非分型流感嗜血杆菌Haps蛋白的分离纯化.pdfVIP

· l880· 南方医科大学学报rJ South Med Univ) 2007；27(12) 非分型流感嗜血杆菌Hap 蛋白的分离纯化 李婉宜，邝 玉，李明远，杨 远，蒋中华，姚 锋，陈长春(四)11大学华西基础医学与法医学院，微生物学教研室 ， 四川 成都 610041) 摘要：目的 优化非分型流感嗜血杆菌Hap。蛋白质片段的分离纯化方法。方法采用盐析、透析脱盐、超滤浓缩、弱阳离子 交换柱HiwapCM SepharoseFastFlow层析纯化HapS蛋白，优化Hap。蛋白质片段的洗脱条件，包括pH值和离子强度， 测定各洗脱液样品在 280 nnl波长处的光吸收值(D )，用折线图显示，SDS．PAGE电泳检测分布图中处于峰值的样 品，观察目的蛋白条带的出现。结果弱阳离子交换柱Hiwap CM Sepharose Fast Flow层析纯化HapS蛋白，缓冲液 l洗 脱液的D280分布为基线，缓冲液 2洗脱液的D280分布折线图中有峰值出现，但峰有拖尾，SDS．PAGE电泳检测该洗脱 峰主要为低分子量的蛋白条带；五种不同离子强度的缓冲液3洗脱液在 100mlno1／LNaCI离子强度时，D 的分布折线 图显示有较高洗脱峰出现，SDS．PAGE电泳检测该洗脱峰有较明显的 l10 000D的目的条带，其余离子强度下均未见 明显洗脱峰出现。结论 SepharoseCMFF层析柱分离纯化Hap。蛋白质片段时，不同pH值的缓冲液对目的蛋白的洗脱 没有明显影响，而主要影响杂蛋白的洗脱；100 mmol／LNaCI离子强度的缓冲液3洗脱液获得较好的洗脱效果。 关键词：Hap 蛋白；阳离子交换层析；蛋白纯化 中图分类号：R378．41 文献标识码：A 文章编号：1673．4254(2007)l2．1880．04 Optimized isolation and purification of non-typeable Haemophilus influenzae Hap protein LI Wan-yi，KUANG Yu，LI Ming-yuan，YANG Yuan，JIANG Zhong-hua，YAO Feng，CHEN Chang-chun Department ofMicrobiology,West China School ofPreclinical and Forensic Medicine，Sichuan University，Chengdu 610041，China Abstract： Objective To optimize the isolation and purification conditions for Hap。protein of non—typeable Haemophilus influenzae．Methods Hap protein was purified by ammonium sulfate precipitation，dialysis desalting and Hiwap weak cation exchange columns of CM Sepharose Fast Flow．The condition of the elution WaS optimized for pH and ionic strength．the absorbance at 280 nnl of the elution samples were detected， and the targeted protein band in the collected samples was observed by SDS．PAGE electrophoresis．Results The Hitrap ion exchange column was eluted with buffer l，which resuked in abaseline dislribution ofabsorbance at 280 nn1．Buffer 2 elution ofthe column resulted in the presence ofpeak absorbance with wails，which WaS identified to be constituted by some 1oW mo