CN115028743B - 一种检测d-2-羟基戊二酸的荧光传感器及其构建方法和应用 - Google Patents
一种检测d-2-羟基戊二酸的荧光传感器及其构建方法和应用 Download PDFInfo
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Abstract
本发明属于生物检测技术领域,具体涉及一种检测D‑2‑羟基戊二酸的荧光传感器及其构建方法和应用。本发明基于荧光共振能量转移技术和来源于反硝化无色杆菌的特异性转录调控因子DhdR开发能检测D‑2‑羟基戊二酸的荧光传感器,其具体是由绿色荧光蛋白Clover、特异性转录调控因子DhdR和红色荧光蛋白Clover组成的融合蛋白,具有灵敏度高、特异性好、制备简单、成分简单、成本较低、易于操作等优点,在D‑2‑羟基戊二酸相关疾病的诊断与治疗中具有广阔的应用前景。
Description
技术领域
本发明属于生物检测技术领域,具体涉及一种检测D-2-羟基戊二酸的荧光传感器及其构建方法和应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
2-羟基戊二酸是一种五碳二元羧酸,因α-碳原子上携带一个羟基,也被称作α-羟基戊二酸。2-羟基戊二酸是2-酮基戊二酸的结构类似物,存在两种手性对映体形式:D-2-羟基戊二酸和L-2-羟基戊二酸。D-2-羟基戊二酸是2-酮基戊二酸的结构类似物,可抑制多种2-酮基戊二酸依赖性双加氧酶、转氨酶的活性,导致基因组范围内的组蛋白和DNA甲基化改变、阻止细胞分化,引起癌症的发生。
D-2-羟基戊二酸是一种存在于许多生物体中的低丰度代谢物,可被D-2-羟基戊二酸脱氢酶D2HGDH催化生成2-酮基戊二酸。D-2-羟基戊二酸尿症是一种罕见的神经系统代谢性疾病,主要临床表现包括发育迟缓、肌张力减退和癫痫的发作,患者的尿液、血浆以及脑脊液中存在大量积累的D-2-羟基戊二酸。此外,异柠檬酸脱氢酶突变也会导致D-2-羟基戊二酸异常积累。目前,研究人员已在多种人类肿瘤中发现异柠檬酸脱氢酶1和异柠檬酸脱氢酶2的突变,包括急性髓性白血病、甲状腺癌、软骨瘤、胆管癌、胶质瘤等。D-2-羟基戊二酸已被视为神经系统代谢性疾病D-2-羟基戊二酸尿症和多种癌症的标志性代谢物。因此,D-2-羟基戊二酸的检测定量对许多D-2-羟基戊二酸相关疾病的诊断与治疗具有重要意义。
目前常用的D-2-羟基戊二酸检测方法主要包括液相色谱-质谱联用技术和气相色谱-质谱联用技术,但发明人发现,质谱法检测的是D-2-羟基戊二酸和L-2-羟基戊二酸浓度的总和,因此可能无法根据检测结果准确对2-羟基戊二酸相关疾病进行诊断。虽然使用合适的衍生试剂或手性柱实现可以实现对D-2-羟基戊二酸和L-2-羟基戊二酸进行有效分离,但该检测方法耗时、繁琐、无法实现高通量、检测成本较高,严重限制了D-2-羟基戊二酸相关疾病诊疗技术的发展。
发明内容
针对上述现有技术的不足,发明人经长期的技术与实践探索,提供一种检测D-2-羟基戊二酸的荧光传感器及其构建方法和应用。本发明基于荧光共振能量转移技术和来源于反硝化无色杆菌的特异性转录调控因子DhdR开发能检测D-2-羟基戊二酸的荧光传感器,具有灵敏度高、特异性好、制备简单、成分简单、成本较低、易于操作等优点。基于上述研究成果,从而完成本发明。
为实现上述技术目的,本发明采用如下技术方案:
本发明的第一个方面,提供一种融合蛋白,所述融合蛋白至少包括一个D-2-羟基戊二酸特异性的转录调控因子,以及,分别与所述D-2-羟基戊二酸特异性的转录调控因子两端连接的两个荧光蛋白。
所述融合蛋白是如下(a1)-(a3)中任意一种:
(a1)SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列组成的蛋白质;
(a2)将(a1)经过一个或多个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的蛋白质;
(a3)其它基因编码的与(a1)所示的氨基酸序列组成具有相似性达到至少50%(包括但不限于50%、60%、70%、80%、85%、90%、95%、99%)并且具有与(a1)所示的融合蛋白相同活性的蛋白。
本发明的第二个方面,提供一种编码上述融合蛋白的核酸分子。
其中,所述核酸分子具有(b1)-(b4)中任一所述核苷酸序列:
(b1)如SEQ ID NO.3或SEQ ID NO.4所示的核苷酸序列;
(b2)如(b1)所示的核苷酸序列经一个或多个核苷酸的替换、缺失或插入形成的序列;
(b3)与(b1)或(b2)限定的核苷酸序列具有至少50%(包括但不限于50%、60%、70%、80%、85%、90%、95%、99%)的同一性,且编码所述融合蛋白的核酸分子;
(b4)在严格条件下能够与如(b1)或(b2)所述的核苷酸序列杂交并编码相同功能融合蛋白的核苷酸序列。
本发明的第三个方面,提供一种重组表达载体,所述重组表达载体含有上述核酸分子。
本发明的第四个方面,提供一种宿主细胞,所述宿主细胞含有上述核酸分子、含有上述核酸分子的重组表达载体或表达上述融合蛋白。
本发明的第五个方面,提供上述融合蛋白、核酸分子、重组表达载体和/或宿主细胞在制备检测D-2-羟基戊二酸的荧光传感器中的应用。
本发明的第六个方面,提供一种检测D-2-羟基戊二酸的荧光传感器,所述荧光传感器包含上述融合蛋白、核酸分子、重组表达载体和/或宿主细胞。
所述荧光传感器还可以包括其他用于D-2-羟基戊二酸检测的试剂、装置和/或设备。
所述试剂包括检测缓冲液(如荧光测定缓冲液:50mM Tris-HCl,pH 7.4)。
所述荧光传感器在实际应用时可包装为试剂盒产品使用。
本发明的第七个方面,提供上述检测D-2-羟基戊二酸的荧光传感器的构建方法,所述构建方法至少包括融合蛋白的构建,具体步骤如下:
分别合成两个荧光蛋白的目的基因以及特异性转录调控因子DhdR的编码基因dhdR插入质粒中获得重组质粒,将重组质粒转入宿主细胞中表达获得。
本发明的第八个方面,提供一种检测D-2-羟基戊二酸的方法,所述方法包括:将待测样品与所述融合蛋白或荧光传感器进行孵育,根据两个荧光蛋白的荧光发射强度比值变化,分析D-2-羟基戊二酸的浓度或有无。
与现有技术方案相比,上述一个或多个技术方案具有如下有益效果:
(1)上述技术方案提供的D-2-羟基戊二酸荧光传感器以来源于反硝化无色杆菌NBRC 15125的特异性转录调控因子DhdR为识别元件,利用DhdR与D-2-羟基戊二酸结合后自身构象改变的特点,结合荧光共振能量转移技术,可将D-2-羟基戊二酸的浓度转换为荧光发射强度比值进行输出,两个荧光蛋白的荧光发射强度比值大小与样品中D-2-羟基戊二酸的浓度有关;
(2)上述技术方案提供的D-2-羟基戊二酸荧光传感器是将特异性转录调控因子DhdR插入至绿色荧光蛋白Clover和红色荧光蛋白mRuby2之间组成的融合蛋白,检测体系中仅包含纯化的D-2-羟基戊二酸荧光传感器和缓冲液,灵敏度高、特异性好、制备简单、成分简单、成本较低、易于操作;
(3)上述技术方案提供的D-2-羟基戊二酸荧光传感器适用于对人血清、尿液、细胞培养基和细胞裂解物等生物样品中D-2-羟基戊二酸的浓度进行定量,定量结果与理论浓度的一致性较高,在D-2-羟基戊二酸尿症与异柠檬酸脱氢酶突变相关的癌症诊疗和小分子抑制剂的鉴定与筛选中具有广阔的应用前景。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为本发明实施例1中DHGFR0N0C表达纯化的SDS-PAGE验证。
图2为本发明实施例1中DHGFR0N0C对D-2-羟基戊二酸的剂量-响应曲线。
图3为本发明实施例2中DHGFR1.0对D-2-羟基戊二酸的剂量-响应曲线。
图4为本发明实施例3中DHGFR1.0的pH稳定性;
其中,图4A为D-2-羟基戊二酸荧光传感器DHGFR1.0在不同pH的50mM Tris-HCl缓冲液中对D-2-羟基戊二酸的荧光强度发射比值;图4B为在pH 10.0的Tris-HCl缓冲液中,DHGFR1.0对不同浓度的D-2-羟基戊二酸的荧光强度发射比值;图4C为使用50mM Tris-HCl缓冲液(pH 10.0)稀释纯化的DHGFR1.0至4/3μM,使用不同pH的缓冲液稀释D-2-羟基戊二酸至0μM、4μM、40μM和400μM,测定DHGFR1.0对不同pH D-2-羟基戊二酸的荧光强度发射比值。
图5为本发明实施例3中DHGFR1.0的光谱学性质分析。
图6为本发明实施例3中DHGFR1.0的特异性分析。
图7为本发明实施例4中DHGFR1.0对人血清中D-2-羟基戊二酸的剂量-响应曲线。
图8为本发明实施例4中DHGFR1.0对尿液中D-2-羟基戊二酸的剂量-响应曲线。
图9为本发明实施例5中DHGFR1.0对细胞培养基和细胞裂解物中D-2-羟基戊二酸的定量结果。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本申请提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本申请所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本申请的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
如前所述,目前常用的D-2-羟基戊二酸检测方法主要包括液相色谱-质谱联用技术和气相色谱-质谱联用技术,但该检测方法耗时、繁琐、无法实现高通量、检测成本较高,严重限制了D-2-羟基戊二酸相关疾病诊疗技术的发展。
基于荧光蛋白和荧光共振能量转移技术的荧光传感器已被广泛应用于检测各种小分子代谢物及研究单细胞或亚细胞区室中各种生理活动,由生物识别元件和一对供、受体荧光蛋白对组成。细菌已进化出多种转录调控因子用于识别各种小分子化合物,可作为生物识别元件构建基于荧光共振能量转移技术的荧光传感器。待测物与生物识别元件结合导致其构象改变,影响生物识别元件两端所融合的供、受体荧光蛋白的相对距离与空间取向,导致荧光蛋白间的荧光发射比值发生变化,该变化可用作相关代谢物检测的定量指标。
有鉴于此,本发明利用荧光共振能量转移技术和来源于反硝化无色杆菌的特异性转录调控因子DhdR开发能检测D-2-羟基戊二酸的荧光传感器。
具体的,本发明的一个典型具体实施方式中,提供一种融合蛋白,所述融合蛋白至少包括一个D-2-羟基戊二酸特异性的转录调控因子,以及,分别与所述D-2-羟基戊二酸特异性的转录调控因子两端连接的两个荧光蛋白。
所述融合蛋白是如下(a1)-(a3)中任意一种:
(a1)SEQ ID NO.1或SEQ ID NO.2所示的氨基酸序列组成的蛋白质;
(a2)将(a1)经过一个或多个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的由其衍生的蛋白质;
(a3)其它基因编码的与(a1)所示的氨基酸序列组成具有相似性达到至少50%(包括但不限于50%、60%、70%、80%、85%、90%、95%、99%)并且具有与(a1)所示的融合蛋白相同活性的蛋白;
上述(a2)中,所述“一个或多个氨基酸残基的取代和/或缺失和/或添加”为不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述(a1)-(a3)中的蛋白质可人工合成,也可先合成其编码基因,再进行生物表达得到。
本发明的又一具体实施方式中,所述D-2-羟基戊二酸特异性的转录调控因子可以为来源反硝化无色杆菌(Achromobacter denitrificans)NBRC 15125的特异性转录调控因子DhdR;其是发明人在前首次发现的调控D-2-羟基戊二酸分解代谢且特异性响应于D-2-羟基戊二酸的转录调控因子。当然,基于本发明的构思,现有已知的其他D-2-羟基戊二酸特异性的转录调控因子同样适用于本申请的技术方案,因此理应属于本申请的保护范围之内。
本发明的又一具体实施方式中,所述荧光蛋白为一类可视化的报告基因编码蛋白,包括绿色荧光蛋白、红色荧光蛋白、黄色荧光蛋白等;在本发明中,第一荧光蛋白可以为绿色荧光蛋白(如绿色荧光蛋白Clover),第二荧光蛋白可以为红色荧光蛋白(如红色荧光蛋白mRuby2)。当D-2-羟基戊二酸存在时,D-2-羟基戊二酸与转录调控因子DhdR结合诱导DhdR构象改变,进而改变其两端连接的两个荧光蛋白在空间上的相对位置与取向,使DhdR所连接的两个荧光蛋白的荧光发射比值改变,从而实现对D-2-羟基戊二酸的检测。
本发明的又一具体实施方式中,提供一种编码上述融合蛋白的核酸分子。
其中,所述核酸分子具有(b1)-(b4)中任一所述核苷酸序列:
(b1)如SEQ ID NO.3或SEQ ID NO.4所示的核苷酸序列;
(b2)如(b1)所示的核苷酸序列经一个或多个核苷酸的替换、缺失或插入形成的序列;
(b3)与(b1)或(b2)限定的核苷酸序列具有至少50%(包括但不限于50%、60%、70%、80%、85%、90%、95%、99%)的同一性,且编码所述融合蛋白的核酸分子;
(b4)在严格条件下能够与如(b1)或(b2)所述的核苷酸序列杂交并编码相同功能融合蛋白的核苷酸序列。
其中,所述核酸分子可以是DNA,如cDNA、基因组DNA或重组DNA。
本发明的又一具体实施方式中,提供一种重组表达载体,所述重组表达载体含有上述核酸分子。
所述重组表达载体通过上述核酸分子有效地连接到表达载体上获得,所述表达载体为病毒载体、质粒、噬菌体、噬菌粒、黏粒、F黏粒、噬菌体或人工染色体中的任意一种或多种;病毒载体可包括腺病毒载体、逆转录病毒载体或腺伴随病毒载体,人工染色体包括细菌人工染色体、噬菌体P1衍生的载体、酵母人工染色体或哺乳动物人工染色体;进一步优选为质粒;所述质粒包括但不限于pETDuet-1和pET28a。
本发明的又一具体实施方式中,提供一种宿主细胞,所述宿主细胞含有上述核酸分子、含有上述核酸分子的重组表达载体或表达上述融合蛋白。
所述宿主细胞是细菌细胞或真菌细胞的任意一种或多种;
其中所述细菌细胞可以为埃希氏菌属、农杆菌属、芽孢杆菌属、链霉菌属、假单胞菌属或葡萄球菌属内的任何种;
更具体的,所述细菌细胞可以为大肠杆菌(如BL21(DE3))。
所述真菌细胞包括酵母菌。
本发明的又一具体实施方式中,提供上述融合蛋白、核酸分子、重组表达载体和/或宿主细胞在制备检测D-2-羟基戊二酸的荧光传感器中的应用。
本发明的又一具体实施方式中,提供一种检测D-2-羟基戊二酸的荧光传感器,所述荧光传感器包含上述融合蛋白、核酸分子、重组表达载体和/或宿主细胞。
所述荧光传感器还可以包括其他用于D-2-羟基戊二酸检测的试剂、装置和/或设备。
所述试剂包括检测缓冲液(如荧光测定缓冲液:50mM Tris-HCl,pH 7.4)。
所述荧光传感器在实际应用时可包装为试剂盒产品使用。
本发明的又一具体实施方式中,提供上述检测D-2-羟基戊二酸的荧光传感器的构建方法,所述构建方法至少包括融合蛋白的构建,具体步骤如下:
分别合成两个荧光蛋白的目的基因以及特异性转录调控因子DhdR的编码基因dhdR插入质粒中获得重组质粒,将重组质粒转入宿主细胞中表达获得。
本发明的又一具体实施方式中,所述步骤如下:
S1、全基因合成绿色荧光蛋白Clover和红色荧光蛋白mRuby2的目的基因,将其分别顺序插入至质粒A中获得重组质粒I;
S2、以步骤S1中重组质粒I为模板,PCR扩增获得Clover-mRuby2基因片段,将其插入至质粒B中,获得重组质粒II;
S3、PCR扩增来源于反硝化无色杆菌NBRC 15125的特异性转录调控因子DhdR的编码基因dhdR,并插入至重组质粒II中,获得重组质粒III;将重组质粒III转入大肠杆菌中,诱导表达,纯化即得。
其中,所述步骤S1中,所述质粒A可以为pETDuet-1;因此步骤S1获得的重组质粒可以为pETDuet-Clover-mRuby2;
所述步骤S2中,所述质粒B可以为pET28a;因此步骤S2获得的重组质粒可以为pET28a-Clover-mRuby2;
所述步骤S3中,所述大肠杆菌可以为大肠杆菌BL21(DE3);所述诱导表达可以采用IPTG进行,所述纯化可以采用镍柱进行亲和层析进行。
本发明中,将采用上述方法获得融合蛋白(氨基酸序列如SEQ ID NO.1所示,核苷酸序列如SEQ ID NO.3所示)进而构建获得的荧光传感器命名为D-2-羟基戊二酸荧光传感器DHGFR0N0C;
为进一步提高荧光传感器对D-2-羟基戊二酸的响应幅度;本发明对特异性转录调控因子DhdR的N末端和/或C末端氨基酸进行截短处理;优选的,所述DhdR截短变体为N末端截短3个氨基酸、C末端截短2个氨基酸的DhdR截短变体,此时获得的所述重组质粒为pET28a-Clover-dhdR3N2C-mRuby2;更进一步的,在截短变体DhdR3N2C的C末端添加人工短肽,以进一步提高荧光传感器对D-2-羟基戊二酸的响应幅度,所述人工短肽可以为4个甘氨酸串联的短肽,此时获得的所述重组质粒为pET28a-Clover-dhdR3N2C-4G-mRuby2,使用该重组质粒纯化获得的融合蛋白(氨基酸序列如SEQ ID NO.2所示,核苷酸序列如SEQ ID NO.4所示)进而构建获得的荧光传感器命名为D-2-羟基戊二酸荧光传感器DHGFR1.0。
本发明的又一具体实施方式中,提供一种检测D-2-羟基戊二酸的方法,所述方法包括:将待测样品与所述融合蛋白或荧光传感器进行孵育,根据两个荧光蛋白的荧光发射强度比值变化,分析D-2-羟基戊二酸的浓度或有无。
本发明的又一具体实施方式中,所述待测样品可以为任意含有D-2-羟基戊二酸或怀疑含有D-2-羟基戊二酸的生物样品或环境样品,所述生物样品包括但不限于受试者血清、尿液、细胞培养基和细胞裂解物;所述受试者可以为人或非人动物,优选为人。
本发明的又一具体实施方式中,提供上述融合蛋白、荧光传感器和/或上述检测方法在D-2-羟基戊二酸相关疾病检测和/或筛选D-2-羟基戊二酸相关药物中的应用。
其中,所述D-2-羟基戊二酸相关疾病包括但不限于神经系统代谢性疾病(如D-2-羟基戊二酸尿症)以及与异柠檬酸脱氢酶突变相关的癌症(如急性髓性白血病、甲状腺癌、软骨瘤、胆管癌、胶质瘤);
所述D-2-羟基戊二酸相关药物包括但不限于D-2-羟基戊二酸抑制剂或D-2-羟基戊二酸激活剂。
以下结合具体实例对本发明作进一步的说明,以下实例仅是为了解释本发明,并不对其内容进行限定。下述实施例中,使用的反硝化无色杆菌(Achromobacterdenitrificans)NBRC 15125购自北京北纳创联生物技术研究院(菌株序号:NCTC 8582);使用的表达载体pETDuet-1和表达载体pET28a购自Novagen公司;其他所使用的材料、试剂等,如无特殊说明,均从商业途径得到。所使用的实验方法,未做具体说明的,均为常规方法。
实施例1:D-2-羟基戊二酸荧光传感器DHGFR0N0C的构建
本实施例中所使用的培养基和试剂如下:
LB培养基:0.5%酵母粉、1%蛋白胨、1%NaCl;
结合缓冲液:20mM Na2HPO4,20mM咪唑,500mM NaCl,pH 7.4;
洗脱缓冲液:20mM Na2HPO4,500mM咪唑,500mM NaCl,pH 7.4;
荧光测定缓冲液:50mM Tris-HCl,pH 7.4。
(1)D-2-羟基戊二酸荧光传感器DHGFR表达质粒的构建
委托通用生物系统(安徽)有限公司对绿色荧光蛋白Clover和红色荧光蛋白mRuby2的目的基因进行全基因合成,分别插入至pETDuet-1的BamHI/SacI酶切位点和SalI/NotI酶切位点之间,并保存于大肠杆菌Top10菌株中。提取质粒后,以pETDuet-Clover质粒为模板,使用引物Clover正向引物和Clover反向引物对不含终止密码子的Clover基因片段进行扩增,使用限制性内切酶BamHI和SacI对重组质粒pETDuet-mRuby2和Clover基因片段的扩增产物双酶切,经T4 DNA连接酶连接后获得重组质粒pETDuet-Clover-mRuby2。其中,所述扩增Clover基因片段的引物序列为:
Clover正向引物5′-CGCGGATCCGATGGTTAGTAAGGGCGAAGAA-3′(SEQ ID NO.5),下划线表示BamHI酶切位点;
Clover反向引物5′-CGAGCTCTTTATACAGTTCATCCATACCAT-3′(SEQ ID NO.6),下划线表示SacI酶切位点。
以重组质粒pETDuet-Clover-mRuby2的为模板,使用CDM正向引物和CDM反向引物PCR扩增获得Clover-mRuby2基因片段,使用限制性内切酶BamHI和NotI对质粒pET28a双酶切使其线性化,通过T5核酸外切酶组装方法将Clover-mRuby2基因片段插入至质粒pET28a。具体方法为向15μL T5核酸外切酶组装体系中加入总体积为5μL的线性化pET28a质粒和Clover-mRuby2基因片段,其中线性化质粒与基因片段的摩尔比为1:3,于30℃条件下连接40分钟,获得重组质粒pET28a-Clover-mRuby2。其中,所述扩增Clover-mRuby2基因片段的引物序列为:
CDM正向引物
5′-AGCAAATGGGTCGCGGATCCATGGTTAGTAAGGGCGAAG-3′(SEQ ID NO.7);
CDM反向引物
5′-TGCTCGAGTGCGGCCGCTTATTTATACAGTTCATCCAT-3′(SEQ ID NO.8)。
以反硝化无色杆菌NBRC 15125的基因组为模板,使用dhdR正向引物和dhdR反向引物通过PCR扩增DhdR的编码基因dhdR,使用限制性内切酶SacI和SalI对重组质粒pET28a-Clover-mRuby2双酶切使其线性化,通过T5核酸外切酶组装方法将dhdR基因片段插入至质粒上述质粒,获得重组质粒pET28a-Clover-dhdR-mRuby2。其中,所述扩增dhdR基因片段的引物序列为:dhdR正向引物
5′-TGAACTGTATAAAGAGCTCATGCTGAGCAAGAGCCTGAC-3′(SEQ ID NO.9);
dhdR反向引物
5′-CTTTACTCACCATGTCGACTGATGTCTGCCTTGCGGCCGG-3′(SEQ ID NO.10)。
(2)D-2-羟基戊二酸荧光传感器表达条件的优化
将重组质粒pET28a-Clover-dhdR-mRuby2通过化学转化法转入大肠杆菌BL21(DE3)中,加入适量LB培养基在37℃条件下复苏1小时,涂布含有50μg/mL卡那霉素抗性的LB固体培养基,在37℃条件下培养12小时后,挑取单克隆进行培养基菌液PCR验证。
将验证正确的单克隆在LB培养基中活化两代后,以1.5%的接种量接种至含有卡那霉素抗性(50μg/mL)的1升LB培养基中,37℃、180rpm振荡培养至OD600nm为0.6~0.8时,向培养基中加入1mM IPTG,23℃、160rpm诱导12小时;于6000rpm离心10分钟收集菌体,使用结合缓冲液洗涤菌体两次后重悬至OD600nm为30,同时加入1mM PMSF和10%甘油。使用高压破碎仪在1200Pa压力下破碎菌体,破碎四次;将破碎液于4℃、12,000rpm离心50分钟以去除细胞碎片,所得上清液经过0.22μm的滤头过滤后,用体积为5mL的镍柱进行纯化,采用不同浓度的洗脱缓冲液洗脱获得纯化的D-2-羟基戊二酸荧光传感器DHGFR0N0C,其基因序列长度为2157个碱基,核苷酸序列如SEQ ID NO.1所示。使用SDS-PAGE检测DHGFR0N0C的纯度,结果如附图1所示。
(3)荧光发射强度比值测定
使用荧光测定缓冲液稀释纯化的DHGFR0N0C至4/3μM,将DHGFR0N0C和待测样品以3:1的体积比混合于黑色的96微孔板中,总体积设置为100μL,每个样品设置三个复孔平行。孵育20分钟后,使用EnSight多功能微孔板检测仪(美国PerkinElmer公司)检测500nm激发时,515nm(Clover)和600nm(mRuby2)处的荧光强度,将600nm处的荧光强度除以515nm处的荧光强度,获得DHGFR0N0C的荧光发射强度比值。
(4)D-2-羟基戊二酸荧光传感器DHGFR0N0C对D-2-羟基戊二酸的响应
使用荧光测定缓冲液配制梯度浓度的D-2-羟基戊二酸标准品溶液,根据上述(3)中所述的荧光发射强度比值测定方法,将DHGFR0N0C和含有不同浓度的D-2-羟基戊二酸标准品溶液混合孵育后,测定每个孔的荧光发射强度比值。将荧光发射强度比值与D-2-羟基戊二酸的浓度相对应,获得DHGFR0N0C的剂量响应曲线,结果如附图2所示,DHGFR0N0C的荧光发射强度比值以浓度依赖的方式响应于所添加的D-2-羟基戊二酸,D-2-羟基戊二酸浓度越大,荧光发射强度比值越小。D-2-羟基戊二酸荧光传感器DHGFR0N0C的最大荧光比值变化ΔRmax为6.05%,半最大效应浓度EC50为4.34μM。
实施例2:D-2-羟基戊二酸荧光传感器DHGFR1.0的构建
本实施例中所使用的培养基和试剂如下:
LB培养基:0.5%酵母粉、1%蛋白胨、1%NaCl;
结合缓冲液:20mM Na2HPO4,20mM咪唑,500mM NaCl,pH 7.4;
洗脱缓冲液:20mM Na2HPO4,500mM咪唑,500mM NaCl,pH 7.4;
荧光测定缓冲液:50mM Tris-HCl,pH 7.4。
(1)D-2-羟基戊二酸荧光传感器DHGFR1.0表达质粒的构建
对特异性转录调控因子DhdR的N末端和/或C末端氨基酸进行截短,以提高荧光传感器对D-2-羟基戊二酸的响应幅度。PCR扩增DhdR截短变体的基因片段,将其插入至重组质粒pET28a-Clover-mRuby2的酶切位点SacI和SalI中间,构建不同传感器变体的编码质粒。
按照实施例1中的方法将不同传感器变体的编码质粒转入大肠杆菌BL21(DE3)中,对传感器变体进行表达与纯化,并测定传感器变体对D-2-羟基戊二酸的响应。以最大荧光比值变化ΔRmax为指标,对传感器变体进行筛选。当DhdR的N末端截短3个氨基酸、C末端截短2个氨基酸时,使用截短变体DhdR3N2C构建的传感器变体具备最大的荧光比值变化,ΔRmax为18.97%。
进一步在截短变体DhdR3N2C的C末端添加人工短肽,以提高荧光传感器对D-2-羟基戊二酸的响应幅度。人工短肽的氨基酸序列如表1所示:
表1人工短肽的氨基酸序列
PCR扩增DhdR3N2C与人工短肽的基因片段,将其插入至重组质粒pET28a-Clover-mRuby2的酶切位点SacI和SalI中间,构建不同传感器变体的编码质粒。
按照实施例1中的方法将传感器变体的编码质粒转入大肠杆菌BL21(DE3)中,对传感器变体进行表达与纯化,并测定传感器变体对D-2-羟基戊二酸的响应。以最大荧光比值变化ΔRmax为指标,对传感器变体进行筛选。其中,在DhdR3N2C的C末端添加4个甘氨酸串联的人工短肽时,传感器具备最大的荧光比值变化,将该传感器变体命名为DHGFR1.0,基因序列长度为2142个碱基,核苷酸序列如SEQ ID NO.2所示对应的重组质粒为
pET28a-Clover-dhdR3N2C-4G-mRuby2。其中,所述扩增dhdR3N2C-4G-基因片段的引物序列为:
dhdR3N2C-4G-正向引物
5′-TGAACTGTATAAAGAGCTCAAGAGCCTGACCTTGACCGA-3′(SEQ ID NO.11);
dhdR3N2C-4G-反向引物
5′-CTTTACTCACCATGTCGACTCCGCCTCCGCCCTGCCTTGCGGCCGGAGAGA-3′(SEQ IDNO.12)。
结果如附图3所示,DHGFR1.0的荧光发射强度比值以浓度依赖的方式响应于所添加的D-2-羟基戊二酸,D-2-羟基戊二酸浓度越大,荧光发射强度比值越小,最大荧光比值变化ΔRmax为25.28%,半最大效应浓度EC50为2.40μM。
实施例3:D-2-羟基戊二酸荧光传感器DHGFR1.0的pH稳定性、光谱学性质和特异性
本实施例中所使用的培养基和试剂如下:
荧光测定缓冲液:50mM Tris-HCl,pH 4.0-10.0。
(1)D-2-羟基戊二酸荧光传感器DHGFR1.0的pH稳定性
配制pH为4.0、5.0、6.0、7.0、7.5、8.0、9.0和10.0的50mM Tris-HCl缓冲液,分别稀释纯化的DHGFR1.0至4/3μM,稀释D-2-羟基戊二酸至0μM、4μM、40μM和400μM,按照实施例1中的方法测定D-2-羟基戊二酸荧光传感器DHGFR1.0在不同pH的50mM Tris-HCl缓冲液中对D-2-羟基戊二酸的荧光强度发射比值,结果如附图4A所示,在缓冲液pH 4.0-7.0的范围内,DHGFR1.0对D-2-羟基戊二酸的响应幅度较小。随着缓冲液pH的逐渐升高(pH 7.0-10.0),DHGFR1.0对D-2-羟基戊二酸的响应幅度逐渐增大,在pH 10.0的Tris-HCl缓冲液中,DHGFR1.0对D-2-羟基戊二酸的响应幅度最大,结果如附图4B所示,最大荧光比值变化ΔRmax为30.37%,半最大效应浓度为3.62μM。使用50mM Tris-HCl缓冲液(pH 10.0)稀释纯化的DHGFR1.0至4/3μM,使用不同pH的缓冲液稀释D-2-羟基戊二酸至0μM、4μM、40μM和400μM,测定DHGFR1.0对不同pH D-2-羟基戊二酸的荧光强度发射比值。结果如附图4C所示,在pH10.0的Tris-HCl缓冲液中,DHGFR1.0检测0μM、1μM、10μM或100μM D-2-羟基戊二酸时荧光比值基本不变,表明DHGFR1.0对D-2-羟基戊二酸的检测不受样品pH的干扰。
(2)D-2-羟基戊二酸荧光传感器DHGFR1.0的光谱学性质
使用50mM Tris-HCl缓冲液(pH 10.0)稀释纯化的DHGFR1.0至4/3μM,稀释D-2-羟基戊二酸至4mM,将稀释后DHGFR1.0分别与0mM和4mM D-2-羟基戊二酸孵育处理,在480nm的激发光条件下,以2nm的步进,连续采集500-700nm的荧光发射值。结果如附图5所示,D-2-羟基戊二酸的添加导致DHGFR1.0在515nm(Clover)处的荧光发射峰升高,而600nm处(mRuby2)的荧光发射峰降低,最终造成DHGFR1.0中mRuby2与Clover的荧光发射强度比值降低。
(3)D-2-羟基戊二酸荧光传感器DHGFR1.0的特异性
使用50mM Tris-HCl缓冲液(pH 10.0)稀释纯化的DHGFR1.0至4/3μM,稀释各化合物至400μM,按照实施例1中的方法检测DHGFR1.0对不同化合物的荧光发射比。结果如附图6所示,表明D-2-羟基戊二酸荧光传感器DHGFR1.0具备很好的特异性。
实施例4:D-2-羟基戊二酸荧光传感器DHGFR1.0在检测含有D-2-羟基戊二酸的人血清和尿液样品中的应用
本实施例中所使用的培养基和试剂如下:
荧光测定缓冲液:50mM Tris-HCl,pH 10.0。
本实施例中,含有D-2-羟基戊二酸的人血清和尿液样品的制备方法为:
静脉采血法收集来自健康志愿者的外周血至含有促凝剂的真空采血管中,4℃条件下静置3h,随后在室温条件下于2,500rpm离心20min,收集分离胶上层的淡黄色液体(血清),配制含有梯度浓度D-2-羟基戊二酸的血清样品,浓度范围为40nM~4mM;收集健康志愿者的尿液样品后,使用孔径为0.22μm对尿液进行过滤,配制含有梯度浓度D-2-羟基戊二酸的尿液样品,浓度范围为40nM~4mM;由于检测体系中,传感器与待测样品的体积比为3:1,即检测时待测样品会被稀释4倍,因此剂量响应曲线中所呈现的D-2-羟基戊二酸的终浓度范围为10nM~1mM。
按照实施1中的方法测定D-2-羟基戊二酸荧光传感器DHGFR1.0对人血清和尿液样品中D-2-羟基戊二酸的剂量响应曲线。结果如附图7-8所示,D-2-羟基戊二酸荧光传感器DHGFR1.0可以响应于不同类型生物样品中的D-2-羟基戊二酸,荧光发射强度比值随D-2-羟基戊二酸浓度的增加而减小。
实施例5:D-2-羟基戊二酸荧光传感器DHGFR1.0在检测含有D-2-羟基戊二酸的细胞样品中的应用
本实施例中所使用的培养基和试剂如下:
荧光测定缓冲液:50mM Tris-HCl,pH 10.0。
本实施例中涉及的HEK293FT细胞和HT1080细胞购买于武汉普诺赛生命科技有限公司,SW1353细胞购买于武汉普诺赛生命科技有限公司,其中,HT1080细胞含有IDH1/R132C突变位点,SW1353细胞含有IDH2/R172S突变位点;GSK864抑制剂和AGI-6780抑制剂购买于美国Sigma-Aldrich公司,其中GSK864抑制剂靶向IDH1突变,AGI-6780抑制剂靶向IDH2/R140Q;细胞培养级PBS购买于美国ThermoFisher Scientific公司,细胞裂解液和BCA蛋白浓度测定试剂盒(增强型)购买于上海碧云天生物技术有限公司。
本实施例中细胞培养基样品和细胞裂解物样品的制备方法为:
对HEK293FT细胞、HT1080细胞和SW1353细胞进行培养,培养过程中分别加入50μMGSK864抑制剂和AGI-6780抑制剂进行处理,以添加DMSO作为对照组。吸取1mL细胞培养基于13,000×g离心5分钟,离心后取上清液得到细胞培养基样品,保存于-80℃冰箱备用;
使用PBS缓冲液对上述已吸取干净培养基上清的细胞培养物洗涤三次,随后加入250μL细胞裂解液处理,裂解5min后,在13,000×g条件下离心5min,离心后吸取上清液得到细胞裂解物样品,使用BCA蛋白浓度测定试剂盒测定细胞裂解物中的蛋白浓度,并将样品保存于-80℃冰箱备用。
所述细胞培养基和细胞裂解物中D-2-羟基戊二酸的定量方法是:
按照实施例1中的方法测定D-2-羟基戊二酸荧光传感器DHGFR1.0对细胞培养基样品和细胞裂解物样品的荧光发射强度比值,同时测定D-2-羟基戊二酸荧光传感器DHGFR1.0在荧光测定缓冲液中对D-2-羟基戊二酸的剂量响应曲线。将细胞培养基样品和细胞裂解物样品的荧光发射强度比值代入所述D-2-羟基戊二酸荧光传感器DHGFR1.0对荧光测定缓冲液中对D-2-羟基戊二酸的剂量响应曲线中,获得荧光发射强度比值所对应的具体D-2-羟基戊二酸浓度,进一步将该结果乘以4及所稀释的倍数,获得对所述细胞培养基样品和细胞裂解物样品中D-2-羟基戊二酸的定量结果,定量结果见附图9。
以上所述仅为本申请的优选实施例而已,并不用于限制本申请,对于本领域的技术人员来说,本申请可以有各种更改和变化。凡在本申请的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
SEQUENCE LISTING
<110> 山东大学
<120> 一种检测 D-2-羟基戊二酸的荧光传感器及其构建方法和应用
<130> 2022804939
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His Val Ala Val Tyr Asp Ala Ile Leu Ala Gly Asp Pro Asp Arg Ala
435 440 445
Arg Leu Ala Ala Thr Arg His Leu Gln Gln Ala Ala Ser Arg Leu Arg
450 455 460
Leu Asp Leu Leu Ser Pro Ala Ala Arg Gln Gly Gly Gly Gly Val Asp
465 470 475 480
Met Val Ser Lys Gly Glu Glu Leu Ile Lys Glu Asn Met Arg Met Lys
485 490 495
Val Val Met Glu Gly Ser Val Asn Gly His Gln Phe Lys Cys Thr Gly
500 505 510
Glu Gly Glu Gly Asn Pro Tyr Met Gly Thr Gln Thr Met Arg Ile Lys
515 520 525
Val Ile Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr
530 535 540
Ser Phe Met Tyr Gly Ser Arg Thr Phe Ile Lys Tyr Pro Lys Gly Ile
545 550 555 560
Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg
565 570 575
Val Thr Arg Tyr Glu Asp Gly Gly Val Val Thr Val Met Gln Asp Thr
580 585 590
Ser Leu Glu Asp Gly Cys Leu Val Tyr His Val Gln Val Arg Gly Val
595 600 605
Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Lys Gly Trp
610 615 620
Glu Pro Asn Thr Glu Met Met Tyr Pro Ala Asp Gly Gly Leu Arg Gly
625 630 635 640
Tyr Thr His Met Ala Leu Lys Val Asp Gly Gly Gly His Leu Ser Cys
645 650 655
Ser Phe Val Thr Thr Tyr Arg Ser Lys Lys Thr Val Gly Asn Ile Lys
660 665 670
Met Pro Gly Ile His Ala Val Asp His Arg Leu Glu Arg Leu Glu Glu
675 680 685
Ser Asp Asn Glu Met Phe Val Val Gln Arg Glu His Ala Val Ala Lys
690 695 700
Phe Ala Gly Leu Gly Gly Gly Met Asp Glu Leu Tyr Lys
705 710 715
<210> 3
<211> 2157
<212> DNA
<213> 人工序列
<400> 3
atggttagta agggcgaaga actgtttacc ggcgtggttc cgattctggt tgaactggat 60
ggtgacgtta atggccataa attttctgtg cgtggtgaag gcgaaggtga cgccaccaat 120
ggtaaactga ccctgaaatt catttgtacc accggcaaac tgccggtgcc gtggccgacc 180
ctggtgacaa cctttggtta tggtgtggcc tgctttagtc gttatccgga tcatatgaaa 240
cagcatgatt tctttaagag tgcaatgccg gaaggctatg ttcaggaacg taccattagt 300
tttaaagatg atggcaccta taagacccgc gcagaagtga aatttgaagg tgacaccctg 360
gttaatcgca ttgaactgaa aggtattgat tttaaagagg atggtaacat tctgggtcat 420
aaactggaat ataatttcaa tagccacaac gtttacatca ccgccgataa acagaaaaat 480
ggtattaagg ccaatttcaa aatccgtcat aatgttgaag acggtagtgt tcagctggcc 540
gatcattatc agcagaatac cccgattggt gacggtccgg tgctgctgcc ggataatcat 600
tatctgagcc atcagagcgc actgagcaaa gatccgaatg aaaaacgtga tcatatggtt 660
ctgctggaat ttgtgaccgc agccggtatt acccatggta tggatgaact gtataaagag 720
ctcatgctga gcaagagcct gaccttgacc gaacaggtcg cccgccagat cgcgggcgac 780
atcgccgaag gcgtccattc cgtgggcgcc aagctgccgc ccggccgtgt cctggcggag 840
cagtacggtg tgagcgccgc ggtcatccgc gaggccaccg agcgcctgcg cgcccagggg 900
ctgatccaga gccgccaggg ctcgggcagc gtggtggtgt cccgcaccgg tgctcagggc 960
ttccaggttt ccgccggcct cgacgatcgc gagcagctgg ccagcgtcta cgaattgcgg 1020
atggaactgg aaggcggcgc ggccgccctg gcggcgaggc gccgcaacgc caccgacctt 1080
gcggccatgg ccgaggccct ggccgcgctg gaagcgaacc tggaccatcc ggaacagggc 1140
gtcgagcacg acatcgcctt ccacgtcgcc atcgccgccg ccacgcacaa ccgttattac 1200
caggacctgc tgcagtacct gaacctgcag ctgcgcctgg ccgtcagcac cgcgcgcacc 1260
aacagccgcc gtcaggaggg cctgaccgcg gtggtgcacc aggaacacgt ggccgtctac 1320
gacgccatcc tcgcgggcga tcccgaccgc gcccgactgg cggcgacccg ccacttgcag 1380
caggcggcca gccgcctgcg tctcgatctc ctctctccgg ccgcaaggca gacatcagtc 1440
gacatggtga gtaaaggtga agaactgatt aaggaaaata tgcgcatgaa agttgttatg 1500
gaaggtagcg tgaatggtca tcagtttaaa tgtaccggcg aaggtgaagg caatccgtat 1560
atgggcaccc agaccatgcg tattaaggtt attgaaggcg gtccgctgcc gtttgccttt 1620
gatattctgg ccaccagctt tatgtatggt agtcgtacct ttattaagta tccgaaaggt 1680
attccggatt tctttaaaca gagttttccg gaaggtttta cctgggaacg cgttacccgc 1740
tatgaagatg gtggtgttgt taccgtgatg caggatacca gcctggaaga tggttgtctg 1800
gtgtatcatg tgcaggtgcg cggcgtgaat tttccgagta atggcccggt tatgcagaaa 1860
aagactaaag gttgggaacc gaataccgaa atgatgtatc cggccgatgg tggcctgcgt 1920
ggttataccc atatggcact gaaagttgat ggtggtggtc atctgagctg cagctttgtg 1980
accacctatc gtagtaaaaa gactgttggt aatatcaaaa tgccgggtat tcatgccgtt 2040
gatcatcgtc tggaacgcct ggaagaaagt gataatgaaa tgtttgtggt gcagcgcgaa 2100
catgccgttg caaaatttgc cggcctgggt ggcggtatgg atgaactgta taaataa 2157
<210> 4
<211> 2142
<212> DNA
<213> 人工序列
<400> 4
atggttagta agggcgaaga actgtttacc ggcgtggttc cgattctggt tgaactggat 60
ggtgacgtta atggccataa attttctgtg cgtggtgaag gcgaaggtga cgccaccaat 120
ggtaaactga ccctgaaatt catttgtacc accggcaaac tgccggtgcc gtggccgacc 180
ctggtgacaa cctttggtta tggtgtggcc tgctttagtc gttatccgga tcatatgaaa 240
cagcatgatt tctttaagag tgcaatgccg gaaggctatg ttcaggaacg taccattagt 300
tttaaagatg atggcaccta taagacccgc gcagaagtga aatttgaagg tgacaccctg 360
gttaatcgca ttgaactgaa aggtattgat tttaaagagg atggtaacat tctgggtcat 420
aaactggaat ataatttcaa tagccacaac gtttacatca ccgccgataa acagaaaaat 480
ggtattaagg ccaatttcaa aatccgtcat aatgttgaag acggtagtgt tcagctggcc 540
gatcattatc agcagaatac cccgattggt gacggtccgg tgctgctgcc ggataatcat 600
tatctgagcc atcagagcgc actgagcaaa gatccgaatg aaaaacgtga tcatatggtt 660
ctgctggaat ttgtgaccgc agccggtatt acccatggta tggatgaact gtataaagag 720
ctcaagagcc tgaccttgac cgaacaggtc gcccgccaga tcgcgggcga catcgccgaa 780
ggcgtccatt ccgtgggcgc caagctgccg cccggccgtg tcctggcgga gcagtacggt 840
gtgagcgccg cggtcatccg cgaggccacc gagcgcctgc gcgcccaggg gctgatccag 900
agccgccagg gctcgggcag cgtggtggtg tcccgcaccg gtgctcaggg cttccaggtt 960
tccgccggcc tcgacgatcg cgagcagctg gccagcgtct acgaattgcg gatggaactg 1020
gaaggcggcg cggccgccct ggcggcgagg cgccgcaacg ccaccgacct tgcggccatg 1080
gccgaggccc tggccgcgct ggaagcgaac ctggaccatc cggaacaggg cgtcgagcac 1140
gacatcgcct tccacgtcgc catcgccgcc gccacgcaca accgttatta ccaggacctg 1200
ctgcagtacc tgaacctgca gctgcgcctg gccgtcagca ccgcgcgcac caacagccgc 1260
cgtcaggagg gcctgaccgc ggtggtgcac caggaacacg tggccgtcta cgacgccatc 1320
ctcgcgggcg atcccgaccg cgcccgactg gcggcgaccc gccacttgca gcaggcggcc 1380
agccgcctgc gtctcgatct cctctctccg gccgcaaggc aggtcgacat ggtgagtaaa 1440
ggtgaagaac tgattaagga aaatatgcgc atgaaagttg ttatggaagg tagcgtgaat 1500
ggtcatcagt ttaaatgtac cggcgaaggt gaaggcaatc cgtatatggg cacccagacc 1560
atgcgtatta aggttattga aggcggtccg ctgccgtttg cctttgatat tctggccacc 1620
agctttatgt atggtagtcg tacctttatt aagtatccga aaggtattcc ggatttcttt 1680
aaacagagtt ttccggaagg ttttacctgg gaacgcgtta cccgctatga agatggtggt 1740
gttgttaccg tgatgcagga taccagcctg gaagatggtt gtctggtgta tcatgtgcag 1800
gtgcgcggcg tgaattttcc gagtaatggc ccggttatgc agaaaaagac taaaggttgg 1860
gaaccgaata ccgaaatgat gtatccggcc gatggtggcc tgcgtggtta tacccatatg 1920
gcactgaaag ttgatggtgg tggtcatctg agctgcagct ttgtgaccac ctatcgtagt 1980
aaaaagactg ttggtaatat caaaatgccg ggtattcatg ccgttgatca tcgtctggaa 2040
cgcctggaag aaagtgataa tgaaatgttt gtggtgcagc gcgaacatgc cgttgcaaaa 2100
tttgccggcc tgggtggcgg tatggatgaa ctgtataaat aa 2142
<210> 5
<211> 31
<212> DNA
<213> 人工序列
<400> 5
cgcggatccg atggttagta agggcgaaga a 31
<210> 6
<211> 30
<212> DNA
<213> 人工序列
<400> 6
cgagctcttt atacagttca tccataccat 30
<210> 7
<211> 39
<212> DNA
<213> 人工序列
<400> 7
agcaaatggg tcgcggatcc atggttagta agggcgaag 39
<210> 8
<211> 38
<212> DNA
<213> 人工序列
<400> 8
tgctcgagtg cggccgctta tttatacagt tcatccat 38
<210> 9
<211> 39
<212> DNA
<213> 人工序列
<400> 9
tgaactgtat aaagagctca tgctgagcaa gagcctgac 39
<210> 10
<211> 40
<212> DNA
<213> 人工序列
<400> 10
ctttactcac catgtcgact gatgtctgcc ttgcggccgg 40
<210> 11
<211> 39
<212> DNA
<213> 人工序列
<400> 11
tgaactgtat aaagagctca agagcctgac cttgaccga 39
<210> 12
<211> 51
<212> DNA
<213> 人工序列
<400> 12
ctttactcac catgtcgact ccgcctccgc cctgccttgc ggccggagag a 51
Claims (5)
1.一种检测D-2-羟基戊二酸的荧光传感器,其特征在于,所述荧光传感器包括融合蛋白;所述融合蛋白的氨基酸序列如SEQ ID NO.2所示。
2.如权利要求1所述的荧光传感器,其特征在于,所述荧光传感器还包括其他用于D-2-羟基戊二酸检测的试剂、装置和/或设备。
3.权利要求1或2所述检测D-2-羟基戊二酸的荧光传感器的构建方法,其特征在于,所述构建方法包括融合蛋白的构建,具体步骤如下:
分别合成两个荧光蛋白的目的基因以及特异性转录调控因子DhdR的编码基因dhdR插入质粒中获得重组质粒,将重组质粒转入宿主细胞中表达获得;
所述编码基因dhdR编码DhdR变体;所述DhdR变体为:在N末端截短3个氨基酸、C末端截短2个氨基酸的DhdR截短变体的C末端添加4个甘氨酸串联的短肽。
4.如权利要求3所述的构建方法,其特征在于,所述步骤如下:
S1、全基因合成绿色荧光蛋白Clover和红色荧光蛋白mRuby2的目的基因,将其分别顺序插入至质粒A中获得重组质粒I;
S2、以步骤S1中重组质粒I为模板,PCR扩增获得Clover-mRuby2基因片段,将其插入至质粒B中,获得重组质粒II;
S3、PCR扩增来源于反硝化无色杆菌NBRC 15125的特异性转录调控因子DhdR的编码基因dhdR,并插入至重组质粒II中,获得重组质粒III;将重组质粒III转入大肠杆菌中,诱导表达,纯化即得;所述编码基因dhdR编码DhdR变体;所述DhdR变体为:在N末端截短3个氨基酸、C末端截短2个氨基酸的DhdR截短变体的C末端添加4个甘氨酸串联的短肽。
5.如权利要求4所述的构建方法,其特征在于,所述步骤S1中,所述质粒A为pETDuet-1;
所述步骤S2中,所述质粒B为pET28a;
所述步骤S3中,所述大肠杆菌为大肠杆菌BL21(DE3);所述诱导表达采用IPTG进行,所述纯化使用镍柱进行亲和层析。
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