CN110684115A - 用于凋亡细胞识别和标记的融合蛋白及其制备方法和应用 - Google Patents
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Abstract
本发明涉及生物化学和检验与检测领域，具体涉及黄色荧光蛋白标记的Annexin A5及其制备方法和在制备凋亡检测试剂中的应用。本发明以pET28a‑Annexin A5‑EGFP‑his重组质粒为模板，构建了重组质粒pET28a‑Annexin A5‑Citrine‑his、pET28a‑Annexin A5‑Venus‑his、pET28a‑Annexin A5‑Ypet‑his。大量表达Annexin A5‑Citrine、Annexin A5‑Venus和Annexin A5‑Ypet融合蛋白，通过Ni柱亲和层析纯化获得了这三种蛋白，纯度约为90％。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3113nmol/L，444.3nmol/L和391.6nmol/L。以上结果显示Annexin A5‑Citrine、Annexin A5‑Venus和Annexin A5‑Ypet融合蛋白均可以用于凋亡细胞的识别和标记。
Description
技术领域
本发明涉及生物化学和检验与检测领域，具体涉及黄色荧光蛋白标记的 AnnexinA5及其制备方法和在制备凋亡检测试剂中的应用。
背景技术
细胞凋亡是细胞程序性死亡最常见的一种形式，它表现为细胞收缩、染色质固缩、细胞膜出泡、细胞凋亡蛋白酶caspase的激活以及细胞膜表面外翻的磷脂酰丝氨酸(PS)呈现出“吞噬我”的信号。凋亡细胞的清除是通过吞噬细胞完成的，吞噬细胞可以识别死亡细胞的上述表型。检测细胞凋亡的方法大多是基于这些形态学的变化和生化指标。
目前，应用最为广泛的凋亡检测探针是Annexin A5。基于Annexin A5的凋亡检测方法是将Annexin A5联结上生物素、荧光素或放射性配体，使之成为一种可检测的标记物，在钙离子存在的情况下，将凋亡细胞与Annexin A5复合物进行混合，凋亡细胞就会迅速结合带有标记的Annexin A5。这种结合可以通过多种技术来测量，如流式细胞术、激光扫描细胞术、共聚焦激光扫描显微术等。正是因为Annexin A5能够与凋亡细胞外翻的PS结合，故它被作为凋亡检测的特异性探针在全世界被广泛应用。为了满足不同的需要，各种标记的Annexin A5 探针相继被开发，且相关产品也实现了商品化。
化学偶联方法标记的Annexin A5被广泛使用，如最常用的FITC(异硫氰酸荧光素)标记的Annexin A5。该探针的制备过程包括蛋白的表达纯化、化学偶联荧光素以及后续游离荧光素的去除，这一过程涉及多步操作并需要精确控制，非常复杂繁琐；此外，氨基介导的化学修饰过程会导致Annexin A5蛋白与细胞膜PS的结合能力下降。另一方面，化学标记Annexin A5的最终产物通常是不同标记程度的混合物，非常不均一，从而限制了它们的使用。而融合了荧光蛋白的 Annexin A5则相对均一，且荧光强度高、光稳定性强。而同样是绿色荧光的 Annexin A5-EGFP荧光蛋白融合分子探针则不涉及化学偶联方法中的多步操作，只需要对融合蛋白进行原核表达和分离纯化，操作简便且成本低，极具应用价值。
黄色荧光蛋白作为绿色荧光蛋白的一种变体，其荧光向红色光谱偏移，这主要是由于蛋白203位苏氨酸变为酪氨酸，它也广泛应用于细胞生物学和分子生物学领域。黄色荧光蛋白最早的变体是EYFP，它虽然仍被广泛使用，但由于其pKa 值高，对卤化物敏感，它的应用并不是很理想。单体形式的变体柠檬黄Citrine 和Venus是目前应用最多的黄色荧光蛋白探针。另一种很有应用潜力的黄色荧光蛋白是能量转移黄色荧光蛋白(yellowfluorescent protein for energy transfer，Ypet)，它是已经开发的亮度最强的黄色荧光蛋白，有很好的光稳定性，对酸性环境的耐受性要比Venus及其他黄色荧光蛋白变体强。考虑到黄色荧光蛋白的亮度很强、光稳定性好等优点，我们将Annexin A5与目前常用的三种黄色荧光蛋白变体 Citrine、Venus和Ypet融合表达来制备凋亡检测探针，以期望获得适合于流式细胞术检测的黄色荧光蛋白标记的Annexin A5探针，作为绿色荧光标记的Annexin A5的替代检测探针，以拓宽细胞凋亡检测探针的应用光谱范围。
本发明构建了三种新的黄色荧光蛋白标记的Annexin A5：Annexin A5-Citrine、Annexin A5-Venus和Annexin A5-Ypet，这三种融合蛋白均可以识别和标记凋亡细胞，可以用于凋亡细胞的识别和标记。
发明内容
本发明本文选择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。以本实验室保存的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。大量表达Annexin A5-Citrine、Annexin A5-Venus 和Annexin A5-Ypet融合蛋白，通过Ni柱亲和层析纯化获得了这三种蛋白，纯度约为90％。本发明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3113nmol/L，444.3nmol/L和391.6nmol/L。本发明的这三种融合蛋白均可以识别和标记凋亡细胞，可以用于凋亡细胞的识别和标记。
本发明提供了Annexin A5-Citrine、Annexin A5-Venus和Annexin A5-Ypet融合蛋白，及其表达和纯化方法；本发明还提供了所获得的重组Annexin A5-Citrine、 AnnexinA5-Venus和Annexin A5-Ypet融合蛋白可以用于凋亡细胞的识别和标记。
附图说明
图1三种黄色荧光蛋白标记的Annexin A5蛋白(the pET28a-Annexin A5-Citrine, pET28a-Annexin A5-Venus and pET28a-Annexin A5-Ypet)表达载体的结构。其中(GS)4作为连接序列，内切酶为点位：NdeⅠ和XhoⅠ。
图2三种蛋白Annexin A5-Citrine,Annexin A5-Venus和Annexin A5-Ypet的SDS-PAGE分析。
图a:37℃诱导表达，各个泳道情况为——M泳道：蛋白Marker；泳道1:含pET28a载体的细菌内总蛋白,IPTG诱导；泳道2:含pET28a-Annexin A5-Citrine载体的细菌内总蛋白, IPTG诱导；泳道3:含pET28a-Annexin A5-Venus载体的细菌内总蛋白,IPTG诱导；泳道4: 含pET28a-Annexin A5-Ypet载体的细菌内总蛋白,IPTG诱导。
图b:20℃诱导表达，各个泳道情况为——M泳道：蛋白Marker；泳道1:含pET28a载体的细菌内总蛋白,IPTG诱导；泳道2:含pET28a-Annexin A5-Citrine载体的细菌内总蛋白, IPTG诱导；泳道3:含pET28a-Annexin A5-Venus载体的细菌内总蛋白,IPTG诱导；泳道4: 含pET28a-Annexin A5-Ypet载体的细菌内总蛋白,IPTG诱导。
图3三种蛋白Annexin A5-Citrine,Annexin A5-Venus和Annexin A5-Ypet的可溶性表达的 SDS-PAGE分析。
图a:左图：37℃诱导表达，各个泳道情况为——M泳道：蛋白Marker；泳道1-2:含
pET28a-Annexin A5-Citrine载体的细菌的上清和沉淀蛋白,IPTG诱导；泳道3-4:含
pET28a-Annexin A5-Venus载体的细菌的上清和沉淀蛋白,IPTG诱导；泳道5-6:含
pET28a-Annexin A5-Ypet载体的细菌的上清和沉淀蛋白,IPTG诱导
右图：可溶性蛋白与总蛋白的比值；
图b:左图：20℃诱导表达，各个泳道情况为——M泳道：蛋白Marker；泳道1-2:含pET28a-Annexin A5-Citrine载体的细菌的上清和沉淀蛋白,IPTG诱导；泳道3-4:含
pET28a-Annexin A5-Venus载体的细菌的上清和沉淀蛋白,IPTG诱导；泳道5-6:含
pET28a-Annexin A5-Ypet载体的细菌的上清和沉淀蛋白,IPTG诱导
右图：可溶性蛋白与总蛋白的比值。
图4三种蛋白Annexin A5-Citrine,Annexin A5-Venus和Annexin A5-Ypet经过Ni柱纯化之后的变性SDS-PAGE分析。各个泳道情况为——M泳道：蛋白Marker；泳道1:AnnexinA5-Venus，泳道2:Annexin A5-Ypet，泳道3:Annexin A5-Citrine。
图5三种蛋白Annexin A5-Citrine,Annexin A5-Venus和Annexin A5-Ypet经过Ni柱纯化之后的非变性SDS-PAGE分析。各个泳道情况为——M泳道：蛋白Marker；泳道1:Annexin A5-Venus，泳道2:Annexin A5-Ypet，泳道3:Annexin A5-Citrine。
图6三种蛋白的粒径大小分布。图a：Annexin A5-Citrine，图b：Annexin A5-Venus.图c： Annexin A5-Ypet。
图7三种蛋白用于流式凋亡检测。图a：Annexin A5-Citrine标记的流式凋亡检测；图b: Annexin A5-Citrine标记的流式凋亡检测的点图分析。
图8三种蛋白对凋亡的检出能力。图a,b,c:分别为AnnexinA5-Citrine,AnnexinA5-Venus和 Annexin A5-Ypet蛋白结合凋亡细胞之后平均荧光强度的分析。图d:三种蛋白与凋亡细胞的亲和力。
图9三种蛋白Citrine,Venus和Ypet的蛋白氨基酸序列分析。
具体实施方式
实施例重组L-asp的表达纯化、性质测定、活性研究和体外安全性研究
材料与方法
实验材料pET28a空载质粒、pET28a-Annexin A5-EGFP-his由本实验室保存。pET28a-Annexin A5-Citrine-his、pET28a-Annexin A5-Venus-his、pET28a-Annexi n A5-Ypet-his均以pET28a-Annexin A5-EGFP-his为模板重新构建。Top10、BL 21菌株保存于本实验室中。Jurkat淋巴瘤细胞由本实验室保存。依托泊苷购自江苏凯基生物技术股份有限公司。
荧光蛋白Citrine、Venus、Ypet cDNA序列的扩增与克隆构建根据Citrine、Venus、Ypet的cDNA序列设计特异性引物，上下游引物中引入NdeⅠ和XhoⅠ酶切位点，具体引物序列如表1所示。PCR扩增条件为94℃预变性5min；94℃变性30s，60℃退火30s，72℃延伸1min，28个循环；72℃延伸7min。
Table 1 Primers of Citrine,Venus and Ypet PCR
将全长的Citrine、Venus、Ypet基因片段和本实验室保存的pET28a-Annexin A5-EGFP-his重组质粒经NdeⅠ和XhoⅠ双酶切并纯化后进行连接，使得Citrine、 Venus、Ypet的N-末端和Annexin A5的C-末端通过Linker蛋白连接，获得了重组质粒pET28a-Annexin A5-Citrine-his、pET28a-Annexin A5-Venus-his、 pET28a-Annexin A5-Ypet-his(图1)。重组质粒转化Top10感受态菌株，涂布卡那霉素抗性平板筛选阳性克隆，菌落PCR鉴定后，送至南京金斯瑞生物科技有限公司进行测序。
Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白的表达分析pET28a空载质粒、pET28a-Annexin A5-Citrine-his、pET28a-Annexin A5-Venus-his、pET28a-Annexin A5-Ypet-his重组质粒分别转化大肠杆菌 BL21(DE3)感受态细胞，分别挑取单克隆接种到3mL含有50μg/mL卡那霉素的 LB液体培养基中，220rpm、37℃振荡培养过夜后，将含有pET28a空载质粒、 pET28a-Annexin A5-Citrine-his、pET28a-Annexin A5-Venus-his、pET28a-Annexin A5-Ypet-his重组质粒的过夜菌按照1:50的比例转接到3mL含有50μg/mL卡那霉素的LB液体培养基中。220rpm、37℃振荡培养至OD600值约为0.6时，加入异丙基-β-D-硫代半乳糖苷诱导剂(isopropyl-β-D-thiogalactoside,IPTG)至终浓度为1mM，分别在37℃和20℃条件下诱导6h和16h。诱导完成后，8000rpm离心5min，收集菌体至1.5mLEP管中，然后用670μL PBS缓冲液重悬菌体，对菌体重悬液进行超声破碎(超声条件为：超声2s/间隔4s，总共2min)，超声结束后，分别取40μL总蛋白于1.5mL EP管中，对含有pET28a-Annexin A5-Citrine-his、pET28a-Annexin A5-Venus-his、pET28a-Annexin A5-Ypet-his的菌体重悬液进行12000rpm离心10min，将上清转移至1.5mL EP管中，沉淀用670 μL PBS重悬，对上述样品进行SDS-PAGE分析。
镍柱亲和层析纯化Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白将3mL含有pET28a-Annexin A5-Citrine-his、pET28a-Annexin A5-Venus-his、pET28a-Annexin A5-Ypet-his重组质粒的过夜活化菌转接至400mL 含有50μg/mL卡那霉素的LB液体培养基中，220rpm、37℃振荡培养3-4h， OD600值达到0.6后，加入IPTG至终浓度为1mM，20℃诱导培养16h。6000rpm 离心5min收集菌体，弃去培养基，然后用40mL 20mM Tris缓冲液(含250 mMNaCl)缓冲液重悬菌体，在冰浴条件下超声破碎菌体(超声条件为：超声4s/ 间隔8s，总共40min)。超声结束后，菌液在4℃条件下，16000rpm离心20min，收集上清。
使用NTA-Ni亲和层析进行蛋白纯化，镍柱使用前先用20mM Tris缓冲液(含 250mMNaCl)进行平衡，平衡体积约为填料体积的5～10倍。上述得到的上清经0.22μm滤膜过滤后上样。上样完毕后，用含有50mM咪唑的20mM Tris缓冲液(含250mM NaCl)清洗未能结合在镍柱上的杂蛋白，然后用含有250mM 咪唑的20mM Tris缓冲液(含250mM NaCl)洗脱目的蛋白，收集最大吸收峰处的洗脱液。Ni柱洗脱收集液中含有高浓度咪唑，使用4℃透析法以除去咪唑。用含不同浓度咪唑的20mM Tris缓冲液(含30mMNaCl)作为透析外液梯度透析目的蛋白24h，除掉目的蛋白中的咪唑。收集样品，进行12％SDS-PAGE，分析目的蛋白纯度。BCA试剂盒测定蛋白浓度，计算产率。
Native-PAGE分析Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白形成聚体的情况上述蛋白溶液测过浓度后，我们各取20μg蛋白进行Native-PAGE。注意Native-PAGE全程需在4℃条件下进行，电泳缓冲液需要预冷，蛋白样品不可在沸水浴中进行变性处理。在配制电泳缓冲液和制胶时不可加入SDS，5×Loading Buffer中不可加入SDS和β-巯基乙醇，因为SDS和β- 巯基乙醇均会导致蛋白质发生变性。
动态光散射实验(DLS)探究Annexin A5-Citrine、Annexin A5-Venus、 AnnexinA5-Ypet融合蛋白的粒径大小及形成聚体的情况将3种融合蛋白分别稀释为3.0mg/mL，然后取300μL蛋白稀释液加入样品池中，使用马尔文粒径分析仪测量目的蛋白的粒径大小并分析其形成聚体的情况。
Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白的凋亡检测Jurkat淋巴瘤细胞用10％胎牛血清的RPMI-1640，在37℃，5％CO2条件下培养。1×106个/mL的细胞用25μM依托泊苷诱导凋亡6h。每5×105个细胞，在4℃条件下800rpm离心5min，弃去培养基，并用PBS缓冲液洗涤细胞两次，再用Binding Buffer缓冲液洗涤细胞两次，最终重悬于200μL的Binding Buffer 缓冲液中。将Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白提前定量稀释到一定浓度，浓度分别为4、10、20、50、100、200、500、1000、 2000、5000nM，分别取200μL不同浓度的Annexin A5-Citrine、Annexin A5-Venus、 Annexin A5-Ypet融合蛋白的稀释液加入到200μL细胞悬液中，使Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白的终浓度分别为2、5、 10、25、50、100、250、500、1000、2500nM。冰上避光孵育30min，然后加入1μL PI，冰上避光孵育5min。流式细胞仪检测：根据融合蛋白的荧光光谱，选择488nm作为激发光波长，选用波长530/30的滤器检测Citrine、Venus和Ypet (FL1)，选用波长大于560nm的滤器检测PI(FL-3)。
Citrine、Venus、Ypet三种荧光蛋白的氨基酸序列比对根据NCBI中提供的Citrine、Venus、Ypet的蛋白质序列，利用DNAMAN软件对这三种荧光蛋白进行多重序列比对，
数据处理采用Graphpad 6软件进行分析。
结果
1 表达载体的构建
根据NCBI中Citrine、Venus和Ypet的cDNA序列设计特异性引物，以本实验室保存的含有Citrine、Venus和Ypet的重组质粒为模板，通过PCR扩增出了 Citrine、Venus和Ypet全长序列。经过NdeⅠ和XhoⅠ双酶切后，将Citrine、Venus 和Ypet片段插入到重组质粒pET28a-Annexin A5-EGFP-his的NdeⅠ和XhoⅠ位点之间，取代原来EGFP的位置，构建了C-末端带有His-Tag的pET28a-Annexin A5-Citrine、pET28a-Annexin A5-Venus、pET28a-Annexin A5-Ypet表达质粒(图1)。菌落PCR鉴定及测序验证重组质粒中Annexin A5-Citrine、Annexin A5-Venus、 Annexin A5-Ypet的序列正确后，转化大肠杆菌BL21(DE3)菌株。
2 Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet的小量表达
将重组质粒pET28a-Annexin A5-Citrine、pET28a-Annexin A5-Venus、 pET28a-Annexin A5-Ypet转化至大肠杆菌BL21(DE3)后，加入1mM IPTG，在 37℃和20℃条件下分别诱导表达，通过SDS-PAGE分析发现在分子量约为65KDa 处有目的蛋白表达条带(图2)，与融合蛋白的理论分子量一致(表2)，而空载质粒在相应的分子量附近未出现明显的蛋白质条带(图2)，这说明这三种融合蛋白在37℃和20℃条件下均成功表达。对37℃和20℃条件下目的蛋白的可溶性产量进行分析，发现20℃诱导表达时，目的蛋白在上清中的可溶性比例明显高于目的蛋白在37℃时的可溶性比例(图3)。结果表明：Annexin A5-Citrine在37℃和20℃条件下的可溶性比例分别为表达蛋白的68.59％和71.57％；Annexin A5-Venus在37℃和20℃条件下的可溶性比例分别为表达蛋白的73.44％和86％；而Annexin A5-Ypet在37℃和20℃条件下的可溶性表达比例分别为表达蛋白的 42.86％和85.42％。
Table 2 The Molecular Weight of Annexin A5-Citrine,Annexin A5-Venusand Annexin A5-Ypet
3 Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet的表达和纯化
根据上述初步表达的结果，我们选择20℃作为目的蛋白的诱导表达温度，诱导时间为16h。Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白表达后，借助融合蛋白的C-末端的His-Tag，利用Ni柱亲和层析纯化Annexin A5-Citrine、AnnexinA5-Venus、Annexin A5-Ypet，纯化获得的蛋白浓度分别为10.37mg/mL、4.85mg/mL、5.37mg/mL，最终纯化产率分别为103.7mg/400mL培养液、40.50mg/400mL培养液和48.33mg/mL培养液，获得的蛋白质纯度均为约 90％左右。
4 Native-PAGE分析Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白形成聚体的情况
Annexin A5蛋白可以形成聚体，荧光蛋白Citrine和Venus主要以单体形式存在，而Ypet可以形成弱二聚体，蛋白是否形成聚体会影响融合蛋白与磷脂酰丝氨酸的结合能力。为了探究Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白是否会形成聚体，我们对这三种融合蛋白进行了Native-PAGE 分析。结果表明这三种融合蛋白均只有一条明显的蛋白质条带，说明没有聚体形成(图5)。
5 Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白的粒径分析
为了进一步探究Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet 形成聚体的情况，我们对这三种融合蛋白进行了动态光散射实验，结果表明这三种融合蛋白的粒径大小均一，而且融合蛋白也不形成聚体(图6)。
6 Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet融合蛋白的凋亡检测分析
将纯化得到的Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet利用流式细胞术观察它与凋亡细胞的结合情况。设置不同的浓度梯度，对细胞进行分群(图7a)，圈选Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet标记的凋亡细胞群(图7b)。对Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet标记的凋亡细胞表面的平均荧光强度进行分析(图8a-c)，结果表明三种融合蛋白的平均荧光强度均随着蛋白浓度的增加而增加，说明纯化得到的 Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet可与凋亡细胞发生特异性结合，能够标记凋亡细胞。
同时，我们对Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet与凋亡细胞的亲和力进行定量分析。最终得出Annexin A5-Citrine与凋亡细胞的亲和力为3113nmol/L(图8d)，Annexin A5-Venus与凋亡细胞的亲和力为444.3 nmol/L(图8d)，Annexin A5-Ypet与凋亡细胞的亲和力为391.6nmol/L(图8d)。三者相比，Annexin A5-Venus和Annexin A5-Ypet与凋亡细胞的亲和力同属纳摩尔级，且属于同一个数量级，说明Annexin A5-Venus和Annexin A5-Ypet均可以用于细胞凋亡检测。而Annexin A5-Citrine与凋亡细胞的亲和力不如另外两个蛋白高，虽然它也可以用于标记凋亡细胞，但就效果而言，却不如Annexin A5-Venus 和Annexin A5-Ypet标记凋亡的效果好。
7 Citrine、Venus、Ypet三种荧光蛋白的氨基酸序列比对
以上结果显示，Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet三种融合蛋白在相对分子量、表达特性、形成聚体的情况、粒径分布等方面均没有明显差异，但是他们与凋亡细胞结合时却表现出了截然不同的亲和力。于是，我们对 Citrine、Venus、Ypet的氨基酸序列进行比对分析以探究这三种荧光蛋白的结构差异对Annexin A5凋亡检测功能的影响。
Citrine、Venus、Ypet均由最初的黄色荧光蛋白EYFP改造而来，通过氨基酸序列比对(图9)，我们发现了三者的氨基酸在多个位点上的差异(表3)。研究发现[25]，F46L突变促进了发色团附近局部结构的改变，因此促进了发色团的氧化，这是发色团成熟的一个限速步骤。此外，也有研究[25]发现：F64L突变造成了β-桶结构内部主要结构的变化，从而阻止了卤化物离子进入到发色团附近的结合腔。M153T，V163A，S175G这三个重要的突变在β-桶外的loop区引入了高度灵活的、更小的侧链，从而促进了荧光蛋白的成熟过程。F46L、F64L、M153T，V163A，S175G是Venus和Ypet区别于最初的EYFP最重要的五个突变，而Citrine在这五个位点的氨基酸并没有改变，这也许可以解释Annexin A5-Citrine与凋亡细胞的亲和力比Annexin A5-Venus、Annexin A5-Ypet与凋亡细胞的亲和力小，说明黄色荧光蛋白的结构会影响Annexin A5与凋亡细胞的结合。与Annexin A5-Venus相比，Annexin A5-Ypet在208、231、234位的差异可能有造成两者亲和力有所差异的结构原因。
Tabel 3 Comparison of sequence difference between Citrine,Venus andYpet.
讨论
Annexin A5是膜联蛋白家族的成员之一，它能够钙依赖性地与凋亡细胞表面的磷脂酰丝氨酸结合，因而可以用于凋亡检测。自从被开发为凋亡探针至今， Annexin A5被广泛用于体外凋亡检测。目前，化学偶联方法标记的Annexin A5 被广泛使用，如FITC(异硫氰酸荧光素)标记的Annexin A5。该探针的制备过程非常复杂繁琐，而且氨基介导的化学修饰过程还会导致Annexin A5与凋亡细胞PS的结合能力下降。另外，化学标记Annexin A5的最终产物通常是不同标记程度的混合物，非常不均一。研究发现，融合了荧光蛋白的AnnexinA5相对均一，且荧光更强、更亮、更稳定[16,17,19,20]。目前市场上只有Annexin A5-EGFP绿色的荧光蛋白融合分子被广泛应用于细胞凋亡检测。
黄色荧光蛋白作为绿色荧光蛋白的一种变体，其荧光向红色光谱偏移，最大激发波长为514nm，最大发射波长为527nm，因此具有比绿色荧光更为广泛的应用面。这主要是由于蛋白203位苏氨酸变为酪氨酸。最初被使用的黄色荧光蛋白是EYFP，目前有三种改良的黄色荧光蛋白：Citrine、Venus、Ypet，三者的氨基酸序列相似性达98％以上。我们选择这三种黄色荧光蛋白与Annexin A5融合制备凋亡检测探针，结果表明Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet与凋亡细胞的结合能力差异很大，其亲和力大小是Annexin A5-Ypet>Annexin A5-Venus>Annexin A5-Citrine。这三种融合蛋白在相对分子量、表达特性、形成聚体的情况、粒径分布等方面均没有明显差异，于是我们猜测荧光蛋白的氨基酸序列可能影响了Annexin A5与凋亡细胞的结合。通过查阅文献，我们发现：与EYFP相比，改良的黄色荧光蛋白有五个重要的突变，分别是F46L、 F64L、M153T、V163A、S175G，这些突变对荧光蛋白的特性有重要的影响。比对结果表明，Venus和Ypet含有这五个位点的突变，而Citrine不含有这五个位点的突变，这也许是Annexin A5-Citrine、Annexin A5-Venus、Annexin A5-Ypet 与凋亡细胞结合能力差异很大的原因，此外，我们还发现AnnexinA5-Venus与 Annexin A5-Ypet在S208P、L231E、D234N位的差异可能有造成两者亲和力有所差异的结构原因。以上结果说明不同黄色荧光蛋白在结构上的细微差异仍让能够较显著地影响Annexin A5与凋亡细胞的结合。
序列表
<110> 南京吉瑞康生物科技有限公司
<120> 用于凋亡细胞识别和标记的融合蛋白及其制备方法和应用
<130> 20180706-2
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1734
<212> DNA
<213> 人工序列(.)
<400> 1
atggcacagg ttctcagagg cactgtgact gacttccctg gatttgatga gcgggctgat 60
gcagaaactc ttcggaaggc tatgaaaggc ttgggcacag atgaggagag catcctgact 120
ctgttgacat cccgaagtaa tgctcagcgc caggaaatct ctgcagcttt taagactctg 180
tttggcaggg atcttctgga tgacctgaaa tcagaactaa ctggaaaatt tgaaaaatta 240
attgtggctc tgatgaaacc ctctcggctt tatgatgctt atgaactgaa acatgccttg 300
aagggagctg gaacaaatga aaaagtactg acagaaatta ttgcttcaag gacacctgaa 360
gaactgagag ccatcaaaca agtttatgaa gaagaatatg gctcaagcct ggaagatgac 420
gtggtggggg acacttcagg gtactaccag cggatgttgg tggttctcct tcaggctaac 480
agagaccctg atgctggaat tgatgaagct caagttgaac aagatgctca ggctttattt 540
caggctggag aacttaaatg ggggacagat gaagaaaagt ttatcaccat ctttggaaca 600
cgaagtgtgt ctcatttgag aaaggtgttt gacaagtaca tgactatatc aggatttcaa 660
attgaggaaa ccattgaccg cgagacttct ggcaatttag agcaactact ccttgctgtt 720
gtgaaatcta ttcgaagtat acctgcctac cttgcagaga ccctctatta tgctatgaag 780
ggagctggga cagatgatca taccctcatc agagtcatgg tttccaggag tgagattgat 840
ctgtttaaca tcaggaagga gtttaggaag aattttgcca cctctcttta ttccatgatt 900
aagggagata catctgggga ctataagaaa gctcttctgc tgctctgtgg agaagatgac 960
ggatccggtt ctggcagcgg ctcaggtcat atggtgagca agggcgagga gctgttcacc 1020
ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg 1080
tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc 1140
accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta cggcctgatg 1200
tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc 1260
gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc 1320
gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac 1380
ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac 1440
gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac 1500
aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc 1560
gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc cctgagcaaa 1620
gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc 1680
actctcggca tggacgagct gtacaagctc gagcaccacc accaccacca ctga 1734
<210> 2
<211> 1734
<212> DNA
<213> 人工序列(.)
<400> 2
atggcacagg ttctcagagg cactgtgact gacttccctg gatttgatga gcgggctgat 60
gcagaaactc ttcggaaggc tatgaaaggc ttgggcacag atgaggagag catcctgact 120
ctgttgacat cccgaagtaa tgctcagcgc caggaaatct ctgcagcttt taagactctg 180
tttggcaggg atcttctgga tgacctgaaa tcagaactaa ctggaaaatt tgaaaaatta 240
attgtggctc tgatgaaacc ctctcggctt tatgatgctt atgaactgaa acatgccttg 300
aagggagctg gaacaaatga aaaagtactg acagaaatta ttgcttcaag gacacctgaa 360
gaactgagag ccatcaaaca agtttatgaa gaagaatatg gctcaagcct ggaagatgac 420
gtggtggggg acacttcagg gtactaccag cggatgttgg tggttctcct tcaggctaac 480
agagaccctg atgctggaat tgatgaagct caagttgaac aagatgctca ggctttattt 540
caggctggag aacttaaatg ggggacagat gaagaaaagt ttatcaccat ctttggaaca 600
cgaagtgtgt ctcatttgag aaaggtgttt gacaagtaca tgactatatc aggatttcaa 660
attgaggaaa ccattgaccg cgagacttct ggcaatttag agcaactact ccttgctgtt 720
gtgaaatcta ttcgaagtat acctgcctac cttgcagaga ccctctatta tgctatgaag 780
ggagctggga cagatgatca taccctcatc agagtcatgg tttccaggag tgagattgat 840
ctgtttaaca tcaggaagga gtttaggaag aattttgcca cctctcttta ttccatgatt 900
aagggagata catctgggga ctataagaaa gctcttctgc tgctctgtgg agaagatgac 960
ggatccggtt ctggcagcgg ctcaggtcat atggtgagca agggcgagga gctgttcacc 1020
ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg 1080
tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagct gatctgcacc 1140
accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgggcta cggcctgcag 1200
tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc 1260
gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc 1320
gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac 1380
ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac 1440
gtctatatca ccgccgacaa gcagaagaac ggcatcaagg ccaacttcaa gatccgccac 1500
aacatcgagg acggcggcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc 1560
gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc cctgagcaaa 1620
gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc 1680
actctcggca tggacgagct gtacaagctc gagcaccacc accaccacca ctga 1734
<210> 3
<211> 1731
<212> DNA
<213> 人工序列(.)
<400> 3
atggcacagg ttctcagagg cactgtgact gacttccctg gatttgatga gcgggctgat 60
gcagaaactc ttcggaaggc tatgaaaggc ttgggcacag atgaggagag catcctgact 120
ctgttgacat cccgaagtaa tgctcagcgc caggaaatct ctgcagcttt taagactctg 180
tttggcaggg atcttctgga tgacctgaaa tcagaactaa ctggaaaatt tgaaaaatta 240
attgtggctc tgatgaaacc ctctcggctt tatgatgctt atgaactgaa acatgccttg 300
aagggagctg gaacaaatga aaaagtactg acagaaatta ttgcttcaag gacacctgaa 360
gaactgagag ccatcaaaca agtttatgaa gaagaatatg gctcaagcct ggaagatgac 420
gtggtggggg acacttcagg gtactaccag cggatgttgg tggttctcct tcaggctaac 480
agagaccctg atgctggaat tgatgaagct caagttgaac aagatgctca ggctttattt 540
caggctggag aacttaaatg ggggacagat gaagaaaagt ttatcaccat ctttggaaca 600
cgaagtgtgt ctcatttgag aaaggtgttt gacaagtaca tgactatatc aggatttcaa 660
attgaggaaa ccattgaccg cgagacttct ggcaatttag agcaactact ccttgctgtt 720
gtgaaatcta ttcgaagtat acctgcctac cttgcagaga ccctctatta tgctatgaag 780
ggagctggga cagatgatca taccctcatc agagtcatgg tttccaggag tgagattgat 840
ctgtttaaca tcaggaagga gtttaggaag aattttgcca cctctcttta ttccatgatt 900
aagggagata catctgggga ctataagaaa gctcttctgc tgctctgtgg agaagatgac 960
ggatccggtt ctggcagcgg ctcaggtcat atgagcaaag gcgaagagct gttcaccggc 1020
gtggtgccca tcctggtgga gctggacggc gacgtgaacg gccacaagtt cagcgtgagc 1080
ggcgagggcg agggcgacgc cacctacggc aagctgaccc tgaagctgct gtgcaccacc 1140
ggcaagctgc ccgtgccctg gcccaccctg gtgaccaccc tgggctacgg cgtgcagtgc 1200
ttcgcccgct accccgacca catgaagcag cacgacttct tcaagagcgc catgcccgag 1260
ggctacgtgc aggagcggac catcttcttc aaggacgacg gcaactacaa gacccgggcc 1320
gaggtgaagt tcgagggcga caccctggtg aaccggatcg agctgaaggg catcgacttc 1380
aaggaggacg gcaacatcct gggccacaag ctggagtaca actacaacag ccacaacgtg 1440
tacatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat ccggcacaac 1500
atcgaggacg gcggcgtgca gctggccgac cactaccagc agaacacccc catcggcgac 1560
ggccccgtgc tgctgcccga caaccactac ctgagctacc agagcgccct gttcaaggac 1620
cccaacgaga agcgggacca catggtgctg ctggagttcc tgaccgccgc cggcatcacc 1680
gagggcatga acgagctcta taagctcgag caccaccacc accaccactg a 1731
<210> 4
<211> 577
<212> PRT
<213> 人工序列(.)
<400> 4
Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp
1 5 10 15
Glu Arg Ala Asp Ala Glu Thr Leu Arg Lys Ala Met Lys Gly Leu Gly
20 25 30
Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala
35 40 45
Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp
50 55 60
Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu
65 70 75 80
Ile Val Ala Leu Met Lys Pro Ser Arg Leu Tyr Asp Ala Tyr Glu Leu
85 90 95
Lys His Ala Leu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu
100 105 110
Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Val
115 120 125
Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp
130 135 140
Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn
145 150 155 160
Arg Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala
165 170 175
Gln Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu
180 185 190
Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys
195 200 205
Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr
210 215 220
Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val
225 230 235 240
Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr
245 250 255
Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val
260 265 270
Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg Lys Glu Phe
275 280 285
Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile Lys Gly Asp Thr
290 295 300
Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp
305 310 315 320
Gly Ser Gly Ser Gly Ser Gly Ser Gly His Met Val Ser Lys Gly Glu
325 330 335
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp
340 345 350
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
355 360 365
Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu
370 375 380
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Phe Gly Tyr Gly Leu Met
385 390 395 400
Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
405 410 415
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
420 425 430
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
435 440 445
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp
450 455 460
Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn
465 470 475 480
Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe
485 490 495
Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His
500 505 510
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
515 520 525
Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu
530 535 540
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
545 550 555 560
Thr Leu Gly Met Asp Glu Leu Tyr Lys Leu Glu His His His His His
565 570 575
His
<210> 5
<211> 577
<212> PRT
<213> 人工序列(.)
<400> 5
Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp
1 5 10 15
Glu Arg Ala Asp Ala Glu Thr Leu Arg Lys Ala Met Lys Gly Leu Gly
20 25 30
Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala
35 40 45
Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp
50 55 60
Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu
65 70 75 80
Ile Val Ala Leu Met Lys Pro Ser Arg Leu Tyr Asp Ala Tyr Glu Leu
85 90 95
Lys His Ala Leu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu
100 105 110
Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Val
115 120 125
Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp
130 135 140
Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn
145 150 155 160
Arg Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala
165 170 175
Gln Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu
180 185 190
Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys
195 200 205
Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr
210 215 220
Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val
225 230 235 240
Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr
245 250 255
Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val
260 265 270
Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg Lys Glu Phe
275 280 285
Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile Lys Gly Asp Thr
290 295 300
Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp
305 310 315 320
Gly Ser Gly Ser Gly Ser Gly Ser Gly His Met Val Ser Lys Gly Glu
325 330 335
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp
340 345 350
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
355 360 365
Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu
370 375 380
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Gln
385 390 395 400
Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
405 410 415
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
420 425 430
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
435 440 445
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp
450 455 460
Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn
465 470 475 480
Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe
485 490 495
Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His
500 505 510
Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp
515 520 525
Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu
530 535 540
Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile
545 550 555 560
Thr Leu Gly Met Asp Glu Leu Tyr Lys Leu Glu His His His His His
565 570 575
His
<210> 6
<211> 576
<212> PRT
<213> 人工序列(.)
<400> 6
Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe Asp
1 5 10 15
Glu Arg Ala Asp Ala Glu Thr Leu Arg Lys Ala Met Lys Gly Leu Gly
20 25 30
Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg Ser Asn Ala
35 40 45
Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu Phe Gly Arg Asp
50 55 60
Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly Lys Phe Glu Lys Leu
65 70 75 80
Ile Val Ala Leu Met Lys Pro Ser Arg Leu Tyr Asp Ala Tyr Glu Leu
85 90 95
Lys His Ala Leu Lys Gly Ala Gly Thr Asn Glu Lys Val Leu Thr Glu
100 105 110
Ile Ile Ala Ser Arg Thr Pro Glu Glu Leu Arg Ala Ile Lys Gln Val
115 120 125
Tyr Glu Glu Glu Tyr Gly Ser Ser Leu Glu Asp Asp Val Val Gly Asp
130 135 140
Thr Ser Gly Tyr Tyr Gln Arg Met Leu Val Val Leu Leu Gln Ala Asn
145 150 155 160
Arg Asp Pro Asp Ala Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala
165 170 175
Gln Ala Leu Phe Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu
180 185 190
Lys Phe Ile Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys
195 200 205
Val Phe Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr
210 215 220
Ile Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val
225 230 235 240
Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu Tyr
245 250 255
Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile Arg Val
260 265 270
Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg Lys Glu Phe
275 280 285
Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile Lys Gly Asp Thr
290 295 300
Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu Cys Gly Glu Asp Asp
305 310 315 320
Gly Ser Gly Ser Gly Ser Gly Ser Gly His Met Ser Lys Gly Glu Glu
325 330 335
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
340 345 350
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
355 360 365
Tyr Gly Lys Leu Thr Leu Lys Leu Leu Cys Thr Thr Gly Lys Leu Pro
370 375 380
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Val Gln Cys
385 390 395 400
Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
405 410 415
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
420 425 430
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
435 440 445
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly
450 455 460
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val
465 470 475 480
Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys
485 490 495
Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Asp His Tyr
500 505 510
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
515 520 525
His Tyr Leu Ser Tyr Gln Ser Ala Leu Phe Lys Asp Pro Asn Glu Lys
530 535 540
Arg Asp His Met Val Leu Leu Glu Phe Leu Thr Ala Ala Gly Ile Thr
545 550 555 560
Glu Gly Met Asn Glu Leu Tyr Lys Leu Glu His His His His His His
565 570 575
Claims (9)
1.用于凋亡细胞识别和标记的融合蛋白Annexin A5-Citrine，其DNA序列如SEQ IDN0:1所示，其蛋白序列如SEQ ID N0:4所示。
2.用于凋亡细胞识别和标记的融合蛋白Annexin A5-Venus，其DNA序列如SEQ ID N0:2所示，其蛋白序列如SEQ ID N0:5所示。
3.用于凋亡细胞识别和标记的融合蛋白Annexin A5-Ypet，其DNA序列如SEQ ID N0:3所示，其蛋白序列如SEQ ID N0:6所示。
4.一种用于凋亡细胞识别和标记的融合蛋白Annexin A5-Citrine的制备方法，其特征为步骤如下：
(1)荧光蛋白Citrine序列的扩增；
(2)将全长的Citrine基因片段通过酶切位点NdeⅠ和XhoⅠ插入到pET28a-Annexin A5-his中，获得了重组质粒pET28a-Annexin A5-Citrine-his；
(3)将重组质粒pET28a-Annexin A5-Citrine-his转化Top10感受态细胞，大量获得重组质粒pET28a-Annexin A5-Citrine-his并转化到表达菌株BL21感受态细胞中，获得表达Annexin A5-Citrine的质粒和菌株；
(4)培养表达Annexin A5-Citrine的菌株；
(5)破菌，提取蛋白获得蛋白Annexin A5-Citrine。
5.一种用于凋亡细胞识别和标记的融合蛋白Annexin A5-Venus的制备方法，其特征为步骤如下：
(1)荧光蛋白Venus序列的扩增；
(2)将全长的Venus基因片段通过酶切位点NdeⅠ和XhoⅠ插入到pET28a-Annexin A5-his中，获得了重组质粒pET28a-Annexin A5-Venus-his；
(3)将重组质粒pET28a-Annexin A5-Venus-his转化Top10感受态细胞，大量获得重组质粒pET28a-Annexin A5-Venus-his并转化到表达菌株BL21感受态细胞中，获得表达Annexin A5-Venus的质粒和菌株；
(4)培养表达Annexin A5-Venus的菌株；
(5)破菌，提取蛋白获得蛋白Annexin A5-Venus。
6.一种用于凋亡细胞识别和标记的融合蛋白Annexin A5-Ypet的制备方法，其特征为步骤如下：
(1)荧光蛋白Ypet序列的扩增；
(2)将全长的Ypet基因片段通过酶切位点NdeⅠ和XhoⅠ插入到pET28a-Annexin A5-his中，获得了重组质粒pET28a-Annexin A5-Ypet-his；
(3)将重组质粒pET28a-Annexin A5-Ypet-his转化Top10感受态细胞，大量获得重组质粒pET28a-Annexin A5-Ypet-his并转化到表达菌株BL21感受态细胞中，获得表达AnnexinA5-Ypet的质粒和菌株；
(4)培养表达Annexin A5-Ypet的菌株；
(5)破菌，提取蛋白获得蛋白Annexin A5-Ypet。
7.融合蛋白Annexin A5-Citrine在凋亡细胞识别和标记中的应用。
8.融合蛋白Annexin A5-Venus在凋亡细胞识别和标记中的应用。
9.融合蛋白Annexin A5-Ypet在凋亡细胞识别和标记中的应用。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113295679A (zh) * | 2021-06-07 | 2021-08-24 | 东南大学 | 检测细胞凋亡的bret活体成像探针 |
| WO2023143464A1 (en) * | 2022-01-30 | 2023-08-03 | Nanjing Reju Therapeutics , Inc. | Fusion protein and application thereof |
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Non-Patent Citations (1)
| Title |
|---|
| 高梦月: "黄色荧光蛋白的结构对AnnexinA5凋亡检测功能的影响探究", 《中国科技论文在线，HTTP://WWW.PAPER.EDU.CN/RELEASEPAPER/CONTENT/201806-60》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113295679A (zh) * | 2021-06-07 | 2021-08-24 | 东南大学 | 检测细胞凋亡的bret活体成像探针 |
| WO2023143464A1 (en) * | 2022-01-30 | 2023-08-03 | Nanjing Reju Therapeutics , Inc. | Fusion protein and application thereof |
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