CN112500465B - 一种sumo化修饰捕获探针的合成方法及应用 - Google Patents
一种sumo化修饰捕获探针的合成方法及应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,涉及一种SUMO化修饰捕获探针、其合成方法及应用。所述的SUMO化修饰捕获探针分子式为SUMO‑C6H8NO3,所述的SUMO为人源SUMO‑1,其核苷酸序列如SEQ ID NO:1所示,其氨基酸序列如SEQ ID NO:2所示。本发明采用基因克隆得到编码人源SUMO‑1蛋白的多肽,通过化学合成方法得到SUMO‑C6H8NO3,SUMO‑C6H8NO3结构稳定,方便化学合成及检测应用。SUMO‑C6H8NO3荧光探针具有高度特异性灵敏性较高。
Description
技术领域
本发明属于生物技术领域,涉及一种SUMO化修饰捕获探针的合成方法及应用,具体涉及一种SUMO化修饰捕获探针(SUMO-(E)4-甲基-2-丁烯酸甲酯)(SUMO-C6H8NO3)、其合成方法及在肿瘤检测中的应用。
背景技术
乳腺癌是发生于乳腺腺体及导管的恶性肿瘤,是女性发病率最高的癌症,是当今世界人类疾病中最常见的发病和死亡原因之一,严重威胁着人类的生命和健康。因此乳腺癌的防治已是世界性的保健问题,是世界卫生组织重点防治的疾病。根据美国癌症协会ACS的数据,在美国,10万人中乳腺癌发病率约为113.3人,在我国,乳腺癌的发病率正逐年上升,在许多大城市已高居女性肿瘤的首位,给社会和家庭带来巨大的财产损失和精神痛苦。
目前,乳腺癌的发病机理还不完全清楚,尚无有效预防措施。众所周知,大多数癌症包括乳腺癌的发生、发展是一个长时间的,慢性的病理变化过程,目前所用乳腺癌的诊断方法只能诊断出瘤体已长到一定体积的癌症,这在许多情况下,已为时过晚。当前乳腺癌的治疗仍以药物治疗、放疗以及手术切除为主,但至今还没有一种行之有效的诊断和治疗方法。手术虽然切除了肿瘤病灶,但也部分甚至全部切除了相应的组织器官,丧失了其正常的功能;而放疗在放射线杀伤肿瘤细胞的同时,也对机体正常组织细胞产生了损伤作用,很大程度限制了肿瘤治疗;药物治疗由于疗效不强和具有较强的毒副作用,而给患者遭成了极大的生理和心理创伤。和其他癌症一样,乳腺癌的有效预防和治疗也有待于弄清和阐明乳腺癌的发病机理,因此基因疗法就显得尤为重要。
小泛素相关修饰物(small ubiquitin-related modifier,SUMO)化修饰是一种重要的动态可逆性蛋白质翻译后修饰,从发现至今已有20余年。迄今已经有超过3000种的SUMO化修饰的蛋白质被发现确认,并且SUMO化修饰对靶蛋白的功能具有重要的调节作用,如细胞亚定位、蛋白质稳定性、信号转导、酶活性、基因转录调控、细胞周期调节、细胞分化等。目前,在哺乳动物中,已经鉴定出4种不同的SUMO蛋白亚型,分别是SUMO-1、SUMO-2、SUMO-3和SUMO-4,SUMO分子在E1活化酶、E2结合酶和E3连接酶的参与下,其C端的双Gly与靶蛋白上赖氨酸侧链ε-NH2通过异肽键共价结合,调控底物蛋白的结构与功能。例如,p53的SUMO化修饰发生在第386位的赖氨酸残基上,并且PIAS(protein inhibitor of activatedSTATs)家族成员可以增强p53的稳定性,同时也有研究证实SUMO化能增强p53的转录活性,进而导致细胞凋亡。PTEN(phosphatidylinositol-3,4,5-trisphosphate-3-phosphatase)蛋白的第254、266和289位的赖氨酸残基可以被SUMO-1和SUMO-2修饰,下调PI3K/AKT通路,进而抑制细胞的增殖和肿瘤的生长。多个与p53相互作用的蛋白质可以被SUMO化,NF-κB、PTEN等信号通路的核心因子在肿瘤发生发展中的作用至少部分依赖SUMO蛋白的活性。因此SUMO化是未来癌症检测和治疗最有潜力的靶标之一。通过研究相关SUMO荧光探针,了解肿瘤发生发展过程中SUMO化水平的动态变化,能够为肿瘤的早期诊断和治疗开辟新的思路。
蛋白质的SUMO化修饰是一个动态且可逆的过程,因此去SUMO化酶被认为是药物治疗靶标。去SUMO化酶(DSP)是由一组SUMO特异性蛋白酶家族SENP(Sentrin/SUMO-specificprotease),SENPs能够调控靶蛋白的SUMO化修饰水平及活性,同时其自身的表达或活性也被一些调控因子所调节。通过序列比对,发现SENPs均具有200个氨基酸左右的酶活性区域,属于C48半胱氨酸蛋白酶,能够切断SUMO与靶蛋白间的异肽键,根据DSP的这一特性,我们设计一种靶向DSP活性中心的半胱氨酸的分子探针,能够特异性地识别并共价结合半胱氨酸,而不切断SUMO与靶蛋白间的异肽键,使其不会发生去SUMO化反应。
由于SUMO化修饰是一个动态的过程,因此对于细胞内SUMO化修饰的定位及动态修饰检测非常困难,目前急需要一种检测手段对SUMO化修饰进行精确定位和动态监测。特别是在一些肿瘤细胞中,SUMO化修饰程度的高低与肿瘤的恶性程度及预后密切相关,因此对于SUMO化修饰的细胞定位,定量分析以及动态修饰变化的追踪将有助于恶性肿瘤的诊断和预后评估。
发明内容
为了解决SUMO化检测中的问题,本发明的目的在于提供了一种SUMO化修饰捕获探针,该探针可快速检测细胞内SUMO化修饰蛋白的亚细胞定位、修饰程度以及动态修饰的变化过程。
本发明的技术方案为:
第一方面,本发明提供了一种SUMO化修饰捕获探针,其分子式为SUMO-C6H8NO3,结构式如下式(I)所示:
进一步地,所述的SUMO为人源SUMO-1,其核苷酸序列如SEQ ID NO:1所示,其氨基酸序列如SEQ ID NO:2所示。
第二方面,本发明提供了一种上述的SUMO化修饰捕获探针的制备方法,其包括以下步骤:
步骤1、通过基因克隆,得到人源SUMO的全长cDNA序列;
步骤2、将步骤1)得到的含有SUMO全长cDNA序列质粒,通过在大肠杆菌中重组表达SUMO蛋白多肽,并通过亲和纯化,得到纯品SUMO蛋白多肽;
步骤3、将步骤2)得到的纯品SUMO蛋白多肽,通过全化学合成方法,得到SUMO-C6H8NO3荧光探针。
具体地,所述的SUMO化修饰捕获探针的制备方法的步骤3中进一步包括:
3.1)将纯品SUMO蛋白多肽,氯甲酸乙酯及N-甲基吗啉于四氢呋喃,降温冷却至-20℃搅拌反应;
3.2)在步骤3.1)反应后,升温到-5℃,孵育后,在此温度下滴加叠氮化钠水溶液,室温反应;
3.3)将步骤3.2)所得产物以醋酸乙酯提取后,加无水硫酸镁干燥,减压浓缩回收得到的白色固体;
3.4)将步骤3.3)所的白色固体,溶于甲苯中,加热至100℃;
3.5)待步骤3.4)的反应液中有气泡放出时,加入烯丙氧羰基,于100℃搅拌反应;
3.6)步骤3.5)中反应结束后,降温到65℃以下即析出产品结晶,抽滤得到粗品,以甲醇为溶剂重结晶,得到晶体;
3.7)将步骤3.6)中所得晶体溶于干燥的三氯甲烷中;
3.8)待步骤3.7)中充分溶解后,冰浴下加入mCPBA,立即置换氮气,冰浴搅拌反应;
3.9)TLC监控至反应完毕,加水淬灭反应,乙酸乙酯萃取,饱和食盐水洗;
3.10)将步骤3.9)中有机相由无水硫酸钠干燥后,减压旋干溶剂,硅胶柱层析分离纯化,得到白色粉末;溶解后,经胰蛋白酶切后,LC-MS鉴定。
第三方面,本发明提供了上述的SUMO化修饰捕获探针以及上述方法制备获得的SUMO化修饰捕获探针检测SUMO化修饰的用途。
进一步的,所述的检测SUMO化修饰包括SUMO化修饰的细胞定位、定量分析和/或动态修饰变化分析。
第四方面,本发明提供了上述的SUMO化修饰捕获探针以及上述方法制备获得的SUMO化修饰捕获探针在制备用于癌症诊断和/或预后的试剂中的用途。
本发明的效果和益处是:
本发明采用基因克隆得到编码人源SUMO-1蛋白的多肽,通过化学合成方法得到SUMO-C6H8NO3,SUMO-C6H8NO3结构稳定,方便化学合成及检测应用。SUMO-C6H8NO3荧光探针具有高度特异性灵敏性较高。
附图说明
图1为人源SUMO-1分子的三维空间结构图。
图2为SUMO蛋白鉴定质谱图
图3为SUMO-1-C6H8NO3荧光探针分子的制备化学全合成方法示意图。
图4为SUMO-1-C6H8NO3荧光探针鉴定质谱图。
图5为SUMO-1-C6H8NO3荧光探针分子在MCF7中的成像效果示意图。
图6为SUMO-1-C6H8NO3荧光探针分子在裸鼠体内成像效果示意图。
具体实施方式
以下结合附图,通过实施例进一步说明本发明,但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是,应当理解的是,这些仅仅是示例性的,并不意图限制本发明,与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
人源SUMO-1基因克隆及表达
人源乳腺癌MCF-7细胞总RNA提取,mRNA纯化,mRNA反转录及cDNA文库构建,设计引物,利用PCR方法筛选人源SUMO1基因。5’端引物为5’-ATGTCTGACCAGGAGGCAAAACCT-3’(SEQID NO:3),3’端引物为其序列为5’–CTAAACTGTTGAATGACCCCCCGT–3’(SEQ ID NO:4)。所获阳性单克隆进行基因核苷酸序列测定。基因测序结果表明编码人源SUMO-1序列由306个核苷酸组成,自5’端至3’端序列为(SEQ ID NO:1):
编码人源SUMO-1蛋白分子,其氨基酸序列为(SEQ ID NO:2;NP_001005781.1):
Met-Ser-Asp-Gln-Glu-Ala-Lys-Pro-Ser-Thr-Glu-Asp-Leu-Gly-Asp-Lys-Lys-Glu-Gly-Glu-Tyr-Ile-Lys-Leu-Lys-Val-Ile-Gly-Gln-Asp-Ser-Ser-Glu-Ile-His-Phe-Lys-Val-Lys-Met-Thr-Thr-His-Leu-Lys-Lys-Leu-Lys-Glu-Ser-Yyr-Cys-Gln-Arg-Gln-Gly-Val-Pro-Met-Asn-Ser-Leu-Arg-Phe-Leu-Phe-Glu-Gly-Gln-Arg-Ile-Ala-Asp-Asn-His-Thr-Pro-Lys-Glu-Leu-Gly-Met-Glu-Glu-Glu-Asp-Val-Ile-Glu-Val-Tyr-Gln-Glu-Gln-Thr-Gly-Gly-His-Ser-Thr-Val(MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHSTV)
具体地,SUMO-1分子的基因的克隆包括:
1)人源MCF7细胞总RNA提取:
①取35mm培养皿培养的MCF7细胞,加入1m1 RNAiso Plus(Trizol,TAKARA,日本),置于脱色摇床上,室温条件下裂解5min。
②将细胞裂解液转移至1.5ml离心管中,并向管中加入200μl的三氯甲烷,剧烈振荡15s,室温静置5min。
③将②中的离心管放入低温冷冻离心机,在4℃条件下,12000rpm离心15min。
④将上清液转移至1.5ml RNase-Free的离心管中,加入500μl的异丙醇,温和翻转离心管使液体混匀,在室温条件下静置10min。
⑤将④中的离心管放入低温冷冻离心机,在4℃条件下,12000rpm离心10min。
⑥去除上清,向离心管中加入1ml 75%乙醇,用轻轻吹打RNA沉淀。
⑦将⑥中的离心管放入低温冷冻离心机中,在4℃条件下,5000rpm离心3min。
⑧用移液器小心除去上清,室温放置数分钟,干燥RNA。
⑨将30μl的RNase-Free水加入⑧中的离心管,溶解RNA。
2)人源cDNA文库构建:
I.cDNA第一链合成(mRNA反转录):
①在去除RNA酶的PCR管中加入1.0μl人源MCF7细胞总RNA、1.0μl Oligo(dT)primer和5.0μl RNase-Free ddH2O,使总体积达到7μl,混匀后短暂离心(2000rpm,30s),离心后于72℃保温10分钟;保温后再将离心管在4℃孵育2分钟。
②在上述离心管中加入以下试剂,2.0μl 5×M-MLV Buffer、0.5μl 10mM dNTPMix、0.5μl 40U/μl RNase Inhibitor和0.25μl RTase M-MLV,混合离心管中试剂并短暂离心(2000rpm,30s),在42℃保温60min,然后70℃保温15s。保温处理后将离心管置于冰上中止合成备用。
II.采用聚合酶链式反应(PCR)方法扩增SUMO1基因:
①将1μl cDNA模板、0.25μl Taq酶、10μl 5×PCR缓冲液、1μl 10mM dNTP、1.0μl5’PCR引物、1.0μl 3’PCR引物以及35.75μl ddH2O在PCR管中进行混合,离心后使混合液聚集于管底。
②在PCR仪中按以下程序扩增:95℃,5min;35个循环:94℃,30sec,56℃,30sec,72℃,1min。循环结束后,于72℃孵育10min,PCR结束后保存于-80℃冰箱。
3)人源SUMO-1基因克隆筛选:
PCR反应结束后,采用1%浓度的琼脂糖凝胶对上述PCR反应产物进行电泳,120V恒压30min后,置于凝胶成像仪观察并拍照,切取含有SUMO-1目的片段的凝胶,利用琼脂糖凝胶DNA回收试剂盒进行回收。将回收的目的片段连接到pGEX-4T-3载体,转化进DH5α感受态细胞。涂板并进行氨苄青霉素和蓝白斑双重筛选,挑取单菌落用M13引物PCR检测插入片段大小。挑取阳性菌落,摇菌提取质粒,送至生工生物工程(上海)股份有限公司进行核苷酸测序,并翻译成氨基酸序列。
4)SUMO蛋白表达及体外纯化
将pGEX-4T-3-SUMO1转化大肠杆菌BL21中,筛选出阳性克隆后,摇瓶培养,带生长到对数生长期,通过IPTG诱导4小时后,继续培养16小时后,超声破碎,并通过亲和纯化柱,纯化蛋白,MS鉴定(如图2)。
实施例2:SUMO-C6H8NO3探针分子合成
SUMO-C6H8NO3荧光探针分子的制备采用化学全合成方法(如图3),目标SUMO蛋白被分成3个片段,其中选择Ala6作为连接点,并以半胱氨酸进行取代以促进连接反应。
①将上述得到的SUMO蛋白0.5mmol,氯甲酸乙酯1.1mmol及N-甲基吗啉1.1mmol于10mL四氢呋喃,降温冷却至-20℃搅拌反应20min;
②上述反应后,升温到-5℃,孵育5min,在此温度下滴加叠氮化钠水溶液(叠氮化钠2.1mmol溶于2mL水中),室温反应15min;
③将上述产物以醋酸乙酯,提取(15mL×3)后,加无水硫酸镁干燥,减压浓缩回收得到的白色固体。
在SUMO-N端片段合成中,以侧链氨基酸被烯丙氧羰基(Alloc)保护的2-氨基丁酸(Dab)取代修饰位点的赖氨酸,并在N端缩合标签分子(如Biotin)。
④将上述白色固体,溶于10mL甲苯中,加热至100℃;
⑤待反应液中有气泡放出时,加入烯丙氧羰基(Alloc),于100℃搅拌反应20min;
⑥反应结束后,降温到65℃以下即析出产品结晶,抽滤得到粗品,以甲醇为溶剂重结晶,得到晶体。
在氨基酸序列缩合完成后,正交脱除Alloc保护基,然后缩合SUMO连接臂。由于连接臂中存在巯基或活化后的不饱和双键,不能兼容于自由基脱硫反应,因此以选择三片段顺序连接的合成策略。首先进行N到C的顺序连接并脱硫得到连接臂修饰的SUMO分子,随后脱除Acm保护基后与SUMO酰肼进行连接,最后活化连接臂而得到SUMO-C6H8NO3探针。
⑦将上述晶体(500mg)溶于干燥的三氯甲烷(5mL)中;
⑧充分溶解后,冰浴下加入mCPBA(98.5mg,0.57mmol),立即置换氮气,冰浴搅拌反应;
⑨TLC监控至反应完毕,加水淬灭反应,乙酸乙酯萃取(20mL),饱和食盐水洗(10mL);
⑩将上述有机相由无水硫酸钠干燥后,减压旋干溶剂,硅胶柱层析(二氯甲烷:乙酸乙酯=5:1)分离纯化,得到白色粉末;取100mg,溶于2.5ml缓冲液中,经胰蛋白酶切后,LC-MS鉴定(如图4)。
实施例3:SUMO-C6H8NO3探针分子在MCF7细胞及小鼠体内应用
将MCF7细胞接种于35mm细胞培养板,待细胞融合度约40%,进行融合实验。将SUMO-C6H8NO3探针分子与MCF7细胞共同孵育,36h后进行荧光显微镜检测,结果如图5所示。
所述探针检测方法为:
由于SUMO蛋白可以特异性识别底物蛋白的赖氨酸残基,因此发生修饰的蛋白通过SUMO化过程相关酶的作用,将SUMO-C6H8NO3探针分子直接与底物结合,由于探针分子可自发荧光,其激发波长为475nm,发射波长为507nm,因此通过对探针分子自发荧光发射光强度及位置进行检测,即可对细胞内SUMO化修饰进行定位,定量,以及动态变化监测。
将含有SUMO-C6H8NO3探针分子的MCF7细胞,通过皮下注射方式,分别注射入裸鼠左右腹侧,通过不同时间进行光强度检测,到注射后7天仍然有很高的发光强度,结果如图6所示。
以上示例性实施方式所呈现的描述仅用以说明本发明的技术方案,并不想要成为毫无遗漏的,也不想要把本发明限制为所描述的精确形式。显然,本领域的普通技术人员根据上述教导做出很多改变和变化都是可能的。选择示例性实施方式并进行描述是为了解释本发明的特定原理及其实际应用,从而使得本领域的其它技术人员便于理解、实现并利用本发明的各种示例性实施方式及其各种选择形式和修改形式。本发明的保护范围意在由所附权利要求书及其等效形式所限定。
序列表
<110> 大连理工大学
<120> 一种SUMO化修饰捕获探针的合成方法及应用
<130> 2020
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 306
<212> DNA
<213> 人类(Homo sapiens)
<400> 1
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acaacacatc tcaagaaact caaagaatca tactgtcaaa gacagggtgt tccaatgaat 180
tcactcaggt ttctctttga gggtcagaga attgctgata atcatactcc aaaagaactg 240
ggaatggagg aagaagatgt gattgaagtt tatcaggaac aaacgggggg tcattcaaca 300
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<213> 人类(Homo sapiens)
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Claims (4)
2.一种如权利要求1所述的SUMO化修饰捕获探针的制备方法,其特征在于,所述的制备方法包括以下步骤:
步骤1、通过基因克隆,得到人源SUMO的全长cDNA序列;
步骤2、将步骤1)得到的含有SUMO全长cDNA序列质粒,通过在大肠杆菌中重组表达SUMO蛋白多肽,并通过亲和纯化,得到纯品SUMO蛋白多肽;
步骤3、将步骤2)得到的纯品SUMO蛋白多肽,通过全化学合成方法,得到SUMO-C6H8NO3荧光探针。
3.根据权利要求2所述的制备方法,其特征在于,步骤3)中进一步包括:
3.1)将纯品SUMO蛋白多肽,氯甲酸乙酯及N-甲基吗啉于四氢呋喃,降温冷却至-20℃搅拌反应;
3.2)在步骤3.1)反应后,升温到-5℃,孵育后,在此温度下滴加叠氮化钠水溶液,室温反应;
3.3)将步骤3.2)所得产物以醋酸乙酯提取后,加无水硫酸镁干燥,减压浓缩回收得到的白色固体;
3.4)将步骤3.3)所的白色固体,溶于甲苯中,加热至100℃;
3.5)待步骤3.4)的反应液中有气泡放出时,加入烯丙氧羰基,于100℃搅拌反应;
3.6)步骤3.5)中反应结束后,降温到65℃以下即析出产品结晶,抽滤得到粗品,以甲醇为溶剂重结晶,得到晶体;
3.7)将步骤3.6)中所得晶体溶于干燥的三氯甲烷中;
3.8)待步骤3.7)中充分溶解后,冰浴下加入mCPBA,立即置换氮气,冰浴搅拌反应;
3.9)TLC监控至反应完毕,加水淬灭反应,乙酸乙酯萃取,饱和食盐水洗;
3.10)将步骤3.9)中有机相由无水硫酸钠干燥后,减压旋干溶剂,硅胶柱层析分离纯化,得到白色粉末;溶解后,经胰蛋白酶切后,LC-MS鉴定。
4.如权利要求1所述的SUMO化修饰捕获探针或者如权利要求2或3所述的方法制备获得的SUMO化修饰捕获探针在制备用于癌症诊断和/或预后的试剂中的用途。
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