非分型流感嗜血杆菌Haps蛋白的分离纯化

目的优化非分型流感嗜血杆菌Haps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Hitrap CM Sepharose Fast Flow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280nm波长处的光吸收值（D280），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Hitrap 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100mmol／L NaCl离子强度时，D280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110000D的目的条带，其余离子强度下均未见明显洗脱峰出现。结论Sepharose CM FF层析柱分离纯化Haps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；100mmol／L NaCl离子强度的缓冲液3洗脱液获得较好的洗脱效果。

Optimized isolation and purification of non-typeable Haemophilus influenzae Haps protein

Objective To optimize the isolation and purification conditions for Haps protein of non-typeable Haemophilus influenzae. Methods Haps protein was purified by ammonium sulfate precipitation, dialysis desalting and Hitrap 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 nm 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 1, which resulted in a baseline distribution of absorbance at 280 nm. Buffer 2 elution of the column resulted in the presence of peak absorbance with trails, which was identified to be constituted by some low molecular weight bands by subsequent SDS-PAGE. In serial column elution with buffer 3 with different ionic strength, a peak absorbance was observed with the ionic strength of 100 mmol/L NaCl, and SDS-PAGE confirmed that the peak was generated by the target protein. No obvious peaks or bands in SDS-PAGE occurred with the other ionic strengths. Conclusion The pH of the buffer only affect the elution of the irrelevant proteins rather than the Haps protein, and elution with the buffer containing100 mmol/L NaCl can be optimal for eluting the Haps protein.