创伤弧菌溶细胞素基因重组蛋白复性条件的优化及溶血活性鉴定

Optimization of fusion protein VVC condition of renaturation and identification of hemolytic activity

目的:对由大肠杆菌表达系统表达的创伤弧菌溶细胞素蛋白包涵体的复性条件进行优化并对其溶血活性进行评价。方法:表达、提取并纯化重组蛋白,并对其包涵体利用PBS透析复性、16种不同复性液透析复性和柱上复性方法进行复性比较,复性蛋白用溶血试验验证其活性。结果:利用三种蛋白复性方法,重组蛋白均不同程度得到复性,其中复性液中盐酸胍、助溶剂及盐离子作用显著。柱上复性效果明显,其复性率高达98%。利用兔红细胞溶血试验检测表明,重组蛋白具有溶血活性,其活性为0.2μg/HU。结论:成功对重组蛋白包涵体复性条件进行优化,并对复性后蛋白质的溶血活性进行鉴定,为今后的免疫学活性和变性蛋白质复性研究奠定了基础。

Objective：To optimize renaturation condition of fusion protein WC and estimate its haemolytic activity. Methods： To express, extract and purify the recombined protein and renaturate the inclusion with PBS dialysis, 16 kinds of different renaturation solutions and purification column were compared with renaturation. The activity of recombination protein was verified by hemolysis test. Results： The purified WC recombination protein was refolded successfully by different renaturation ways. Renaturation rate of recombination protein on purification column was nearlly 98%. The haemolysis activity was 0.2 μg/HU. Conclusions： WC recombination protein is refolded successfully. Based on these, the renaturation protein and WC immunology study can be carried out.