CN113373200A - 一种生物传感器检测转录因子NF-κB p50的方法 - Google Patents
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Abstract
本发明公开了一种生物传感器检测转录因子NF‑κB p50的方法，该方法将带有PAM结构域的双链DNA作为激活序列，与测试样品和反应缓冲液混合孵育后，将核酸外切酶III(Exo III)及反应缓冲液添加到溶液中，孵育，再将配置好的CRISPR报告溶液(CRM)加入溶液中以启动CRISPR反应，测量所得的样品的荧光，最后根据不同浓度的测试样品的荧光值绘制浓度与荧光强度之间的关系图，检测样品的荧光值并代入回归曲线，计算出NF‑κB p50的浓度。本发明的NF‑κB p50检测方法具有灵敏度高，选择性强，成本低的优点，能够实现肿瘤中的NF‑κB p50高效检测及对于NF‑κB p50抑制性药物的筛选和评估。
Description
技术领域
本发明涉及一种生物传感器检测某种目标物的方法，具体为一种生物传感器检测转录因子NF-κB p50的方法。
背景技术
生物标志物在诊断和预后中的关键作用已经得到普遍的认可，而其进行准确检测的优势在精密医学的发展中也具有重要的意义。nuclear factor-kappa B(NF-κB)是哺乳动物细胞中普遍存在的一种核转录因子，它可以调节超过150个基因的表达并参与炎症应激、病毒感染、细胞增殖和凋亡等反应。由于NF-κB的水平变化与类风湿关节炎、癌症和神经退行性疾病紧密相关，因此，对NF-κB的特异性检测可以间接反映人体对于这些疾病的易感程度，这让它具有成为一种在临床诊断和治疗中有效生物标志物的潜力。但当前，用于检测转录因子的分析技术发展并不完善，包括电泳迁移率变化分析法和免疫分析法在内的多种常规检测方法缺点明显，因此，非常需要能够提高性能和降低成本的新的检测技术的发明。
荧光法是将转录因子和适体的特异性结合与荧光基因链的某些特定变化构成联系，从而利用荧光信号表征转录因子存在的一种方法。CRISPR基因剪切技术是一种利用原核生物天然存在的免疫系统定向切除特定基因位点的靶向性技术。
发明内容
发明目的：本发明的目的在于提供一种生物传感器检测转录因子NF-κB p50的方法，该生物传感器由核酸外切酶保护，且为CRISPR/Cas12a整合的生物传感器，该方法能够特异性识别目标物NF-κB p50，增强检测的灵敏度和准确性。
技术方案：本发明所述的生物传感器检测转录因子NF-κB p50的方法，具体包括如下步骤：
(a)将Cas12a、crRNA、NE缓冲液进行混合，然后加入荧光报告基因并在冰浴下混合以制备CRISPR报告组合CRM混合液；
(b)将带有PAM结构域的双链DNA作为激活序列，和反应缓冲液进行混合，再加入测试样品，孵育；
(c)将ExoIII核酸外切酶及(b)中所述的反应缓冲液添加到(b)所得的溶液中，孵育；
(d)将(a)中配制的CRM混合液加入(c)中所得的最终溶液中，孵育，再在60-70℃下灭活；
(e)将(d)中所得的最终溶液在读板仪上进行快速和高通量检测，在450-570nm之间进行激发和发射；
(f)将(d)中所得的最终溶液用荧光光谱仪测量荧光；
(g)重复上述步骤，检测不同浓度的测试样品的荧光值，通过绘制关系图，计算NF-κB p50浓度与荧光强度之间的关系；
(h)将待测样品作为测试样品，进行步骤(a)-(f)，将测得的荧光值与步骤(g)获得的关系图进行对比，计算待测样品中NF-κB p50的浓度。
所述的方法，步骤(a)中Cas12a浓度为40-80nM。
所述的方法，步骤(a)中crRNA浓度为450-600nM。
所述的方法，步骤(a)所述的crRNA的序列为：
斜体字母表示与激活序列结合的区域。
所述的方法，步骤(b)所述的带有PAM结构域的双链DNA的序列为：
斜体字母表示crRNA识别序列，有下划线的字母表示PAM结构域；加粗字母表示与NF-κB结合的区域。
所述的方法，步骤(b)中反应缓冲液为含有Na2HPO4/NaH2PO4、CH3COONa、Mg(CH3COOH)2、glycerol、poly(dI-dC)的溶液，pH 7-8。
所述的方法，步骤(b)中激活序列的浓度为30-60nM。
所述的方法，步骤(c)中ExoIII核酸外切酶的浓度为2-6单位/L。
所述的方法，步骤(e)中分别在510nm和566nm处进行激发和发射。
所述的方法，步骤(f)中荧光光谱仪在λ＝510nm的波长下测量荧光。
本发明的反应原理如图1所示。
有益效果：与现有技术相比，本发明具有以下显著优点：(1)将CRISPR技术融入荧光法检测转录因子的方案中，较大程度地增加了生物传感器的便捷性和选择性。本方法首次使用基于CRISPR技术的CRM传感元件切割荧光报告基因方法，不但增强了对于NF-κB p50识别特异性，而且提高NF-κB p50检测的灵敏度，此方法的检测限已经达到了femtomolar的水平。(2)本方法在一定波长范围内的荧光强度和NF-κB p50浓度存在线性关系，因此可以由荧光强度定量计算NF-κB p50浓度。(3)该生物传感器的整个过程主要包括样品添加和恒温培养，且不包含复杂的核酸反应电路，具有简便易行的优点；此外，所有检测过程中的操作都是在一个管子中进行的，从而避免了引入杂质和实验样品进入外界环境中。(4)本方法实验材料易于获得且成本较现有其他方法显著降低，每次检测平均成本仅为ELISA技术的1/11。(5)本方法使用的CRISPR传感元件可以在冷冻干燥条件下长期储存，在-20℃环境下至少可以储存2个月而不丧失传感性能。(6)本方法不仅为实现NF-κB p50的定量检测提供了一种更好的方法，并且具有筛选和评估靶向转录因子的药物的应用潜力。
附图说明
图1为本发明的原理图；
图2为ExoIII消化时间与总反应的荧光之间的关系；
图3为CRISPR报告反应时间与总反应的荧光之间的关系；
图4为CRM在不同环境下配制与总反应的荧光之间的关系；
图5为系统NF-κB p50浓度与荧光强度的线性关系；
图6为该方法对NF-κB p50的选择性；
图7为用这种方法和商用ELISA试剂盒给出的肿瘤样品中NF-κB p50的定量结果的比较；
图8为该方法的抑制实验。
具体实施方式
药品和试剂：所有HPLC纯化的DNA序列是由生工生物工程(中国上海)提供的。用于制备缓冲液的化学试剂由普迈生物科技有限公司(中国上海)获得。FEN1由BBI(中国上海)提供。人重组NF-κB p50是通过Enzo生物科技(美国纽约)购买的，
Cas12a和NEBuffer 2.1是由NEB(美国马萨诸塞州)获取的。人血清白蛋白(HSA)和牛血清白蛋白(BSA)是从普迈生物科技有限公司(中国上海)购买的。人类干扰素γ(IFN-γ)和冬凌草甲素(CAS No.28957-04-2)来自MedChemExpress(美国新泽西州)。本实验用到的超纯水由生工生物工程(中国上海)提供。
实施例1
本发明检测方法的条件优化实验
具体方法如下：
(1)将设计好的5μL，浓度为50nM的激活序列、30μL测试样品和15μL反应缓冲液混合，在35-40℃下孵育10-20min。然后，将1μL，4U/μLExoIII外切酶以及1×反应缓冲液添加到溶液中，在37℃下孵育30min。反应缓冲液为：10mM Na2HPO4/NaH2PO4、100mM CH3COONa、10mM Mg(CH3COOH)2、10％glycerol、0.05mg/mL poly(dI-dC)，pH＝7.2。30μL的测试样品为可能含有目标物的溶液，溶剂为水或缓冲液。
(2)将50μL CRM混合物(50nM Cas12a，450-600nM crRNA，10×NE
2.1)和5-15μM荧光报告基因滴入(1)所得的样品中以启动CRISPR反应。反应条件为：在37℃下孵育30min，再在65℃下灭活10min。步骤(1)中激活序列如下表所示：
斜体字母表示crRNA识别序列，有下划线的字母表示PAM结构域；加粗字母表示与NF-κB结合的区域。
crRNA序列如下表所示：
斜体字母表示与激活序列结合的区域。
(3)再在Synergy H-1读板仪上进行快速和高通量检测，分别在510nm和566nm处进行激发和发射。所得的样品用F-7100荧光光谱仪在λ＝510nm的波长下测量荧光，记录荧光值。
通过分别改变步骤(1)和步骤(2)中ExoIII外切酶消化时间、CRISPR报告反应时间以及CRM配制环境，观察荧光强度，确认最佳反应条件。由于检测中同时加入NF-κB p50和ExoIII外切酶时的荧光曲线与只加入CRM和激活序列的荧光曲线几乎完全相同，整个过程无需加入NF-κB p50。
具体操作为：(1)将2.5nM ExoIII外切酶与5nM激活序列混合，在37℃下孵育不同的时间间隔，然后添加50μL CRM预混液，并在37℃孵育30分钟，再在65℃下灭活10分钟。(2)将添加了NE缓冲液2.1的CRM预混液直接与5nM激活序列混合并在37℃下孵育不同的时间间隔，再在65℃下灭活10分钟。(3)将添加了超纯水或不同缓冲液的CRM预混液直接与5nM激活序列混合并在37℃下孵育30分钟，再在65℃下灭活10分钟。ExoIII消化时间与总反应的荧光之间的关系如图2所示，可以看出，最佳消化时长在30min左右。CRISPR报告反应时间与荧光强度的关系如图3所示，可以看出，最佳报告反应时长在30min左右。CRM在不同环境下配制与总反应的荧光之间的关系如图4所示，可以看出，最佳反应环境为NE缓冲液2.1。
实施例2
将实施例1中步骤(1)中的测试样品更换成不同浓度的NF-κB p50，其他条件不变，重复实施例1中的步骤1)-3)，检测荧光。其中，不同浓度的NF-κB p50具体配置方法为：将NF-κB p50标准品用反应缓冲液进行稀释，得到浓度为0.5、20、60、150、400、600、900、1200、1600、2000、2500pM的NF-κB p50溶液。
根据对应的不同浓度的NF-κB p50时传感系统的荧光光谱图，绘制NF-κB p50浓度与荧光信号之间的线性关系，如图5所示。根据线性进行拟合，得到的线性范围是0.5-1600pM，依照信噪比＝3的计算规则，检测限为0.2pM。
实施例3
本发明检测方法的选择性实验
将实施例1中步骤1)中的测试样品更换为相应浓度的BSA、HSA、c-Myc、p53、MITF、AP-1、STAT以及NF-kB p105，其他条件不变，重复实施例1中的步骤1)-3)，检测荧光，得到本发明方法检测目标NF-κB p50的选择性结果，如图4所示。从图6中可以看出本发明的方法对目标NF-κB p50具有良好的选择性。
实施例4
本发明的方法和商用ELISA试剂盒给出的肿瘤样品中NF-κB p50的定量结果的比较，检测方法根据Biomatik试剂盒提供的说明书进行操作，如图7所示。
实施例5
本发明的方法和最近发表的NF-κB p50的定量结果的比较，如表1所示。
表1.本发明的方法与最近发表的NF-κB p50的定量结果的比较
实施例6
本发明检测方法的回收率实验
将样品换成DLD-1细胞，并向样品加入相同量的目标分子，进行加样检测，其他条件不变，重复实施例1中的步骤(1)-(3)，获得本方法在实际样品中的回收率，结果见表2。可以看出，本检测方法的回收率良好。
表2本发明所制备的传感器检测DLD-1cells的检测结果
实施例7
本发明检测方法的抑制实验
将样品换成不同浓度的冬凌草甲素(一种抑制NF-κBp50的DNA结合的天然产物)与1nM NF-κBp50的混合物，并向样品加入相同量的目标分子，进行加样检测，其他条件不变，重复实施例1中的步骤(1)-(3)，检测荧光，得到加入不同浓度的冬凌草甲素时传感系统的荧光光谱图，如图8所示。可以看出随着冬凌草甲素浓度增加，系统的荧光信号逐渐减弱。这表明本发明的检测方法可以反映抑制性药物对NF-κBp50的抑制作用强度。因此，本发明可以实现对于NF-κB p50抑制性药物的筛选和评估。
Claims (10)
1.一种生物传感器检测转录因子NF-κB p50的方法，其特征在于，具体包括如下步骤：
(a)将Cas12a、crRNA、NE缓冲液进行混合，然后加入荧光报告基因并在冰浴下混合以制备CRISPR报告组合CRM混合液；
(b)将带有PAM结构域的双链DNA作为激活序列，和反应缓冲液进行混合，再加入测试样品，孵育；
(c)将ExoIII核酸外切酶及(b)中所述的反应缓冲液添加到(b)所得的溶液中，孵育；
(d)将(a)中配制的CRM混合液加入(c)中所得的最终溶液中，孵育，再在60-70℃下灭活；
(e)将(d)中所得的最终溶液在读板仪上进行快速和高通量检测，在450-570nm之间进行激发和发射；
(f)将(d)中所得的最终溶液用荧光光谱仪测量荧光；
(g)重复上述步骤，检测不同浓度的测试样品的荧光值，通过绘制关系图，计算NF-κBp50浓度与荧光强度之间的关系；
(h)将待测样品作为测试样品，进行步骤(a)-(f)，将测得的荧光值与步骤(g)获得的关系图进行对比，计算待测样品中NF-κB p50的浓度。
2.根据权利要求1所述的方法，其特征在于，步骤(a)中Cas12a浓度为40-80nM。
3.根据权利要求1所述的方法，其特征在于，步骤(a)中crRNA浓度为450-600nM。
4.根据权利要求1所述的方法，其特征在于，步骤(a)所述的crRNA的序列为：
斜体字母表示与激活序列结合的区域。
5.根据权利要求1所述的方法，其特征在于，步骤(b)所述的带有PAM结构域的双链DNA的序列为：
斜体字母表示crRNA识别序列，有下划线的字母表示PAM结构域；加粗字母表示与NF-κB结合的区域。
6.根据权利要求1所述的方法，其特征在于，步骤(b)中反应缓冲液为含有Na2HPO4/NaH2PO4、CH3COONa、Mg(CH3COOH)2、glycerol、poly(dI-dC)的溶液，pH 7-8。
7.根据权利要求1所述的方法，其特征在于，步骤(b)中激活序列的浓度为30-60nM。
8.根据权利要求1所述的方法，其特征在于，步骤(c)中ExoIII核酸外切酶的浓度为2-6单位/L。
9.根据权利要求1所述的方法，其特征在于，步骤(e)中分别在510nm和566nm处进行激发和发射。
10.根据权利要求1所述的方法，其特征在于，步骤(f)中荧光光谱仪在λ＝510nm的波长下测量荧光。
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