【作者】 高超；

【导师】 华子春；

【作者基本信息】 南京大学 ， 生物化学与分子生物学（专业学位）， 2012， 硕士

【摘要】 人膜联蛋白V(Annexin Ⅴ)具有钙依赖性的结合细胞膜上带负电荷的磷脂的特性。细胞凋亡时,磷脂酰丝氨酸(phosphatidylserine, PS)由细胞膜内侧翻转到细胞膜外侧。经荧光标记或放射性标记的Annexin Ⅴ可以特异性结合磷脂酰丝氨酸,与染色质的荧光染料碘化丙啶(PI)联用可以用来检测细胞凋亡。细胞凋亡时细胞膜完整PI无法进入细胞内呈阴性,而标记的Annexin Ⅴ可以和细胞膜结合呈阳性,区别于细胞坏死时的双阳性。此外,在体内放射元素性标记Annexin Ⅴ可用于动脉粥样硬化、癌症等疾病的诊断检测和治疗评估。Annexin Ⅴ相对分子质量为36.5kDa,是包含320个氨基酸残基组成的单链蛋白。Annexin Ⅴ由N端的前导序列(约20个氨基酸残基)和C端核心区组成,核心区包括四个重复序列,每个重复序列大概70个氨基酸残基。每个重复序列又由5个a螺旋组成,a螺旋间由loop组成,其中AB-loop和DE-loop结合Ca2+时发挥重要作用。Annexin Ⅴ的四个重复片段构成圆盘状结构,钙离子结合位点位于圆盘状的凸面,氨基末端和羧基末端位于圆盘状的凹面。在钙离子存在条件下,Annexin Ⅴ圆盘状的凸面与带负电的磷脂结合。野生型Annexin Ⅴ常用于早期细胞凋亡检测,本实验旨在降低Annexin Ⅴ分子量,构建Annexin Ⅴ变体以期发现更灵敏的检测效果。基于生物信息学分析,在本实验中共构建了四个变体：变体一,根据国外文献报导去掉氮端10个氨基酸；变体二,去掉整个氮端前导序列；变体三,去掉同源性最小的重复片段三；变体四：去掉最后一个重复片段。Annexin Ⅴ及其变体诱导表达、分离纯化后均获得目的蛋白。流式细胞术分析表明,变体一仍保持和野生型Annexin Ⅴ相近的细胞凋亡检测功能；变体二、变体三在流式图双标记检测时未出现特异性的细胞凋亡检测峰,但是在单标记检测时却出现细胞凋亡检测峰；变体四与野生型相比较,细胞凋亡检测能力有所下降。在荧光显微镜下观察四种变体均与凋亡细胞相结合。更多还原

【Abstract】 Human annexin Ⅴ can combine negatively charged phospholipids on the membrance through calcium ion. When apoptosis occurs, phosphatidyl serine (phosphatidylserine, PS) flips from the inner side of membrance to the lateral. Fluorescence or radioactivity marked Annexin Ⅴ specifically combines PS, which can be used to detect apoptosis together with chromatin fluorescent dye propidium iodide (PI).When apoptosis occurs, cell membrane keeps integrity and PI can not enter cell, but labeled Annexin V has membrane-bound characteristics, which is different from the double positive results during cell necrosis. In addition, radioactive Annexin V can be used for diagnosis and treatment assessment of atherosclerosis, cancer and other diseases in vivo.Annexin Ⅴ has the molecular weight of36.5kDa, contains320amino acid residues in one single-chain. Annexin Ⅴ is composited by the N-terminal leader sequence and the C-terminal core area, which contains four repeat sequences and each repeat contains about70amino acid residues, consisting of five alpha helix and four loops.The AB and DE loops play important roles in combination of Ca2+. Four repeats forma disc-shaped structure, where calcium binding sites is located in the convex surface and two terminals are located in the concave side. Convex side bounds negatively charged phospholipids in the presence of calcium ion. The wild-type Annexin Ⅴ is commonly used for early detection of apoptosis, this study was designed to investigate the structure and function relationship of Annexin Ⅴ with the aim to develop a better apoptotic detector.In the current research, four mutants were constructed based on bio informatics analysis:variant Ⅰ-remove the N-terminal10amino acids; variantⅡ-remove the entire N-terminal leader sequence; variantⅢ-remove the third repeat with minimal homology; variantⅣ-remove the last repeat. Four target proteins were expressed by induction,isolated and purified. Flow cytometric analysis showed that variant Ⅰ remains similar to wild-type Annexin Ⅴ, variants Ⅱ and Ⅲ did not show apoptotic detection peak during double labeling with Annexin Ⅴ and PI, but displayed specific apoptotic peak when using Annexin Ⅴ alone. Mean while variant Ⅳ gave specific but declined detections compared to wild-type. All four variants could detected apoptotic cells observed under the fluorescent microscope.更多还原