基因工程犬干扰素α的制备及纯化工艺

Preparation and Purification of Recombinant Canine Interferon Alpha

目的:运用基因工程技术制备高活性的重组犬α干扰素。方法:用发酵罐大量培养工程菌,经超声破碎菌体获粗制包涵体,用1%TritonX-100洗涤去除部分杂蛋白后,以8mol/L尿素溶解包涵体,稀释复性并超滤浓缩,用阴离子柱层析法纯化目的蛋白,测定重组犬干扰素α的活性。结果:得到的重组犬干扰素α的相对分子质量为19×103,蛋白含量为0.55mg/mL,纯度95.65%,目的蛋白回收率为13.75%,活性1.78×107U/mL,比活性3.24×107U/mg。结论:制备了高纯度且具有高活性的重组犬α干扰素。

Objective： To obtain recombinant canine interferon α（CalFN-α） with high activity, the purification craft was investigated. Methods： Engineering strain was fermented, and the CalFN-α was expressed insolubly. The inclusion bodies were collected and further washed with 1% TritonX-100. Subsequently, they were denatured in solution including 8 mol/L urea and refolded by diluted process, concentrated by dialysis. Negative ion exchange chromatography was used to purify the protein of interest, and the bioactivity of which was tested. Results： Molecular of CalFN-α was 19 kD detected by the SDS-PAGE. Final protein concentration was 0.55 mg/mL, the purity was 95.65%, and recovery rate reached 13.75%. The activity and specific activity of CalFN-α was 1.78×10^7 U/mL and 3.24×10^7 U/mg, respectively. Conclusion： High purity as well as high activity recombinant CalFN-α was prepared successfully.