IGF-1 fusion protein was constructed and transformed into Escherichia coli Origami B(DE3) strain to obtain the recombinant strain. After induction with IPTG, the target protein was purified from the soluble fractions of the bacteria cell lysate by affinity chromatography, desalination, thrombin digestion and affinity chromatography of the enzyme digested products. An activity evaluation system was established by 3T3 cell proliferation method and the activity of the obtained IGF-1 was measured. The results showed that the sequence of the IGF-1 fusion protein prokaryotic expression vector was correct and the fusion protein was soluble upon 0.05 mmol/L IPTG induction at 25℃ for 16 h. After preliminary purification, thrombin digestion and re-purification, IGF-1 target protein with purity over 90% was obtained. Using the established activity evaluation system, the specific activity of IGF-1 was 2.47×10⁵ U/mg, which was close to the standard product available at the market. The preparation technology of IGF-1 developed in this study may facilitate the development and industrial production of IGF-1 drugs.

Key words:insulin like growth factor-1 (IGF-1);virtual prediction;genetic recombination;activity evaluation