HSP65-MUC1融合蛋白中试生产工艺的优化及生物学活性检测方法的鉴定

Optimization of HSP65-MUC1 Purification in Pilot Scale and Identification of Methods to Detect Biological Function

目的优化HSP65-MUC1融合蛋白的中试生产工艺,并建立稳定的生物学活性检测方法。方法通过发酵技术获得大量表达HSP65-MUC1的大肠杆菌,高压均质机破菌获得上清液,经不同浓度的饱和硫酸铵、疏水层析、离子交换层析三步纯化后,以期获得高纯度、高质量的融合蛋白HSP65-MUC1;利用流式细胞仪检测HSP65-MUC1作用后,人外周血来源的树突状细胞(DC)表达CD86分子的能力。结果含有pET28a-HSP65-MUC1重组质粒的大肠杆菌BL21(DE3)能有效表达目的蛋白,10 g发酵菌体经饱和硫酸铵沉淀、phenyl sepharose FF层析柱和Q FF离子交换层析柱纯化后,可获得413.7 mg、纯度高达96%的目的蛋白;同时与阴性对照相比(10.13%±0.89%),纯化的HSP65-MUC1能显著提高DC细胞表面CD86分子的表达(29.98%±1.02%)。结论 HSP65-MUC1的中试生产工艺得到了有效的优化,同时鉴定了其生物学活性,从而为临床研究提供基础。

Objective To optimize HSP65-MUC1 fusion protein purification in pilot scale through protein purifi cation techniques and identify the methods for biological activity detection. Methods E. coli expressing HSP65-MUC1 was obtained by fermentation, then homogenized to obtain the supernatant. To acquire high-purity, high-quality HSP65-MUC1, the supernatant was treated with saturated ammonium sulfate, phenyl sepharose FF column and Q FF ionexchange chromatography column purifi cation. The expression of CD86 on the surface of DC cells treated with HSP65-MUC1 was determined with flow cytometry. Results E. coli containing pET28a-HSP65-MUC1 recombinant plasmid can effectively express target protein. A total of 413.7 mg of HSP65-MUC1 was obtained after 10 g of fermented cells was treated with saturated ammonium sulfate, phenyl sepharose FF column and Q FF ion-exchange chromatography column, and the purity was nearly 96%. Compared with negative control（10.13% ± 0.89%）, purifi ed HSP65-MUC1 could signifi cantly improve the expression of CD86 on the surface of DC cells（29.98% ± 1.02%）. Conclusion The pilot scale production of purifi ed HSP65-MUC1 has been effectively optimized, and the methods of its biological activity detection have been identifi ed, which simultaneously provides the basis for clinical studies.