CN114057891B - 柠檬酸光学探针及其制备方法和应用 - Google Patents
柠檬酸光学探针及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及柠檬酸光学探针及其制备方法和应用。一方面,本发明涉及光学探针,包含柠檬酸敏感多肽B或C或其功能变体和光学活性多肽A或其功能变体,其中光学活性多肽A或其功能变体位于柠檬酸敏感多肽B或C或其功能变体的串联体之间。本发明也涉及上述探针的制备方法及其在检测柠檬酸中的应用。
Description
技术领域
本发明涉及光学探针技术领域,尤其涉及柠檬酸光学探针及其制备方法和应用。
背景技术
柠檬酸是三羧酸(TCA)循环中的重要代谢物。在线粒体中,乙酰辅酶A和草酰乙酸生成柠檬酸的反应是三羧酸循环中十分重要的一环,可以说是整个TCA循环启动的第一步。而柠檬酸随后形成异柠檬酸使TCA循环继续进行,维持了细胞的正常生理功能。
除了参与三羧酸循环外,柠檬酸在哺乳动物细胞能量代谢方面有着关键作用。细胞内的柠檬酸盐可以通过上调或下调代谢途径上的酶,来影响糖酵解,TCA循环,糖异生和脂肪酸合成,可以改变能量的走向。例如,柠檬酸可通过抑制磷酸果糖激酶1(PFK1)和6-磷酸果糖激酶-2/果糖-2,6-二磷酸酶(PFK2)对糖酵解产生负反馈。柠檬酸通过降低果糖-1,6-二磷酸果糖(F1,6P)的水平,也间接抑制了丙酮酸激酶。柠檬酸可以通过丙酮酸脱氢酶和琥珀酸脱氢酶抑制TCA循环。除了会抑制TCA循环,柠檬酸还会刺激消耗ATP的途径(如糖异生和脂质合成),进一步减少ATP的水平。此外,它通过刺激乙酰辅酶A羧化酶,增加丙二酰辅酶A的形成,一方面,丙二酰辅酶A会诱导脂肪酸的生物合成,另一方面,丙二酰辅酶A通过肉碱棕榈酰转移酶-Ⅰ来抑制脂肪酸的线粒体运输,继而抑制β-氧化。
有研究表明柠檬酸和癌症代谢、免疫细胞的活化和组蛋白乙酰化息息相关。胞外柠檬酸盐通过质膜-柠檬酸转运蛋白提供给癌细胞,如果使质膜-柠檬酸转运蛋白失活可以让癌细胞失去细胞外柠檬酸供给,肿瘤生长会减慢。但当癌细胞内柠檬酸盐在很高的水平时,柠檬酸盐会抑制PFK2,减慢了癌细胞的生长,并干扰其他细胞功能。柠檬酸和免疫细胞的活化和乙酰化有关:在巨噬细胞和树突状神经细胞的代谢中,三羧酸循环发生了变化,其结果表现为柠檬酸盐和琥珀酸盐的积累。在异柠檬酸脱氢酶催化环节发生断裂,柠檬酸在TCA循环中得到积累。大量产生的柠檬酸通过线粒体柠檬酸盐载体被输出到胞质溶胶中,随后被代谢为乙酰辅酶A。而乙酰化过程受乙酰辅酶A浓度的动态调节,柠檬酸代谢得到的乙酰辅酶A可以用于组蛋白和非组蛋白的乙酰化。在对柠檬酸转运蛋白进行突变后会削弱柠檬酸从线粒体的运输,从而导致组蛋白乙酰化的显著降低,验证了这一观点。同时,柠檬酸代谢产生的乙酰辅酶A也会上调脂质的生物合成,这对于促炎介质和消炎介质的产生都非常重要。线粒体中柠檬酸盐的代谢还与巨噬细胞中几种重要的促炎介质因子的产生有关。此外,柠檬酸盐衍生的衣康酸酯具有直接的抗菌作用,并且还显示出其作为抗炎剂的作用。
柠檬酸目前的检测方法有很多种:比如如核磁共振法、高效液相色谱法、近红外光谱法、离子色谱法、分光光度法等。这些检测分析方法或需要专业分析仪器设备,对样品的完整性造成破坏,而且大多只能应用于食品或者发酵液中的柠檬酸的检测,很难用于细胞或者体内检测,无法实时监控活细胞的柠檬酸浓度变化。因此,亟待开发新的检测方法,实现在细胞内、外,简便、快捷、特异性高的实时、定位、定量、高通量检测柠檬酸。
发明内容
本发明的目的在于提供在细胞内、外实时定位、高通量、定量检测柠檬酸的探针和方法。
为了实现上述发明目的,本发明提供以下技术方案:
本发明第一方面提供了一种融合蛋白,包括柠檬酸敏感多肽B和光学活性多肽A,其中光学活性多肽A位于两个或多个柠檬酸敏感多肽B或者C之间,形成B1-L1-A-L2-B2式的融合蛋白结构,B1和B2分别独立选自CitA或与其具有至少90%序列相同性并且保留柠檬酸敏感功能的变体和CcpE或与其具有至少90%序列相同性并且保留柠檬酸敏感功能的变体,L1和L2是接头。
在一个或多个实施方案中,CitA具有SEQ ID NO:1所示的序列,CcpE具有SEQ IDNO:2所示的序列。
在一个或多个实施方案中,光学活性多肽A选自黄色荧光蛋白、绿色荧光蛋白、蓝色荧光蛋白、红色荧光蛋白。在一个实施方式中,光学活性多肽A选自以下的任意一种或多种:具有SEQ ID NO:3的cpYFP、具有SEQ ID NO:4的cpGFP、具有SEQ ID NO:5的cpBFP、具有SEQ ID NO:6的cpmApple,和与其任一具有至少90%序列相同性并且保留荧光显色功能的变体。在一个或多个实施方案中,光学活性多肽A在N端或C端具有截短或突变。优选地,A的N端或C端具有1-5个氨基酸的截短,例如1-4个、1-3个。
在一个或多个实施方案中,L1和L2分别独立选自无、P、H、N、S、Q、A、D、Y、M、T、E、C、L、V、W、I、F、K、R、G、GS、GGS、GGGS、GSGGS、GGSGGS、GGGSGGS、RSE、GGS、PAP、PDA、RVR、RED、PER、RNA、PDP、RGA、RPP、TWD、TLE。
在一个或多个实施方案中,B1和B2相同。
在第一方面的一个或多个实施方案中,B1和B2是CitA,A选自cpYFP、cpGFP、cpBFP、cpmApple,L1和L2选自无、G、GS、GGS、RSE、PAP、PDA、RVR、RED、PER、RNA、PDP、RGA、RPP、TWD、TLE。优选地,A是cpYFP,L1选自无、G、GS、GGS、TWD、TLE,L2选自无、G、GS、GGS、RSE、GGS、PAP、PDA、RVR、RED、PER、RNA、PDP、RGA、RPP。
在一个或多个实施方案中,B1和B2是CitA,A是cpYFP,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、无/RSE、无/PAP、无/PDA、无/RVR、无/RED、无/PER、无/RNA、无/PDP、无/RGA、无/RPP、G/GS、G/GGS、GS/GS、GS/GGS、GGS/GS、GGS/GGS、GS/无、GGS/无。
优选地,A是cpYFP,L1和L2的组合选自:GS/无、GGS/无。
优选地,A是cpYFP,L1和L2的组合选自:无/G、无/GS、无/GGS、无/RSE、无/GGS、无/PAP、无/PDA、无/RVR、无/RED、无/PER、无/RNA、无/PDP、无/RGA、无/RPP、G/GS、G/GGS、GS/GS、GS/GGS、GGS/GS、GGS/GGS;
更优选地,A是cpYFP,L1和L2的组合选自:无/G、无/GS、无/GGS、无/RSE、无/GGS、无/PAP、无/PDA、无/RVR、无/RED、无/PER、无/RNA、无/PDP、无/RGA、无/RPP、G/GGS、TWD/RPP、TLE/RPP。
在一个或多个实施方案中,B1和B2是CitA,A是cpGFP,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、G/无、G/G、G/GS、G/GGS、GS/无、GS/G、GS/GS、GS/GGS、GGS/无、GGS/G、GGS/GS、GGS/GGS。
优选地,A是cpGFP,L1和L2的组合选自:无/G、无/GS、无/GGS、G/GS、GGS/无、GGS/GS。
更优选地,A是cpGFP,L1和L2的组合选自:无/GGS。
在一个或多个实施方案中,B1和B2是CitA,A是cpBFP,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、G/无、G/G、G/GS、G/GGS、GS/无、GS/G、GS/GS、GS/GGS、GGS/无、GGS/G、GGS/GS、GGS/GGS。
优选地,A是cpBFP,L1和L2的组合选自:GS/G、GS/GS、GGS/无、GGS/G。
更优选地,A是cpBFP,L1和L2的组合选自:GS/GS。
在一个或多个实施方案中,B1和B2是CitA,A是cpmApple,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、G/无、G/G、G/GS、G/GGS、GS/无、GS/G、GS/GS、GS/GGS、GGS/无、GGS/G、GGS/GS、GGS/GGS。
优选地,A是cpmApple,L1和L2的组合选自:无/GS、G/无、G/GS、G/GGS、GGS/G。
更优选地,A是cpmApple,L1和L2的组合选自:G/GS、G/GGS。
在第一方面的一个或多个实施方案中,B1和B2是CcpE,A选自cpYFP、cpGFP、cpBFP、cpmApple,L1和L2选自无、G、P、H、N、S、Q、A、D、Y、M、T、E、C、L、V、W、I、F、K、R、GS、GGS、GGGS、GSGGS、GGSGGS、GGGSGGS。优选地,A是cpYFP,L1选自无、G、P、H、N、S、Q、A、D、Y、M、T、E、C、L、V、W、I、F、K、R、GS、GGS、GGGS、GSGGS、GGSGGS、GGGSGGS,L2选自无、G、GS、GGS、GGGS、GSGGS、GGSGGS、GGGSGGS。
在一个或多个实施方案中,B1和B2是CcpE,A是cpYFP,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、无/GGGS、无/GSGGS、无/GGSGGS、无/GGGSGGS、G/无、G/G、G/GS、G/GGS、G/GGGS、G/GSGGS、G/GGSGGS、G/GGGSGGS、GS/G、GS/GS、GS/GGS、GS/GGGS、GS/GSGGS、GS/GGSGGS、GS/GGGSGGS、GGS/无、GGS/G、GGS/GS、GGS/GGS、GGS/GGGS、GGS/GSGGS、GGS/GGSGGS、GGS/GGGSGGS、GGGS/无、GGGS/G、GGGS/GS、GGGS/GGS、GGGS/GGGS、GGGS/GSGGS、GGGS/GGSGGS、GGGS/GGGSGGS、GSGGS/无、GSGGS/G、GSGGS/GS、GSGGS/GGS、GSGGS/GGGS、GSGGS/GSGGS、GSGGS/GGSGGS、GSGGS/GGGSGGS、GGSGGS/G、GGSGGS/GS、GGSGGS/GGS、GGSGGS/GGGS、GGSGGS/GSGGS、GGSGGS/GGSGGS、GGSGGS/GGGSGGS、GGGSGGS/无、GGGSGGS/G、GGGSGGS/GS、GGGSGGS/GGS、GGGSGGS/GGGS、GGGSGGS/GSGGS、GGGSGGS/GGSGGS、GGGSGGS/GGGSGGS、P/GGSGGS、H/GGSGGS、N/GGSGGS、S/GGSGGS、Q/GGSGGS、A/GGSGGS、D/GGSGGS、Y/GGSGGS、M/GGSGGS、T/GGSGGS、E/GGSGGS、C/GGSGGS、L/GGSGGS、V/GGSGGS、W/GGSGGS、I/GGSGGS、F/GGSGGS、K/GGSGGS、R/GGSGGS。
优选地,A是cpYFP,L1和L2的组合选自:无/GGGS、无/GSGGS、无/GGSGGS、无/GGGSGGS、G/GGGS、G/GSGGS、G/GGSGGS、G/GGGSGGS、GS/GGGS、GS/GSGGS、GS/GGSGGS、GS/GGGSGGS、GGS/GGGS、GGS/GSGGS、GGS/GGSGGS、GGS/GGGSGGS、GGGS/GGSGGS、GGGS/GGGSGGS、GSGGS/GSGGS、GSGGS/GGSGGS、GSGGS/GGGSGGS、GGSGGS/GSGGS、GGSGGS/GGSGGS、GGSGGS/GGGSGGS、P/GGSGGS、H/GGSGGS、N/GGSGGS、S/GGSGGS、Q/GGSGGS、A/GGSGGS、D/GGSGGS、Y/GGSGGS、M/GGSGGS、T/GGSGGS、E/GGSGGS、C/GGSGGS、L/GGSGGS、K/GGSGGS、V/GGSGGS、W/GGSGGS、I/GGSGGS、R/GGSGGS。
优选地,A是cpYFP,L1和L2的组合选自:无/GGGSGGS、G/GSGGS、G/GGSGGS、G/GGGSGGS、GS/GSGGS、GS/GGSGGS、GS/GGGSGGS或GGS/GGGSGGS、P/GGSGGS、H/GGSGGS、N/GGSGGS、S/GGSGGS、Q/GGSGGS、A/GGSGGS、D/GGSGGS、Y/GGSGGS、M/GGSGGS、T/GGSGGS、E/GGSGGS、C/GGSGGS。
在一个或多个实施方案中,B1和B2是CcpE,A是cpGFP,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、无/GGGS、无/GSGGS、无/GGSGGS、无/GGGSGGS、G/无、G/G、G/GS、G/GGGS、G/GSGGS、G/GGSGGS、G/GGGSGGS、GS/无、GS/G、GS/GS、GS/GGS、GS/GGGS、GS/GSGGS、GS/GGSGGS、GS/GGGSGGS、GGS/无、GGS/G、GGS/GS、GGS/GGS、GGS/GGGS、GGS/GSGGS、GGS/GGSGGS、GGS/GGGSGGS、GGGS/G、GGGS/GS、GGGS/GGS、GGGS/GGGS、GGGS/GSGGS、GGGS/GGSGGS、GGGS/GGGSGGS、GSGGS/GS、GSGGS/GGS、GSGGS/GGGS、GSGGS/GSGGS、GSGGS/GGSGGS、GSGGS/GGGSGGS、GGSGGS/无、GGSGGS/G、GGSGGS/GS、GGSGGS/GGS、GGSGGS/GGGS、GGSGGS/GGSGGS、GGSGGS/GGGSGGS、、GGGSGGS/G、GGGSGGS/GS、GGGSGGS/GGS、GGGSGGS/GGGS、GGGSGGS/GSGGS、GGGSGGS/GGSGGS、GGGSGGS/GGGSGGS。
优选地,A是cpGFP,L1和L2的组合选自:无/GSGGS、无/GGSGGS、无/GGGSGGS、G/GSGGS、G/GGSGGS、G/GGGSGGS、GS/GGSGGS、GS/GGGSGGS、GGS/G、GGS/GS、GGS/GGGS、GGS/GSGGS、GGS/GGSGGS、GGS/GGGSGGS、GGGS/GSGGS、GSGGS/GSGGS、GGGSGGS/GS、GGGSGGS/GGS、GGGSGGS/GGGS、GGGSGGS/GSGGS。
更优选地,A是cpGFP,L1和L2的组合选自:无/GGGSGGS、G/GSGGS、G/GGSGGS、G/GGGSGGS。
在一个或多个实施方案中,B1和B2是CcpE,A是cpBFP,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、无/GGGS、无/GSGGS、无/GGSGGS、G/无、G/G、G/GS、G/GGS、G/GGGS、G/GSGGS、G/GGGSGGS、GS/无、GS/G、GS/GS、GS/GGS、GS/GGGS、GS/GSGGS、GS/GGSGGS、GS/GGGSGGS、GGS/无、GGS/G、GGS/GS、GGS/GGS、GGS/GGGS、GGS/GSGGS、GGS/GGSGGS、GGS/GGGSGGS、GGGS/无、GGGS/G、GGGS/GS、GGGS/GGS、GGGS/GGGS、GGGS/GSGGS、GGGS/GGSGGS、GSGGS/无、GSGGS/GS、GSGGS/GGGS、GSGGS/GSGGS、GSGGS/GGSGGS、GSGGS/GGGSGGS、GGSGGS/G、GGSGGS/GS、GGSGGS/GGS、GGSGGS/GGGS、GGSGGS/GSGGS、GGSGGS/GGSGGS、GGSGGS/GGGSGGS、GGGSGGS/无、GGGSGGS/GS、GGGSGGS/GGS、GGGSGGS/GSGGS、GGGSGGS/GGSGGS、GGGSGGS/GGGSGGS。
优选地,A是cpBFP,L1和L2的组合选自:无/无、无/GGSGGS、G/GGGS、GS/GGS、GS/GGGS、GGS/G、GGS/GS、GGS/GGS、GGS/GGSGGS、GGS/GGGSGGS、GGGS/G、GGGS/GS、GGSGGS/GGGS、GGGSGGS/GGS。
更优选地,A是cpBFP,L1和L2的组合选自:GGGS/GS。
在一个或多个实施方案中,B1和B2是CcpE,A是cpmApple,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、无/GGGS、无/GSGGS、无/GGSGGS、无/GGGSGGS、G/G、G/GGS、G/GGGS、G/GSGGS、G/GGSGGS、G/GGGSGGS、GS/无、GS/GS、GS/GGGS、GS/GSGGS、GS/GGSGGS、GS/GGGSGGS、GGS/无、GGS/G、GGS/GS、GGS/GGS、GGS/GGGS、GGS/GSGGS、GGS/GGSGGS、GGS/GGGSGGS、GGGS/G、GGGS/GS、GGGS/GGS、GGGS/GGGS、GGGS/GGGSGGS、GSGGS/G、GSGGS/GS、GSGGS/GGS、GSGGS/GGGS、GSGGS/GSGGS、GSGGS/GGSGGS、GSGGS/GGGSGGS、GGSGGS/无、GGSGGS/G、GGSGGS/GS、GGSGGS/GGS、GGSGGS/GSGGS、GGSGGS/GGSGGS、GGSGGS/GGGSGGS、GGGSGGS/G、GGGSGGS/GS、GGGSGGS/GGGS、GGGSGGS/GSGGS、GGGSGGS/GGSGGS、GGGSGGS/GGGSGGS。
优选地,A是cpmApple,L1和L2的组合选自:无/GGSGGS、无/GGGSGGS、G/GSGGS、G/GGSGGS、G/GGGSGGS。
更优选地,A是cpmApple,L1和L2的组合选自:无/GGSGGS、无/GGGSGGS。
在一个或多个实施方案中,所述融合蛋白中,B1、A和B2的变体是在B1、A和B2的N端或者C端具有一个或多个(优选1-5个)氨基酸的截短和/或突变的变体。优选地,B1和B2是CitA,A是cpYFP,并且所述融合蛋白具有选自以下的一个或多个特征:
L1为无,L2为PDP;
B1的C端具有1-5个氨基酸的截短,例如1-4个、1-3个;
B1的C端具有1-3个氨基酸的截短并且C端第4个氨基酸是甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸;
A的N端具有1-5个氨基酸的截短,例如1-4个、1-3个;
A的N端具有1个氨基酸的截短并且N端第2个氨基酸是赖氨酸、精氨酸、酪氨酸、苯丙氨酸、组氨酸;
A的N端具有1个氨基酸的截短并且N端第2个氨基酸是甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、第3个氨基酸是赖氨酸、精氨酸、组氨酸;
在一个或多个实施方案中,所述融合蛋白具有SEQ ID NO:7-14中任一所示的序列。
在一个或多个实施方案中,本文所述融合蛋白包含氨基酸序列SEQ ID NO:7-14中任一或其变体。在一个实施方式中,本发明提供的光学探针包含与氨基酸序列SEQ ID NO:7-14中任一有35%、40%、50%、60%、70%、80%、85%、90%、95%、99%序列相同性的序列。在优选实施方式中,本发明提供的融合蛋白包含与氨基酸序列SEQ ID NO:7-14中任一实质上相似或相同的序列。在更优选的实施方式中,本发明提供的融合蛋白包含SEQ IDNO:12或由其组成。
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。
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:7-14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。所述试剂盒还包含检测柠檬酸所需的其他试剂,例如缓冲液、对照。
本发明还提供检测样品中柠檬酸的方法,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柠檬酸的方法,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柠檬酸。
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柠檬酸接触,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。所述化合物调节细胞对柠檬酸的摄取能力。
本发明还提供筛选影响细胞柠檬酸代谢的化合物的方法,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影响细胞柠檬酸代谢的化合物。
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柠檬酸细胞内/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:本发明提供的柠檬酸光学探针易于成熟,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、定量检测柠檬酸,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柠檬酸光学探针对柠檬酸的最高响应达到对照的13.6倍以上,并且可以在细胞浆、线粒体、细胞核、内质网、核排阻和过氧化物酶体等亚细胞结构中对细胞进行定位、定性、定量检测,并且可以进行高通量的化合物筛选以及血液中柠檬酸定量检测。
附图说明
图1为实施例1所述的示例性柠檬酸光学探针的SDS-PAGE图;
图2为实施例6所述的示例性的对柠檬酸结合蛋白CitA(图2,A和B)或CcpE(图2,C)两侧的连接肽进行随机突变获得的光学探针对柠檬酸响应变化图;
图3为实施例7所述的示例性的对柠檬酸结合蛋白CitA两侧的连接肽进行随机突变获得的光学探针对不同浓度柠檬酸的滴定曲线;
图4为实施例8所述的示例性的柠檬酸光学探针的荧光光谱性质图;
图5为实施例9所述的示例性柠檬酸光学探针的特异性检测的柱状图;
图6为实施例10所述的示例性柠檬酸光学探针在哺乳动物细胞中的亚细胞定位照片;
图7为实施例11所述的对示例性柠檬酸光学探针在哺乳动物细胞中对柠檬酸响应的示意图;
图8为实施例12所述的示例性柠檬酸光学探针在活细胞水平进行高通量化合物筛选的点图;
图9为实施例13所述的示例性柠檬酸光学探针对小鼠和人血液中的丙酮酸进行定量的柱状图。
具体实施方式
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“柠檬酸敏感多肽”或“柠檬酸响应多肽”指对柠檬酸产生响应的多肽,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。本文所述柠檬酸敏感多肽还可包括其功能变体。柠檬酸敏感多肽的功能变体包括但不限于可以与柠檬酸相互作用从而发生与亲本柠檬酸敏感多肽相同或相似变化的变体。
本发明所述柠檬酸敏感多肽包括但不限于柠檬酸结合蛋白CitA或CcpE或与其有90%以上同源性的变体。本发明所述示例性柠檬酸结合蛋白CitA来源于肺炎克雷伯菌Klebsiella pneumoniae,柠檬酸结合蛋白CcpE源自金黄色葡萄球菌Staphylococcusaureus。示例性CitA蛋白如SEQ ID NO:1所示,CcpE蛋白如SEQ ID NO:2所示。柠檬酸结合蛋白可以感应柠檬酸浓度的变化,在柠檬酸浓度动态变化的过程中柠檬酸结合蛋白的空间构象也会发生改变。
本文所用术语“光学探针”或“融合蛋白”是指与光学活性多肽融合的柠檬酸敏感多肽。发明人发现,柠檬酸敏感多肽例如柠檬酸结合蛋白专一性地对生理浓度的柠檬酸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。借助不同柠檬酸浓度下测定的荧光蛋白的荧光绘制标准曲线,可以检测并分析柠檬酸的存在和/或水平。在本发明的光学探针中,光学活性多肽A(例如荧光蛋白)可操作地插入两个或多个柠檬酸敏感多肽B之间。光学活性多肽A位于两个或多个柠檬酸敏感多肽的序列之间,形成B1-A-B2的探针结构;柠檬酸敏感多肽B和柠檬酸相互作用导致光学活性多肽A的光学信号变强。在一个或多个实施方案中,B1和B2相同。
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包括其功能变体。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:3所示;cpGFP如SEQ ID NO:4所示;cpBFP如SEQ ID NO:5所示;cpmApple如SEQ ID NO:6所示。
在本发明的融和蛋白中,光学活性多肽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L1和L2与两端的B连接,形成顺序为B1-L1-A-L2-B2的结构。
“接头”、“连接区”或“连接肽”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。示例性地,本发明中柠檬酸敏感多肽CitA与光学活性多肽的连接区氨基端的氨基酸数目选择的是0-3个,羧基端的氨基酸数目选择的是0-3;柠檬酸敏感多肽CcpE与光学活性多肽的连接区氨基端的氨基酸数目选择的是0-7个,羧基端的氨基酸数目选择的是0-7个;连接肽为1个氨基酸时为G,2个氨基酸时为GS,3个氨基酸时为GGS,4个氨基酸时为GGGS,5个氨基酸时为GSGGS,6个氨基酸时为GGSGGS,7个氨基酸时为GGGSGGS。接头可具有突变,所以L1和L2还可分别独立选自P、H、N、S、Q、A、D、Y、M、T、E、C、L、V、W、I、F、K、R、RSE、GGS、PAP、PDA、RVR、RED、PER、RNA、PDP、RGA、RPP、TWD、TLE。当重组光学探针作为基本单元与功能蛋白连接时,可以融合在重组光学探针的氨基酸或羧基端。接头序列可为一个或多个柔性氨基酸组成的短肽链,如G和/或S。
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。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、脯氨酸、苯丙氨酸、甲硫氨酸、柠檬酸)、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、柠檬酸、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。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柠檬酸敏感多肽。突变可以是氨基酸种类的突变,也可以是柠檬酸敏感多肽的截短。在示例性实施方式中,在C(0.PDP)的基础上,对光学活性多肽的N端与柠檬酸敏感多肽CitA的C端的连接氨基酸处进行截短与随机突变。例如,在B1和B2是CitA,A是cpYFP时,并且所述融合蛋白可具有选自以下的一个或多个特征:L1为无,L2为PDP;B1的C端具有1-5个氨基酸的截短,例如1-4个、1-3个;B1的C端具有1-3个氨基酸的截短并且C端第4个氨基酸是甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸;A的N端具有1-5个氨基酸的截短,例如1-4个、1-3个;A的N端具有1个氨基酸的截短并且N端第2个氨基酸是赖氨酸、精氨酸、酪氨酸、苯丙氨酸、组氨酸;A的N端具有1个氨基酸的截短并且N端第2个氨基酸是甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、第3个氨基酸是赖氨酸、精氨酸、组氨酸。优选地,所述融合蛋白具有SEQ ID NO:7-14中任一所示的序列。
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等(1977)Nucleic Acids Res.25:3389和Altschul等(1990)J.Mol.Biol.215:403。
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“功能变体”、“衍生物”和“类似物”是指基本上保持与原始多肽或蛋白(例如CitA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CitA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。所述类似物还包括具有不同于天然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,本发明的柠檬酸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。
本发明还提供了上述柠檬酸光学探针的制备方法,包括以下步骤:1)将编码本文所述柠檬酸光学探针的核酸序列纳入表达载体;2)将表达载体转移到宿主细胞中;2)在适合所述表达载体表达的条件下培养所述宿主细胞,3)分离柠檬酸光学探针。
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”或“核苷酸”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。DNA可以是编码链或非编码链。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。如本领域所知的,等位变体是一个核酸的替换形式,它可能是一个或多个核苷酸的取代、缺失或插入,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。在本发明中适用的重组载体包括但不限于细菌质粒。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。在一个实施方式中,表达载体可采用市售的pCDF载体,无其他特殊要求。示例性地,采用HindIII和XhoI分别对编码所述光学探针的核苷酸序列和表达载体进行双酶切,然后将二者的酶切产物连接得到重组表达载体。本发明对酶切和连接的具体步骤和参数没有特殊限定,采用本领域常规的步骤和参数即可。
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。在本发明实施例中所用的示例性宿主细胞为大肠杆菌BL21-DE3菌株。本领域一般技术人员都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。
本发明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,分离得到柠檬酸光学探针。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。本发明对分离所述柠檬酸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。这些方法是本领域技术人员所熟知的,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。在一个实施方式中,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柠檬酸光学探针在柠檬酸实时定位、定量检测以及高通量化合物筛选中的应用。在一个方面,所述的柠檬酸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,进行柠檬酸的实时定位;通过柠檬酸标准滴加曲线结合荧光信号的变化进行相应柠檬酸的定量检测。荧光信号的变化通过例如标准化后的荧光信号比值展示,即样品的485纳米荧光信号与420纳米荧光信号之比与对照的相应之比的比值。本发明所述的柠檬酸标准滴加曲线是根据柠檬酸光学探针在不同浓度柠檬酸的情况下的标准化后的荧光信号比值绘制而成。本发明柠檬酸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,测定柠檬酸含量的变化,从而筛选出对柠檬酸含量变化有影响的化合物。在本发明中所述的柠檬酸光学探针在柠檬酸实时定位、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。所述试剂盒还包含检测柠檬酸所需的其他试剂。所述其他试剂本领域周知,例如缓冲液、对照柠檬酸标品。示例性缓冲液例如100mM HEPES和100mM NaCl,pH 7.4。
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柠檬酸光学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。本领域普通技术人员按照以下实施例,不难根据具体情况略作修改和变换而成功实施本发明,这些修改和变换均落在本申请权利要求的范围内。
实施例中所用的基于pCDF-cpYFP,pCDF-柠檬酸结合蛋白质粒由华东理工大学蛋白质实验室构建,pCDF质粒载体购自Invitrogen公司。克隆菌株Mach1、BL21-DE3购自Invitrogen公司。其余材料、试剂和仪器参见CN201810215698.7,其所有内容通过引用纳入本文。
II.分子生物学方法和细胞实验方法
II.1蛋白质的表达,纯化和荧光检测
1.将表达载体(例如以pCDF为基础的柠檬酸光学探针表达载体)转化到BL21(DE3)细胞中,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24-36小时。
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。9600rpm,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100mM HEPES缓冲液(pH 7.4)中的蛋白。
4.纯化的蛋白经过SDS-PAGE鉴定后,使用测定缓冲液(100mM HEPES,100mM NaCl,pH 7.4)稀释探针成终浓度为0.2-5μM的蛋白溶液。用测定缓冲液(100mM HEPES,100mMNaCl,pH 7.4)将柠檬酸配制成终浓度为50mM的储液。
5.取100μl 1μM的蛋白溶液,37℃温育10分钟,加入柠檬酸滴定,测定蛋白的420nm光激发后528nm发射和485nm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。
6.取100μl 1μM的蛋白溶液,37℃温育10分钟,加入柠檬酸,测定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通过分光光度计和荧光分光光度计完成。
II.2哺乳动物细胞的转染和荧光检测
1.将pCDNA3.1+为基础的柠檬酸光学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。
II.3其余实验
实施例所涉其他实验过程参见CN201810215698.7,其所有内容通过引用纳入本文。
实施例1:柠檬酸结合蛋白质粒
通过PCR扩增肺炎克雷伯菌Klebsiella pneumoniae基因中的柠檬酸敏感多肽CitA与金黄色葡萄球菌Staphylococcus aureus基因中的柠檬酸敏感多肽CcpE。PCR产物凝胶电泳后利用胶回收试剂盒进行回收,通过同源重组的方式克隆到pCDF-duet1载体的多克隆位点区域,连接产物转化DH5α菌株,转化后的DH5α涂布于LB平板(链霉素100ug/mL),置于37℃培养过夜。将生长DH5α转化子进行菌落PCR鉴定,对阳性克隆进行接菌、质粒抽提后进行测序。
实施例2:不同连接肽的cpYFP光学探针的表达和检测
本实施例中,以pCDF-CitA为基础进行线性化,将cpYFP与CitA的基因通过overlapPCR连接在一起,然后将cpYFP-CitA的基因序列插入到pCDF-CitA载体中,获得含不同连接肽的目的质粒pCDF-CitA-(0/1/2/3AA)-cpYFP-(0/1/2/3AA)-CitA。以同样的方式获得含不同连接肽的质粒pCDF-CcpE-(0/1/2/3/4/5/6/7AA)-cpYFP-(0/1/2/3/4/5/6/7AA)-CcpE。将重组质粒转化到BL21(DE3)菌株中,添加诱导剂IPTG低温诱导目的蛋白表达,然后纯化目标蛋白。通过SDS-PAGE验证目标蛋白的大小。实验结果表明pCDF-CitA-(0/1/2/3AA)-cpYFP-(0/1/2/3AA)-CitA与pCDF-CcpE-(0/1/2/3/4/5/6/7AA)-cpYFP-(0/1/2/3/4/5/6/7AA)-CcpE表达出的含His-tag纯化标签的融合蛋白分别为58.2和77.7kDa附近,与目标蛋白大小一致。结果如图1所示。
使用表达CitA-(0/1/2/3AA)-cpYFP-(0/1/2/3AA)-CitA和CcpE-(0/1/2/3/4/5/6/7AA)-cpYFP-(0/1/2/3/4/5/6/7AA)-CcpE融合蛋白质的大肠杆菌的破碎上清进行柠檬酸响应筛选,将含有10mM柠檬酸的融合荧光蛋白质的检测信号除以无柠檬酸的融合荧光蛋白质的检测信号,结果如表1所示,C(N1.N2)与E(N1.N2)中C代表CitA,E代表CcpE,N1和N2分别代表cpYFP的N端和C端连接肽的个数,连接肽为1个氨基酸时为G,2个氨基酸时为GS,3个氨基酸时为GGS,4个氨基酸时为GGGS,5个氨基酸时为GSGGS,6个氨基酸时为GGSGGS,7个氨基酸时为GGGSGGS。表中的变化倍数是标准化后的荧光信号比值,计算方法如下:
荧光信号=样品荧光值-背景荧光值
检测结果显示,基于敏感多肽CitA构建的探针中:除C(1.0)、C(1.1)、C(2.0)、C(2.1)、C(3.0)、C(2.1)之外的探针均具有超过对照(1倍以上)的响应;对柠檬酸响应超过1.2倍的光学探针有C(0.1)、C(0.2)、C(0.3)、C(1.2)、C(1.3)、C(2.2)、C(2.3)、C(3.2)与C(3.3);对柠檬酸响应超过1.5倍的光学探针有C(0.1)、C(0.2)、C(0.3)与C(1.3)。基于敏感多肽CcpE构建的探针:所有探针均具有超过对照(1倍以上)的响应;对柠檬酸响应超过1.5倍的光学探针有E(0.4)、E(0.5)、E(0.6)、E(0.7)、E(1.4)、E(1.5)、E(1.6)、E(1.7)、E(2.4)、E(2.5)、E(2.6)、E(2.7)、E(3.4)、E(3.5)、E(3.6)、E(3.7)、E(4.6)、E(4.7)、E(5.5)、E(5.6)、E(5.7)、E(6.5)、E(6.6)、E(6.7)、;对柠檬酸响应超过2倍的光学探针有E(0.7)、E(1.5)、E(1.6)、E(1.7)、E(2.5)、E(2.6)、E(2.7)和E(3.7)。
表1
CitA | 变化倍数 | CcpE | 变化倍数 | 变化倍数 | 变化倍数 | 变化倍数 | ||||
C(0.0) | 1.14 | E(0.0) | 1.01 | E(2.0) | 1.00 | E(4.0) | 1.09 | E(6.0) | 0.97 | |
C(0.1) | 1.60 | E(0.1) | 1.21 | E(2.1) | 1.17 | E(4.1) | 1.14 | E(6.1) | 1.17 | |
C(0.2) | 1.29 | E(0.2) | 1.47 | E(2.2) | 1.21 | E(4.2) | 1.26 | E(6.2) | 1.15 | |
C(0.3) | 1.73 | E(0.3) | 1.01 | E(2.3) | 1.24 | E(4.3) | 1.18 | E(6.3) | 1.15 | |
C(1.0) | 0.93 | E(0.4) | 1.57 | E(2.4) | 1.78 | E(4.4) | 1.43 | E(6.4) | 1.39 | |
C(1.1) | 0.99 | E(0.5) | 1.70 | E(2.5) | 2.03 | E(4.5) | 1.37 | E(6.5) | 1.57 | |
C(1.2) | 1.31 | E(0.6) | 1.81 | E(2.6) | 2.02 | E(4.6) | 1.66 | E(6.6) | 1.53 | |
C(1.3) | 1.49 | E(0.7) | 2.04 | E(2.7) | 2.06 | E(4.7) | 1.56 | E(6.7) | 1.50 | |
C(2.0) | 0.72 | E(1.0) | 1.01 | E(3.0) | 1.02 | E(5.0) | 1.01 | E(7.0) | 1.02 | |
C(2.1) | 0.92 | E(1.1) | 1.32 | E(3.1) | 1.14 | E(5.1) | 1.24 | E(7.1) | 1.18 | |
C(2.2) | 1.35 | E(1.2) | 1.20 | E(3.2) | 1.16 | E(5.2) | 1.20 | E(7.2) | 1.15 | |
C(2.3) | 1.21 | E(1.3) | 1.42 | E(3.3) | 1.21 | E(5.3) | 1.18 | E(7.3) | 1.19 | |
C(3.0) | 0.62 | E(1.4) | 1.57 | E(3.4) | 1.53 | E(5.4) | 1.47 | E(7.4) | 1.23 | |
C(3.1) | 0.81 | E(1.5) | 2.72 | E(3.5) | 1.74 | E(5.5) | 1.65 | E(7.5) | 1.37 | |
C(3.2) | 1.37 | E(1.6) | 2.89 | E(3.6) | 1.94 | E(5.6) | 1.71 | E(7.6) | 1.40 | |
C(3.3) | 1.05 | E(1.7) | 2.58 | E(3.7) | 2.36 | E(5.7) | 1.65 | E(7.7) | 1.39 | |
cpYFP | 1.00 | cpYFP | 1.00 |
实施例3:不同连接肽的cpGFP光学探针的表达和检测
按照实施例2中的方法将cpYFP替换为cpGFP,构建柠檬酸绿色荧光蛋白荧光探针。检测结果如表2所示。基于敏感多肽CitA构建的探针:所有探针均具有超过对照(1倍以上)的响应;对柠檬酸响应超过1.2倍的光学探针有C(0.1)、C(0.2)、C(0.3)、C(1.2)、C(3.0)、C(3.2)。对柠檬酸响应超过1.5倍的光学探针有C(0.3)。基于敏感多肽CcpE构建的探针:除E(1.3)、E(4.0)、E(5.0)、E(5.1)、E(6.5)、E(7.0)之外的探针均具有超过对照(1倍以上)的响应;对柠檬酸响应超过1.2倍的光学探针有E(0.5)、E(0.6)、E(0.7)、E(1.5)、E(1.6)、E(1.7)、E(2.6)、E(2.7)、E(3.1)、E(3.2)、E(3.4)、E(3.5)、E(3.6)、E(3.7)、E(4.5)、E(5.5)、E(7.2)、E(7.3)、E(7.4)、E(7.5);对柠檬酸响应超过1.5倍的光学探针有E(0.7)、E(1.5)、E(1.6)和E(1.7)。
表2
CitA | 变化倍数 | CcpE | 变化倍数 | 变化倍数 | 变化倍数 | 变化倍数 | ||||
C(0.0) | 1.18 | E(0.0) | 1.19 | E(2.0) | 1.02 | E(4.0) | 0.96 | E(6.0) | 1.06 | |
C(0.1) | 1.21 | E(0.1) | 1.17 | E(2.1) | 1.02 | E(4.1) | 1.10 | E(6.1) | 1.00 | |
C(0.2) | 1.24 | E(0.2) | 1.12 | E(2.2) | 1.11 | E(4.2) | 1.14 | E(6.2) | 1.00 | |
C(0.3) | 1.57 | E(0.3) | 1.16 | E(2.3) | 1.04 | E(4.3) | 1.08 | E(6.3) | 1.00 | |
C(1.0) | 1.14 | E(0.4) | 1.10 | E(2.4) | 1.08 | E(4.4) | 1.05 | E(6.4) | 1.13 | |
C(1.1) | 1.13 | E(0.5) | 1.40 | E(2.5) | 1.13 | E(4.5) | 1.48 | E(6.5) | 0.97 | |
C(1.2) | 1.28 | E(0.6) | 1.47 | E(2.6) | 1.45 | E(4.6) | 1.11 | E(6.6) | 1.14 | |
C(1.3) | 1.19 | E(0.7) | 1.58 | E(2.7) | 1.37 | E(4.7) | 1.05 | E(6.7) | 1.01 | |
C(2.0) | 1.01 | E(1.0) | 1.13 | E(3.0) | 1.02 | E(5.0) | 1.00 | E(7.0) | 0.82 | |
C(2.1) | 1.05 | E(1.1) | 1.16 | E(3.1) | 1.23 | E(5.1) | 0.99 | E(7.1) | 1.03 | |
C(2.2) | 0.91 | E(1.2) | 1.17 | E(3.2) | 1.22 | E(5.2) | 1.18 | E(7.2) | 1.22 | |
C(2.3) | 1.03 | E(1.3) | 0.90 | E(3.3) | 1.19 | E(5.3) | 1.05 | E(7.3) | 1.22 | |
C(3.0) | 1.25 | E(1.4) | 1.08 | E(3.4) | 1.24 | E(5.4) | 1.13 | E(7.4) | 1.24 | |
C(3.1) | 1.11 | E(1.5) | 1.90 | E(3.5) | 1.36 | E(5.5) | 1.26 | E(7.5) | 1.30 | |
C(3.2) | 1.21 | E(1.6) | 1.86 | E(3.6) | 1.30 | E(5.6) | 1.01 | E(7.6) | 1.15 | |
C(3.3) | 1.00 | E(1.7) | 1.70 | E(3.7) | 1.45 | E(5.7) | 1.04 | E(7.7) | 1.07 | |
cpGFP | 1.00 | cpGFP | 1.00 |
实施例4:不同连接肽的cpBFP光学探针的表达和检测
按照实施例2中的方法将cpYFP替换为cpBFP,构建柠檬酸蓝色荧光蛋白荧光探针。检测结果如表3所示。基于敏感多肽CitA构建的探针:除C(0.1)、C(1.3)之外的探针均具有超过对照(1倍以上)的响应;对柠檬酸响应超过1.2倍的光学探针有C(2.1)、C(2.2)、C(3.0)、C(3.1);对柠檬酸响应超过1.5倍的光学探针有C(2.2)。基于敏感多肽CcpE构建的探针:除E(0.7)、E(1.6)、E(4.7)、E(5.1)、E(5.3)、E(6.0)、E(7.1)、E(7.4)之外的探针均具有超过对照(1倍以上)的响应;对柠檬酸响应超过1.2倍的光学探针有E(0.0)、E(0.6)、E(1.4)、E(2.3)、E(2.4)、E(3.1)、E(3.2)、E(3.3)、E(3.6)、E(3.7)、E(4.1)、E(4.2)、E(6.4)、E(7.3);对柠檬酸响应超过1.5倍的光学探针有E(4.2)。
表3
CitA | 变化倍数 | CcpE | 变化倍数 | 变化倍数 | 变化倍数 | 变化倍数 | ||||
C(0.0) | 1.08 | E(0.0) | 1.22 | E(2.0) | 1.19 | E(4.0) | 1.01 | E(6.0) | 0.98 | |
C(0.1) | 0.90 | E(0.1) | 1.12 | E(2.1) | 1.07 | E(4.1) | 1.20 | E(6.1) | 1.09 | |
C(0.2) | 1.18 | E(0.2) | 1.07 | E(2.2) | 1.14 | E(4.2) | 1.58 | E(6.2) | 1.05 | |
C(0.3) | 1.02 | E(0.3) | 1.04 | E(2.3) | 1.30 | E(4.3) | 1.10 | E(6.3) | 1.02 | |
C(1.0) | 1.18 | E(0.4) | 1.07 | E(2.4) | 1.27 | E(4.4) | 1.04 | E(6.4) | 1.22 | |
C(1.1) | 1.13 | E(0.5) | 1.17 | E(2.5) | 1.04 | E(4.5) | 1.07 | E(6.5) | 1.03 | |
C(1.2) | 1.06 | E(0.6) | 1.25 | E(2.6) | 1.02 | E(4.6) | 1.14 | E(6.6) | 1.15 | |
C(1.3) | 0.95 | E(0.7) | 0.94 | E(2.7) | 1.15 | E(4.7) | 0.92 | E(6.7) | 1.03 | |
C(2.0) | 1.07 | E(1.0) | 1.05 | E(3.0) | 1.17 | E(5.0) | 1.18 | E(7.0) | 1.09 | |
C(2.1) | 1.30 | E(1.1) | 1.09 | E(3.1) | 1.32 | E(5.1) | 1.00 | E(7.1) | 0.92 | |
C(2.2) | 1.56 | E(1.2) | 1.03 | E(3.2) | 1.22 | E(5.2) | 1.18 | E(7.2) | 1.19 | |
C(2.3) | 1.07 | E(1.3) | 1.20 | E(3.3) | 1.25 | E(5.3) | 0.87 | E(7.3) | 1.25 | |
C(3.0) | 1.25 | E(1.4) | 1.26 | E(3.4) | 1.12 | E(5.4) | 1.09 | E(7.4) | 0.97 | |
C(3.1) | 1.24 | E(1.5) | 1.04 | E(3.5) | 1.13 | E(5.5) | 1.04 | E(7.5) | 1.02 | |
C(3.2) | 1.01 | E(1.6) | 0.87 | E(3.6) | 1.27 | E(5.6) | 1.19 | E(7.6) | 1.16 | |
C(3.3) | 1.06 | E(1.7) | 1.06 | E(3.7) | 1.26 | E(5.7) | 1.05 | E(7.7) | 1.04 | |
cpBFP | 1.00 | cpBFP | 1.00 |
实施例5:不同连接肽的cpmApple光学探针的表达和检测
按照实施例2中的方法将cpYFP替换为cpmApple,构建柠檬酸红色荧光蛋白荧光探针。检测结果如表4所示,检测结果显示基于敏感多肽CitA构建的探针:除C(2.0)、C(2.1)、C(2.3)之外的探针均具有超过对照(1倍以上)的响应;对柠檬酸响应超过1.2倍的光学探针有C(0.2)、C(1.0)、C(1.2)、C(1.3)、C(3.1);对柠檬酸响应超过1.5倍的光学探针有C(1.2)和C(1.3);基于敏感多肽CcpE构建的探针:除E(1.0)、E(1.2)、E(2.1)、E(2.3)、E(4.0)、E(4.5)、E(4.6)、E(5.0)、E(6.4)、E(7.0)、E(7.3)之外的探针均具有超过对照(1倍以上)的响应;对柠檬酸响应超过1.5倍的光学探针有E(0.6)、E(0.7)、E(1.5)、E(1.6)、E(1.7);对柠檬酸响应超过2倍的光学探针有E(0.6)、E(0.7)。
表4
CitA | 变化倍数 | CcpE | 变化倍数 | 变化倍数 | 变化倍数 | 变化倍数 | ||||
C(0.0) | 1.16 | E(0.0) | 1.17 | E(2.0) | 1.16 | E(4.0) | 0.91 | E(6.0) | 1.05 | |
C(0.1) | 1.04 | E(0.1) | 1.30 | E(2.1) | 0.99 | E(4.1) | 1.01 | E(6.1) | 1.21 | |
C(0.2) | 1.38 | E(0.2) | 1.14 | E(2.2) | 1.19 | E(4.2) | 1.16 | E(6.2) | 1.18 | |
C(0.3) | 1.01 | E(0.3) | 1.36 | E(2.3) | 0.94 | E(4.3) | 1.08 | E(6.3) | 1.22 | |
C(1.0) | 1.25 | E(0.4) | 1.12 | E(2.4) | 1.18 | E(4.4) | 1.23 | E(6.4) | 0.95 | |
C(1.1) | 1.04 | E(0.5) | 1.36 | E(2.5) | 1.26 | E(4.5) | 0.87 | E(6.5) | 1.16 | |
C(1.2) | 1.55 | E(0.6) | 2.13 | E(2.6) | 1.41 | E(4.6) | 0.95 | E(6.6) | 1.06 | |
C(1.3) | 1.54 | E(0.7) | 2.00 | E(2.7) | 1.21 | E(4.7) | 1.19 | E(6.7) | 1.00 | |
C(2.0) | 1.00 | E(1.0) | 0.98 | E(3.0) | 1.16 | E(5.0) | 0.85 | E(7.0) | 0.99 | |
C(2.1) | 0.83 | E(1.1) | 1.04 | E(3.1) | 1.07 | E(5.1) | 1.13 | E(7.1) | 1.14 | |
C(2.2) | 1.13 | E(1.2) | 0.92 | E(3.2) | 1.18 | E(5.2) | 1.12 | E(7.2) | 1.11 | |
C(2.3) | 1.00 | E(1.3) | 1.17 | E(3.3) | 1.24 | E(5.3) | 1.00 | E(7.3) | 1.00 | |
C(3.0) | 1.15 | E(1.4) | 1.44 | E(3.4) | 1.09 | E(5.4) | 1.09 | E(7.4) | 1.09 | |
C(3.1) | 1.26 | E(1.5) | 1.74 | E(3.5) | 1.14 | E(5.5) | 1.27 | E(7.5) | 1.14 | |
C(3.2) | 1.14 | E(1.6) | 1.78 | E(3.6) | 1.15 | E(5.6) | 1.04 | E(7.6) | 1.13 | |
C(3.3) | 1.06 | E(1.7) | 1.95 | E(3.7) | 1.03 | E(5.7) | 1.04 | E(7.7) | 1.08 | |
cpmApple | 1.00 | cpmApple | 1.00 |
实施例6:突变的cpYFP光学探针的表达和检测
在C(0,3)的基础上构建光学探针突变体。首先对cpYFP荧光蛋白C端的3个氨基酸的连接肽进行随机突变,通过反向PCR线性化质粒C(0.3),对得到的PCR产物在PNK、T4 DNA连接酶和PEG4000的作用下加磷连接,将连接产物转化BL21(DE3)菌株,然后进行筛选。将含有10mM柠檬酸的融合荧光蛋白质的检测信号除以无柠檬酸的融合荧光蛋白质的检测信号,挑选检测结果显示对柠檬酸响应超过2.5倍的样品进行测序,得到突变体C(0.RSE)、C(0.GGS)、C(0.PAP)、C(0.PDA)、C(0.RVR)、C(0.RED)、C(0.PER)、C(0.RNA)、C(0.PDP)、C(0.RGA)、C(0.RPP),其中C(0.PDP)对柠檬酸响应最大,约为3.3倍,如图2,A所示。在C(0.PDP)的基础上,对cpYFP荧光蛋白N端与敏感多肽CitA的连接处进行截短与随机突变,进行筛选,检测结果显示对柠檬酸响应超过2.5倍的样品进行测序,得到突变体C1-2A6、C2-2F6、C7-1F3、C7-1G1(同C(0.3))、C7-1G4、C8-2E6、C13-2F4、C14-1H2(分别如SEQ ID NO:7-14所示)。结果如图2,B所示,对cpYFP荧光蛋白N端与敏感多肽CitA的连接处进行截短或突变的样品对柠檬酸响应增强,表明cpYFP蛋白N端与敏感多肽CitA的连接处对柠檬酸的结合非常重要。
基于敏感多肽CcpE构建的探针对柠檬酸响应超过2.5倍的光学探针有E(1.5)、E(1.6)、和E(1.7),在E(1.6)的基础上构建光学探针突变体,首先对cpYFP荧光蛋白N端的1个氨基酸的连接肽进行饱和突变,通过反向PCR线性化质粒E(1.6),对得到的PCR产物在PNK、T4 DNA连接酶和PEG4000的作用下加磷连接,将连接产物转化BL21(DE3)菌株,然后进行筛选。将含有10mM这些柠檬酸的融合荧光蛋白质的检测信号除以无柠檬酸的融合荧光蛋白质的检测信号。结果如图2,C所示,虽然响应倍数低于突变之前的E(1.6)(E(G.6)),但是所有突变体对柠檬酸均具有超过1倍的响应;除E(F.6)之外的突变体对柠檬酸都具有超过1.5倍的响应;E(P.6)、E(H.6)、E(N.6)、E(S.6)、E(Q.6)、E(A.6)、E(D.6)、E(Y.6)、E(M.6)、E(T.6)、E(E.6)、E(C.6)、E(R.6)对柠檬酸都具有超过2倍的响应;E(P.6)对柠檬酸具有超过2.5倍的响应。
实施例7:光学探针的性能
对实施例2和实施例6中所得的部分柠檬酸光学探针,即C1-2A6、C2-2F6、C7-1F3、C7-1G1、C7-1G4、C8-2E6、C13-2F4、C14-1H2和E(1.6)进行浓度梯度的柠檬酸检测,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。C7-1G4、C8-2E6、C13-2F4、C1-2A6、C14-1H2、C7-1G1、C2-2F6、C7-1F3和E(1.6)的Kd(结合常数)分别为15μM、59μM、200μM、204μM、206μM、237μM、269μM、408μM、和1125μM,变化幅度分别为3.3倍、5.2倍、13.6倍、8.2倍、6.9倍、3.1倍、2.5倍、4.0倍和2.8倍,结果如图3所示。
实施例8:柠檬酸光学探针的光谱性能和特异性
示例性地,将纯化的柠檬酸光学探针C13-2F4和E(1.6)分别进行0mM和10mM柠檬酸处理10分钟后,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:以350nm至500nm的激发范围和530nm的发射波长记录激发光谱,每2nm读取一次。结果显示,探针C13-2F4和E(1.6)在约420和490nm处分别有两个激发峰,如图4所示。
对纯化的柠檬酸光学探针C13-2F4和E(1.6)测定其特异性,结果表明探针具有很好的特异性,如图5所示。
实施例9:光学探针的亚细胞定位和光学探针在亚细胞内的性能
本实施例中,使用不同的定位信号肽与光学探针C13-2F4融合,将光学探针定位到不同的细胞器中。
用融合不同定位信号肽的光学探针质粒转染HeLa细胞36小时后,使用PBS冲洗,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。结果如图6所示。柠檬酸光学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、内质网、核排阻、过氧化物酶体等亚细胞。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。
细胞浆内柠檬酸主要由线粒体中TCA循环产生,虽然细胞质膜存在柠檬酸运载体,但主要表达于人的肝脏和睾丸组织,在其他种类细胞系中表达受限。由于柠檬酸的胞质转运蛋白表达有较高的组织特异性,外源补充柠檬酸探针响应不显著,因此,尝试使用活性小分子化合物,对柠檬酸代谢进行调控,进而观察表达在胞浆内的柠檬酸探针对胞内柠檬酸动态水平的响应情况。胞质内柠檬酸主要来自线粒体TCA循环,并在胞浆中参与脂肪酸合成。针对脂肪酸代谢途径,我们选择了其关键代谢酶ACC(乙酰辅酶A羧化酶),以及CPT1(肉碱脂酰转移酶)抑制剂。
用胞浆表达的光学探针质粒C13-2F4转染HEK293细胞36小时后,使用HBSS清洗两遍,并置于HBSS溶液中,在补充或不补充葡萄糖的条件下,基于胞浆内表达柠檬酸探针的细胞,检测脂肪酸代谢抑制剂对柠檬酸代谢的影响。结果如图7所示,对照组在检测的30分钟内,荧光比率下降约50%。假设ACC被抑制,则脂肪酸的合成被阻断,可能导致柠檬酸积累。加入ACC处理的实验组中,探针测定结果显示,柠檬酸探针荧光比率相对于对照组降低变缓,30分钟时荧光比率接近0分钟时的荧光比率,说明ACC抑制,增加了胞内柠檬酸水平,与预期相符。假设CPT1被抑制,则进入线粒体发生β氧化的脂肪酸减少,TCA循环原料不足,柠檬酸产生降低,可能会导致胞浆内柠檬酸水平降低。加CPT1抑制剂的实验组中,探针结果显示,加入CPT1抑制剂,30分钟时,荧光比率明显低于对照组,表明胞浆内柠檬酸水平降低,与预期相符。上述实验实例表明,在活细胞内表达柠檬酸探针,测定的荧光比率可以有效的反应细胞内柠檬酸水平的动态情况。
实施例10:在活细胞中基于光学探针进行高通量化合物筛选
本实施例中,我们使用胞浆表达C13-2F4的HEK293细胞进行高通量化合物筛选。经转染的HEK293细胞使用HBSS冲洗,置于HBSS溶液中处理1小时,然后使用10μM的化合物处理1小时。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作为对照进行标准化。结果如图8所示,在使用的2000种化合物中,绝大部分的化合物对柠檬酸代谢影响极小,部分化合物能够调控柠檬酸代谢,有8种化合物能够提高细胞浆中柠檬酸的含量,另外有7种化合物能够明显降低细胞浆中柠檬酸的含量。
实施例11:光学探针定量检测血液中的柠檬酸
在本实施中,使用纯化的C13-2F4对小鼠和人的血液上清中的柠檬酸进行分析。
将C13-2F4与稀释的血液上清混合处理10分钟后,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9所示,小鼠血液中的柠檬酸含量在279μM左右,人血液中的柠檬酸含量在160μM左右。
由以上实施例可知,本发明提供的柠檬酸光学探针,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、定量检测柠檬酸;并且能够进行高通量的化合物筛选。
其它实施方式
本说明书描述了许多实施方式。然而应理解,本领域技术人员通过阅读本说明书获知的不背离本发明的构思和范围的各种改进,也应包括在所附权利要求书的范围内。
序列表
<110> 华东理工大学
<120> 柠檬酸光学探针及其制备方法和应用
<130> 204707
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 133
<212> PRT
<213> Klebsiella pneumoniae
<400> 1
Met Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
1 5 10 15
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
20 25 30
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
35 40 45
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
50 55 60
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
65 70 75 80
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
85 90 95
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
100 105 110
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
115 120 125
Ile Glu Gln Leu Glu
130
<210> 2
<211> 211
<212> PRT
<213> Staphylococcus aureus
<400> 2
Met Phe Asp Lys Met Gln Ala His Ile Gly Glu Val Asn Gly Thr Ile
1 5 10 15
Ser Ile Gly Cys Ser Ser Leu Ile Gly Gln Thr Leu Leu Pro Glu Val
20 25 30
Leu Ser Leu Tyr Asn Ala Gln Phe Pro Asn Val Glu Ile Gln Val Gln
35 40 45
Val Gly Ser Thr Glu Gln Ile Lys Ala Asn His Arg Asp Tyr His Val
50 55 60
Met Ile Thr Arg Gly Asn Lys Val Met Asn Leu Ala Asn Thr His Leu
65 70 75 80
Phe Asn Asp Asp His Tyr Phe Ile Phe Pro Lys Asn Arg Arg Asp Asp
85 90 95
Val Thr Lys Leu Pro Phe Ile Glu Phe Gln Ala Asp Pro Ile Tyr Ile
100 105 110
Asn Gln Ile Lys Gln Trp Tyr Asn Asp Asn Leu Glu Gln Asp Tyr His
115 120 125
Ala Thr Ile Thr Val Asp Gln Val Ala Thr Cys Lys Glu Met Leu Ile
130 135 140
Ser Gly Val Gly Val Thr Ile Leu Pro Glu Ile Met Met Lys Asn Ile
145 150 155 160
Ser Lys Glu Gln Phe Glu Phe Glu Lys Val Glu Ile Asp Asn Glu Pro
165 170 175
Leu Ile Arg Ser Thr Phe Met Ser Tyr Asp Pro Ser Met Leu Gln Leu
180 185 190
Pro Gln Val Asp Ser Phe Val Asn Leu Met Ala Ser Phe Val Glu Gln
195 200 205
Pro Lys Ala
210
<210> 3
<211> 246
<212> PRT
<213> Artificial Sequence
<400> 3
Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly
1 5 10 15
Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val
20 25 30
Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
35 40 45
Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu Ser
50 55 60
Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val
65 70 75 80
Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val Asp
85 90 95
Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr Gly
100 105 110
Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys
115 120 125
Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu
130 135 140
Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
145 150 155 160
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> 4
<211> 241
<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 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 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
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Gln
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> 5
<211> 243
<212> PRT
<213> Artificial Sequence
<400> 5
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> 6
<211> 242
<212> PRT
<213> Artificial Sequence
<400> 6
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> 7
<211> 512
<212> PRT
<213> Artificial Sequence
<400> 7
Met Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
1 5 10 15
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
20 25 30
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
35 40 45
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
50 55 60
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
65 70 75 80
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
85 90 95
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
100 105 110
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
115 120 125
Ile Glu Gln Leu Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln
130 135 140
Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp
145 150 155 160
Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
165 170 175
Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser
180 185 190
Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
195 200 205
Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
210 215 220
Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu
225 230 235 240
Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn
245 250 255
Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr
260 265 270
Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val
275 280 285
Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe
290 295 300
Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala
305 310 315 320
Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp
325 330 335
Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu
340 345 350
Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn
355 360 365
Ile Leu Gly His Lys Leu Glu Tyr Asn Pro Asp Pro Asp Ile Thr Glu
370 375 380
Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala Leu Ile Gln Ala Met
385 390 395 400
Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala Val Gln Lys Arg Asp
405 410 415
Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met Arg Ser Phe Ser Asp
420 425 430
Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly Gln Arg Leu Tyr His
435 440 445
Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu Gly Gly Asp Ser Asp
450 455 460
Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser Val Arg Lys Gly Ser
465 470 475 480
Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile Gln Asp Ala Thr Gly
485 490 495
Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr Ile Glu Gln Leu Glu
500 505 510
<210> 8
<211> 512
<212> PRT
<213> Artificial Sequence
<400> 8
Met Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
1 5 10 15
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
20 25 30
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
35 40 45
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
50 55 60
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
65 70 75 80
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
85 90 95
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
100 105 110
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
115 120 125
Ile Glu Gln Leu His Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln
130 135 140
Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp
145 150 155 160
Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
165 170 175
Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser
180 185 190
Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
195 200 205
Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
210 215 220
Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu
225 230 235 240
Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn
245 250 255
Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr
260 265 270
Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val
275 280 285
Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe
290 295 300
Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala
305 310 315 320
Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp
325 330 335
Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu
340 345 350
Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn
355 360 365
Ile Leu Gly His Lys Leu Glu Tyr Asn Pro Asp Pro Asp Ile Thr Glu
370 375 380
Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala Leu Ile Gln Ala Met
385 390 395 400
Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala Val Gln Lys Arg Asp
405 410 415
Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met Arg Ser Phe Ser Asp
420 425 430
Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly Gln Arg Leu Tyr His
435 440 445
Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu Gly Gly Asp Ser Asp
450 455 460
Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser Val Arg Lys Gly Ser
465 470 475 480
Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile Gln Asp Ala Thr Gly
485 490 495
Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr Ile Glu Gln Leu Glu
500 505 510
<210> 9
<211> 510
<212> PRT
<213> Artificial Sequence
<400> 9
Met Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
1 5 10 15
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
20 25 30
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
35 40 45
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
50 55 60
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
65 70 75 80
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
85 90 95
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
100 105 110
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
115 120 125
Ile Asp Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
130 135 140
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser
145 150 155 160
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
165 170 175
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu
180 185 190
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
195 200 205
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val
210 215 220
Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr
225 230 235 240
Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
245 250 255
Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys
260 265 270
Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
275 280 285
Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg
290 295 300
Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro
305 310 315 320
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn
325 330 335
Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
340 345 350
Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu
355 360 365
Gly His Lys Leu Glu Tyr Asn Pro Asp Pro Asp Ile Thr Glu Glu Arg
370 375 380
Leu His Tyr Gln Val Gly Gln Arg Ala Leu Ile Gln Ala Met Gln Ile
385 390 395 400
Ser Ala Met Pro Glu Leu Val Glu Ala Val Gln Lys Arg Asp Leu Ala
405 410 415
Arg Ile Lys Ala Leu Ile Asp Pro Met Arg Ser Phe Ser Asp Ala Thr
420 425 430
Tyr Ile Thr Val Gly Asp Ala Ser Gly Gln Arg Leu Tyr His Val Asn
435 440 445
Pro Asp Glu Ile Gly Lys Ser Met Glu Gly Gly Asp Ser Asp Glu Ala
450 455 460
Leu Ile Asn Ala Lys Ser Tyr Val Ser Val Arg Lys Gly Ser Leu Gly
465 470 475 480
Ser Ser Leu Arg Gly Lys Ser Pro Ile Gln Asp Ala Thr Gly Lys Val
485 490 495
Ile Gly Ile Val Ser Val Gly Tyr Thr Ile Glu Gln Leu Glu
500 505 510
<210> 10
<211> 514
<212> PRT
<213> Artificial Sequence
<400> 10
Met Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
1 5 10 15
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
20 25 30
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
35 40 45
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
50 55 60
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
65 70 75 80
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
85 90 95
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
100 105 110
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
115 120 125
Ile Glu Gln Leu Glu Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp
130 135 140
Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val
145 150 155 160
Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
165 170 175
Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe
180 185 190
Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val
195 200 205
Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
210 215 220
Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu
225 230 235 240
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp
245 250 255
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
260 265 270
Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu
275 280 285
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys
290 295 300
Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
305 310 315 320
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
325 330 335
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
340 345 350
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp
355 360 365
Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Gly Ser Asp Ile
370 375 380
Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala Leu Ile Gln
385 390 395 400
Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala Val Gln Lys
405 410 415
Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met Arg Ser Phe
420 425 430
Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly Gln Arg Leu
435 440 445
Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu Gly Gly Asp
450 455 460
Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser Val Arg Lys
465 470 475 480
Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile Gln Asp Ala
485 490 495
Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr Ile Glu Gln
500 505 510
Leu Glu
<210> 11
<211> 517
<212> PRT
<213> Artificial Sequence
<400> 11
Met Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
1 5 10 15
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
20 25 30
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
35 40 45
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
50 55 60
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
65 70 75 80
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
85 90 95
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
100 105 110
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
115 120 125
Ile Glu Gln Leu Glu Thr Trp Asp Tyr Asn Ser Asp Asn Val Tyr Ile
130 135 140
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
145 150 155 160
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
165 170 175
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
180 185 190
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
195 200 205
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
210 215 220
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
225 230 235 240
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
245 250 255
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
260 265 270
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
275 280 285
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
290 295 300
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
305 310 315 320
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
325 330 335
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
340 345 350
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe
355 360 365
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Pro Asp
370 375 380
Pro Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
385 390 395 400
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
405 410 415
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
420 425 430
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
435 440 445
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
450 455 460
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
465 470 475 480
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
485 490 495
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
500 505 510
Ile Glu Gln Leu Glu
515
<210> 12
<211> 510
<212> PRT
<213> Artificial Sequence
<400> 12
Met Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
1 5 10 15
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
20 25 30
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
35 40 45
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
50 55 60
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
65 70 75 80
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
85 90 95
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
100 105 110
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
115 120 125
Ile Glu Gln Leu Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
130 135 140
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser
145 150 155 160
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
165 170 175
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu
180 185 190
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
195 200 205
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val
210 215 220
Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr
225 230 235 240
Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
245 250 255
Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys
260 265 270
Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
275 280 285
Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg
290 295 300
Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro
305 310 315 320
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn
325 330 335
Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
340 345 350
Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu
355 360 365
Gly His Lys Leu Glu Tyr Asn Pro Asp Pro Asp Ile Thr Glu Glu Arg
370 375 380
Leu His Tyr Gln Val Gly Gln Arg Ala Leu Ile Gln Ala Met Gln Ile
385 390 395 400
Ser Ala Met Pro Glu Leu Val Glu Ala Val Gln Lys Arg Asp Leu Ala
405 410 415
Arg Ile Lys Ala Leu Ile Asp Pro Met Arg Ser Phe Ser Asp Ala Thr
420 425 430
Tyr Ile Thr Val Gly Asp Ala Ser Gly Gln Arg Leu Tyr His Val Asn
435 440 445
Pro Asp Glu Ile Gly Lys Ser Met Glu Gly Gly Asp Ser Asp Glu Ala
450 455 460
Leu Ile Asn Ala Lys Ser Tyr Val Ser Val Arg Lys Gly Ser Leu Gly
465 470 475 480
Ser Ser Leu Arg Gly Lys Ser Pro Ile Gln Asp Ala Thr Gly Lys Val
485 490 495
Ile Gly Ile Val Ser Val Gly Tyr Thr Ile Glu Gln Leu Glu
500 505 510
<210> 13
<211> 512
<212> PRT
<213> Artificial Sequence
<400> 13
Met Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
1 5 10 15
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
20 25 30
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
35 40 45
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
50 55 60
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
65 70 75 80
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
85 90 95
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
100 105 110
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
115 120 125
Ile Glu Gln Leu Gln Arg Asp Asn Val Tyr Ile Met Ala Asp Lys Gln
130 135 140
Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp
145 150 155 160
Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
165 170 175
Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser
180 185 190
Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu
195 200 205
Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr
210 215 220
Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu
225 230 235 240
Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn
245 250 255
Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr
260 265 270
Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val
275 280 285
Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe
290 295 300
Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala
305 310 315 320
Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp
325 330 335
Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu
340 345 350
Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn
355 360 365
Ile Leu Gly His Lys Leu Glu Tyr Asn Pro Asp Pro Asp Ile Thr Glu
370 375 380
Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala Leu Ile Gln Ala Met
385 390 395 400
Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala Val Gln Lys Arg Asp
405 410 415
Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met Arg Ser Phe Ser Asp
420 425 430
Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly Gln Arg Leu Tyr His
435 440 445
Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu Gly Gly Asp Ser Asp
450 455 460
Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser Val Arg Lys Gly Ser
465 470 475 480
Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile Gln Asp Ala Thr Gly
485 490 495
Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr Ile Glu Gln Leu Glu
500 505 510
<210> 14
<211> 517
<212> PRT
<213> Artificial Sequence
<400> 14
Met Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
1 5 10 15
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
20 25 30
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
35 40 45
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
50 55 60
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
65 70 75 80
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
85 90 95
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
100 105 110
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
115 120 125
Ile Glu Gln Leu Glu Thr Leu Glu Tyr Asn Ser Asp Asn Val Tyr Ile
130 135 140
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
145 150 155 160
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
165 170 175
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
180 185 190
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
195 200 205
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
210 215 220
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
225 230 235 240
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
245 250 255
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
260 265 270
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
275 280 285
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
290 295 300
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
305 310 315 320
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
325 330 335
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
340 345 350
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe
355 360 365
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Pro Asp
370 375 380
Pro Asp Ile Thr Glu Glu Arg Leu His Tyr Gln Val Gly Gln Arg Ala
385 390 395 400
Leu Ile Gln Ala Met Gln Ile Ser Ala Met Pro Glu Leu Val Glu Ala
405 410 415
Val Gln Lys Arg Asp Leu Ala Arg Ile Lys Ala Leu Ile Asp Pro Met
420 425 430
Arg Ser Phe Ser Asp Ala Thr Tyr Ile Thr Val Gly Asp Ala Ser Gly
435 440 445
Gln Arg Leu Tyr His Val Asn Pro Asp Glu Ile Gly Lys Ser Met Glu
450 455 460
Gly Gly Asp Ser Asp Glu Ala Leu Ile Asn Ala Lys Ser Tyr Val Ser
465 470 475 480
Val Arg Lys Gly Ser Leu Gly Ser Ser Leu Arg Gly Lys Ser Pro Ile
485 490 495
Gln Asp Ala Thr Gly Lys Val Ile Gly Ile Val Ser Val Gly Tyr Thr
500 505 510
Ile Glu Gln Leu Glu
515
Claims (9)
1.一种融合蛋白,包括柠檬酸敏感多肽B和光学活性多肽A,其中光学活性多肽A位于两个柠檬酸敏感多肽B之间,形成B1-L1-A-L2-B2式的融合蛋白结构,其中,L1和L2是接头,B1和B2分别独立选自如SEQ ID NO: 1所示的CitA和如SEQ ID NO: 2所示的CcpE,所述光学活性多肽A选自以下的任意一种:如SEQ ID NO: 3的cpYFP、如SEQ ID NO: 4的cpGFP、如SEQID NO: 5的cpBFP、如SEQ ID NO: 6的cpmApple,
其中,
B1和B2是CitA,A是cpYFP,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、无/RSE、无/PAP、无/PDA、无/RVR、无/RED、无/PER、无/RNA、无/PDP、无/RGA、无/RPP、G/GS、G/GGS、GS/GS、GS/GGS、GGS/GS、GS/无、GGS/无,或者
B1和B2是CitA,A是cpGFP,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、G/无、G/G、G/GS、G/GGS、GGS/无、GGS/G、GGS/GS,或者
B1和B2是CitA,A是cpBFP,L1和L2的组合选自:无/GS、G/无、G/G、GS/G、GS/GS、GGS/无、GGS/G,或者
B1和B2是CitA,A是cpmApple,L1和L2的组合选自:无/无、无/GS、G/无、G/GS、G/GGS、GS/GS、GGS/无、GGS/G、GGS/GS,或者
B1和B2是CcpE,A是cpYFP,L1和L2的组合选自:无/G、无/GS、无/GGGS、无/GSGGS、无/GGSGGS、无/GGGSGGS、G/G、G/GS、G/GGS、G/GGGS、G/GSGGS、G/GGSGGS、G/GGGSGGS、GS/G、GS/GS、GS/GGS、GS/GGGS、GS/GSGGS、GS/GGSGGS、GS/GGGSGGS、GGS/G、GGS/GS、GGS/GGS、GGS/GGGS、GGS/GSGGS、GGS/GGSGGS、GGS/GGGSGGS、GGGS/G、GGGS/GS、GGGS/GGS、GGGS/GGGS、GGGS/GSGGS、GGGS/GGSGGS、GGGS/GGGSGGS、GSGGS/G、GSGGS/GS、GSGGS/GGS、GSGGS/GGGS、GSGGS/GSGGS、GSGGS/GGSGGS、GSGGS/GGGSGGS、GGSGGS/G、GGSGGS/GS、GGSGGS/GGS、GGSGGS/GGGS、GGSGGS/GSGGS、GGSGGS/GGSGGS、GGSGGS/GGGSGGS、GGGSGGS/G、GGGSGGS/GS、GGGSGGS/GGS、GGGSGGS/GGGS、GGGSGGS/GSGGS、GGGSGGS/GGSGGS、GGGSGGS/GGGSGGS、P/GGSGGS、H/GGSGGS、N/GGSGGS、S/GGSGGS、Q/GGSGGS、A/GGSGGS、D/GGSGGS、Y/GGSGGS、M/GGSGGS、T/GGSGGS、E/GGSGGS、C/GGSGGS、L/GGSGGS、V/GGSGGS、W/GGSGGS、I/GGSGGS、F/GGSGGS、K/GGSGGS、R/GGSGGS,或者
B1和B2是CcpE,A是cpGFP,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、无/GGGS、无/GSGGS、无/GGSGGS、无/GGGSGGS、G/无、G/G、G/GS、G/GSGGS、G/GGSGGS、G/GGGSGGS、GS/GS、GS/GSGGS、GS/GGSGGS、GS/GGGSGGS、GGS/G、GGS/GS、GGS/GGS、GGS/GGGS、GGS/GSGGS、GGS/GGSGGS、GGS/GGGSGGS、GGGS/G、GGGS/GS、GGGS/GSGGS、GGGS/GGSGGS、GSGGS/GS、GSGGS/GGGS、GSGGS/GSGGS、GGSGGS/GGGS、GGSGGS/GGSGGS、GGGSGGS/GS、GGGSGGS/GGS、GGGSGGS/GGGS、GGGSGGS/GSGGS、GGGSGGS/GGSGGS,或者
B1和B2是CcpE,A是cpBFP,L1和L2的组合选自:无/无、无/G、无/GSGGS、G/GGS、G/GGGS、GS/无、GS/GS、GS/GGS、GS/GGGS、GS/GGGSGGS、GGS/无、GGS/G、GGS/GS、GGS/GGS、GGS/GGGS、GGS/GSGGS、GGS/GGSGGS、GGS/GGGSGGS、GGGS/G、GGGS/GS、GGGS/GGS、GGGS/GGSGGS、GSGGS/无、GSGGS/GS、GSGGS/GGSGGS、GGSGGS/GGGS、GGSGGS/GGSGGS、GGGSGGS/GS、GGGSGGS/GGS、GGGSGGS/GGSGGS,或者
B1和B2是CcpE,A是cpmApple,L1和L2的组合选自:无/无、无/G、无/GS、无/GGS、无/GGGS、无/GSGGS、无/GGSGGS、无/GGGSGGS、G/GGS、G/GGGS、G/GSGGS、G/GGSGGS、G/GGGSGGS、GS/无、GS/GS、GS/GGGS、GS/GSGGS、GS/GGSGGS、GS/GGGSGGS、GGS/无、GGS/GS、GGS/GGS、GGS/GSGGS、GGS/GGSGGS、GGGS/GS、GGGS/GGGS、GGGS/GGGSGGS、GSGGS/G、GSGGS/GS、GSGGS/GSGGS、GGSGGS/G、GGSGGS/GS、GGSGGS/GGS、GGSGGS/GSGGS、GGGSGGS/G、GGGSGGS/GS、GGGSGGS/GSGGS、GGGSGGS/GGSGGS,或者
所述融合蛋白如SEQ ID NO: 7-14所示。
2.一种核酸分子,其包含
(1)权利要求1所述的多肽的编码序列,
(3)(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所述的多肽;
(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所述的多肽;
(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
检测柠檬酸所需的其他试剂。
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柠檬酸中的用途。
8.检测样品中柠檬酸的方法,包括:使权利要求1所述的融合蛋白或权利要求5所述的宿主细胞与样品接触,和检测光学活性多肽的变化,所述宿主细胞表达和/或分泌所述融合蛋白。
9.筛选影响细胞柠檬酸代谢的化合物方法,包括:使权利要求1所述的融合蛋白的细胞与候选化合物接触,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影响细胞柠檬酸代谢的化合物。
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