犬α干扰素真核表达条件优化纯化及活性研究

Study on the optimization of the eukaryotic expression conditions of canine interferon alpha and the purification and activity of its target protein

为了对高效表达犬α干扰素的毕赤酵母重组菌株进行表达条件的优化,对表达后的目的蛋白进行纯化及活性研究,研究通过单因素试验方法对影响犬α干扰素的真核表达摇瓶培养条件进行了优化,利用Phenyl Sphareose High Performance疏水层析介质,Q Sepharose High performance强阴离子交换层析介质对表达后目的蛋白进行纯化,期间采用Sephadex TM G-25 Fine介质脱盐,采用MDCK-VSV微量病变抑制法对纯化后蛋白的活性进行检测。结果表明:得出最佳的诱导表达条件为诱导温度28℃,诱导时间72 h,诱导p H值6.0,诱导甲醇浓度1.0%;优化后目的蛋白表达量达到0.18 mg/m L,比优化前提高了54%;纯化后获得纯度达90%以上的目的蛋白,测得目的蛋白的比活为1.08×108U/mg。说明摇瓶优化可显著提高目的蛋白的表达量,毕赤酵母重组菌株能够获得较高具有较高活性的目的蛋白,可用于犬α干扰素的大规模生产及临床应用。

To optimize the expression conditions of Pichia pastoris recombinant strains efficiently expressing canine interferon alpha and study the purification and activity of the expressed target protein, the culture conditions of shaking flasks affecting the eukaryotic expression of canine interferon alpha were optimized by single factor test method. The expressed target protein was purified using Phenyl Sphareose High Performance hydrophobic chromatography medium and Q Sepharose High performance strong anion exchange chromatography media, and the activity of purified protein was detected using SephadexTM G - 25 Fine medium for desalinization and MDCK - VSV micro - cytopathic effect inhibition. The results showed that the optimal conditions for inducing the expression were the induction temperature of 28 ℃, induction time of 72 h, pH value of 6.0 for induction, and the induced concentration of methanol of 1.0%. The expression level of target protein reached 0.18 mg/mL after optimization,which was increased by 54% than before optimization. The final purity of the target protein reached more than 90% after optimization, and the specific activity of the purified target protein was 1.08 × 10^8 U/mg. The results indicate that flask optimization significantly increase the expression level of the target protein, and Pichia pastoris recombinant strains can obtain higher level of proteins with higher activity, which can be used for large - scale production and clinical application of canine interferon alpha.