CN113777295A - 用于检测肿瘤标志物pd-l1的高灵敏度量子点探针、制备方法及应用 - Google Patents
用于检测肿瘤标志物pd-l1的高灵敏度量子点探针、制备方法及应用 Download PDFInfo
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- CN113777295A CN113777295A CN202111083304.5A CN202111083304A CN113777295A CN 113777295 A CN113777295 A CN 113777295A CN 202111083304 A CN202111083304 A CN 202111083304A CN 113777295 A CN113777295 A CN 113777295A
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Abstract
本发明公开了一种检测细胞表面肿瘤标志物PD‑L1的量子点荧光探针、制备方法及应用,该探针荧光强度高,提高了相关检测的灵敏度。包括步骤:1)识别PD‑L1的纳米抗体重组蛋白的设计、识别PD‑L1的纳米抗体重组蛋白的原核表达和纯化;2)含有生物素基团多肽的制备;3)识别PD‑L1的纳米抗体重组蛋白与生物素化多肽的体外酶法融合;生物素化的识别PD‑L1的纳米抗体重组蛋白的纯化;4)生物素化的识别PD‑L1的纳米抗体重组蛋白与链酶亲和素标记量子点的偶联探针制备。本发明的探针荧光强度高,结合流式细胞术、免疫荧光、酶联免疫吸附技术能有效检测细胞表面肿瘤标志物PD‑L1的表达,方法简便,检测灵敏度高。
Description
技术领域
本发明属于生物医学/抗原检测技术领域,具体涉及一种用于检测细胞表面肿瘤标志物PD-L1的基于纳米抗体重组蛋白和荧光量子点的探针、制备方法及应用。
背景技术
基于程序性死亡受体1(PD-1)和程序性死亡受体配体1(PD-L1)的免疫检查点抑制原理的肿瘤治疗策略在多种肿瘤的临床治疗中展现出了巨大应用前景,因此准确对该标志物进行检测是能否开展相关治疗的关键。PD-1属于B7免疫球蛋白家族,是T细胞上的一种抑制性跨膜受体。PD-L1是其主要配体,广泛表达于免疫细胞以及多种恶性肿瘤细胞,包括恶性黑色素瘤、肺癌、肝癌、肾细胞癌、卵巢癌和结直肠癌等。在机体正常免疫负性调节过程中,PD-1/PD-L1对于调控免疫稳态发挥重要作用。然而,肿瘤细胞表面高表达的PD-L1能够阻断T细胞免疫应答,产生免疫抑制性肿瘤微环境,是肿瘤免疫逃逸的一个重要机制。肿瘤细胞表面过度表达的PD-L1不仅是理想的肿瘤有效治疗靶点,也是肿瘤精准诊断的一个重要标志物分子,开发PD-L1的准确检测方法对开展相关的肿瘤治疗具有非常重要的意义。
现有PD-L1的临床检测方法主要是基于单克隆抗体的免疫组化(IHC)技术,该方法特异性和灵敏度均不高,假阴性常见,所使用的单克隆抗体组织穿透性差,尤其对实体瘤及较厚样本组织难以准确检测,严重制约了基于PD-L1/PD-1的肿瘤精准诊疗的疗效和进一步推广应用。此外,也有单克隆抗体标记同位素的PD-L1探针报道,但由于其分子量大,半衰期长,不能迅速从血液中清除,需要较长时间的等待才能获取较低背景的放射性成像,增加病灶部位的成像显著性,耗时比较长。基于同位素的探针也存在一些无法回避的缺陷,如放射性辐射的危害,空间分辨率低等,且该类技术涉及到同位素或者PET-CT等大型设备,对设备环境等要求很高,目前尚难以临床推广。
发明内容
发明目的:为了克服现有肿瘤标志物PD-L1检测技术中存在的缺陷和技术瓶颈,并提高PD-L1检测技术的灵敏度和准确度,本发明提供了一种基于纳米抗体重组蛋白和荧光量子点的用于PD-L1检测的高灵敏度探针、制备方法及应用。纳米抗体,具有体积小、稳定性好、亲和能力强、易修饰、组织穿透能力强和免疫原性低等优势,是非常理想的肿瘤标志物和靶点识别分子。量子点是一种新型纳米荧光染料,具有量子产率高、稳定性好、粒径小等特点,其产生的近红外光生物组织穿透能力强且受生物体自发荧光干扰小,是生物医学分子检测研究的热点荧光分子。
为实现上述目的,本发明采用的技术方案为:
本发明是一种高效的肿瘤标志物PD-L1的检测新技术,首先外源表达识别PD-L1的纳米抗体重组蛋白RNB-MSH,然后固相合成制备含有生物素的多肽序列GK-Bio,利用SortaseA酶介导的体外酶法将该重组蛋白与生物素多肽进行融合,得到生物素化识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio,最后将重组蛋白RNB-MS-Bio与链酶亲和素标记量子点偶联形成探针,结合流式细胞术、免疫荧光、酶联免疫吸附用于细胞表面肿瘤标志物PD-L1的检测。
所述的外源表达,是在原核大肠杆菌E.coli BL21(DE3)中进行表达。
本发明的一个目的在于,提供一种用于检测细胞表面肿瘤标志物PD-L1的基于纳米抗体重组蛋白和量子点荧光的探针,该探针由以下两个部分组成:
a、生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio,
b、链霉亲和素标记量子点;
本发明的又一个目的在于,提供一种制备上述生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio的方法,该重组蛋白由两部分经偶联所得:
c、识别PD-L1的纳米抗体重组蛋白RNB-MSH,
d、含有生物素的多肽序列,即生物素化多肽GK-Bio;
其中,所述的识别PD-L1的纳米抗体重组蛋白RNB-MSH由能够识别PD-L1的纳米抗体羧基端修饰所得,所述的修饰为:在所述的能够识别PD-L1的纳米抗体的羧基端加入MYC标签、转肽酶A(SortaseA)识别位点、His标签序列。
所述的生物素化多肽为含有生物素基团和甘氨酸重复的多肽序列,生物素基团可以以任何方式形式存在于多肽的任何位置。
在本发明实施例中,该生物素化多肽的氨基端含有1-3个甘氨酸重复序列,该生物素化多肽的羧基端含有生物素侧链修饰的赖氨酸组成。所述的生物素化多肽也可以是基于所述生物素化短肽的序列的改变具有类似目的的多肽,即基于该生物素化短肽基本特征获得的具有类似功能的多肽。
在本发明实施例中,所述的生物素化多肽选取其中一种如SEQ ID No.4所示。
在本发明实施例中,所述识别PD-L1纳米抗体重组蛋白RNB-MSH的氨基酸序列如SEQ ID No.2所示,编码所述能识别PD-L1的纳米抗体重组蛋白RNB-MSH的核苷酸序列如SEQID No.3所示。
本发明的一个目的在于,提供一种用于检测肿瘤标志物PD-L1的高灵敏度量子点探针的制备方法,包括以下步骤:
1)识别PD-L1的纳米抗体重组蛋白RNB-MSH的原核表达和纯化;
2)生物素化多肽GK-Bio的制备;
3)所述识别PD-L1的纳米抗体重组蛋白RNB-MSH与生物素化多肽偶联,得生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio;
4)所述生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio与链酶亲和素标记量子点进行偶联,制备得所述探针。
在本发明实施例中,步骤1)中,所述能识别PD-L1的纳米抗体重组蛋白RNB-MSH的表达包括以下步骤:将所述的编码能识别PD-L1的纳米抗体重组蛋白RNB-MSH的核苷酸序列克隆至pET22b表达载体,转化至大肠杆菌E.coli BL21(DE3),诱导剂的浓度为0.2-1.0mM/L,诱导的温度为16-37℃,最后经镍离子亲和柱层析纯化所得。
在本发明实施例中,步骤2)中,所述生物素的多肽序列GK-Bio经固相合成所得,包括以下步骤:以5-100mM氨基树脂为固相合成载体,使用固相合成仪按照SEQ No.3的序列进行合成,其中赖氨酸的侧链含有生物素基团,其它氨基酸均为标准氨基酸,所有氨基酸的氨基端均含有Fmoc保护基团,合成完成后在强酸条件下脱去保护基团,经半制备HPLC纯化所得。
在本发明实施例中,步骤3)中,所述的生物素化的识别PD-L1的重组蛋白RNB-MS-Bio的体外酶法偶联制备方法为:酶反应缓冲体系为50mM Tris,150mM NaCl,5mM CaCl2,将所述酶反应体系与10-150μM所述的识别PD-L1纳米抗体重组蛋白RNB-MSH、50-500μM生物素化多肽序列GK-Bio、1-10μM转肽酶A酶(Sortase A酶)混合,于4-37℃反应1-12h。然后,将反应液和镍离子磁珠孵育10-90min,获得上清溶液即为所述的生物素化的识别PD-L1的重组蛋白RNB-MS-Bio。
在本发明实施例中,步骤4)中,所述基于生物素化的识别PD-L1的重组蛋白RNB-MS-Bio和荧光量子点的探针Strep-QDs的制备方法为:反应的缓冲液体为pH6.0-8.0PBS缓冲液,在所述的缓冲液中加入两种物质,形成0.1-10μM的生物素化的识别PD-L1的重组蛋白RNB-MS-Bio、1-100μM的链酶亲和素标记量子点的偶联探针反应体系,于4-37℃下避光,10-200RPM的转速振荡反应30min-2h,制得所述探针。
本发明的另一目的在于,提供一种应用于检测肿瘤标志物PD-L1的基于纳米抗体和荧光量子点的探针的应用,用于细胞表面肿瘤标志物PD-L1的检测。
在本发明实施例中,所述细胞表面肿瘤标志物PD-L1的检测包括对细胞表面PD-L1的流式细胞术检测、对细胞表面PD-L1的酶联免疫吸附检测及对细胞表面PD-L1的免疫荧光检测。
在本发明实施例中,所述的探针用于对细胞表面PD-L1的流式细胞术检测,包括以下步骤:将10-100μL含10-100nM生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio与待测细胞混合于4-37℃孵育30-90min,PBS清洗后加10-50μL含有20-100nM的Strep-QDs溶液重悬细胞于4-37℃孵育30-90min,PBS洗涤细胞后重悬细胞,进行流式细胞仪检测,发射光谱605nm±5nm;。
在本发明实施例中,所述的探针应用于对细胞表面PD-L1的酶联免疫吸附检测,包括以下步骤:用4%多聚甲醛室温将细胞固定于多孔培养板10-30min,PBST洗细胞三次。后封闭液室温封闭1-2小时,PBST洗细胞三次,相应每孔中加入浓度为10-50nM的100μL的生物素化的识别PD-L1的重组蛋白RNB-MS-Bio,于4-37℃孵育0.5-2小时,完成后用PBST洗板三次,然后每孔加入50μL含1-20nM的所述的链霉亲和素标记的量子点,4-37℃孵育0.5-2小时,PBST洗孔三次,后用酶标仪在激发波长405nm,发射波长605nm下对每孔的吸收值进行检测。
在本发明实施例中,所述的探针应用于对细胞表面PD-L1的免疫荧光检测,包括以下步骤:将肿瘤组织切片在65℃放置45-60min进行脱蜡,用二甲苯和100%-50%乙醇浸泡5-10min,PBS洗组织切片三次,后将组织切片置于0.01M,pH6.0的柠檬酸钠缓冲溶液中,煮沸10min,保温10min,PBS洗组织切片三次,完成后用封闭液于湿盒内37℃封闭2小时,PBS洗组织切片三次,在相应的组织切片上滴加20-50μL浓度为25-100nM的生物素化的识别PD-L1的重组蛋白RNB-MS-Bio,于4-37℃孵育1-12h,完成后用PBS洗组织切片三次,然后在切片上滴加20-50μL含1-20nM的所述的链霉亲和素标记的量子点,37℃避光孵育30min,PBS洗组织切片三次,后在组织切片上滴加一滴抗荧光猝灭剂,用激光共聚焦显微镜在不同激发光下观察组织切片并拍照。
有益效果:本发明提供的用于检测细胞表面肿瘤标志物PD-L1的基于纳米抗体重组蛋白和荧光量子点的探针、制备方法及应用,与现有技术相比,具有以下优势:本发明成功构建了检测灵敏度高的细胞表面肿瘤标志物PD-L1检测方法,该方法具有稳定性好,操作简便,检测信号强度高、灵敏度高等优点。本发明所述的生物素化的识别PD-L1的重组蛋白RNB-MS-Bio的制备发方法,操作简单、成本低、转化效率高、绿色环保、无有毒有害物质排放。本发明所述的探针为基于PD-L1肿瘤标志物的肿瘤精准诊断和治疗提供了可靠有效的技术支撑。
附图说明
图1为本发明技术路线示意图。
图2是纳米抗体重组蛋白RNB-MSH的表达与纯化后的SDS-PAGE电泳图。
其中,泳道M为分子量标准蛋白样品,泳道H为全菌液,泳道S为发酵液上清,泳道1为50mM咪唑浓度洗脱样品,泳道2-4为100mM咪唑浓度洗脱样品,泳道5-8为150mM咪唑浓度洗脱样品,泳道9-10为250mM咪唑浓度洗样品,泳道11为500mM咪唑浓度洗脱样品。
图3为含有生物素化多肽GK-Bio的质谱图。
图4为含有生物素化多肽GK-Bio纯化后的HPLC图谱。
图5是酶法制备生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio流程中的关键SDS-PAGE电泳图。
其中,泳道M为分子量标准蛋白样品,泳道1为纯化后纳米抗体重组蛋白RNB-MSH的对照,泳道2为SortaseA酶蛋白对照,泳道3为酶反应0时样品,泳道4为酶反应3小时后的样品,泳道5为镍磁珠纯化后的生物素化的识别PD-L1纳米抗体重组蛋白RNB-MS-Bio,泳道6为镍磁珠洗杂样品。
图6是验证生物素化的纳米抗体重组蛋白RNB-MS-Bio的Western Blot图;泳道M为标准蛋白样品,泳道1为与HRP-Strep孵育后的RNB-MS-Bio。
图7为用于检测细胞表面肿瘤标志物PD-L1的基于纳米抗体重组蛋白和量子点荧光的探针的酶联免疫吸附应用及其特异性的关键数据。
其中,CHO为不表达PD-L1的中国仓鼠卵巢细胞、CHO-PD-L1为高表达PD-L1的中国仓鼠卵巢细胞。同时以PBS作为空白对照组。RNB-MS-Bio+Strep-QDs为生物素化后的纳米抗体重组蛋RNB-MS-Bio和荧光量子点的探针处理实验组、PBS+Strep-QDs为荧光量子点的单独处理对照。
图8为比较本发明提供探针和基于传统荧光染料FITC探针的检测效果的关键数据。
其中,CHO为不表达PD-L1的中国仓鼠卵巢细胞、CHO-PD-L1为高表达PD-L1的中国仓鼠卵巢细胞、MDA-MB-231为表达PD-L1的肿瘤细胞。同时以PBS为空白对照组。RNB-MS-Bio+Strep-FITC为传统荧光染料FITC组,RNB-MS-Bio+Strep-QDs为本发明探针组,PBS+Strep-FITC、PBS+Strep-QDs为对应的阴性对照组(传统荧光染料的单独处理对照组、荧光量子点的单独处理对照组)。
图9为用于检测细胞表面肿瘤标志物PD-L1的基于纳米抗体重组蛋白和量子点荧光探针的流式细胞术应用关键数据。
其中,CHO为不表达PD-L1的中国仓鼠卵巢细胞、CHO-PD-L1为高表达PD-L1的中国仓鼠卵巢细胞、MDA-MB-231为表达PD-L1的肿瘤细胞。Control为以PBS为空白对照组、RNB-MS-Bio+Strep-QDs为本发明所述的探针。
图10为用于检测细胞表面肿瘤标志物PD-L1的基于纳米抗体重组蛋白和量子点荧光探针的免疫荧光应用关键数据。
其中,25-100nM RNB-MS-Bio为不同浓度本发明所述的生物素化后的纳米抗体重组蛋白实验组,PBS为对照组。Strep-QDs为量子点荧光图,DAPI为细胞核荧光图,Merge为两张图的共定位图。
具体实施方式
本发明公开了一种用于检测细胞表面肿瘤标志物PD-L1的探针、制备方法及应用,具体涉及一种基于生物素化的能识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio和链霉亲和素标记的荧光量子组成的探针、制备方法及应用,如图1所示,制备发明的探针及其应用包括步骤:
1)识别PD-L1的纳米抗体重组蛋白RNB-MSH的表达;
2)含有生物素基团的多肽即生物素化多肽GK-Bio的制备;
3)所述能识别PD-L1的纳米抗体重组蛋白RNB-MSH与含有生物素基团的多肽即生物素化多肽GK-Bio之间的体外酶法融合,得到生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio;
4)生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio与链酶亲和素标记量子点的偶联探针制备。
5)利用上述探针通过流式细胞术、酶联免疫吸附、免疫荧光等对细胞表面的肿瘤标志物PD-L1进行检测。
其中,步骤1)中,识别PD-L1的纳米抗体重组蛋白RNB-MSH的制备,步骤如下:
1.1)设计编码上述重组蛋白的基因序列,在其羧基端加入MYC标签、转肽酶A(SortaseA)识别位点、His标签。
1.2)利用大肠杆菌BL21(DE3)表达上述重组蛋白;
1.3)采用镍离子亲和层析柱纯化得到上述的重组蛋白;
其中,步骤3)中,生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio的制备,步骤如下:
2.1)将能识别PD-L1的纳米抗体重组蛋白RNB-MSH与含有生物素基团的多肽GK-Bio在转肽酶A的催化作用下进行偶联;
2.2)镍离子亲和层析磁珠去处未反应物料,纯化得到生物素化的能识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio。
本发明所提供的探针,结合酶联免疫吸附技术、流式细胞术、免疫荧光能够有效检测细胞表面肿瘤标志物PD-L1的表达,可以应用于肿瘤标志物PD-L1相关肿瘤的临床诊断、治疗、及愈后监测。所述的肿瘤标志物PD-L1检测方法比用传统荧光染料制备的探针荧光强度高,灵敏度高、专一性好、制备成本较低。
下面结合附图和实施例对本发明作更进一步的说明。但应当理解这些实施例并非限制本发明的范围。根据下述实施例,可以更好的理解本发明。然而,本领域的技术人员容易理解,实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
在本发明中所使用的术语,除非另有说明,一般具有本领域普通技术人员通常理解的含义。
在以下实施例中,未详细描述的各种过程和方法是本领域中公知的常规方法。
实施例1能识别PD-L1的纳米抗体重组蛋白RNB-MSH的设计
本实施例中,纳米抗体重组蛋白RNB-MSH序列中能够识别PD-L1的部分蛋白质氨基酸序列来源于专利CN 106397592 A(原始氨基酸序列为该专利中的SEQ ID NO.28),氨基酸序列如SEQ ID No.1所示。在其他实施例中,只要能满足可识别PD-L1的蛋白质氨基酸序列均可以使用,均在本发明的保护范围内。
对SEQ ID No.1所示序列的羧基末端添加MYC标签、转肽酶A识别序列、镍离子亲和层析纯化组氨酸标签后得到本发明中具备额外新功能的能识别PD-L1的纳米抗体重组蛋白RNB-MSH(SEQ ID No.2)。具体方法如下:
首先,在纳米抗体的蛋白质序列羧基末端加入MYC标签(EQKLISEEDLNGAA)、转肽酶A酶切的识别位点LPETGG(L:亮氨酸;P:脯氨酸;E:谷氨酸;T:苏氨酸;G:甘氨酸);然后再加入组氨酸纯化标签HHHHHH(H:组氨酸),得到本发明所述的能识别PD-L1的纳米抗体重组蛋白RNB-MSH的氨基酸序列(SEQ ID No.2)。将上述重组蛋白质序列按照大肠杆菌核苷酸密码子反推得到识别PD-L1的纳米抗体重组蛋白RNB-MSH的核苷酸序列,如SEQ ID No.3所示。合成核苷酸序列(SEQ ID No.3)并亚克隆插入表达载体pET22b(+)质粒中,并将该质粒转入大肠杆菌E.coli BL21(DE3)。该菌株即为生产能识别PD-L1的纳米抗体重组蛋白RNB-MSH的发酵菌株E.coli BL21(DE3)-RNB-MSH。
实施例2能识别PD-L1的纳米抗体重组蛋白RNB-MSH的表达与纯化
将E.coli BL21(DE3)-RNB-MSH甘油菌接种至5mL含有氨苄抗性的LB发酵培养基(蛋白胨10g/L,酵母粉5g/L,氯化钠10g/L),于摇床37℃,200rpm过夜培养。第二天按照2%的接种量转接TB发酵培养基(蛋白胨12g/L,酵母粉24g/L,甘油4g/L,KH2PO423.1g/L,K2HPO4125.4g/L),当发酵菌体密度达到OD600约为0.6-0.8时,加入终浓度为0.8mM的诱导剂IPTG,发酵的条件为摇床37℃,200rpm转速,时间24h。
发酵完毕后,9000rpm离心5min收集菌体,弃置上清。菌体用破壁缓冲液(10mMTris,500mM NaCl,pH 8.0)重悬后,用超声破碎仪进行破碎,破碎条件为:冰浴,运行2s,停3s,运行时长20-60min。破碎完毕后,9000rpm离心10min,重复三次,以彻底除去细胞碎片,得到澄清的破壁上清。破壁上清用镍离子亲和层析柱进行纯化,用含50mM咪唑的洗涤缓冲液冲洗层析柱以去除杂蛋白。用含50mM-500mM咪唑的洗涤缓冲液采用梯度方式将重组蛋白RNB-MSH洗下。纯化后的蛋白经脱盐后冻干,置于-20℃备用。
图2为纯化后的重组蛋白SDS-PAGE电泳表征图,经Image J软件灰度分析表明,纯化后RNB-MSH的纯度在95%以上。
实施例3含有生物素基团的多肽GK-Bio的制备
本申请实施例中选用的生物素化多肽GK-Bio的氨基酸序列如SEQ ID No.4所示。采用基于Fmoc保护策略的固相合成技术进行GK-Bio的制备。所有Fmoc保护的标准氨基酸、含有生物素基团的赖氨酸均用DMF溶解,浓度为0.2M,取0.22g Rink Resin固相树脂,采用CEM Liberty BlueTM自动多肽合成仪进行合成。Fmoc脱保护试剂为20%的哌啶溶液、缩合试剂为0.25M的TBTU。合成结束后将含有多肽的树脂转入圆底烧瓶,加入5-10mL切割试剂,所述的切割试剂为TFA/H2O/TIPS(95:2.5:2.5,v/v/v),于室温磁力搅拌2-4小时。然后过滤去除树脂,收集上清并用冰乙醚进行沉淀和离心,干燥后得到粗品。将上述粗肽样品使用半制备高效液相色谱法HPLC及反向C18柱进行纯化,流动相A为100%的乙腈,流动相B为100%的超纯水,收集目标组分得到纯化的多肽,冻干并称重。
称取1mg上述多肽纯品,加入500μL乙腈和水的混合溶液进行溶解,过滤后通过RP-HPLC进行检测,通过峰面积归一法对所得GK-Bio多肽的纯度进行计算,纯度大于95%。
图3为纯化后的含有生物素基团的多肽GK-Bio的质谱分析图,分子量与预期值相符为999.1Da。
图4为纯化后的含有生物素基团的多肽GK-Bio的HPLC分析图,数据分析表明其纯度达到95%以上。
实施例4生物素化识别PD-L1纳米抗体重组蛋白RNB-MS-Bio的制备
将20μM识别PD-L1纳米抗体重组蛋白RNB-MSH、100μM含有生物素化多肽GK-Bio、5μM Sortase A酶溶解混合于1mL含有50mM Tris,150mM NaCl,5mM CaCl2的酶反应体系,于4-37℃反应1-12h,定时取样进行分析。然后,将最终的反应液和镍离子磁珠孵育30min,获得上清溶液即为所述的生物素化的识别PD-L1的重组蛋白RNB-MS-Bio。将所得的重组蛋白及不同时间的粗反应液通过SDS-PAGE凝胶电泳分析。
图5为SDS-PAGE电泳分析的数据图,RNB-MS-Bio较RNB-MSH分子量有明显减少,符合预期的分子量变化。经过纯化后的RNB-MS-Bio纯度大于95%。
图6为RNB-MS-Bio的western blot分析结果图。利用链霉亲和素标记的辣根过氧化物酶分析结果显示该重组蛋白成功进行了生物化的修饰。
实施例5生物素化识别PD-L1纳米抗体重组蛋白RNB-MS-Bio与荧光量子点探针用于酶联免疫吸附对细胞表面PD-L1的检测
将待测细胞接种于96孔板内培养24小时,去除上清后用4%多聚甲醛于30℃下固定20min,PBST洗细胞三次。然后用酶联免疫吸附ELISA封闭液于30℃条件下封闭2小时,PBST洗细胞三次,相应每孔中加入100μL浓度为25nM的RNB-MS-Bio,37℃条件下孵育1小时,对照组加入100μLPBS,孵育完成后用PBST洗板三次。然后每孔加入50μL浓度为20nM的链霉亲和素标记的量子点(QDs),37℃孵育1小时,PBST洗板三次,对照组则加入50μL的PBS。最后用酶标仪进行荧光检测,激发波长405nm,发射波长605m。
图7为所述探针对细胞表面PD-L1检测特异性的关键数据。所述探针在细胞无PD-L1表达(即CHO Cell)时其本底荧光强度非常低,而当细胞表面表达PD-L1(即CHO/PD-L1Cell)时探针荧光强度显著的高于细胞无PD-L1表达的细胞。仅有链霉亲和素标记的量子点(Strep-QDs)的对照组的荧光强度则非常低。表明本发明提供的探针具有显著提高的灵敏度。
图8为所述探针与传统荧光检测方法的对比关键数据。以表达PD-L1的肿瘤细胞MDA-MB-231为参照组,在PD-L1无表达时(CHO Cell),本发明提供的探针(RNB-MS-Bio+Strep-QDs)本身的非特异性吸附比传统荧光染料(RNB-MS-Bio+Strep-FITC)的探针较低,与空白对照组(PBS+Strep-FITC、PBS+Strep-QDs)无明显差异;
在PD-L1高表达时(CHO-PD-L1 Ce1l),本发明提供的探针(RNB-MS-Bio+Strep-QDs)其信号强度远远强于传统荧光染料(RNB-MS-Bio+Strep-FITC)及空白对照组(PBS+Strep-FITC、PBS+Strep-QDs)的探针。表明本发明提供的探针具有显著提高的灵敏度。
实施例6生物素化识别PD-L1纳米抗体重组蛋白RNB-MS-Bio与荧光量子点探针用于流式细胞术对细胞表面PD-L1的检测
将待测细胞转移至流式管内,离心去上清,PBS洗涤细胞2次,每管中加入100μL含50nM生物素化识别PD-L1纳米抗体重组蛋白RNB-MS-Bio的PBS溶液,混合均匀后于冰上孵育30min,对照组则加入等量流式液。孵育完成后加入500μL流式液,1000g,4℃离心5min后去上清,加50μL浓度为20nM的链霉亲和素标记的量子点QDs重悬细胞,冰育30min后加入500μL流式液,1000g,4℃离心5min后去上清,PBS洗涤细胞2次,离心5min;每管加入200μLPBS重悬细胞,流式细胞仪进行检测。
图9为所述探针用于流式细胞术检测PD-L1的关键数据。在不表达PD-L1的细胞CHO样品中,采用所述探针RNB-MS-Bio+Strep-QDs检测结果与对照组Control相比几乎没有差别。
在高表达PD-L1的CHO-PD-L1和MDA-MB-231样品中,所述探针RNB-MS-Bio+Strep-QDs检测的信号位移明显高于对照组Control,尤其是在高表达PD-L1的CHO-PD-L1中,检测的信号位移高达两个次方级,表明本发明所述的探针具有高灵敏度和特异性。
实施例7生物素化识别PD-L1纳米抗体重组蛋白RNB-MS-Bio与荧光量子点探针用于免疫荧光对细胞表面PD-L1的检测
将肿瘤组织切片放在置片架上后放入65℃烘箱中脱蜡45min,用二甲苯浸泡两次,每次10min,再用100%-50%乙醇浸泡5min,后将组织切片置于0.01M,pH6.0的柠檬酸钠缓冲溶液中,煮沸10min,保温10min,PBS洗组织切片三次,完成后在切片上肿瘤组织处滴加10%BSA封闭液于湿盒内37℃封闭2小时,PBS洗组织切片三次,在相应切片的肿瘤组织处滴加50μL浓度为25-100nM的生物素化的识别PD-L1的重组蛋白RNB-MS-Bio,于4℃孵育12小时,成后用PBS洗组织切片三次,然后在切片上肿瘤组织处滴加20-50μL含10nM的所述的链霉亲和素标记的量子点,37℃避光孵育30min,PBS洗组织切片三次,后在切片的肿瘤组织处滴加一滴抗荧光猝灭剂,用激光共聚焦显微镜在不同激发光下观察组织切片并拍照。
图10为所述探针用于免疫荧光检测PD-L1的关键数据。在本发明探针的实验组中,不同浓度的探针量子点发光位置均与DAPI染的细胞核发光位置重叠,而在PBS对照组中则检测不到量子点的荧光,表明本发明探针能高效检测到肿瘤细胞表面PD-L1的表达。
以上实施例说明,本发明提供的基于生物素化识别PD-L1纳米抗体重组蛋白RNB-MS-Bio与荧光量子点的探针,结合酶联免疫吸附、流式细胞术、免疫荧光可特异性地与细胞表面的肿瘤标志物PD-L1受体结合,其荧光强度显著高于现有的常规荧光探针。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 江南大学
<120> 用于检测肿瘤标志物PD-L1的高灵敏度量子点探针、制备方法及应用
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Claims (10)
1.一种用于检测肿瘤标志物PD-L1的高灵敏度量子点探针,其特征在于,该探针由以下两个部分偶联形成:
a、生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio,
b、链酶亲和素标记的量子点;
其中,所述生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio由以下组分偶联获得:
c、识别PD-L1的纳米抗体重组蛋白RNB-MSH,
d、生物素化多肽GK-Bio;
其中,所述识别PD-L1的纳米抗体重组蛋白RNB-MSH由识别PD-L1纳米抗体经羧基端修饰获得,所述羧基端修饰为:在所述识别PD-L1的纳米抗体的羧基端修饰Myc标签、转肽酶A识别位点、His标签序列;
所述生物素化多肽GK-Bio为含有生物素基团和甘氨酸重复的多肽序列。
2.根据权利要求1所述的用于检测肿瘤标志物PD-L1的高灵敏度量子点探针,其特征在于,所述识别PD-L1的纳米抗体重组蛋白RNB-MSH的氨基酸序列如SEQ ID No.2所示。
3.根据权利要求1所述的用于检测肿瘤标志物PD-L1的高灵敏度量子点探针,其特征在于,所述生物素化多肽为氨基酸序列中含有1-3个甘氨酸序列,或是基于所述短肽的序列的改变具有类似目的的多肽。
4.根据权利要求3所述的用于检测肿瘤标志物PD-L1的高灵敏度量子点探针,其特征在于,所述生物素化多肽的氨基酸序列如SEQ ID No.4所示。
5.根据权利要求1-4任一所述的用于肿瘤标志物PD-L1的高灵敏度量子点探针的制备方法,其特征在于,包括以下步骤:
1)识别PD-L1的纳米抗体重组蛋白RNB-MSH的原核表达和纯化;
2)生物素化多肽GK-Bio的制备;
3)所述识别PD-L1的纳米抗体重组蛋白RNB-MSH与生物素化多肽偶联,得生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio;
4)所述生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio与链酶亲和素标记量子点进行偶联,制备得所述探针。
6.根据权利要求5所述的用于检测肿瘤标志物PD-L1的高灵敏度量子点探针的制备方法,其特征在于,步骤2)中,所述生物素化多肽GK-Bio的制备采用多肽固相合成方法,包括以下步骤:以5-100mM氨基树脂为固相合成载体,使用固相合成仪器按照SEQ No.3的序列进行合成,其中赖氨酸的侧链含有生物素基团,其它氨基酸均为标准氨基酸,所有氨基酸的氨基端含有Fmoc保护基团,合成完成后在强酸条件下脱去保护基团,经半制备HPLC纯化所得。
7.根据权利要求5所述的用于检测肿瘤标志物PD-L1的高灵敏度量子点探针的制备方法,其特征在于,步骤3)中,所述偶联采用体外酶法融合修饰的方法,包括以下步骤:酶反应体系为包含50mM Tris,150mM NaCl,5mM CaCl2的pH 6.0~8.0PBS缓冲液,将所述酶反应体系与10~150μM识别PD-L1的纳米抗体重组蛋白RNB-MSH、50~500μM生物素化多肽GK-Bio、1~10μM转肽酶A酶混合,于4~37℃震荡反应1~12h得反应液;将所述反应液和镍离子磁珠孵育10~90min,获得上清溶液即为所述生物素化的识别PD-L1的纳米抗体重组蛋白RNB-MS-Bio。
8.根据权利要求5所述的用于检测肿瘤标志物PD-L1的高灵敏度量子点探针的制备方法,其特征在于,步骤4)中,所述探针的制备方法为:在pH 6.0~8.0PBS缓冲液中,形成0.1~10μM生物素化的识别PD-L1的重组蛋白RNB-MS-Bio、1~100μM链酶亲和素标记量子点的偶联探针反应体系,4~37℃下避光,10~200rpm振荡反应0.5h~2h。
9.根据权利要求1-4任一所述的用于检测肿瘤标志物PD-L1的高灵敏度量子点探针,或根据权利要求5-8任一所述方法制备的的用于检测肿瘤标志物PD-L1的高灵敏度量子点探针的应用,其特征在于,用于细胞表面肿瘤标志物PD-L1的检测。
10.根据权利要求9所述的用于检测肿瘤标志物PD-L1的高灵敏度量子点探针的应用,其特征在于,所述细胞表面肿瘤标志物PD-L1的检测包括对细胞表面PD-L1的流式细胞术检测、对细胞表面PD-L1的酶联免疫吸附检测及对细胞表面PD-L1的免疫荧光检测。
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CN117147830B (zh) * | 2023-10-26 | 2024-01-12 | 德州国科医疗科技有限公司 | 一种特异性真菌d-葡聚糖检测荧光染色液 |
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