包涵体中重组人神经生长因子的纯化、复性及鉴定

Purification, Renaturation and Characterization of Recombinant Human Nerve Growth Factor from Inclusion Body in E. coli

目的利用简单的纯化工艺，获得具有生物活性的重组人神经生长因子（rhNGF）。方法利用PET－11d－NGF／BL21工程菌表达rhNGF，通过改变培养基成份优化发酵条件。建立了一种在提取包涵体后经一步强阳离子柱层析的简单、快速、重复性好的rhNGF纯化方法。纯化产物用鸡胚背根神经节伸长法测定其生物学活性。结果优化发酵条件后，表达量提高至约占总菌体的10．9％。经一步层析，重组蛋白的纯度可达到80％以上。1BU（生物活性单位）约为0．5μg／mL。结论包涵体复性表明，包涵体的复性与复性液中氧化型与还原型含疏基化合物的比例密切相关。抑制杂菌生长，可明显提高rhNGF的表达量。纯化的rhNGF为分析NGF结构与功能及探讨其临床应用提供了材料。

Using a simple purification process. We obtained the bioactive recombinant human nerve growth factor (rhNGF ). Methods We used PET-11dNGF/B121 to express hNGF. By means of optimization of culture conditions, we got a increased expression level.We set up a simple, fast and efficient purificantion method, after isolating the Inclusion bodies (IBS), using a strong anion exchanger for one step chromatography, The purified products was bioassayed with chick dorsal root ganglia. Results The recombinant protein amounted to 10. 9% of total proteins in bacterial cells by optimizing culture conditions. We obtained a purity up to 80% after one-step chromatograhy. The 1 biological unit (BU) of the purified protein is about 0. 5ug. Conclusion Inhibition of bacteria heterosis can lead to higher yields of the rhNGF. Renaturation analysis indicates that the optimum ratios of re duced and oxidized glutathione were related to the efficiency of renaturation. Purified rhNGF is necessary for studying the structure-function relationship and clinical use.