黄色荧光蛋白的结构对Annexin A5凋亡检测功能的影响探究  

The Influence of Structrue of Yellow Fluorescent Proteins on the Apoptotic Detection Ability of Annexin A5

作　　者：高梦月 王云轲 黄先杰 张晶[1] 华子春[1,2,3] Gao Mengyue;Wang Yunke;Huang Xianjie;Zhang Jing;Hua Zichun(School of Life Sciences,the State Key Laboratoly of Pharmaceutieal Biotechnology,Nanjing University,Nanjing 210023,China;Reserach Institute of Pharmaceutical Bioteehnology,Jiangsu Industrial Technology Research Institute and High-Teeh Research Institute of Nanjing University at Changzhou,Changzhou 213164,China;Shenzhen Research Institute of Nanjing University,Shenzhen 518057,China)

机构地区：[1]南京大学生命科学学院,医药生物技术国家重点实验室,南京210023 [2]江苏省产业技术研究院医药生物技术研究所,常州南京大学高新技术研究院,常州213164 [3]南京大学深圳研究院,深圳518057

出　　处：《中国细胞生物学学报》2018年第11期1894-1904,共11页Chinese Journal of Cell Biology

基　　金：国家重点研发计划(批准号:2017YFA0104301、2016YFC0902700、2017YFA0506002);国家自然科学基金(批准号:81630092、81773099、81570790、81573338);深圳市科技创新委员会(批准号:JCYJ20160331152141936、KQTD20140630165057031)资助的课题~~

摘　　要：细胞凋亡早期,磷脂酰丝氨酸(PS)从细胞膜内侧翻转到细胞膜外侧, Annexin A5可与PS高亲和力结合,这是其检测凋亡的原理。绿色荧光探针标记的Annexin A5和碘化丙啶联合标记应用于流式细胞术进行细胞凋亡检测,该方法灵敏度高、特异性好、效率高,已成为目前全世界通用的细胞凋亡检测方法。除FITC-Annexin A5外, Annexin A5-EGFP融合蛋白是另一个常用的简单、可信、易于制备的凋亡检测探针。黄色荧光蛋白是绿色荧光蛋白的一种突变体,其荧光向红色光谱偏移。目前,有三种改良的黄色荧光蛋白:Citrine、Venus、Ypet,这三种改良的蛋白荧光更亮、更稳定、成熟更快。该文选择Citrine、Venus、Ypet来制备Annexin A5凋亡检测探针,通过原核表达和分离纯化Annexin A5-Citrine、Annexin A5-Venus和Annexin A5-Ypet融合蛋白,获得了高产量的可溶性蛋白,探究这三种融合蛋白与凋亡细胞的结合能力,筛选出适用于流式检测的黄色荧光蛋白标记的Annexin A5,为后续研究奠定基础。以pET28a-Annexin A5-EGFP-his重组质粒为模板,利用分子生物学手段又构建了重组质粒pET28a-Annexin A5-Citrine-his、pET28a-Annexin A5-Venus-his、pET28a-Annexin A5-Ypet-his,然后通过Ni柱亲和层析纯化获得了这三种蛋白,纯度约为90%。该文重点比较了三者检测细胞凋亡的能力,流式细胞仪检测结果显示, Annexin A5-Citrine、Annexin A5-Venus和Annexin A5-Ypet融合蛋白均可以识别和标记凋亡细胞,它们与凋亡细胞的亲和力分别是3 113.0 nmol/L、444.3 nmol/L和391.6 nmol/L。三者与凋亡细胞的亲和力差异很大,通过对Citrine、Venus、Ypet的氨基酸序列进行分析,该文初步发现了决定三个黄色荧光蛋白与凋亡细胞亲和力的关键氨基酸。At the early stage of apoptosis, phosphatidylserine(PS) will turn over from the inner cell membrane to the outer cell membrane. When calcium ions are present, Annexin A5 can combine to PS at high affinity and this is the principle of apoptosis detection. Apoptosis detection based on Annexin A5 labeled with green fluorescent probes/iodide by FCM(flow cytometry) is sensitive, efficient and specific. Annexin A5-EGFP fusion protein is a simple, reliable apoptosis detection probe and it is easy to prepare. Yellow fluorescent protein is a mutant of green fluorescent protein and its fluorescence spectra shifts to red spectra. Currently, there are three modified yellow fluorescent proteins: Citrine, Venus, Ypet and the three modified proteins’ fluorescence are brighter, more stable and mature faster. In this paper, Citrine, Venus and Ypet were selected to prepare Annexin A5 apoptosis detection probes. Annexin A5-Citrine, Annexin A5-Venus and Annexin A5-Ypet were expressed in the prokaryotic expression system with a high yield of soluble protein. Also, we explored the combination ability of the three fusion proteins and apoptotic cells and then screened out Annexin A5 labeled by yellow fluorescent proteins, which was suitable for apoptosis detection based on FCM in order to lay the foundation for further study. Three new recombinant plasmids pET28 a-Annexin A5-Citrine-his, pET28 a-Annexin A5-Venus-his, pET28 a-Annexin A5-Ypet-his were constructed for the expression. Three recombinant proteins were purified by the Ni column affinity chromatography and their purity is about 90%. Also, this paper compared the three fusion proteins’ ability to detect apoptosis and flow cytometric analysis showed that Annexin A5-Citrine, Annexin A5-Venus, Annexin A5-Ypet could recognize and bind to apoptotic cells and their affinity with apoptotic cells was respectively 3 113.0 nmol/L, 444.3 nmol/L, 391.6 nmol/L. The affinity between the three fusion proteins and apoptotic cells was very different. The paper analyzed the amino acid sequenc

关 键 词：膜联蛋白A5 纯化 凋亡检测 流式细胞术 序列比对 

分 类 号：Q255[生物学—细胞生物学]