犬干扰素α制备工艺的优化

Optimization of Process for Production of Canine Interferon Alpha

关键词:  犬干扰素α  原核表达  纯化  质量

Key words:  canine interferon α  prokaryotic expression  purification  quality

摘要:

根据GenBank的犬干扰素α序列,经密码子优化后,设计并合成了编码CaIFN-α蛋白的基因片段。CaIFN-α基因与pBV220温度诱导表达载体连接,转化大肠杆菌BL21。经培养、升温诱导表达,目的蛋白以包涵体的形式存在,蛋白分子量约为18 000,蛋白表达量为25% 。菌体经超声破碎、变性、复性后目的蛋白纯度为69%,收率为28%；疏水层析后目的蛋白纯度87%,收率为81%；分子筛层析后目的蛋白纯度为96％,收率为90%。纯化的CaIFN-α蛋白的生物学比活性为3?10₇IU/mg,内毒素为≤10EU/mg,符合兽用药要求。此制备工艺总产率高达20%、重现性好,适用于大规模生产。

Abstract:

According to the canine interferon alpha gene in GenBank, optimized, designed and synthesised the CaIFN-α gene fragments. CaIFN-α gene was cloned into pBV220 vector and was expressed in E. coli BL21. The molecular weight of CaIFN-α was 18000 and the expression of CaIFN -α was 25%.Then the inclusion bodies was denaturated and renaturated,the purity was 69%,and recovery rate reached 28%.Hydrophobic interaction chromatography was used to purify the protein,the purity was 87%,and recovery rate reached 81%.Then the protein was purified by molecular sieving chromatography,the purity was 96%, and recovery rate reached 90%.The specific activity of CaIFN - a was 3?10₇IU/mg. The endotoxin was ≤10EU / mg. The yield was 20% .This process for production was suitable for large-scale production.