用阴离子交换层析纯化牛皮蝇素A蛋白的研究  

ISOLATE HYPODERMOTIN A BY NEGATIVE ION EXCHANGE CHROMATOGRAPHY

作　　者：徐梅倩[1] 顾越星[1] 孙怡[1] 曹杰[1] 朱顺海[1] 孟秀珍[1] 何国声[1] N.Moire C.Boulard 

机构地区：[1]中国农业科学院上海家畜寄生虫病研究所,上海200232

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

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(S2 cell) was cultured for 3 days, the supernatant was collected and concentrated, then dialysed against 25mM pH8.5 TrisHCl buffer. The recombinant HA was isolated by MonoQ column from the dialysed supernatant. The protein peaks were detected at 280nm. SDSPAGE demonstrated that the molecular weight of the first protein was similar to that of natural HA. 0.1% geltin substrate SDSPAGE showed the enzyme activity of recombinant HA. Dotimmunogold verifid the antigen activity. The approach of Negative ion exchange chromatography has many advantages such as easy operation and high efficiency.

关 键 词：阴离子交换层析 纯化 牛皮蝇素A蛋白 SDS-PAGE 斑点免疫金渗滤法 牛皮蝇蛆病 免疫诊断 

分 类 号：S858.235.9[农业科学—临床兽医学]