摘要
建立了用阴离子交换层析AKTAFPLC纯化果蝇S2细胞分泌的重组牛皮蝇素A(HypodermotinA,HA)的方法。将诱导培养3天的S2细胞上清液浓缩后,经25mMpH8.5Tris-HCl缓冲液透析,在MonoQ柱上以Tris-HCl+NaCl为流动相进行分离。在检测波长280nm处获得蛋白峰。经SDS-PAGE分析,第一峰的分子量与原重组蛋白HA分子量相符,并对明胶大分子具有酶活性;用斑点免疫金渗滤法检测证实对牛皮蝇抗体具有免疫原性。用阴离子交换层析纯化HA的方法具有获得的蛋白质纯度高、操作简便、生产效率高等特点。
An approach of isolating recombinant Hypodertin(HA), which was secreted into the supernatant of Drosophila expression system(DES), was established by Negative ion exchange chromatography. After the drosophila cells(S2 cell) was cultured for 3 days, the supernatant was collected and concentrated, then dialysed against 25mM pH8.5 TrisHCl buffer. The recombinant HA was isolated by MonoQ column from the dialysed supernatant. The protein peaks were detected at 280nm. SDSPAGE demonstrated that the molecular weight of the first protein was similar to that of natural HA. 0.1% geltin substrate SDSPAGE showed the enzyme activity of recombinant HA. Dotimmunogold verifid the antigen activity. The approach of Negative ion exchange chromatography has many advantages such as easy operation and high efficiency.
出处
《中国兽医寄生虫病》
2003年第1期1-3,F002,共4页
Chinese Journal of Veterinary Parasitology
基金
欧盟资助项目:INCO-DEVN0ICA4-CT200030036
