融合蛋白P11-A1的构建、表达及生物活性研究  

Construction,expression and bioactivity study of fusion protein P11-A1

作　　者：龙民慧[1] 王园园[1] 朱贺[1] 王曼 邹民吉[1] 马百坤[2] 王嘉玺[1] 徐东刚[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]南京军区总医院,南京210002

出　　处：《军事医学科学院院刊》2008年第6期511-514,518,共5页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的:获得P11-A1融合蛋白并对融合蛋白的生物活性进行分析。方法:将人类载脂蛋白A1和P11基因进行拼接,然后连接在表达载体pBV220上,将表达载体转化到大肠杆菌BL21进行表达,SDS-PAGE分析其表达情况。通过离子交换层析对融合蛋白进行纯化、Western印迹检测纯化产物。用液体闪烁计数器检测融合蛋白体外合成高密度脂蛋白(HDL)的能力来测定转酯活性,同时用溶圈法检测融合蛋白的溶栓活性。结果:融合蛋白的表达量为29%;离子交换层析获得纯度约为90%的蛋白;Western印迹显示该重组蛋白能够特异地识别载脂蛋白A1抗体,并且该融合蛋白在浓度为0.28 mg/ml时转酯活性为21.93 nmoL/(h·ml),溶栓活性为22.2 U/mg。结论:成功获得具有转酯活性和溶栓功能的P11-A1融合蛋白。Objective：To study the function of P11-A1 fusion gene, produce purified protein of P11-A1 and test its biological activity. Methods： The recombinant plasmids （pBV22-Pll-A1） harboring human apolipoprotein A1 and SI00 （P11 ）gene were constructed and were transformed into E. coli BL21. The fusion protein was expressed by temperature induction, and was purified by ion exchange chromatography. The quantity of expression was analyzed by SDS-PAGE. The biological activity of fusion protein was tested with liquid scintillation counter and the fibrin plate method. Results： The ex- pression levels of proteins reached up to 29% of the total proteins. Purity exceeded to 90% with ion exchange chromatogra- phy. The protein could specially recognize the antibody to ApoA1. Its transesterification activity was 21.93 nmol/（h · ml） and fibrinolytic activity was 22.2 U/mg at the concentration of 0.28 mg/ml. Conclusion ： The recombination protein has transesterification and fibrinolytic activities.

关 键 词：重组融合蛋白质类 表达 纯化 转酯活性 纤溶活性 

分 类 号：Q78[生物学—分子生物学]