CN112798796B - 一种基于dna-paint的细胞膜上pd-l1蛋白检测方法及序列 - Google Patents
一种基于dna-paint的细胞膜上pd-l1蛋白检测方法及序列 Download PDFInfo
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Abstract
本发明公开了一种基于DNA‑PAINT的细胞膜上PD‑L1蛋白检测方法及序列，利用设计的DNA锚链和DNA成像链，通过细胞固定、抗体偶联、孵育，采用全内反射照明模式进行DNA‑PAINT成像，用于检测乳腺癌细胞表面上PD‑L1蛋白。利用DNA‑PAINT超分辨技术不受荧光淬灭影响，空间分辨率高等优点，检测到了MB‑231细胞和MCF‑7细胞上PD‑L1蛋白在膜上的分布，为乳腺癌的免疫治疗提供一定的帮助。
Description
技术领域
本发明涉及单分子定位与荧光免疫染色的领域，特别涉及一种基于DNA-PAINT的细胞膜上PD-L1蛋白检测方法及序列。
背景技术
在全球范围内，乳腺癌是女性中最常见的癌症，仅2018年就确诊了210万例乳腺癌病例。目前，使用检查点抑制剂的免疫疗法正在作为一种新的乳腺癌治疗手段出现。在乳腺癌的免疫治疗中，PD-L1蛋白是重要的免疫检查点分子。乳腺癌细胞会过表达PD-L1蛋白，它与T细胞上的PD-1蛋白结合，从而抑制T细胞的免疫功能，实现癌细胞的免疫逃逸。免疫治疗是利用免疫检查点抗体阻断癌细胞上免疫检查点，从而增加T细胞效应，延长细胞耐受性，达到清除肿瘤细胞的目的。
为了更好的了解癌细胞的免疫检查点蛋白，利用超分辨光学成像技术(DNA-PAINT)对乳腺癌细胞表面的PD-L1蛋白进行精确的空间定位和定量分析。DNA-PAINT通过两条互补DNA链(即锚链和成像链)之间的瞬时相互作用来实现对多个目标的顺序检测。在DNA-PAINT中，染料标记的成像链(8–10nt)瞬时与锚链结合。成像链和锚链之间的瞬时杂交和解离分别代表荧光的开启和关闭状态。通过全内反射荧光(TIRF)显微镜检测荧光的开和关，从而产生“闪烁”事件。通过采集多张荧光闪烁的图片进行超分辨图像的重建可达到约25nm的空间分辨率。对于目标区域，DNA探针之间的结合率与目标分子的浓度成正比。可以进一步调整这种可编程的结合动力学(即闪烁频率和持续时间)以定量分析分布在衍射极限以下区域内的密集堆积靶标，这被称为定量PAINT(或qPAINT)。
利用DNA-PAINT的高分辨率有望在单分子层面上，对乳腺癌细胞表面的蛋白进行空间的定位分析和定量分析，从而更加准确的表征乳腺癌细胞的生物标志物，为将来更好的免疫治疗乳腺癌提供支持。
发明内容
本发明为了更加准确的检测乳腺癌细胞表面的PD-L1蛋白，提供了一种基于DNA-PAINT的细胞膜上PD-L1蛋白检测方法及序列，可用于对乳腺癌细胞表面的PD-L1蛋白进行空间的定位分析和定量分析，以更好的指导肿瘤免疫治疗的发展。
作为本方面的一个方面，本发明的技术方案：一种基于DNA-PAINT的细胞膜上PD-L1蛋白检测方法，其包括以下步骤，(1)将PD-L1抗体与DBCO-sulfo-NHS进行偶联、孵育、提纯；，得到DBCO-sulfo-NHS偶联的抗体；(2)将DNA锚链与所述DBCO-sulfo-NHS偶联的抗体进行共孵育，提纯后获得DNA偶联的抗体，所述DNA锚链的序列如SEQ NO.1所示；(3)将所述DNA偶联的抗体加入预处理的细胞中进行孵育，之后进行冲洗，冲洗结束后加入DNA成像链并孵育，进行DNA-PAINT超分辨成像，所述DNA成像链的序列如SEQ NO.2所示。
优选的，步骤(1)中，所述将PD-L1抗体与DBCO-sulfo-NHS进行偶联、孵育、提纯，其为将1μL 0.443mg/mL的PD-L1抗体和8μL 100mM的DBCO-sulfo-NHS加到400μL的PBS溶液中，铝箔包裹后放入摇床中孵育2小时，设置孵育温度为4℃，所述提纯，其为将孵育后的溶液离心，然后加入PBS溶液重悬提纯的溶液中，以同样的参数再次离心。
优选的，步骤(2)中，所述将DNA锚链与所述DBCO-sulfo-NHS偶联的抗体进行共孵育，其为用铝箔包裹放入摇床中在37℃下孵育90min。
优选的，步骤(3)中，所述预处理的细胞包括MCF-7细胞、MB-231细胞的一种或几种，所述将所述DNA偶联的抗体加入预处理的细胞中进行孵育的条件为4℃环境下孵育12小时以上。
优选的，步骤(3)中，所述进行DNA-PAINT超分辨成像，其采用的激光为405nm、642nm，曝光时间为100ms，采集5000帧以上。
作为本发明的另一方面，本发明提供一种用于PD-L1蛋白检测的序列，其包括DNA锚链和DNA成像链，所述DNA锚链的序列如SEQ NO.1所示，所述DNA成像链的序列如SEQ NO.2所示。
本发明的有益效果如下：
本发明基于DNA-PAINT的细胞膜PD-L1蛋白质检测用于检测乳腺癌细胞表面上PD-L1蛋白。利用DNA-PAINT超分辨技术不受荧光淬灭影响，空间分辨率高等优点，检测到了MB-231细胞和MCF-7细胞上PD-L1蛋白在膜上的分布，并对它们进行了定量的分析，其中MCF-7细胞的平均强度为52.62795，MB-231细胞的平均强度为23.21649，定量分析的结果可以反应细胞膜表面PD-L1蛋白的多少，为乳腺癌的免疫治疗提供一定的参考帮助。
附图说明
图1为MCF-7细胞上PD-L1蛋白的DNA-PAINT成像，其中，左：MCF-7细胞的细胞核染色图片，中：PD-L1蛋白的DNA-PAINT成像,右：两个通道的重合，其中细胞区域的平均强度为52.62795；
图2为MB-231细胞上PD-L1蛋白的DNA-PAINT成像，其中，左：MB-231细胞的细胞核染色图片，中：PD-L1蛋白的DNA-PAINT成像,右：两个通道的重合，其中细胞区域的平均强度为23.21649。
图3为本发明的机理示意图。
具体实施方式
实验所用的原料：1、PBS缓冲液为pH＝7.4，浓度为10mM的PBS缓冲液；2、DNA锚链和DNA成像链均由生工生物工程(上海)公司合成；3、其余材料均为市售所得。
实施例1：
取1μL的PD-L1抗体与8μL的DBCO-sulfo-NHS在4℃下摇床孵育2个小时，之后抽取到50KD的超滤管中，配平离心，转速为5600rpm，温度为4℃，时间为5min。同样的参数设置离心两次，之后加入400μL的PBS重悬提取到新的试管中备用。向偶联PD-L1抗体的溶液中加入4μL、100uM的DNA锚链，碱基序列为SEQ NO.1：C-3Azide-TCCTCCTCCTCCTCCTCCT-3’。将其放在摇床上孵育90min，温度为37℃。反应结束抽取至50KD的超滤管中，配平离心，转速为5600rpm，时间为10min，温度为26℃。同样的参数设置离心两次，最后加入400μL的PBS重悬抽取至新的试管中备用。
从细胞培养箱中取出细胞，弃去细胞培养液，用PBS冲洗3次，每次5min。取5mL4％的多聚甲醛和10μL50％的戊二醛配置固定液，将固定液加入细胞中孵育20min。然后利用PBS冲洗，加入1mg/mL的NaBH4摇床反应7min，PBS冲洗之后加入山羊血清封闭，摇床室温反应90min，以防止抗体的非特异性的吸附。反应结束后用PBS冲洗，加入之前制备好的DNA锚链偶联的PD-L1抗体，用铝箔包裹放入4℃冰箱中孵育12h以上。
将反应结束的细胞取出，弃去抗体溶液，用PBS冲洗结束后加100μL 5μg/mL的DAPI溶液孵育15min。最后用PBS冲洗。
实施例2：
上述基于DNA-PAINT的细胞膜上PD-L1蛋白质检测成像为：
向实施例1中加入DNA成像链(浓度为1nM，SEQ NO.2，序列为5’-AGGAGGA-Cy5)，孵育10min后对PD-L1蛋白进行DNA-PAINT成像。具体的，采用全内反射照明模式，100倍油镜，打开405nm的激光器对细胞核进行成像以确定细胞的具体位置，之后切换到642nm的激光器采集Cy5荧光团的DNA成像链的荧光信号，曝光时间为100ms，采集5000帧以上，以对PD-L1蛋白进行准确的空间定位和定量分析。
DNA-PAINT成像参数设置为：405nm激光强度为2％；642nm激光强度为4％；曝光时间为100ms；成像张数为5000张。
本发明提供的一种基于DNA-PAINT的细胞膜上PD-L1蛋白质检测方法，使用的细胞是经过预处理的，具体为：首先用固定液固定需要检测的细胞，之后冲洗加入NaBH4以还原固定液中的醛基，然后加入适量的山羊血清对细胞进行封闭，防止抗体的非特异性吸附。具体的，利用5mL 4％的多聚甲醛和10μL 50％的戊二醛配置固定液，将固定液加入细胞中孵育20min以上。然后利用PBS冲洗，加入1mg/mL的NaBH4摇床反应7min以上,再用PBS冲洗之后加入山羊血清封闭，摇床室温反应90min。
本发明的实验探索优化过程如下：
本发明最初的检测效果不佳，无法在细胞区域观察到DNA-PAINT的闪烁现象，对此不断调整PD-L1抗体与DNA锚链的偶联的实验步骤。其中比较关键的是两次偶联的实验温度，在PD-L1抗体与DBCO-sulfo-NHS偶联时，孵育温度设置为4℃，在DNA锚链与PD-L1抗体偶联时，孵育温度设置为37℃。在成像过程中，DNA-PAINT的闪烁现象杂乱无章，并不集中于细胞区域。对此将前期封闭细胞的1％BSA换成山羊血清，来防止抗体的非特异性吸附。在成像过程中，DNA成像链的浓度也至关重要，DNA成像链浓度过稀则无法检测到全部的PD-L1蛋白，无法采集足够多的闪烁点，成像时间过长；DNA成像链浓度过浓则会造成背景信号太强，成像效果不佳，最终在尝试多个浓度的DNA成像链之后选出最佳的浓度为1nM。在TIRF模式下成像，倾斜角度需要调整以实现最佳的成像效果，角度过大或者过小均会导致DNA-PAINT的闪烁现象不佳甚至消失。
本发明公开了一种基于DNA-PAINT的细胞膜上PD-L1蛋白质检测，该方法利用DNA-PAINT检测乳腺癌细胞表面的PD-L1蛋白。首先将DNA通过DBCO-sulfo-NHS偶联至PD-L1抗体上，之后将偶联了DNA锚链的PD-L1抗体与细胞共孵育，最后加入DNA成像链进行DNA-PAINT超分辨成像。该方法可对细胞膜表面的PD-L1蛋白进行准确的空间定位和定量的分析，具有分辨率高、灵敏度好等优点。
应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110> 东南大学
<120> 一种基于DNA-PAINT的细胞膜上PD-L1蛋白检测方法及序列
<141> 2020-12-24
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Claims (6)
1.一种基于DNA-PAINT的细胞膜上PD-L1蛋白检测方法，其特征在于：包括以下步骤：
(1)将PD-L1抗体与DBCO-sulfo-NHS于4℃下进行偶联、孵育、提纯，得到DBCO-sulfo-NHS偶联的抗体；
(2)将DNA锚链与所述DBCO-sulfo-NHS偶联的抗体进行37℃下共孵育，提纯后获得DNA偶联的抗体，所述DNA锚链的序列如SEQ NO.1所示；
(3)将所述DNA偶联的抗体加入预处理的细胞中进行孵育，之后进行冲洗，冲洗结束后加入DNA成像链并孵育，进行DNA-PAINT超分辨成像，所述DNA成像链的序列如SEQ NO.2所示；
上述使用的细胞是经过预处理的，具体为：首先用固定液固定需要检测的细胞，之后冲洗加入NaBH4以还原固定液中的醛基，然后加入适量的山羊血清对细胞进行封闭，防止抗体的非特异性吸附；具体的，利用5mL 4％的多聚甲醛和10μL 50％的戊二醛配置固定液，将固定液加入细胞中孵育20min以上；然后利用PBS冲洗，加入1mg/mL的NaBH4摇床反应7min以上,再用PBS冲洗之后加入山羊血清封闭，摇床室温反应90min。
2.根据权利要求1所述基于DNA-PAINT的细胞膜上PD-L1蛋白检测方法，其特征在于：步骤(1)中，所述将PD-L1抗体与DBCO-sulfo-NHS进行偶联、孵育、提纯，其为将1μL 0.443mg/mL的PD-L1抗体和8μL100mM的DBCO-sulfo-NHS加到400μL的PBS溶液中，铝箔包裹后放入摇床中孵育2小时，设置孵育温度为4℃，所述提纯，其为将孵育后的溶液离心，然后加入PBS溶液重悬提纯的溶液中，以同样的参数再次离心。
3.根据权利要求1所述基于DNA-PAINT的细胞膜上PD-L1蛋白检测方法，其特征在于：步骤(2)中，所述将DNA锚链与所述DBCO-sulfo-NHS偶联的抗体进行共孵育，其为用铝箔包裹放入摇床中在37℃下孵育90min。
4.根据权利要求1所述基于DNA-PAINT的细胞膜上PD-L1蛋白检测方法，其特征在于：步骤(3)中，所述预处理的细胞包括MCF-7细胞、MB-231细胞的一种或几种，所述将所述DNA偶联的抗体加入预处理的细胞中进行孵育的条件为4℃环境下孵育12小时以上。
5.根据权利要求1或4所述基于DNA-PAINT的细胞膜上PD-L1蛋白检测方法，其特征在于：步骤(3)中，所述进行DNA-PAINT超分辨成像，其采用的激光为405nm、642nm，曝光时间为100ms，采集5000帧以上。
6.一种用于PD-L1蛋白检测的序列，其特征在于：包括DNA锚链和DNA成像链，所述DNA锚链的序列如SEQ NO.1所示，所述DNA成像链的序列如SEQ NO.2所示。
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| US5279952A (en) * | 1991-08-09 | 1994-01-18 | Board Of Regents, The University Of Texas System | PCR-based strategy of constructing chimeric DNA molecules |
| EP2154144A1 (en) * | 2008-08-06 | 2010-02-17 | Changchun Huapu Biotechnology Co., Ltd. | Oligonucleotides and use thereof |
| WO2010122110A1 (en) * | 2009-04-22 | 2010-10-28 | Basf Plant Science Company Gmbh | Whole seed specific promoter |
| CN106456801A (zh) * | 2014-02-07 | 2017-02-22 | Sbi生物技术有限公司 | 用于治疗肿瘤的抑制性寡核苷酸 |
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| US5279952A (en) * | 1991-08-09 | 1994-01-18 | Board Of Regents, The University Of Texas System | PCR-based strategy of constructing chimeric DNA molecules |
| EP2154144A1 (en) * | 2008-08-06 | 2010-02-17 | Changchun Huapu Biotechnology Co., Ltd. | Oligonucleotides and use thereof |
| WO2010122110A1 (en) * | 2009-04-22 | 2010-10-28 | Basf Plant Science Company Gmbh | Whole seed specific promoter |
| CN106456801A (zh) * | 2014-02-07 | 2017-02-22 | Sbi生物技术有限公司 | 用于治疗肿瘤的抑制性寡核苷酸 |
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