CN113817067B - 一种环二鸟苷酸光学探针及其制备方法和应用 - Google Patents
一种环二鸟苷酸光学探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了c‑di‑GMP荧光探针,包含(A)光学活性多肽或与其具有至少70%序列相同性并具有光学活性的变体,和(B)c‑di‑GMP敏感多肽或与其具有至少70%序列相同性并具有c‑di‑GMP敏感性的变体,其中,A位于B的序列内,将B分为B1和B2两个部分,形成氨基端到羧基端方向上具有B1‑A‑B2的结构,或一个或多个A位于两个或更多个B之间,通过接头与两端的B连接。本发明提供的c‑di‑GMP荧光探针蛋白分子量相对较小且易于表达,荧光动态变化大,特异性好,并且能够通过基因操作的在细胞和大肠杆菌中进行相关的应用研究,能够高通量、实施、定量检测c‑di‑GMP。
Description
技术领域
本发明涉及光学探针的技术领域,尤其涉及一种环二鸟苷酸(c-di-GMP)光学探针及其制备方法和应用。
背景技术
C-di-GMP(环二鸟苷酸)最初是由Moshe Benziman和他的同事在1987年发现的,他们发现c-di-GMP可以别构激活α-葡萄变形杆菌中的纤维素合成。c-di-GMP已被证明可以调节细菌的生物膜的形成、细菌运动、细菌毒力、细胞周期、分化和其他过程。此外,c-di-GMP还可以作为核糖开关发挥作用。调节细菌中的c-di-GMP信号通路可能是一种控制医疗和工业环境中生物膜形成和扩散的新方法。
正是由于c-di-GMP具有如此重要的作用,因此准确检测其变化对于研究c-di-GMP介导的信号通路具有重要意义。C-di-GMP的传统检测方法是色谱-质谱-核磁共振波谱连用法:Franziska等人将色谱、质谱、核磁共振三种技术连用对c-di-GMP进行定量分析,从而确定DGC酶催化产生c-di-GMP的含量(Zahringer,Massa et al.2011);该研究先通过HPLC-MS(高效液相色谱-质谱)连用的技术对纯化的c-di-GMP进行分析鉴定,随后又用1H-NMR(核磁共振波谱)的方法对c-di-GMP进行定量(Zahringer,Massa et al.2011)。能够应用于活细胞或或细菌中的方法目前有RNA探针法、FRET型荧光探针、BRET型荧光探针。MingC.Hammond等利用RNA适配体GEMM-1/Spinach和DFHBI染料组成了Vc2-Spinach小分子核酸生物传感器,用于检测c-di-GMP和cAMP-GMP(Kellenberger,Wilson et al.2013)。Samuel.Miller等将CFP和YFP与PilZ蛋白融合构建了一种FRET型的c-di-GMP生物传感器,用于研究c-di-GMP对铜绿假单胞菌细胞分裂的影响(Christen,Kulasekara et al.2010);Zhao-Xun Liang等人将一种青色荧光蛋白mCerulean与黄色荧光蛋白mVenus与MrkH-VCA0042结合蛋白融合构建了一种FRET型生物传感器,用于研究生物膜分散剂或在巨噬细胞的恶劣环境条件下的大肠杆菌中c-di-GMP水平变化(Ho,Chong et al.2013)MingC.Hammond等应用CSL-BRET(分裂荧光素酶与生物发光共振能量转移)开发了第一个用于c-di-GMP检测的化学发光生物传感器(Dippel,Anderson et al.2018)。
传统的核磁、色谱方法在活细胞或细菌研究中这些检测方法存在很大的缺陷,需要经过耗时的样品处理过程:细胞或细菌破碎、分离提取纯化等,不能在活细菌和细胞中进行原位、实时、动态、高通量和高时空分辨率的检测。而应用于活细胞或活菌中的RNA方法受到内源性的c-di-GMP干扰,荧光依靠外源添加染料来产生。因此,还需要考虑染料在细菌中的渗透性问题。FRET型探针包括两种荧光蛋白,存在两种蛋白的光谱重叠易干扰,共同表达的效率低和水溶性差等问题。在BRET方法中,荧光素酶Rluc需要外源添加底物才能产生荧光信号,检测方法较为繁琐。此外,体内外广泛存在的c-di-GMP类似物GTP、GDP、ATP、AMP、ADP、NAD+、NADP+严重影响现有技术中检测方法的准确性。因此,本领域仍需要能在活细胞或活细菌内、外实时原位、定量、准确高效检测c-di-GMP的方法。
发明内容
有鉴于此,本发明的目的在于提供一种在活细胞或活细菌内、外实时原位、定量、准确高效检测c-di-GMP的方法。
为了实现上述发明目的,本发明提供以下技术方案:
本发明提供一种c-di-GMP光学探针,其是融合蛋白,包含光学活性多肽A或与其具有至少70%序列相同性并具有光学活性的变体,和c-di-GMP敏感多肽B或与其具有至少70%序列相同性并具有c-di-GMP敏感性的变体,
其中,A位于B的序列内,将B分为B1和B2两个部分,形成氨基端到羧基端方向上具有B1-A-B2的结构,或
一个或多个A位于两个或更多个B的序列之间,通过接头与两端的B连接,优选地,一个A位于两个B的序列之间,通过接头与两端的B连接,形成氨基端到羧基端方向上具有B-A-B的结构。
本发明提供一种c-di-GMP光学探针,其是具有选自以下序列的融合蛋白,
(I)光学活性多肽A位于c-di-GMP敏感多肽B之间形成的氨基端到羧基端方向上具有B1-A-B2的结构的氨基酸序列,或
一个或多个光学活性多肽A位于两个或更多个c-di-GMP敏感多肽B之间,通过接头与两端的B连接的序列,和
(II)与(I)有至少35%、40%、50%、60%、70%、80%、85%、90%、95%、99%序列相同性并具有检测c-di-GMP的功能的变体。
在一个实施方案中,c-di-GMP敏感多肽包括c-di-GMP结合蛋白的c-di-GMP结合域。
在一个实施方案中,所述敏感多肽源自天蓝色链霉菌。在一个实施方案中,所述敏感多肽是c-di-GMP结合蛋白或其具有与c-di-GMP结合功能的片段。
在一个或多个实施方案中,所述c-di-GMP结合蛋白是BldD蛋白。
在一个实施方案中,c-di-GMP敏感多肽具有SEQ ID NO:1所示的序列,或与其有至少35%、40%、50%、60%、70%、80%、85%、90%、95%、99%序列相同性并保留c-di-GMP结合功能的变体。
在一个或多个实施方案中,所述变体是截短的变体。
在一个实施方案中,光学活性多肽是荧光蛋白或其功能片段或变体。
在一个实施方案中,荧光蛋白选自黄色荧光蛋白(如cpYFP,优选具有SEQ ID NO:2所示序列或与其具有至少70%序列相同性的序列)、绿色荧光蛋白(如cpGFP,优选具有SEQID NO:3所示序列或与其具有至少70%序列相同性的序列)、蓝色荧光蛋白(如cpBFP,优选具有SEQ ID NO:4所示序列或与其具有至少70%序列相同性的序列)、红色荧光蛋白(如cpmApple,优选具有SEQ ID NO:5所示序列或与其具有至少70%序列相同性的序列)中任一种或多种。
在一个实施方案中,荧光蛋白如SEQ ID NO:2-5中任一所示。
在一些实施方案中,所述融合蛋白还包含位于其N端和/或C端的其他多肽。在一些实施方案中,其他多肽是将融和蛋白定位到不同细胞器或亚细胞器的多肽、用于纯化的标签或者用于免疫印迹的标签。
在第一方面的一些实施方案中,光学活性多肽位于c-di-GMP敏感多肽的选自以下的位置中:氨基酸9-19和/或35-51。
在一个实施方案中,光学活性多肽位于c-di-GMP敏感多肽的选自以下的一个或多个位点:9/10,10/11,11/12,16/17,17/18,18/19,35/36,36/37,36/38,36/39,36/40,36/41,36/42,37/38,37/39,37/40,37/41,37/42,38/39,38/40,38/41,38/42,39/40,39/41,39/42,40/41,40/42,41/42,42/43,43/44,44/45,45/46,46/47,47/48,48/49,49/50,50/51。
在一个实施方案中,光学活性多肽位于c-di-GMP敏感多肽的选自以下的一个或多个位点:9/10,10/11,11/12,17/18,35/36,36/37,36/38,36/39,36/40,36/41,36/42,37/38,37/41,38/40,38/41,38/42,39/40,39/41,39/42,40/41,43/44,44/45,45/46,48/49,50/51。在一个实施方案中,光学活性多肽位于c-di-GMP敏感多肽的选自以下的一个或多个位点:36/37,36/38,36/39,36/40,36/41,36/42,37/38。
在一个实施方案中,光学活性多肽位于c-di-GMP敏感多肽的选自以下的一个或多个位点:35/36,36/38,37/38,37/39,37/40,37/41,37/42,38/39,38/40,39/40,40/41,41/42。在一个实施方案中,光学活性多肽位于c-di-GMP敏感多肽的选自以下的一个或多个位点:37/38,37/40,37/41,39/40。
在一个实施方案中,光学活性多肽位于c-di-GMP敏感多肽的选自以下的一个或多个位点:16/17,35/36,36/38,36/39,37/38,37/39,37/40,37/41,37/42,38/39,39/40,40/41。在一个实施方案中,光学活性多肽位于c-di-GMP敏感多肽的选自以下的一个或多个位点:37/39,37/40,37/41,37/42。
在一个实施方案中,光学活性多肽位于c-di-GMP敏感多肽的选自以下的一个或多个位点:36/39,38/39,38/40,38/41,39/40,39/41。
在一个或多个实施方案中,光学活性多肽是黄色荧光蛋白,其位于c-di-GMP敏感多肽的选自以下的一个或多个位点:9/10,10/11,11/12,17/18,35/36,36/37,36/38,36/39,36/40,36/41,36/42,37/38,37/41,38/40,38/41,38/42,39/40,39/41,39/42,40/41,43/44,44/45,45/46,48/49,50/51。更优选地,光学活性多肽是黄色荧光蛋白,其位于c-di-GMP敏感多肽的选自以下的一个或多个位点:36/37,36/38,36/39,36/40,36/41,36/42,37/38。优选地,光学活性多肽具有如SEQ ID NO:2所示序列。优选地,c-di-GMP敏感多肽具有SEQ ID NO:1所示的序列。
在一个或多个实施方案中,光学活性多肽是绿色荧光蛋白,其位于c-di-GMP敏感多肽的选自以下的一个或多个位点:35/36,36/38,37/38,37/39,37/40,37/41,37/42,38/39,38/40,39/40,40/41,41/42;更优选地,光学活性多肽是绿色荧光蛋白,其位于c-di-GMP敏感多肽的选自以下的一个或多个位点:37/38,37/40,37/41,39/40。优选地,光学活性多肽具有如SEQ ID NO:3所示序列。优选地,c-di-GMP敏感多肽具有SEQ ID NO:1所示的序列。
在一个或多个实施方案中,光学活性多肽是蓝色荧光蛋白,其位于c-di-GMP敏感多肽的选自以下的一个或多个位点:16/17,35/36,36/38,36/39,37/38,37/39,37/40,37/41,37/42,38/39,39/40,40/41;更优选地,光学活性多肽是蓝色荧光蛋白,其位于c-di-GMP敏感多肽的选自以下的一个或多个位点:37/39,37/40,37/41,37/42。优选地,光学活性多肽具有如SEQ ID NO:4所示序列。优选地,c-di-GMP敏感多肽具有SEQ ID NO:1所示的序列。
在一个或多个实施方案中,光学活性多肽是红色荧光蛋白,其位于c-di-GMP敏感多肽的选自以下的一个或多个位点:36/39,38/39,38/40,38/41,39/40,39/41。优选地,光学活性多肽具有如SEQ ID NO:5所示序列。优选地,c-di-GMP敏感多肽具有SEQ ID NO:1所示的序列。
在一个或多个实施方案中,光学探针具有SEQ ID NO:6-12所示序列或由其组成。在一个实施方案中,本发明提供的光学探针包含与SEQ ID NO:6-12中任一有至少35%、至少40%、至少50%、至少60%、至少70%、至少80%、至少85%、至少90%、至少95%、至少99%序列相同性的序列。
在一个实施方案中,所述融合蛋白还包含侧接所述光学活性多肽A的任一端或两端的一个或多个接头,所述融合蛋白具有B1-接头1-A-接头2-B2的结构。
在第一方面的其他实施方案中,光学活性多肽A位于两个或多个c-di-GMP敏感多肽B的序列之间,通过接头X和Y与两端的B连接,形成顺序为B-X-A-Y-B的结构。
在一个或多个实施方案中,c-di-GMP敏感多肽具有SEQ ID NO:1所示的序列,或与其有至少70%、80%、85%、90%、95%、99%序列相同性并保留c-di-GMP结合功能的变体。优选地,c-di-GMP敏感多肽如SEQ ID NO:1所示。在一个或多个实施方案中,光学活性多肽具有如SEQ ID NO:2-5中任一所示的序列或与其具有至少70%序列相同性的序列。
在一个或多个实施方案中,X和Y分别独立选自无或由G和/或S组成的接头肽。在一个或多个实施方案中,所述接头肽的长度为0-10个氨基酸。在一个或多个实施方案中,X为0-10、0-9、0-8、0-7、0-6、0-5、0-4、0-3、0-2、0-1、0个氨基酸,优选0-7个氨基酸,更优选0-5个氨基酸。在一个或多个实施方案中,Y为0-10、1-10、2-10、2-9、2-8、2-7、2-6、2-5、2-4、2-3、2个氨基酸,优选2-9个氨基酸,更优选2-7个氨基酸。
在一个或多个实施方案中,X和Y分别独立选自无、G、S、GS、SG、GGS、GSG、SGG、GSS、SGS、SSG、GGGS、GGSG、GSGG、SGGG、GGSS、GSGS、GSSG、SGGS、SGSG、SSGG、GSSS、SGSS、SSGS、SSSG、GSGGS、GSGGGS、GSGSGGS。
在一个或多个实施方案中,X选自无、G、GS、GGS、GGGS、GSGGS。
在一个或多个实施方案中,Y选自无、GS、GSG、GSGS、GSGGS、GSGGGS、GSGSGGS。
在一个实施方案中,X和Y选自以下的一种或多种X/Y组合:0/GS,0/GSG,0/GSGS,0/GSGGS,0/GSGGGS,0/GSGSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,G/GSGGGS,G/GSGSGGS,GS/GS,GS/GSG,GS/GSGS,GS/GSGGS,GS/GSGGGS,GS/GSGSGGS,GGS/GS,GGS/GSG,GGS/GSGS,GGS/GSGGS,GGS/GSGGGS,GGS/GSGSGGS,GGGS/GS,GGGS/GSG,GGGS/GSGS,GGGS/GSGGS,GGGS/GSGGGS,GGGS/GSGSGGS,GSGGS/GS,GSGGS/GSG,GSGGS/GSGS,GSGGS/GSGGS,GSGGS/GSGGGS,GSGGS/GSGSGGS。
在一个或多个实施方案中,光学活性多肽是黄色荧光蛋白,优选具有SEQ ID NO:2所示的序列或与其具有至少70%序列相同性的序列。X和Y的氨基酸数为:X为0个氨基酸且Y为3、4、5、6或7个氨基酸,X为1个氨基酸且Y为2、3、4、5、6或7个氨基酸,X为2个氨基酸且Y为4、5、6或7个氨基酸,X为3个氨基酸且Y为3、5、6或7个氨基酸,X为4个氨基酸且Y为3、4、5、6或7个氨基酸,或X为5个氨基酸且Y为2、3、4、5、6或7个氨基酸。优选地,X和Y的氨基酸数为:X为0个氨基酸且Y为3、4或5个氨基酸,X为1个氨基酸且Y为2、3、4或5个氨基酸,X为2个氨基酸且Y为3个氨基酸,X为3个氨基酸且Y为5个氨基酸,或X为5个氨基酸且Y为3个氨基酸。在一个实施方案中,X和Y选自以下的一种或多种X/Y组合:0/GSG、0/GSGS、0/GSGGS、0/GSGGGS、0/GSGSGGS、G/GS、G/GSG、G/GSGS、G/GSGGS、G/GSGGGS、G/GSGSGGS、GS/GSGS、GS/GSGGS、GS/GSGGGS、GS/GSGSGGS、GGS/GSG、GGS/GSGGS、GGS/GSGGGS、GGS/GSGSGGS、GGGS/GSG、GGGS/GSGS、GGGS/GSGGS、GGGS/GSGGGS、GGGS/GSGSGGS、GSGGS/GS、GSGGS/GSG、GSGGS/GSGS、GSGGS/GSGGS、GSGGS/GSGGGS、GSGGS/GSGSGGS。在优选实施方案中,X和Y选自以下的X/Y组合:0/GSG,0/GSGS,0/GSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,GS/GSG,GGS/GSGGS,GSGGS/GSG。
在一个或多个实施方案中,光学活性多肽是绿色荧光蛋白,优选具有SEQ ID NO:3所示的序列或与其具有至少70%序列相同性的序列。X和Y的氨基酸数为:X为0个氨基酸且Y为2、3、4、6或7个氨基酸,X为1个氨基酸且Y为2、3、4、5、6或7个氨基酸,X为2个氨基酸且Y为2、3、4、6或7个氨基酸,X为3个氨基酸且Y为2、3、4、5、6或7个氨基酸,X为4个氨基酸且Y为2、3、4、5、6或7个氨基酸,或X为5个氨基酸且Y为2、3、4、5、6或7个氨基酸。优选地,X和Y的氨基酸数为:X为0个氨基酸且Y为6个氨基酸,X为1个氨基酸且Y为3或5个氨基酸,X为2个氨基酸且Y为2、3、4或7个氨基酸,X为3个氨基酸且Y为3个氨基酸,X为4个氨基酸且Y为3、4或6个氨基酸,或X为5个氨基酸且Y为2个氨基酸。在一个实施方案中,X和Y选自以下的一种或多种X/Y组合:0/GS,0/GSG,0/GSGS,0/GSGGGS,0/GSGSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,G/GSGGGS,G/GSGSGGS,GS/GS,GS/GSG,GS/GSGS,GS/GSGGGS,GS/GSGSGGS,GGS/GS,GGS/GSG,GGS/GSGS,GGS/GSGGS,GGS/GSGGGS,GGS/GSGSGGS,GGGS/GS,GGGS/GSG,GGGS/GSGS,GGGS/GSGGS,GGGS/GSGGGS,GGGS/GSGSGGS,GSGGS/GS,GSGGS/GSG,GSGGS/GSGS,GSGGS/GSGGS,GSGGS/GSGGGS,GSGGS/GSGSGGS。在优选实施方案中,X和Y选自以下的X/Y组合:0/GSGGGS,G/GSG,G/GSGGS,GS/GS,GS/GSG,GS/GSGS,GS/GSGSGGS,GGS/GSG,GGGS/GSG,GGGS/GSGS,GGGS/GSGGGS,GSGGS/GS。
在一个或多个实施方案中,光学活性多肽是蓝色荧光蛋白,优选具有SEQ ID NO:4所示的序列或与其具有至少70%序列相同性的序列。X和Y的氨基酸数为:X为0个氨基酸且Y为2、3、4、5、6或7个氨基酸,X为1个氨基酸且Y为2、3、4、5、6或7个氨基酸,X为2个氨基酸且Y为3、4、6或7个氨基酸,X为3个氨基酸且Y为2、3、4、5、6或7个氨基酸,X为4个氨基酸且Y为2、3、4、5、6或7个氨基酸,或X为5个氨基酸且Y为2、3、4、5、6或7个氨基酸。优选地,X和Y的氨基酸数为:X为0个氨基酸且Y为6个氨基酸,X为1个氨基酸且Y为3个氨基酸,X为2个氨基酸且Y为4、6、或7个氨基酸,X为3个氨基酸且Y为3或6个氨基酸,X为4个氨基酸且Y为2、6或7个氨基酸,或X为5个氨基酸且Y为7个氨基酸。在一个实施方案中,X和Y选自以下的一种或多种X/Y组合:0/GS,0/GSG,0/GSGS,0/GSGGS,0/GSGGGS,0/GSGSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,G/GSGGGS,G/GSGSGGS,GS/GSG,GS/GSGS,GS/GSGGGS,GS/GSGSGGS,GGS/GS,GGS/GSG,GGS/GSGS,GGS/GSGGS,GGS/GSGGGS,GGS/GSGSGGS,GGGS/GS,GGGS/GSG,GGGS/GSGS,GGGS/GSGGS,GGGS/GSGGGS,GGGS/GSGSGGS,GSGGS/GS,GSGGS/GSG,GSGGS/GSGS,GSGGS/GSGGS,GSGGS/GSGGGS,GSGGS/GSGSGGS。在优选实施方案中,X和Y选自以下的X/Y组合:0/GSGGGS,G/GSG,GS/GSGS,GS/GSGGGS,GS/GSGSGGS,GGS/GSG,GGS/GSGGGS,GGGS/GS,GGGS/GSGGGS,GGGS/GSGSGGS,GSGGS/GSGSGGS。
在一个或多个实施方案中,光学活性多肽是红色荧光蛋白,优选具有SEQ ID NO:5所示的序列或与其具有至少70%序列相同性的序列。X和Y的氨基酸数为:X为0个氨基酸且Y为5个氨基酸,X为1个氨基酸且Y为2、3、6或7个氨基酸,X为2个氨基酸且Y为2个氨基酸。在一个实施方案中,X和Y选自以下的一种或多种X/Y组合:0/GSGGS,G/GS,G/GSG,G/GSGGGS,G/GSGSGGS,GS/GS。
在一个或多个实施方案中,光学探针具有SEQ ID NO:13所示序列或由其组成。在一个实施方案中,本发明提供的光学探针包含与SEQ ID NO:13中任一有至少35%、至少40%、至少50%、至少60%、至少70%、至少80%、至少85%、至少90%、至少95%、至少99%序列相同性的序列。
本发明还提供核酸分子,其包含本文所述多肽、探针或蛋白的编码序列或其互补序列或片段。在一个实施方案中,本发明的核酸分子具有选自以下的序列:(1)SEQ ID NO:6-13中任一所示氨基酸序列的编码序列或其互补序列,(2)与(1)具有至少99%、95%、90%、80%、70%或50%相同性的序列,(3)(1)或(2)的片段。
本发明还涉及上述核酸分子的变体,包括编码本发明光学探针或融合蛋白的片段、类似物、衍生物、可溶性片段和变体的核酸序列或其互补序列。
本发明还提供包含本文所述核酸分子的核酸构建物。该核酸序列编码本发明所述融和蛋白。在一个或多个实施方案中,所述核酸分子的序列与表达控制序列操作性连接。在一个或多个实施方案中,所述核酸构建物是克隆载体、表达载体或重组载体。在一些实施方案中,表达载体选自原核表达载体、真核表达载体和病毒载体。
本发明还提供包含本发明所述核酸分子或核酸构建物的细胞。在一个或多个实施方案中,所述细胞表达本文所述融和蛋白。
本发明提供制备本文所述融和蛋白的方法,包括:提供表达本文所述融和蛋白或含有本文所述核酸分子或核酸构建物的细胞,在所述融和蛋白表达的条件下培养所述细胞,和分离所述融和蛋白。
本发明还提供包括本文所述融和蛋白、核酸分子和/或核酸构建物或如本文所述方法制备的融和蛋白的检测试剂盒。
本发明还提供检测样品中c-di-GMP的方法,包括:使本文所述融和蛋白或如本文所述方法制备的融和蛋白与样品接触,和检测光学活性多肽的变化。所述检测可以在体内、体外、亚细胞或原位进行。所述样品例如活的大肠杆菌细胞。
本文还提供定量样品中c-di-GMP的方法,包括:使本文所述融和蛋白或如本文所述方法制备的融和蛋白与样品接触,检测光学活性多肽的变化,和根据光学活性多肽的变化定量样品中的c-di-GMP。
本发明还提供筛选化合物(例如药物)的方法,包括:使本文所述融和蛋白或如本文所述方法制备的融和蛋白与候选化合物接触,检测光学活性多肽的变化,和根据光学活性多肽的变化筛选化合物。所述方法可以高通量地筛选化合物。
本发明还提供筛选化合物的方法,包括:使表达本文所述融和蛋白的细胞与候选化合物和任选的c-di-GMP接触,检测光学活性多肽的变化,和根据光学活性多肽的变化筛选化合物。所述化合物调节细胞对c-di-GMP的摄取能力。
本发明还提供本文所述融和蛋白或如本文所述方法制备的融和蛋白在c-di-GMP细胞内/外定位中的应用。在一个或多个实施方案中,所述定位是实时定位。
本发明的有益效果:本发明提供的c-di-GMP荧光探针,包括对c-di-GMP敏感的多肽B和荧光蛋白A;所述的荧光蛋白A插入到多肽B中,将B分为多肽B1和多肽B2两个部分,形成B1-A-B2式的探针结构;所述的荧光蛋白A插入两个串联的完整敏感多肽B中间,形成B-A-B式的探针结构。本发明提供的B1-A-B2式c-di-GMP荧光探针,易于成熟,荧光动态变化大,特异性好,并且能够通过基因操作的方法在细胞中表达,可在细胞内外实时定位、高通量检测c-di-GMP,省去了耗时的处理样品步骤。实验效果表明本申请所提供的的c-di-GMP荧光探针对c-di-GMP的最高响应达到5倍以上,可以在细胞浆、线粒体、细胞核、内质网、细胞膜等亚细胞结构中对细胞进行定位检测,在细菌中实现探针的可视化研究,并且可以进行高通量的化合物筛选与大肠杆菌中c-di-GMP定量检测。
附图说明
下面结合附图和实施例对本发明作进一步说明。
图1为示例性(A)插入型荧光探针(插入位点36/40,36/41)及(B)串联型荧光探针BldD-cpYFP-0/GSG、BldD-cpYFP-0/GSGS、BldD-cpYFP-0/GSGGS的SDS-PAGE分析图。所述的G、S为甘氨酸、丝氨酸的缩写,0代表无氨基酸接头。
图2,A为串联不同接头形成的c-di-GMP串联型黄色荧光探针对c-di-GMP响应变化图。图2,B为黄色荧光蛋白cpYFP在c-di-GMP结合蛋白不同插入位点的插入型荧光探针对c-di-GMP响应变化图。
图3,A为串联不同接头形成的c-di-GMP串联型绿色荧光探针对c-di-GMP响应变化图。图3,B为绿色荧光蛋白cpGFP在c-di-GMP结合蛋白不同插入位点的插入型荧光探针对c-di-GMP响应变化图。
图4,A串联不同接头形成的c-di-GMP串联型蓝色荧光探针对c-di-GMP响应变化图。图4,B为蓝色荧光蛋白cpBFP在c-di-GMP结合蛋白不同插入位点的插入型荧光探针对c-di-GMP响应变化图。
图5,A串联不同接头形成的c-di-GMP串联型红色荧光探针对c-di-GMP响应变化图。图5,B为红色荧光蛋白cpmApple在c-di-GMP结合蛋白不同插入位点的插入型荧光探针对c-di-GMP响应变化图。
图6为不同插入位点36/37,36/38,36/39,36/40,36/41,36/42,37/38的c-di-GMP插入型黄色荧光探针对不同浓度c-di-GMP的滴定曲线。
图7为不同插入位点36/37,36/38,36/39,36/40,36/41,36/42,37/38的c-di-GMP插入型黄色荧光探针对不同核苷酸底物的特异性测试图。
图8为不同插入位点37/38,39/40,37/40,37/41的c-di-GMP插入型绿色荧光探针对不同浓度c-di-GMP的滴定曲线。
图9为不同插入位点37/38,39/40,37/40,37/41的c-di-GMP插入型绿色荧光探针对不同核苷酸底物的特异性测试图。
图10为不同插入位点36/37,36/38,36/39,36/40,36/41,36/42,37/38的c-di-GMP插入型黄色荧光探针的荧光光谱性质图。
图11为不同插入位点37/38,37/40,37/41,39/40的c-di-GMP插入型绿色荧光探针的荧光光谱性质图。
图12为示例性c-di-GMP插入型荧光探针cpYFP-36/41在哺乳动物细胞中亚细胞器定位分析图。
图13为示例性c-di-GMP插入型荧光探针cpYFP-36/41在大肠杆菌中对c-di-GMP跨膜运输进行动态监测。
图14为示例性c-di-GMP插入型荧光探针在大肠杆菌JM109(DE3)细胞中的成像图。
图15为示例性c-di-GMP插入型荧光探针在活细胞水平进行高通量化合物筛选分析图。
图16为图15中的c-di-GMP插入型荧光探针对大肠杆菌中的c-di-GMP定量分析图。
具体实施方案
在给出数值或范围时,本文所用术语“约”指该数值或范围在给定数值或范围的20%以内、10%以内和5%以内。
本文所用术语“包含”、“包括”和其等同形式包括“含有”以及“由……组成”的含义,例如“包含”X的组合物可仅由X组成或可含有其它物质,例如X+Y。
本文所用术语“c-di-GMP敏感多肽”或“c-di-GMP响应多肽”指对c-di-GMP产生响应的多肽,所述响应包括与敏感多肽的相互作用相关的多肽的化学,生物学,电学或生理学参数的任何响应。响应包括小的变化,例如,多肽的氨基酸或肽片段的方向的变化以及例如多肽的一级,二级或三级结构的变化,包括例如质子化,电化学势和/或构象的变化。可以理解的是,只要荧光蛋白部分的荧光被改变,可检测到的改变不需要是构象改变。本文所述c-di-GMP敏感多肽还可包括其功能变体。C-di-GMP敏感多肽的功能变体包括但不限于可以与c-di-GMP相互作用从而发生与亲本c-di-GMP敏感多肽相同或相似变化的变体。
本发明所述c-di-GMP敏感多肽包括但不限于来源于天蓝色链霉菌(Streptomycescoelicolor)的c-di-GMP结合蛋白BldD或与其具有70%以上同源性并且保留c-di-GMP结合功能的变体。C-di-GMP结合蛋白可以感应c-di-GMP浓度的变化,在c-di-GMP浓度动态变化的过程中c-di-GMP结合蛋白的空间构象也会发生很大改变。BldD由c-di-GMP结合/调节域和DNA结合域组成。示例性BldD蛋白如SEQ ID NO:1所示。在一个或多个实施方案中,c-di-GMP敏感多肽包含c-di-GMP蛋白的c-di-GMP结合域,而不包括DNA结合域。
本文所用术语“光学探针”是指与光学活性多肽融合的c-di-GMP敏感多肽。发明人发现,c-di-GMP敏感多肽例如c-di-GMP结合蛋白专一性地对生理浓度的c-di-GMP结合后所产生的构象变化会引起光学活性多肽(例如荧光蛋白)的构象变化,进而导致光学活性多肽的光学性质发生改变。借助不同c-di-GMP浓度下测定的荧光蛋白的荧光绘制标准曲线,可以检测并分析c-di-GMP的存在和/或水平。当描述本发明光学探针时(例如描述插入位点或突变位点时),提及氨基酸残基编号均参考SEQ ID NO:1。
在本发明的光学探针中,光学活性多肽(例如荧光蛋白)可操作地插入c-di-GMP敏感多肽中。基于蛋白质的“光学活性多肽”是具有发射荧光能力的多肽。优选地,选择蛋白质底物以具有在未激活和活化的构象状态下容易区分的荧光特性。本文所述光学活性多肽还可为其功能变体。光学活性多肽的功能变体包括但不限于可以发生与亲本光学活性多肽相同或相似荧光性质变化的变体。
本文所用术语“荧光蛋白”指在激发光照射下发出荧光的蛋白质。荧光蛋白作为生物科学领域的基础检测手段,例如绿色荧光蛋白GFP及由该蛋白突变衍生出的环状重排的绿色荧光蛋白(cpGFP)、环状重排的黄色荧光蛋白(cpYFP)、环状重排的蓝色荧光蛋白(cpBFP)等;还有红色荧光蛋白RFP,及由该蛋白衍生出来的环状重排的蛋白,如cpmApple。本领域知晓可用于本发明的荧光蛋白及其序列。示例性地,cpYFP如SEQ ID NO:2所示;cpGFP如SEQ ID NO:3所示;cpBFP如SEQ ID NO:4所示;cpmApple如SEQ ID NO:5所示。
本发明所述的c-di-GMP光学探针是融合蛋白,包括(A)光学活性多肽或与其具有至少70%序列相同性并具有光学活性的变体,和(B)c-di-GMP敏感多肽或与其具有至少70%序列相同性并具有c-di-GMP敏感性的变体。所述多肽B和c-di-GMP的特异性结合导致光学活性多肽A的信号改变。本文融合蛋白有两种实施形式,一种形式是A位于B的序列内,将B分为B1和B2两个部分,形成顺序为B1-A-B2的结构;另一种形式是一个或多个A位于两个或更多个B的序列之间,通过接头与两端的B连接。
在本发明的光学活性多肽A位于c-di-GMP敏感多肽B的序列内所形成的插入型重组融和蛋白光学探针中,光学活性多肽可以位于c-di-GMP敏感多肽的任何位置。本文的插入型重组光学探针有时表示为Bl-光学活性多肽-dD,例如Bl-cpYFP-dD。在一个实施方案中,光学活性多肽以N-C方向位于N-C方向的c-di-GMP敏感多肽的任何位置,如氨基酸残基9-12,16-19和35-51区域。在一个实施方案中,光学活性多肽位于c-di-GMP敏感多肽的选自以下的一个或多个位点:9/10,10/11,11/12,16/17,17/18,18/19,35/36,36/37,36/38,36/39,36/40,36/41,36/42,37/38,37/39,37/40,37/41,37/42,38/39,38/40,38/41,38/42,39/40,39/41,39/42,40/41,40/42,41/42,42/43,43/44,44/45,45/46,46/47,47/48,48/49,49/50,50/51。本文中,如果以“数字/数字”形式表示的位点中的两个数字是连续的整数,则表示光学活性多肽位于该数字所述的氨基酸之间。例如插入位点36/37表示光学活性多肽位于c-di-GMP敏感多肽的氨基酸36与37之间。如果以“数字/数字”形式表示的位点中的两个数字不是连续的整数,则表示光学活性多肽置换该数字所示氨基酸之间的氨基酸。例如插入位点36/41表示光学活性多肽置换c-di-GMP敏感多肽的氨基酸37-40。光学活性多肽与两端的敏感多肽部分之间可以具有接头。“接头”或“连接区”指在本发明多肽、蛋白质或核酸中连接两个部分的氨基酸或核苷酸序列。接头可为0个或多个柔性氨基酸组成的短肽链,如G、S、Y。在一个实施方案中,本发明光学探针具有SEQ ID NO:6-12所示序列或由其组成。
在本发明的一个或多个光学活性多肽A位于两个或更多个c-di-GMP敏感多肽B的序列之间所形成的串联型重组融和蛋白光学探针中,A可以是一个、两个、三个或更多个,B可以是两个、三个、四个、五个或更多个。例如,所述融合蛋白中的组件可以具有选自以下任一种顺序:B-A-B、B-B-A-B、B-A-B-B、B-A-A-B、B-B-A-B-B、B-B-B-A-B、B-A-B-B-B、B-A-B-A-B、B-B-A-A-B、B-A-A-B-B。在一个或多个实施方案中,A位于两个B的序列之间,通过接头X和Y与两端的B连接,形成顺序为B-X-A-Y-B的结构。X和Y分别独立选自无或由G和/或S组成的接头肽。X可为0-10、0-9、0-8、0-7、0-6、0-5、0-4、0-3、0-2、0-1、0个氨基酸,优选0-7个氨基酸,更优选0-5个氨基酸。Y可为0-10、1-10、2-10、2-9、2-8、2-7、2-6、2-5、2-4、2-3、2个氨基酸,优选2-9个氨基酸,更优选2-7个氨基酸。例如,X和Y分别独立选自无、G、S、GS、SG、GGS、GSG、SGG、GSS、SGS、SSG、GGGS、GGSG、GSGG、SGGG、GGSS、GSGS、GSSG、SGGS、SGSG、SSGG、GSSS、SGSS、SSGS、SSSG、GSGGS、GSGGGS、GSGSGGS。X可选自无、G、GS、GGS、GGGS、GSGGS。Y可选自无、GS、GSG、GSGS、GSGGS、GSGGGS、GSGSGGS。本文中,接头X和Y的选择组合称为X/Y,选自以下的一种或多种:0/GS,0/GSG,0/GSGS,0/GSGGS,0/GSGGGS,0/GSGSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,G/GSGGGS,G/GSGSGGS,GS/GS,GS/GSG,GS/GSGS,GS/GSGGS,GS/GSGGGS,GS/GSGSGGS,GGS/GS,GGS/GSG,GGS/GSGS,GGS/GSGGS,GGS/GSGGGS,GGS/GSGSGGS,GGGS/GS,GGGS/GSG,GGGS/GSGS,GGGS/GSGGS,GGGS/GSGGGS,GGGS/GSGSGGS,GSGGS/GS,GSGGS/GSG,GSGGS/GSGS,GSGGS/GSGGS,GSGGS/GSGGGS,GSGGS/GSGSGGS。在优选实施方案中,X/Y选自:0/GSG,0/GSGS,0/GSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,GS/GSG,GGS/GSGGS,GSGGS/GSG。在一个或多个实施方案中,光学探针具有SEQ ID NO:13所示序列或由其组成。
本文所述光学探针作为基本单元与其他蛋白或多肽连接。其他蛋白或多肽不影响光学探针的性质。其他蛋白或多肽可位于所述光学探针的N端和/或C端。其他多肽包括将光学探针定位到不同细胞器或亚细胞器的多肽、用于纯化的标签或者用于免疫印迹的标签。本文所述亚细胞器包括细胞浆、线粒体、细胞核、内质网、细胞膜等。在一些实施方案中,用于纯化的标签或者用于免疫印迹的标签包括6组氨酸(6*His)、谷胱甘肽硫转移酶(GST)、Flag。光学探针和其它蛋白或多肽之间可具有接头,接头序列可为0个或多个柔性氨基酸组成的短肽链,如G、S、Y。
提到某多肽或蛋白时,本发明所用术语“变体”或“突变体”包括具有所述多肽或蛋白相同功能、但序列不同的变体。多肽或蛋白的变体可包括:同源序列、保守性变体、等位变体、天然突变体、诱导突变体。这些变体包括但并不限于:在所述多肽或蛋白的序列中缺失、插入和/或取代一个或多个(通常为1-30个,较佳地1-20个,更佳地1-10个,最佳地1-5个)氨基酸,以及在其羧基末端和/或氨基末端添加一个或数个(通常为20个以内,较佳地为10个以内,更佳地为5个以内)氨基酸获得的序列。这些变体还可包含与所述多肽或蛋白的序列相同性为至少约70%、至少约75%、至少约80%、至少约85%、至少约90%、至少约95%、至少约98%、至少约99%或100%的多肽或蛋白。不希望受理论限制,氨基酸残基发生改变而不改变多肽或蛋白质的总体构型和功能,即功能保守突变。例如,在本领域中,用性能相近或相似的氨基酸进行取代时,通常不会改变多肽或蛋白的功能。在本领域中,性能相似的氨基酸往往指具有相似侧链的氨基酸家族,在本领域已有明确定义。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、乳酸、苯丙氨酸、甲硫氨酸、色氨酸)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。又比如,在氨基末端和/或羧基末端添加一个或数个氨基酸通常也不会改变多肽或蛋白的功能。对于许多常见已知非遗传性编码氨基酸的保守氨基酸取代本领域已知。其他非编码氨基酸的保守取代可基于其物理性质与遗传上编码的氨基酸的性质的比较来确定。本发明光学探针可包含具有突变的c-di-GMP敏感多肽。突变可以是氨基酸种类的突变,也可以是c-di-GMP敏感多肽的截短。在本发明中主要是指c-di-GMP敏感多肽的截短。
在两种或多种多肽或核酸分子序列中,术语“相同性”或“相同性百分数”指在比较窗口或指定区域上,采用本领域已知方法如序列比较算法,通过手工比对和目测检查来比较和比对最大对应性时,两个或多个序列或子序列相同或其中在指定区域有一定百分数的氨基酸残基或核苷酸相同(例如,60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%相同)。例如,适合测定序列相同性百分数和序列相似性百分数的优选算法是BLAST和BLAST 2.0算法,分别可参见Altschul(1997)等(Altschul,Madden et al.1997)和Altschul(1990)等(Altschul,Gish et al.1990)。
本领域技术人员公知,在基因克隆操作中因为需要引入酶切位点而在表达的多肽或蛋白末端引入了一个或多个不相干的残基并不影响目的多肽或蛋白的活性。此外,为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化,可将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内,例如,包括但不限于,适合的接头肽、信号肽、前导肽、末端延伸、谷胱甘肽S-转移酶(GST)、麦芽糖E结合蛋白、蛋白A、如6His或Flag的标签,或Xa因子或凝血酶或肠激酶的蛋白水解酶位点。
本文所用术语“功能片段”、“衍生物”和“类似物”是指基本上保持与原始多肽或蛋白(例如c-di-GMP结合蛋白或荧光蛋白)相同的生物学功能或活性的蛋白。本发明的多肽或蛋白(例如c-di-GMP结合蛋白或荧光蛋白)的功能变体、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的蛋白,而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的,或(ii)在一个或多个氨基酸残基中具有取代基团的蛋白,或(iii)成熟蛋白与另一个化合物(比如延长蛋白半衰期的化合物,例如聚乙二醇)融合所形成的蛋白,或(iv)附加的氨基酸序列融合到此蛋白序列而形成的蛋白(如分泌序列或用来纯化此蛋白的序列或蛋白原序列,或与抗原IgG片段的形成的融合蛋白)。根据本文的教导,这些功能变体、衍生物和类似物属于本领域熟练技术人员公知的范围。所述类似物可以是具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物,以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解,本发明的c-di-GMP敏感多肽并不限于上述列举的代表性蛋白、变体、衍生物和类似物。修饰(通常不改变一级结构)形式包括:体内或体外的蛋白的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化,如那些在蛋白的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的蛋白。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸,磷酸丝氨酸,磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的蛋白。
本发明包含编码本发明所述光学探针的核酸分子。本发明所用术语“核酸分子”或“核苷酸”或“多核苷酸”或“核酸序列”可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的、编码链或非编码链。提到核酸时,本文所用术语“变体”可以是天然发生的等位变体或非天然发生的变体。这些核苷酸变体包括简并变体、取代变体、缺失变体和插入变体,但不会从实质上改变其编码的蛋白的功能。本发明核酸可包含与所述核酸序列的序列相同性为至少约70%、至少约75%、至少约80%、至少约85%、至少约90%、至少约95%、至少约98%、至少约99%或100%的核苷酸序列。本发明还涉及与上述的序列杂交的核酸片段。如本文所用,“核酸片段”的长度至少含15个核苷酸,较好是至少30个核苷酸,更好是至少50个核苷酸,最好是至少100个核苷酸以上。核酸片段可用于核酸的扩增技术(如PCR),例如引物或探针。
本发明光学探针或融合蛋白的全长序列或其片段通常可以用PCR扩增法、人工合成法或重组法获得。本领域知晓常规PCR、合成法、重组法的步骤和所用试剂。此外,可通过突变PCR或化学合成等方法将突变引入本发明蛋白序列中。
本发明也涉及核酸构建物,该核酸构建物含有本文所述的多核苷酸,以及与这些序列操作性连接的一个或多个调控序列。本发明所述的多核苷酸可以多种方式被操作以保证所述多肽或蛋白的表达。在将核酸构建物插入载体之前可根据表达载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。
在某些实施方案中,所述核酸构建物是载体。载体可以是克隆载体,也可以是表达载体,或者是同源重组载体。本发明的多核苷酸可被克隆入许多类型的载体,例如,质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。克隆载体可用于提供本发明蛋白或多肽的编码序列。表达载体可以以细菌载体或病毒载体形式提供给细胞。通常通过可操作地连接本发明的多核苷酸至启动子,并将构建体并入表达载体,实现本发明多核苷酸的表达。该载体对于复制和整合真核细胞可为合适的。在一个或多个实施方案中,克隆载体和表达载体是一种载体,即克隆表达载体。同源重组载体用于将本文所述的表达框整合到宿主基因组中。
典型的表达载体包含可用于调节期望核酸序列表达的表达控制序列,与本发明所述的核酸序列或其互补序列操作性连接。本文所用术语“表达控制序列”指调控目的基因的转录、翻译和表达的可以与目的基因操作性连接的元件,可以是复制起点、启动子、标记基因或翻译控制元件,包括增强子、操纵子、终止子、核糖体结合位点等,表达控制序列的选择取决于所用的宿主细胞。在重组表达载体中,“操作性连接”是指目的的核苷酸序列与调节序列以允许核苷酸序列表达的方式连接。本领域的技术人员熟知能用于构建含本发明融合蛋白编码序列和合适的转录/翻译控制信号的表达载体的方法。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上,以指导mRNA合成。这些启动子的代表性例子有:大肠杆菌的lac或trp启动子;λ噬菌体PL启动子;真核启动子包括CMV立即早期启动子、HSV胸苷激酶启动子、早期和晚期SV40启动子、反转录病毒的LTR和其他一些已知的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
在获得重组表达载体后,将该载体转化到宿主细胞中,以产生包括融合蛋白的蛋白或肽。此种转移过程可用转化或转染等本领域技术人员熟知的常规技术进行。本发明所述的宿主细胞是指能够接收和容纳重组DNA分子的细胞,是重组基因扩增的场所,理想的受体细胞应该满足易于获取和增殖两个条件。本发明的“宿主细胞”可包括原核细胞和真核细胞,具体包括细菌细胞、酵母细胞、昆虫细胞和哺乳动物细胞。具体的可为大肠杆菌,链霉菌属,鼠伤寒沙门氏菌的细菌细胞,真菌细胞如酵母,植物细胞,果蝇S2或Sf9的昆虫细胞,CHO、COS、HEK293、HeLa细胞、或Bowes黑素瘤细胞的动物细胞等,其中包括但不限于上述的那些宿主细胞。所述宿主细胞优选各种利于基因产物表达或发酵生产的细胞,此类细胞已为本领域熟知并常用。本领域一般技术人员清楚如何选择适当的载体、启动子、增强子和宿主细胞。
本发明所述的转移到宿主细胞的方法为本领域常规的方法,包括磷酸钙或氯化钙共沉淀、DEAE-甘露聚糖-介导的转染、脂转染、天然感受态、化学介导的转移或电穿孔。当宿主为原核生物如大肠杆菌时,所述方法优选的为CaCl2法或MgCl2法处理,所用的步骤为本领域公知。当宿主细胞是真核细胞时,可选用如下的DNA转染方法:磷酸钙共沉淀法,常规机械方法如显微注射、电穿孔、脂质体包装等。
在将表达载体转入宿主细胞后,对转入表达载体的宿主细胞进行扩增表达培养,分离得到c-di-GMP光学探针。所述宿主细胞扩增表达培养采用常规的方法即可。根据所用的宿主细胞种类,培养中所用的培养基可以是各种常规培养基。本领域技术人员知晓适于宿主细胞生长的条件。
在本发明中,光学探针在细胞内、细胞膜上表达、或分泌到细胞外。如果需要,可利用其物理的、化学的和其它特性通过各种分离方法分离或纯化重组的蛋白。本发明对分离所述c-di-GMP荧光蛋白的方法没有特殊限定,采用本领域常规的融合蛋白的分离方法即可,包括但并不限于:常规的复性处理、盐析方法、离心、渗透破菌、超声处理、超离心、分子筛层析、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。在一个实施方案中,利用His标签的亲和层析法进行光学探针的分离。
本发明还提供了所述c-di-GMP光学探针在c-di-GMP实时定位、定量检测以及高通量化合物筛选中的应用。在一个方面,所述的c-di-GMP光学探针优选与细胞不同部位的信号肽连接,转入到细胞中,通过检测细胞中荧光信号的强弱,进行c-di-GMP的实时定位;通过c-di-GMP标准滴加曲线进行相应c-di-GMP的定量检测。本发明所述的c-di-GMP标准滴加曲线是根据c-di-GMP光学探针在不同浓度c-di-GMP的情况下的荧光信号绘制而成。本发明所述c-di-GMP光学探针直接转入细胞中,在c-di-GMP实时定位和定量检测过程中,不需要耗时的样品处理过程,更加准确。本发明c-di-GMP光学探针在进行高通量化合物筛选时,将不同的化合物添加到细胞培养液中,测定c-di-GMP含量的变化,从而筛选出对c-di-GMP含量变化有影响的化合物。在本发明中所述的c-di-GMP光学探针在c-di-GMP实时定位、定量检测以及高通量化合物筛选中的应用,均是非诊断和治疗目的,不涉及疾病的诊断和治疗。本发明还提供进行上述检测的试剂盒,包括本文所述融和蛋白、核酸分子核酸构建物和/或细胞。所述试剂盒还包含使用本文所述方法检测c-di-GMP所需的其他试剂,如LB培养基、Tris缓冲液等。本领域知晓所述其他试剂的种类和用量。
在本文中,浓度、含量、百分数和其它数值均可用范围的形式表示。也应理解,使用这种范围形式只是为了方便和简洁,应该被弹性地解读为包括范围上下限所明确提及的数值,还应包括该范围内包括的所有单个数值或子范围。
实施例
下面结合实施例对本发明提供的c-di-GMP荧光探针进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。
I.实验材料和试剂
实施例中主要采用常规的基因工程分子生物学克隆方法和细胞培养以及成像方法等,这些方法是本领域普通技术人员所熟知的,例如:简·罗斯凯姆斯等的《分子生物学实验参考手册》,J.萨姆布鲁克,D.W.拉塞尔著,黄培堂等译:《分子克隆实验指南》(第三版,2002年8月,科学出版社出版,北京);费雷谢尼等的《动物细胞培养:基本技术指南》(第五版),章静波,徐存拴等译;J.S.博尼费斯农,M.达索等的《精编细胞生物学实验指南》,章静波等译。
实施例中所用的基于pCDFduet-cpYFP,pCDFduet-c-di-GMP结合蛋白质粒由华东理工大学蛋白质实验室构建,pCDFduet质粒载体购自Invitrogen公司。所有用于PCR的引物均由上海捷瑞生物工程技术有限公司合成、纯化和经质谱法鉴定正确。实施例中构建的表达质粒都经过序列测定,序列测定由华大基因公司和杰李测序公司完成。各实施例所用的Taq DNA聚合酶购自东盛生物,pfu DNA聚合酶购自天根生化科技(北京)有限公司,primeSTAR DNA聚合酶购自TaKaRa公司,三种聚合酶购买时都附带赠送对应聚合酶缓冲液和dNTP。BamHI、BglII、HindIII、NdeI、XhoI、EcoRI、SpeI等限制性内切酶、转染试剂Lip2000Kit购于Invitrogen公司。c-di-GMP等均购自Sigma公司。除非特别声明,无机盐类等化学试剂均购自sigma-aldrich公司。HEPES盐,氨苄青霉素(Amp)和链霉素(Str)购自Ameresco公司;96孔检测黑板、384孔荧光检测黑板购自Grenier公司。
实施例中所用的DNA纯化试剂盒购自BBI公司(加拿大),普通质粒小抽试剂盒购自天根生化科技(北京)有限公司。克隆菌株Mach1购自Invitrogen公司。镍柱亲和层析柱和脱盐柱填料均来自GE healthcare公司。
实施例中用到的主要仪器:Biotek Synergy 2多功能酶标仪(美国Bio-Tek公司),X-15R高速冷冻离心机(美国Beckman公司),Microfuge22R台式高速冷冻离心机(美国Beckman公司),PCR扩增仪(德国Biometra公司),超声破碎仪(宁波新芝公司),核酸电泳仪(申能博彩公司),荧光分光光度计(美国Varian公司),CO2恒温细胞培养箱(SANYO),倒置荧光显微镜(日本尼康公司)。
II.分子生物学方法和细胞实验方法
II.1聚合酶链式反应(PCR):
1.目的片段扩增PCR:
该方法主要用于基因片段扩增和菌落PCR鉴定阳性克隆。所述PCR扩增的反应体系如下:模板序列0.5-1μl,正向引物(25μM)0.5μl,反向引物(25μM)0.5μl,10×pfu缓冲液5μl,pfu DNA聚合酶0.5μl,dNTP(10mM)1μl,灭菌超纯水(ddH2O)41.5-42μl,总体积50μl。PCR扩增程序如下:95℃变性2-10分钟,30轮循环(94-96℃持续30-45秒,50-65℃持续30-45秒,72℃持续一定时间(600bp/min)),72℃延伸10分钟。
2.长片段(>2500bp)扩增PCR:
本发明中使用的长片段扩增,主要是反向PCR扩增载体,在下述实施例中用于获得定点突变的一种技术。在变异部位设计反向PCR引物,其中一条引物的5’端包含变异的核苷酸序列。扩增后的产物就含有相应的突变位点。长片段扩增PCR反应体系如下:模板序列(10pg-1ng)1μl,正向引物(25μM)0.5μl,反向引物(25μM)0.5μl,5×PrimerSTAR缓冲液10μl,PrimerSTAR DNA聚合酶0.5μl,dNTP(2.5mM)4μl,灭菌超纯水(ddH2O)33.5μl,总体积50μl。PCR扩增程序如下:95℃变性5分钟,30轮循环(98℃持续10秒,50-68℃持续5-15秒,72℃持续一定时间(1000bp/min)),72℃延伸10分钟;或者95℃变性5分钟,30轮循环(98℃持续10秒,68℃持续一定时间(1000bp/min)),72℃延伸10分钟。
II.2核酸内切酶酶切反应:
对质粒载体进行双酶切的体系如下:质粒载体20μl(约1.5μg),10×缓冲液5μl,限制性内切酶1 1-2μl,限制性内切酶2 1-2μl,用灭菌超纯水补至总体积50μl。反应条件37℃,1小时。
II.4目的片段和载体的连接反应
不同的片段和载体之间的连接方法有所差异,本发明中使用了三种连接方法
1.平末端短片段和线性化载体的平末端连接
该方法的原理是PCR获得的平末端产物在T4 PNK作用下对DNA片段的5’末端进行磷酸化反应后,与线性化的载体在PEG4000和T4 DNA连接酶的作用下连接获得重组质粒。同源重组连接体系如下:T4 PNK处理的DNA片段4μl,线性化载体片段4μl,PEG4000 1μl,10×T4连接酶缓冲液1μl,T4 DNA连接酶1μl,总计10μl。反应条件22℃,30分钟。
2.含有粘性末端的DNA片段和含有粘性末端载体片段的连接
通过限制性内切酶切割的DNA片段通常会产生突出的粘性末端,因此可以和含有序列互补的粘性末端载体片段连接,形成重组质粒。连接反应体系如下:酶切后的PCR产物片段DNA 1-7μl,酶切后的质粒0.5-7μl,10×T4连接酶缓冲液1μl,T4 DNA连接酶1μl,灭菌超纯水补至总体积10μl。反应条件16℃,4-8小时。
3.含有同源DNA片段的载体与片段之间的连接
通过PCR技术在片段上引入15-30bp与载体同源的DNA,利用Infusion同源重组酶将片段与载体连接,形成重组质粒。连接反应体系如下:载体1-10μl,片段0.5-10μl,Infusion同源重组酶试剂10μl,灭菌超纯水补至总体积20μl。反应条件50℃,20-30分钟。
II.5感受态细胞的制备与转化
感受态细胞的制备:
1.挑取单菌落(如Mach1)接种于5mL LB培养基中,37℃摇床过夜。
2.取0.5-1mL过夜培养的菌液转种到50mL LB培养基中,37℃,220rpm培养3至5小时,直到OD600达到0.5。
3.冰浴预冷细胞2小时。
4.4℃,4000rpm离心10分钟。
5.弃上清,用5mL预冷的缓冲液重悬细胞,待均匀后再加入重悬缓冲液至终体积为50mL。
6.冰浴45分钟。
7.4℃ 4000rpm离心10分钟,用5mL冰预冷的储存缓冲液重悬细菌。
8.每个EP管中放100μL菌液,-80℃或液氮冻存。
重悬缓冲液:CaCl2(100mM)、MgCl2(70mM)、NaAc(40mM)
储存缓冲液:0.5mL DMSO、1.9mL 80%甘油、1mL 10×CaCl2(1M)、1mL10×MgCl2(700mM)、1mL 10×NaAc(400mM)、4.6mL ddH2O
感受态细胞的转化:
1.取100μl感受态细胞于冰浴上融化。
2.加入适当体积的连接产物,轻轻吹打混匀,冰浴30分钟。通常加入的连接产物的体积少于感受态细胞体积的1/10。
3.将菌液放入42℃水浴中热激90秒,迅速转移至冰浴中放置5分钟。
4.加入500μl LB,于37℃恒温摇床上200rpm培养1小时。
5.将菌液4000rpm离心3分钟,留200μl上清将菌体吹匀,均匀涂布于含适当抗生素的琼脂平板表面,平板于37℃恒温培养箱内倒置过夜。
II.6蛋白质的表达,纯化和荧光检测
1.将pCDFduet-BldD为基础的c-di-GMP探针质粒转化到JM109(DE3)中,倒置培养过夜,从平板上挑取克隆到250ml锥形瓶中,置于37℃摇床,220rpm培养至OD=0.4-0.8,加入1/1000(v/v)的IPTG(1M),18℃诱导表达24-36h。
2.诱导表达完成后,4000rpm,30分钟离心收菌,加入50mM的磷酸盐缓冲液重悬菌体沉淀,超声破碎至菌体澄清。10000rpm,4℃离心20分钟。
3.离心上清通过自装的镍柱亲和层析柱纯化获得蛋白,镍柱亲和层析后的蛋白再通过自装的脱盐柱获得溶解在50mM Tris缓冲液(pH 7.4)或者磷酸盐缓冲液PBS中的蛋白。
4.纯化的c-di-GMP荧光蛋白探针蛋白经过SDS-PAGE鉴定后,使用测定缓冲液(50mM Tris,200mM NaCl,5%glycerol,pH=7.5)或者磷酸盐缓冲液PBS稀释探针成终浓度为1~5μM的蛋白溶液。用测定缓冲液(50mM Tris,pH 7.5)或者磷酸盐缓冲液PBS将c-di-GMP配制成终浓度为1mM的储液。
5.取50μl 1μM的蛋白溶液,37℃温育5min,分别加入c-di-GMP混匀后至终浓度为50μM,利用多功能荧光酶标仪测定蛋白在340nm下的光吸收。
6.取50μl 1μM的荧光探针溶液,37℃温育5min,加入c-di-GMP滴定,测定蛋白的485nm荧光激发后528nm发射的荧光强度。对样品的荧光激发、发射测定利用多功能荧光酶标仪完成。
7.取50μl 1μM的荧光探针溶液,37℃温育5min,加入c-di-GMP,测定探针蛋白的吸收光谱和荧光光谱。对样品的吸收光谱和荧光光谱的测定是通过分光光度计和荧光分光光度计完成。
II.7哺乳动物细胞的转染和荧光检测
1.将PAAV为基础的c-di-GMP探针质粒通过转染试剂Lipofectamine2000(Invitrogen)转染到HeLa中,置于37℃,5%CO2的细胞培养箱中培养。待外源基因充分表达24~36h后进行荧光检测。
2.诱导表达完成后,将贴壁的HeLa细胞,用PBS冲洗三次,置于HBSS溶液中分别进行荧光显微镜和酶标仪检测。
实施例1:c-di-GMP结合蛋白质粒
通过PCR扩增天蓝色链霉菌基因中的BldD基因,PCR产物凝胶电泳后纯化回收,同时对pCDFduet载体进行PCR扩增。采用同源重组的方法连接,将连接产物转化DH5α(TransGen Biotech)感受态细胞,转化的DH5α涂布于LB平板(链霉素100ug/mL),置于37℃培养过夜。将生长DH5α转化子进行质粒抽提后,进行PCR鉴定。阳性质粒经过测序正确后进行后续的质粒构建。
实施例2:不同插入位点及串联接头的cpYFP光学探针的表达和检测
本实施例首先以pCDFduet-BldD为基础选择了下述位点插入cpYFP,得到包含单态光学探针的编码序列的相应质粒:9/10,10/11,11/12,16/17,17/18,18/19,35/36,36/37,36/38,36/39,36/40,36/41,36/42,37/38,37/39,37/40,37/41,37/42,38/39,38/40,38/41,38/42,39/40,39/41,39/42,40/41,40/42,41/42,42/43,43/44,44/45,45/46,46/47,47/48,48/49,49/50,50/51。示例性的插入型光学探针的氨基酸序列如表1所示。示例性的插入型光学探针的核酸序列如SEQ ID NO:14所示(BldD-36/41-cpYFP)。
表1
序列 | 插入位点 |
SEQ ID NO:6 | 36/37 |
SEQ ID NO:7 | 36/38 |
SEQ ID NO:8 | 36/39 |
SEQ ID NO:9 | 36/40 |
SEQ ID NO:10 | 36/41 |
SEQ ID NO:11 | 36/42 |
SEQ ID NO:12 | 37/38 |
通过PCR扩增cpYFP的DNA片段,同时通过反向PCR扩增产生含有不同断裂位点的pCDFduet-c-di-GMP结合蛋白线性化载体(cpYFP与pCDFduet-BldD载体扩增的引物含有15-25bp的同源片段)。将线性化的pCDFduet-BldD和cpYFP片段在Infusion同源重组酶的作用下连接产生重组质粒,将这些平板在Kodak多功能活体成像系统,挑取在FITC通道激发下有黄色荧光的克隆,由北京六合华大基因科技股份有限公司上海分公司完成测序。
经过测序正确后,将重组质粒转化到JM109(DE3)中诱导表达,并纯化蛋白质,通过SDS-PAGE电泳大小在40kDa附近。该大小符合pCDFduet-Bl-cpYFP-dD表达出的含His-tag纯化标签的Bl-cpYFP-dD融合蛋白质的大小。结果如图1所示。
将纯化的Bl-cpYFP-dD融合蛋白质进行c-di-GMP响应筛选,将含有50μM c-di-GMP的融合荧光蛋白质的检测信号除以无c-di-GMP的融合荧光蛋白质的检测信号。结果如图2,A所示,检测结果显示对c-di-GMP响应超过2倍的有36/37,36/38,36/39,36/40,36/41,36/42,37/38。检测结果显示对c-di-GMP响应超过1.2倍的有:9/10,10/11,11/12,17/18,35/36,36/37,36/38,36/39,36/40,36/41,36/42,37/38,37/41,38/40,38/41,38/42,39/40,39/41,39/42,40/41,43/44,44/45,45/46,48/49,50/51。
本实施例中,还构建含不同串联接头的cpYFP多重光学探针的质粒。以pCDFduet-BldD作为含EcoRI及BamHI酶切位点的线性化载体。将BldD片段与cpYFP片段进行PCR扩增后回收,回收好的BldD与cpYFP片段进行overlap PCR连接在一起,得到带有BamHI-BldD-(0AA/1AA/2AA/3AA/4AA/5AA)-cpYFP-(0AA/1AA/2AA/3AA/4AA/5AA)-EcoRI的片段,利用EcoRI及BamHI双酶将载体pCDFduet及片段BamHI-BldD-(0AA/1AA/2AA/3AA/4AA/5AA)-cpYFP-EcoRI进行双酶切,酶切后直接回收,最后将回收的载体与片段利用T4 DNA连接酶连接。连接产物转化DH5α,转化的DH5α涂布于LB平板(链霉素100ug/mL),置于37℃培养过夜。将生长DH5α转化子进行质粒抽提后,进行PCR鉴定。阳性质粒经过测序正确后转化JM109(DE3)并纯化蛋白质,通过SDS-PAGE电泳大小在54kDa附近。该大小符合pCDFduet-BldD-cpYFP-BldD表达出的含His-tag纯化标签的BldD-cpYFP-BldD融合蛋白质的大小。蛋白电泳结果如图1所示。
根据多重荧光探针中cpYFP两端的下述接头氨基酸数目与种类,得到包含多重光学探针编码序列的相应质粒:0/GS,0/GSG,0/GSGS,0/GSGGS,0/GSGGGS,0/GSGSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,G/GSGGGS,G/GSGSGGS,GS/GS,GS/GSG,GS/GSGS,GS/GSGGS,GS/GSGGGS,GS/GSGSGGS,GGS/GS,GGS/GSG,GGS/GSGS,GGS/GSGGS,GGS/GSGGGS,GGS/GSGSGGS,GGGS/GS,GGGS/GSG,GGGS/GSGS,GGGS/GSGGS,GGGS/GSGGGS,GGGS/GSGSGGS,GSGGS/GS,GSGGS/GSG,GSGGS/GSGS,GSGGS/GSGGS,GSGGS/GSGGGS,GSGGS/GSGSGGS。荧光探针编号和列表如表2所示。编号中的两位数字表示光学活性多肽前/后的接头所含氨基酸数目,如“02”表示光学活性多肽前/后的氨基酸接头为0/GS。
将纯化的串联式融合蛋白质进行c-di-GMP响应筛选,将含有50μM c-di-GMP的融合荧光蛋白质的检测信号除以无c-di-GMP的融合荧光蛋白质的检测信号。结果如图2,B所示,检测结果显示对c-di-GMP响应超过1倍的有Y03,Y04,Y05,Y06,Y07,Y12,Y13,Y14,Y15,Y16,Y17,Y24,Y25,Y26,Y27,Y33,Y35,Y36,Y37,Y43,Y44,Y45,Y46,Y47,Y52,Y53,Y54,Y55,Y56,Y57;对c-di-GMP响应超过1.2倍的有Y03,Y04,Y05,Y12,Y13,Y14,Y15,Y23,Y35,Y53。示例性多重光学探针的序列如表2所示。作为示例,SEQ ID NO:13显示Y15(BldD-G-cpYFP-GSGGS-BldD)的氨基酸序列。
表2
编号 | 接头序列 | 编号 | 接头序列 |
Y02 | 0/GS | Y32 | GGS/GS |
Y03 | 0/GSG | Y33 | GGS/GSG |
Y04 | 0/GSGS | Y34 | GGS/GSGS |
Y05 | 0/GSGGS | Y35 | GGS/GSGGS |
Y06 | 0/GSGGGS | Y36 | GGS/GSGGGS |
Y07 | 0/GSGSGGS | Y37 | GGS/GSGSGGS |
Y12 | G/GS | Y42 | GGGS/GS |
Y13 | G/GSG | Y43 | GGGS/GSG |
Y14 | G/GSGS | Y44 | GGGS/GSGS |
Y15 | G/GSGGS | Y45 | GGGS/GSGGS |
Y16 | G/GSGGGS | Y46 | GGGS/GSGGGS |
Y17 | G/GSGSGGS | Y47 | GGGS/GSGSGGS |
Y22 | GS/GS | Y52 | GSGGS/GS |
Y23 | GS/GSG | Y53 | GSGGS/GSG |
Y24 | GS/GSGS | Y54 | GSGGS/GSGS |
Y25 | GS/GSGGS | Y55 | GSGGS/GSGGS |
Y26 | GS/GSGGGS | Y56 | GSGGS/GSGGGS |
Y27 | GS/GSGSGGS | Y57 | GSGGS/GSGSGGS |
实施例3,不同插入位点及串联接头的cpGFP光学探针的表达和检测
按照实施例1中的方法将cpYFP替换为绿色荧光蛋白cpGFP,融合到c-di-GMP结合蛋白中构建c-di-GMP绿色荧光蛋白荧光探针,按照实施例2中的方法表达并检测。结果如图3,A所示,荧光检测结果显示对c-di-GMP响应超过2倍的有37/38,37/40,37/41,39/40位点。
按照实施例2中的方法获得含不同串联接头的cpGFP多重光学探针。结果如图3,B所示,检测结果显示对c-di-GMP响应超过1倍的有G02,G03,G04,G06,G07,G12,G13,G14,G15,G16,G17,G22,G23,G24,G26,G27,G32,G33,G34,G35,G36,G37,G42,G43,G44,G45,G46,G47,G52,G53,G54,G55,G56,G57;对c-di-GMP响应超过1.2倍的有G06,G13,G15,G22,G23,G24,G27,G33,G43,G44,G46,G52。
实施例4,不同插入位点及串联接头的cpBFP光学探针的表达和检测
按照实施例1中的方法将cpYFP替换为蓝色荧光蛋白cpBFP,融合到c-di-GMP结合蛋白中构建c-di-GMP蓝色荧光蛋白荧光探针,按照实施例2中的方法表达并检测。结果如图4,A所示,荧光检测结果显示对c-di-GMP响应超过2倍的有37/39,37/40,37/41,37/42位点。
按照实施例2中的方法获得含不同串联接头的cpBFP多重光学探针。结果如图4,B所示,检测结果显示对c-di-GMP响应超过1倍的有B02,B03,B04,B05,B06,B07,B12,B13,B14,B15,B16,B17,B23,B24,B26,B27,B32,B33,B34,B35,B36,B37,B42,B43,B44,B45,B46,B47,B52,B53,B54,B55,B56,B57;对c-di-GMP响应超过1.2倍的有B06,B13,B24,B26,B27,B33,B36,B42,B46,B47,B57。
实施例5,不同插入位点及串联接头的cpmApple光学探针的表达和检测
按照实施例1中的方法将cpYFP替换为蓝色荧光蛋白cpmApple,融合到c-di-GMP结合蛋白中构建c-di-GMP红色荧光蛋白荧光探针,按照实施例2中的方法表达并检测。结果如图5,A所示,荧光检测结果显示对c-di-GMP响应超过1.2倍的有36/39,38/39,38/40,38/41,39/40,39/41位点。
按照实施例2中的方法获得含不同串联接头的cpmApple多重光学探针。结果如图5,B所示,对c-di-GMP响应超过1倍的有A05,A12,A13,A16,A17,A22。
实施例6,对c-di-GMP响应倍数超过2倍的cpYFP光学探针的滴定曲线和特异性
选择7个Bl-cpYFP-dD融合蛋白质36/37,36/38,36/39,36/40,36/41,36/42,37/38进行浓度梯度的c-di-GMP检测,检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值的变化,不同插入位点36/37,36/38,36/39,36/40,36/41,36/42,37/38c-di-GMP探针的Kd(结合常数)分别为7.02μM、2.13μM、1.97μM、4.13μM、1.80μM、1.93μM、7.82μM,变化幅度分别为2.04倍、2.30倍、2.46倍、3.48倍、4.98倍、1.92倍、2.21倍,结果如图6所示。同时,对探针进行特异性检测,结果表明探针对c-di-GMP具有很好的专一性,如图7所示。
实施例7,对c-di-GMP响应倍数超过2倍的cpGFP光学探针的滴定曲线和特异性
选择4个Bl-cpYFP-dD融合蛋白质37/38,37/40,37/41,39/40进行浓度梯度的c-di-GMP检测,检测400nm激发525nm发射处荧光强度和488nm激发525nm发射处荧光强度比值的变化,不同插入位点37/38,37/40,37/41,39/40c-di-GMP探针的Kd(结合常数)分别为12.26μM、76.40μM、86.36μM、56.90μM,变化幅度分别为2.60倍、4.14倍、6.56倍、4.12倍,结果如图8所示。同时,对探针进行特异性检测,结果表明探针对c-di-GMP具有很好的专一性,如图9所示。
实施例8,c-di-GMP黄色荧光蛋白探针的光谱性能
选择7个Bl-cpYFP-dD融合蛋白质36/37,36/38,36/39,36/40,36/41,36/42,37/38进行浓度梯度的c-di-GMP分别进行0μM和50μM c-di-GMP处理10min后,使用荧光分光光度计进行荧光谱的检测。对激发光谱的测定:固定发射波长为530nm,记录350-500nm的激发光谱,每5nm读取一次。光谱曲线如图10所示。
实施例8,c-di-GMP绿色荧光蛋白探针的光谱性能
选择4个Bl-cpGFP-dD融合蛋白质37/38,37/40,37/41,39/40进行浓度梯度的c-di-GMP分别进行0μM和50μM c-di-GMP处理10min后,使用荧光分光光度计进行荧光谱的检测。对激发光谱的测定:固定发射波长为530nm,记录350-500nm的激发光谱,每5nm读取一次。光谱曲线如图11所示。
实施例9,探针的亚细胞器定位和在亚细胞器内的性能
本实施例中,我们使用不同的定位信号肽与c-di-GMP荧光探针BldD-36/41-cpYFP的C端或N端进行融合,并且将c-di-GMP荧光探针BldD-36/41-cpYFP定位到不同的细胞器中。
将融合不同定位信号肽的c-di-GMP荧光探针BldD-36/40-cpYFP基因的质粒转染HeLa细胞36小时后,使用PBS冲洗之后,置于HBSS溶液中使用倒置荧光显微镜进行FITC通道下进行荧光检测。我们发现c-di-GMP荧光探针通过与不同的特异定位信号肽融合能够定位到包括细胞浆、线粒体、细胞核、高尔基体、内质网和细胞膜等亚细胞器中。结果如图12所示,不同的亚细胞结构中都显示有荧光,并且荧光的分布和强度各不相同。
实施例10:c-di-GMP跨膜运输的动态监测
将转化BldD-36/41-cpYFP基因的大肠杆菌JM109(DE3)细胞,诱导表达24小时后,使用PBS冲洗之后,置于Tris溶液中检测5min时间段内420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值的变化。结果如图13所示,在饥饿2个小时之后,添加不同浓度的c-di-GMP后检测30min,ratio 420/485逐渐增加,最高可以达到2倍。
实施例11:探针在大肠杆菌JM109(DE3)细胞中的成像
将转化BldD-36/41-cpYFP基因的大肠杆菌JM109(DE3)细胞在18℃表达蛋白24小时后进行离心:4000rpm离心5min。弃去培养基,加入原培养基0.1倍含量的PBS缓冲液重悬细菌。重悬后的细菌溶液在室温下孵育5min后,吸取2μl加入到载玻片上,缓慢地盖上盖玻片,轻柔压片后,置于激光共聚焦显微镜下进行成像分析,成像图见图14。
实施例12,活菌水平基于探针进行高通量化合物筛选
本实施例中,我们使用c-di-GMP探针BldD-36/41-cpYFP表达的大肠杆菌JM109(DE3)细菌进行了高通量化合物筛选。
将表达BldD-36/40-cpYFP探针的大肠杆菌使用PBS冲洗之后,置于PBS溶液中(无c-di-GMP)处理1hours后,使用10μM的化合物进行处理1hours。分别滴加c-di-GMP。使用酶标仪记录420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值变化。以未用任何化合物处理的样品为标准。结果如图15所示,我们发现使用2400种化合物处理的细胞中,绝大部分的化合物对c-di-GMP进入细胞影响极小。有2种化合物能够提高细胞对c-di-GMP的摄取能力,另外有6种化合物能够明显降低细胞对c-di-GMP的摄取。
实施例13,探针对大肠杆菌中c-di-GMP进行定量检测
在本实施例中,使用纯化的c-di-GMP荧光探针BldD-36/41-cpYFP蛋白质对大肠杆菌中c-di-GMP进行了分析。
将c-di-GMP荧光探针BldD-36/41-cpYFP荧光蛋白质与稀释处理的大肠杆菌细胞进行混合处理10min后,使用酶标仪检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度比值。结果如图16所示,发现大肠杆菌内的c-di-GMP含量在3.56μM左右。
由以上实施例可知,本发明提供的c-di-GMP荧光探针,蛋白分子量相对较小且易于成熟,荧光动态变化大,特异性好,并且能够通过基因操作的方法在细胞中表达,可在细胞内外实时定位、定量检测细菌内的c-di-GMP;并且能够进行高通量的化合物筛选。
以上所述仅是本发明的优选实施方案,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> 华东理工大学
<120> 一种环二鸟苷酸光学探针及其制备方法和应用
<130> 204706
<141> 2020-06-18
<160> 14
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Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg Tyr
165 170 175
Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu
180 185 190
Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr
195 200 205
Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg
210 215 220
Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly
225 230 235 240
His Lys Leu Glu Tyr Asn
245
<210> 3
<211> 241
<212> PRT
<213> Artificial Sequence
<400> 3
Asn Val Tyr Ile Lys Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
1 5 10 15
Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln Leu Ala Tyr
20 25 30
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
35 40 45
Asp Asn His Tyr Leu Ser Val Gln Ser Lys Leu Ser Lys Asp Pro Asn
50 55 60
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
65 70 75 80
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser
85 90 95
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
100 105 110
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
115 120 125
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
130 135 140
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
145 150 155 160
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
165 170 175
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Ile Gln Glu
180 185 190
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
195 200 205
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
210 215 220
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
225 230 235 240
Asn
<210> 4
<211> 243
<212> PRT
<213> Artificial Sequence
<400> 4
Asn Val Tyr Ile Lys Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn
1 5 10 15
Phe Lys Ile Arg His Asn Ile Glu Gly Gly Gly Val Gln Leu Ala Tyr
20 25 30
His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro
35 40 45
Asp Asn His Tyr Leu Ser Val Gln Ser Ile Leu Ser Lys Asp Pro Asn
50 55 60
Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly
65 70 75 80
Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser
85 90 95
Glu Ser Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
100 105 110
Ile Gln Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val
115 120 125
Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys
130 135 140
Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
145 150 155 160
Thr Thr Leu Ser His Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His
165 170 175
Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Gly Gly Tyr Ile
180 185 190
Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg
195 200 205
Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu
210 215 220
Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu
225 230 235 240
Glu Tyr Asn
<210> 5
<211> 242
<212> PRT
<213> Artificial Sequence
<400> 5
Val Ser Glu Arg Met Tyr Pro Glu Asp Gly Ala Leu Lys Ser Glu Ile
1 5 10 15
Lys Lys Gly Leu Arg Leu Lys Asp Gly Gly His Tyr Ala Ala Glu Val
20 25 30
Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr
35 40 45
Ile Val Asp Ile Lys Leu Asp Ile Val Ser His Asn Glu Asp Tyr Thr
50 55 60
Ile Val Glu Gln Cys Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly
65 70 75 80
Met Asp Glu Leu Tyr Lys Gly Gly Thr Gly Gly Ser Leu Val Ser Lys
85 90 95
Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe Met Arg Phe Lys
100 105 110
Val His Met Glu Gly Ser Val Asn Gly His Glu Phe Glu Ile Glu Gly
115 120 125
Glu Gly Glu Gly Arg Pro Tyr Glu Ala Phe Gln Thr Ala Lys Leu Lys
130 135 140
Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro
145 150 155 160
Gln Phe Met Tyr Gly Ser Lys Ala Tyr Ile Lys His Pro Ala Asp Ile
165 170 175
Pro Asp Tyr Phe Lys Leu Ser Phe Pro Glu Gly Phe Arg Trp Glu Arg
180 185 190
Val Met Asn Phe Glu Asp Gly Gly Ile Ile His Val Asn Gln Asp Ser
195 200 205
Ser Leu Gln Asp Gly Val Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr
210 215 220
Asn Phe Pro Pro Asp Gly Pro Val Met Gln Lys Lys Thr Met Gly Trp
225 230 235 240
Glu Ala
<210> 6
<211> 368
<212> PRT
<213> Artificial Sequence
<400> 6
Met Gly Ser Ser His His His His His His Ser Gln Asp Pro Met Glu
1 5 10 15
Pro Pro Pro Lys Leu Val Leu Asp Leu Glu Arg Leu Ala Thr Val Pro
20 25 30
Ala Glu Lys Ala Gly Pro Leu Gln Arg Tyr Ala Ala Thr Ile Gln Ser
35 40 45
Gln Arg Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
50 55 60
Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly
65 70 75 80
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
85 90 95
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val
100 105 110
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
115 120 125
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn
130 135 140
Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe
145 150 155 160
Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
165 170 175
His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly
180 185 190
Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro
195 200 205
Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala
210 215 220
Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met
225 230 235 240
Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly
245 250 255
Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val
260 265 270
Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn Ile
275 280 285
Leu Gly His Lys Leu Glu Tyr Asn Gly Asp Tyr Asn Gly Lys Val Leu
290 295 300
Ser Ile Arg Gln Asp Asp Leu Arg Thr Leu Ala Val Ile Tyr Asp Gln
305 310 315 320
Ser Pro Ser Val Leu Thr Glu Gln Leu Ile Ser Trp Gly Val Leu Asp
325 330 335
Ala Asp Ala Arg Arg Ala Val Ala Ser His Asp Glu Leu Leu Gln Tyr
340 345 350
Pro Tyr Asp Val Pro Glu Phe Gly Gly Thr Lys Leu Cys Cys His Arg
355 360 365
<210> 7
<211> 367
<212> PRT
<213> Artificial Sequence
<400> 7
Met Gly Ser Ser His His His His His His Ser Gln Asp Pro Met Glu
1 5 10 15
Pro Pro Pro Lys Leu Val Leu Asp Leu Glu Arg Leu Ala Thr Val Pro
20 25 30
Ala Glu Lys Ala Gly Pro Leu Gln Arg Tyr Ala Ala Thr Ile Gln Ser
35 40 45
Gln Arg Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
50 55 60
Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly
65 70 75 80
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
85 90 95
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val
100 105 110
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
115 120 125
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn
130 135 140
Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe
145 150 155 160
Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
165 170 175
His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly
180 185 190
Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro
195 200 205
Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala
210 215 220
Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met
225 230 235 240
Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly
245 250 255
Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val
260 265 270
Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn Ile
275 280 285
Leu Gly His Lys Leu Glu Tyr Asn Asp Tyr Asn Gly Lys Val Leu Ser
290 295 300
Ile Arg Gln Asp Asp Leu Arg Thr Leu Ala Val Ile Tyr Asp Gln Ser
305 310 315 320
Pro Ser Val Leu Thr Glu Gln Leu Ile Ser Trp Gly Val Leu Asp Ala
325 330 335
Asp Ala Arg Arg Ala Val Ala Ser His Asp Glu Leu Leu Gln Tyr Pro
340 345 350
Tyr Asp Val Pro Glu Phe Gly Gly Thr Lys Leu Cys Cys His Arg
355 360 365
<210> 8
<211> 366
<212> PRT
<213> Artificial Sequence
<400> 8
Met Gly Ser Ser His His His His His His Ser Gln Asp Pro Met Glu
1 5 10 15
Pro Pro Pro Lys Leu Val Leu Asp Leu Glu Arg Leu Ala Thr Val Pro
20 25 30
Ala Glu Lys Ala Gly Pro Leu Gln Arg Tyr Ala Ala Thr Ile Gln Ser
35 40 45
Gln Arg Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
50 55 60
Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly
65 70 75 80
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
85 90 95
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val
100 105 110
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
115 120 125
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn
130 135 140
Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe
145 150 155 160
Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
165 170 175
His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly
180 185 190
Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro
195 200 205
Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala
210 215 220
Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met
225 230 235 240
Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly
245 250 255
Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val
260 265 270
Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn Ile
275 280 285
Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Gly Lys Val Leu Ser Ile
290 295 300
Arg Gln Asp Asp Leu Arg Thr Leu Ala Val Ile Tyr Asp Gln Ser Pro
305 310 315 320
Ser Val Leu Thr Glu Gln Leu Ile Ser Trp Gly Val Leu Asp Ala Asp
325 330 335
Ala Arg Arg Ala Val Ala Ser His Asp Glu Leu Leu Gln Tyr Pro Tyr
340 345 350
Asp Val Pro Glu Phe Gly Gly Thr Lys Leu Cys Cys His Arg
355 360 365
<210> 9
<211> 365
<212> PRT
<213> Artificial Sequence
<400> 9
Met Gly Ser Ser His His His His His His Ser Gln Asp Pro Met Glu
1 5 10 15
Pro Pro Pro Lys Leu Val Leu Asp Leu Glu Arg Leu Ala Thr Val Pro
20 25 30
Ala Glu Lys Ala Gly Pro Leu Gln Arg Tyr Ala Ala Thr Ile Gln Ser
35 40 45
Gln Arg Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
50 55 60
Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly
65 70 75 80
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
85 90 95
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val
100 105 110
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
115 120 125
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn
130 135 140
Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe
145 150 155 160
Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
165 170 175
His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly
180 185 190
Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro
195 200 205
Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala
210 215 220
Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met
225 230 235 240
Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly
245 250 255
Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val
260 265 270
Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn Ile
275 280 285
Leu Gly His Lys Leu Glu Tyr Asn Asn Gly Lys Val Leu Ser Ile Arg
290 295 300
Gln Asp Asp Leu Arg Thr Leu Ala Val Ile Tyr Asp Gln Ser Pro Ser
305 310 315 320
Val Leu Thr Glu Gln Leu Ile Ser Trp Gly Val Leu Asp Ala Asp Ala
325 330 335
Arg Arg Ala Val Ala Ser His Asp Glu Leu Leu Gln Tyr Pro Tyr Asp
340 345 350
Val Pro Glu Phe Gly Gly Thr Lys Leu Cys Cys His Arg
355 360 365
<210> 10
<211> 364
<212> PRT
<213> Artificial Sequence
<400> 10
Met Gly Ser Ser His His His His His His Ser Gln Asp Pro Met Glu
1 5 10 15
Pro Pro Pro Lys Leu Val Leu Asp Leu Glu Arg Leu Ala Thr Val Pro
20 25 30
Ala Glu Lys Ala Gly Pro Leu Gln Arg Tyr Ala Ala Thr Ile Gln Ser
35 40 45
Gln Arg Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
50 55 60
Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly
65 70 75 80
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
85 90 95
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val
100 105 110
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
115 120 125
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn
130 135 140
Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe
145 150 155 160
Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
165 170 175
His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly
180 185 190
Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro
195 200 205
Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala
210 215 220
Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met
225 230 235 240
Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly
245 250 255
Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val
260 265 270
Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn Ile
275 280 285
Leu Gly His Lys Leu Glu Tyr Asn Gly Lys Val Leu Ser Ile Arg Gln
290 295 300
Asp Asp Leu Arg Thr Leu Ala Val Ile Tyr Asp Gln Ser Pro Ser Val
305 310 315 320
Leu Thr Glu Gln Leu Ile Ser Trp Gly Val Leu Asp Ala Asp Ala Arg
325 330 335
Arg Ala Val Ala Ser His Asp Glu Leu Leu Gln Tyr Pro Tyr Asp Val
340 345 350
Pro Glu Phe Gly Gly Thr Lys Leu Cys Cys His Arg
355 360
<210> 11
<211> 363
<212> PRT
<213> Artificial Sequence
<400> 11
Met Gly Ser Ser His His His His His His Ser Gln Asp Pro Met Glu
1 5 10 15
Pro Pro Pro Lys Leu Val Leu Asp Leu Glu Arg Leu Ala Thr Val Pro
20 25 30
Ala Glu Lys Ala Gly Pro Leu Gln Arg Tyr Ala Ala Thr Ile Gln Ser
35 40 45
Gln Arg Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys
50 55 60
Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly
65 70 75 80
Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
85 90 95
Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val
100 105 110
Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
115 120 125
Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn
130 135 140
Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe
145 150 155 160
Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly
165 170 175
His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly
180 185 190
Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro
195 200 205
Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala
210 215 220
Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met
225 230 235 240
Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly
245 250 255
Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val
260 265 270
Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn Ile
275 280 285
Leu Gly His Lys Leu Glu Tyr Asn Lys Val Leu Ser Ile Arg Gln Asp
290 295 300
Asp Leu Arg Thr Leu Ala Val Ile Tyr Asp Gln Ser Pro Ser Val Leu
305 310 315 320
Thr Glu Gln Leu Ile Ser Trp Gly Val Leu Asp Ala Asp Ala Arg Arg
325 330 335
Ala Val Ala Ser His Asp Glu Leu Leu Gln Tyr Pro Tyr Asp Val Pro
340 345 350
Glu Phe Gly Gly Thr Lys Leu Cys Cys His Arg
355 360
<210> 12
<211> 368
<212> PRT
<213> Artificial Sequence
<400> 12
Met Gly Ser Ser His His His His His His Ser Gln Asp Pro Met Glu
1 5 10 15
Pro Pro Pro Lys Leu Val Leu Asp Leu Glu Arg Leu Ala Thr Val Pro
20 25 30
Ala Glu Lys Ala Gly Pro Leu Gln Arg Tyr Ala Ala Thr Ile Gln Ser
35 40 45
Gln Arg Gly Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln
50 55 60
Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp
65 70 75 80
Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
85 90 95
Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser
100 105 110
Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
115 120 125
Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
130 135 140
Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu
145 150 155 160
Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn
165 170 175
Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr
180 185 190
Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val
195 200 205
Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe
210 215 220
Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala
225 230 235 240
Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp
245 250 255
Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu
260 265 270
Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn
275 280 285
Ile Leu Gly His Lys Leu Glu Tyr Asn Asp Tyr Asn Gly Lys Val Leu
290 295 300
Ser Ile Arg Gln Asp Asp Leu Arg Thr Leu Ala Val Ile Tyr Asp Gln
305 310 315 320
Ser Pro Ser Val Leu Thr Glu Gln Leu Ile Ser Trp Gly Val Leu Asp
325 330 335
Ala Asp Ala Arg Arg Ala Val Ala Ser His Asp Glu Leu Leu Gln Tyr
340 345 350
Pro Tyr Asp Val Pro Glu Phe Gly Gly Thr Lys Leu Cys Cys His Arg
355 360 365
<210> 13
<211> 466
<212> PRT
<213> Artificial Sequence
<400> 13
Met Gly Ser Ser His His His His His His Ser Gln Asp Pro Met Glu
1 5 10 15
Pro Pro Pro Lys Leu Val Leu Asp Leu Glu Arg Leu Ala Thr Val Pro
20 25 30
Ala Glu Lys Ala Gly Pro Leu Gln Arg Tyr Ala Ala Thr Ile Gln Ser
35 40 45
Gln Arg Gly Asp Tyr Asn Gly Lys Val Leu Ser Ile Arg Gln Asp Asp
50 55 60
Leu Arg Thr Leu Ala Val Ile Tyr Asp Gln Ser Pro Ser Val Leu Thr
65 70 75 80
Glu Gln Leu Ile Ser Trp Gly Val Leu Asp Ala Asp Ala Arg Arg Ala
85 90 95
Val Ala Ser His Asp Glu Leu Leu Gln Tyr Pro Tyr Asp Val Pro Glu
100 105 110
Phe Gly Gly Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln
115 120 125
Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp
130 135 140
Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
145 150 155 160
Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser
165 170 175
Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
180 185 190
Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
195 200 205
Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu
210 215 220
Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn
225 230 235 240
Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr
245 250 255
Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val
260 265 270
Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe
275 280 285
Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala
290 295 300
Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp
305 310 315 320
Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu
325 330 335
Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn
340 345 350
Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Ser Gly Gly Ser Met Glu
355 360 365
Pro Pro Pro Lys Leu Val Leu Asp Leu Glu Arg Leu Ala Thr Val Pro
370 375 380
Ala Glu Lys Ala Gly Pro Leu Gln Arg Tyr Ala Ala Thr Ile Gln Ser
385 390 395 400
Gln Arg Gly Asp Tyr Asn Gly Lys Val Leu Ser Ile Arg Gln Asp Asp
405 410 415
Leu Arg Thr Leu Ala Val Ile Tyr Asp Gln Ser Pro Ser Val Leu Thr
420 425 430
Glu Gln Leu Ile Ser Trp Gly Val Leu Asp Ala Asp Ala Arg Arg Ala
435 440 445
Val Ala Ser His Asp Glu Leu Leu Gln Tyr Pro Tyr Asp Val Pro Glu
450 455 460
Phe Gly
465
<210> 14
<211> 1095
<212> DNA
<213> Artificial Sequence
<400> 14
atgggcagca gccatcacca tcatcaccac agccaggatc caatggaacc gcccccaaaa 60
ctggtcctgg atctggaacg cctggccact gtgcctgcag agaaggctgg accactgcag 120
cgttatgcag caaccattca gtctcagcgg tacaacagcg acaacgtcta tatcatggcc 180
gacaagcaga agaacggcat caaggccaac ttcaagatcc gccacaacgt cgaggacggc 240
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg 300
ctgcccgaca accactacct gagcttccag tccgtcctga gcaaagaccc caacgagaag 360
cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac 420
gagctgtaca acgtggatgg cggtagcggt ggcaccggca gcaagggcga ggagctgttc 480
accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc 540
gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gctgatctgc 600
accaccggca agctgcccgt gccctggccc accctcgtga ccaccctcgg ctacggcctg 660
aagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg 720
cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc 780
cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc 840
ggcttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacgg taaagtgctg 900
agtattcgtc aggatgacct gcgcaccctg gccgtgatct atgaccagag cccatccgtt 960
ctgacagaac agctgatctc atggggcgtt ctggatgcag acgctcgccg tgcagtggca 1020
tcccacgacg agctgctgca gtacccatac gatgttccag aatttggggg taccaagctt 1080
tgctgccacc gctga 1095
Claims (10)
1.一种融合蛋白,包含:(A)光学活性多肽,和(B)如SEQ ID NO:1所示的c-di-GMP敏感多肽,其中,
(1)A位于B的序列内,将B分为B1和B2两个部分,形成氨基端到羧基端方向上具有B1-A-B2的结构,其中,
光学活性多肽是cpYFP,其位于c-di-GMP敏感多肽的选自以下任一位点:9/10,10/11,11/12,16/17,17/18,18/19,35/36,36/37,36/38,36/39,36/40,36/41,36/42,37/38,37/39,37/41,38/39,38/40,38/41,38/42,39/40,39/41,39/42,40/41,41/42,43/44,44/45,45/46,48/49,49/50,50/51,或
光学活性多肽是cpGFP,其位于c-di-GMP敏感多肽的选自以下任一位点:10/11,11/12,16/17,17/18,18/19,35/36,36/38,36/40,36/42,37/38,37/39,37/41,37/42,38/39,38/40,38/41,38/42,39/40,39/41,40/41,41/42,48/49,或
光学活性多肽是cpBFP,其位于c-di-GMP敏感多肽的选自以下任一位点:9/10,10/11,11/12,16/17,17/18,18/19,35/36,36/37,36/38,36/39,36/40,36/41,36/42,37/38,37/39,37/40,37/41,37/42,38/39,38/40,38/41,39/40,39/41,39/42,40/41,41/42,45/46,48/49,49/50,或
光学活性多肽是cpmApple,其位于c-di-GMP敏感多肽的选自任一位点:9/10,10/11,11/12,16/17,17/18,18/19,35/36,36/37,36/38,36/39,36/40,36/41,36/42,37/38,37/39,37/40,37/41,37/42,38/39,38/40,38/41,38/42,39/40,39/41,39/42,40/41,41/42,42/43,43/44,44/45,45/46,46/47,47/48,48/49,49/50,
或者
(2)A位于两个B之间,通过接头与两端的B连接,形成氨基端到羧基端方向上具有B-A-B的结构,其中,
光学活性多肽cpYFP位于两个c-di-GMP敏感多肽之间,通过接头X和Y与两端的c-di-GMP敏感多肽连接,其中,X/Y选自:0/GS,0/GSG,0/GSGS,0/GSGGS,0/GSGGGS,0/GSGSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,G/GSGGGS,G/GSGSGGS,GS/GS,GS/GSG,GS/GSGS,GS/GSGGS,GS/GSGGGS,GS/GSGSGGS,GGS/GS,GGS/GSG,GGS/GSGS,GGS/GSGGS,GGS/GSGGGS,GGS/GSGSGGS,GGGS/GS,GGGS/GSG,GGGS/GSGS,GGGS/GSGGS,GGGS/GSGGGS,GGGS/GSGSGGS,GSGGS/GS,GSGGS/GSG,GSGGS/GSGS,GSGGS/GSGGS,GSGGS/GSGGGS,GSGGS/GSGSGGS,或
光学活性多肽cpGFP位于两个c-di-GMP敏感多肽之间,通过接头X和Y与两端的c-di-GMP敏感多肽连接,并且X/Y选自:0/GS,0/GSG,0/GSGS,0/GSGGGS,0/GSGSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,G/GSGGGS,G/GSGSGGS,GS/GS,GS/GSG,GS/GSGS,GS/GSGGS,GS/GSGGGS,GS/GSGSGGS,GGS/GS,GGS/GSG,GGS/GSGS,GGS/GSGGS,GGS/GSGGGS,GGS/GSGSGGS,GGGS/GS,GGGS/GSG,GGGS/GSGS,GGGS/GSGGS,GGGS/GSGGGS,GGGS/GSGSGGS,GSGGS/GS,GSGGS/GSG,GSGGS/GSGS,GSGGS/GSGGS,GSGGS/GSGGGS,GSGGS/GSGSGGS,或
光学活性多肽cpBFP位于两个c-di-GMP敏感多肽之间,通过接头X和Y与两端的c-di-GMP敏感多肽连接,并且X/Y选自:0/GS,0/GSG,0/GSGS,0/GSGGS,0/GSGGGS,0/GSGSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,G/GSGGGS,G/GSGSGGS,GS/GS,GS/GSGS,GS/GSGGS,GS/GSGGGS,GS/GSGSGGS,GGS/GS,GGS/GSG,GGS/GSGS,GGS/GSGGS,GGS/GSGGGS,GGS/GSGSGGS,GGGS/GS,GGGS/GSG,GGGS/GSGS,GGGS/GSGGS,GGGS/GSGGGS,GGGS/GSGSGGS,GSGGS/GS,GSGGS/GSG,GSGGS/GSGS,GSGGS/GSGGS,GSGGS/GSGGGS,GSGGS/GSGSGGS,或
光学活性多肽cpmApple位于两个c-di-GMP敏感多肽之间,通过接头X和Y与两端的c-di-GMP敏感多肽连接,并且X/Y选自:0/GS,0/GSGGS,0/GSGGGS,0/GSGSGGS,G/GS,G/GSG,G/GSGS,G/GSGGS,G/GSGGGS,G/GSGSGGS,GS/GS,GS/GSG,GS/GSGS,GS/GSGGS,GS/GSGGGS,GGS/GS,GGS/GSG,GGS/GSGS,GGS/GSGGGS,GGS/GSGSGGS,GGGS/GS,GGGS/GSGS,GGGS/GSGGS,GGGS/GSGSGGS,GSGGS/GSG,GSGGS/GSGS,GSGGS/GSGGS,GSGGS/GSGGGS。
2. 一种核酸分子,其序列选自:
(1)权利要求1所述的融合蛋白的编码序列,或
(2)(1)的互补序列。
3.一种核酸构建物,包含权利要求2所述的核酸分子。
4.如权利要求3所述的核酸构建物,其特征在于,所述核酸构建物是克隆载体、表达载体或重组载体。
5.一种宿主细胞,所述宿主细胞
(1)表达权利要求1所述的融合蛋白;
(2)包含权利要求2所述的核酸分子;或
(3)包含权利要求3或4所述的核酸构建物。
6.一种检测试剂盒,其包含选自以下的一项或任意多项:
(1)权利要求1所述的融合蛋白;
(2)权利要求2所述的核酸分子;
(3)权利要求3或4所述的核酸构建物;
(4)权利要求5所述的宿主细胞;和
检测c-di-GMP所需的其他试剂。
7.权利要求1所述的融合蛋白、权利要求2所述的核酸分子、权利要求3或4所述的核酸构建物或权利要求5所述的宿主细胞在检测样品中的c-di-GMP或筛选对c-di-GMP含量变化有影响的化合物中的应用,所述检测包括c-di-GMP定性、定位或定量检测。
8.一种检测样品中c-di-GMP的方法,包括:使权利要求1所述的融合蛋白与样品接触,和检测光学活性多肽的变化。
9.一种筛选对c-di-GMP含量变化有影响的化合物的方法,包括:使权利要求1所述的融合蛋白与候选化合物接触,检测光学活性多肽的变化,和根据光学活性多肽的变化筛选化合物。
10.如权利要求9所述的方法,其特征在于,所述方法包括:使权利要求1所述的融合蛋白与候选化合物和c-di-GMP接触,检测光学活性多肽的变化,和根据光学活性多肽的变化筛选化合物。
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