CN1476481A - 分离的荧光素酶及其用途 - Google Patents
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Abstract
本发明涉及荧光素酶LuAL、Lu164和Lu22的核苷酸和氨基酸序列，以及它们的活性和用途。
Description
本发明涉及荧光素酶LuAL、Lu164、Lu16、Lu39、Lu45、Lu52和Lu22的核苷酸和氨基酸序列以及它们的活性和用途。荧光素酶
发光表示发射可见光谱范围内的光子，这种发射是通过激发发光分子产生的。与荧光不同，这种光的能量不是以较短波长的辐射形式从外部提供的。
在化学发光和生物发光之间存在差别。化学发光表示一种化学反应，这种反应导致分子受到激发，当被激发的电子回到基态时，该分子自身发出光线。如果该反应是由酶催化时就叫做生物发光。参与所述反应的酶通常被称为荧光素酶。
有关发光生物的综述可以参见Hastings等1995。
荧光素酶是过氧化物酶或单加氧酶和双加氧酶。形成所述发光产物的原始物质的酶底物被称为荧光素。不同物种之间的荧光素有所不同。所述系统的量子产率为0.1-0.9个光子/转化底物分子(Idelgaufts，1993)。
可以根据其来源或酶促特性对荧光素酶进行分类。下面提供了某些类型荧光素酶的概述：细菌荧光素酶
表1：细菌荧光素酶Coelenterazine-依赖型真核荧光素酶
表2：Coelenterazine-依赖型真核荧光素酶Coelenterazine-非依赖型真核荧光素酶
表3：Coelenterazine-非依赖型真核荧光素酶
| 基因产物 | 生物 | 底物 | λ | 表达 | 参考文献/专利 |
| Lux基因 | Vibrio fischerii | FMN，十二烷醛NADH | 495nm | 细胞质 | Apley et al.，1985Gustafson G.，US 5196524 |
| 基因产物 | 生物 | 底物 | λ | 表达 | 参考文献/专利 |
| Renilla荧光素酶 | Renillareniformis | Coelenterazine | 480nm | 细胞质 | Mathews et al.，1977Lorenz et al，1991Lorenz et al.1996Alan，P；WO 0020619Milton J.，US 5418155Roelant C.，WO 9938999 |
| Vargula/Cypridia荧光素酶 | Vargulahilgendorferii | Vargula荧光素 | 460nm | 分泌 | Thomspon et al.，1989Thompson et al.，1990Tora，JP 05064583Tora，JP 08027200Renard et al.，WO 9520653 |
| Watasemia荧光素酶 | Wataseniascintillans | Watasemia荧光素 | ？ | 细胞质 | Inoue et al.，1976 |
| Olophorus荧光素酶 | Olophorusgracilirostris | Coelenterazine | 454 | 分泌 | Inouye et al，2000 |
| 水母蛋白 | Aequoriaaequoria | Coelenterazine(Ca2+激活的) | 470nm | 细胞质 | Head et al.2000Shimomura et al.，2000Jones et al.，1999Kendall et al.，1998Inouye et al.，1985Shimomura et al.，1969Cormier et al.，US 5798441Cormier et al.，US 5422266 |
| Obelin | Obelia | Coelenterazine | 470nm | 细胞质 | Matveev et al.，1999Berestovskaya，1999 |
| 基因产物 | 生物 | 底物 | λ | 表达 | 参考文献 |
| 萤火虫荧光素酶 | Photinuspyralis | 萤火虫荧光素，ATP | 550nm | 细胞质 | Webster et al.，1980Gould et al.，1988Sala-Newby et al，1992Bonin et al.，1994Sherf B.，US 5670356KIKK，JP 09187281 |
还可以根据某些荧光素酶的底物专一性区分各种荧光素酶。最重要的底物包括Coelenterazine(Jones等1999)和荧光素，以及这两种物质的衍生物。下面示出了这两种底物及其通过荧光素酶进行的转化的示意图：荧光素酶底物
下面通过举例形式示出了某些荧光素酶底物及其转化。本文所示出的所有底物是通过酶促转化的，同时释放出光和二氧化碳(CO2)并且消耗氧气(O2)。所述反应对辅因子或能量载体(例如，对于萤火虫荧光素酶来说是ATP)的依赖性是酶专一性的。
水母蛋白在将Coelenterazine转化成Coelenteramide时，会发出波长为470纳米的光线。荧光素
荧光素 羟基荧光素
萤火虫荧光素在将荧光素转化成羟基荧光素时，会发出波长为560纳米的光线。Vargula-荧光素
Vargula-荧光素 Vargula-羟基荧光素
Vargula荧光素酶在将Vargula荧光素转化成Vargula-羟基荧光素时会发出波长为460纳米的光线。报导系统
报导基因或指示基因通常是一种基因，其基因产物可以通过简单的生物化学或组织化学方法方便地检测到。至少有两种类型的报导基因是可以区别的。
1.抗性基因。抗性基因是一种基因，其表达会使细胞产生对抗生素或其他物质的抗性。在缺乏该抗性基因时，所述抗生素或其他物质在生长培养基中的存在会导致细胞死亡。
2.报导基因。在基因工程技术中，报导基因的产物是作为融合的或未融合的指示剂使用的。最常见的报导基因包括β-半乳糖苷酶(Alam等1990)，碱性磷酸酶(Yang等1997；Cullen等1992)，荧光素酶和其他发光蛋白(Shinomura，1985；Phillips GN，1997；Snowdowne等，1984)。Metridia longa物种的分类节肢动物门→甲壳纲→挠足亚纲
Metridia longa种属于甲壳纲，特别是挠足亚纲或浮游动物。分离cDNA
为了研究Metridia longa种的生物发光活性，从白海(Biologische Station Kartesh，俄罗斯)获得样品，并且保存在液氮中。为了制备Metridia longa的cDNA库，通过Krieg(Krieg等1996)的方法使用异硫氰酸盐分离RNA。
进行RT-PCR以便制备cDNA。为此，按照以下方案将10微克RNA与逆转录酶(Superscribt Gold II)一起培养：
PCR 1. 30秒钟 95℃
2. 6分钟 68℃
3. 10秒钟 95℃
4. 6分钟 68℃
在步骤3之后进行步骤4，进行17个循环
为了使聚合酶失活，在37℃下用蛋白酶K培养反应产物30分钟，并且用乙醇使cDNA沉淀。将cDNA溶解在水中，并且在37℃用SfiI培养1小时。对反应产物进行凝胶过滤，以便分离出小片段。然后将分离的cDNA连接到SfiI裂解过的并且脱磷酸化的λTriplEx2载体上。然后为了制备λ噬菌体表达库，使用体外包装系统中的SMART cDNA库构建试剂盒(Clontech)将克隆的cDNA片段包封到λ噬菌体中。
通过实施库筛选鉴定含有具有表达Colenterazin依赖型荧光素酶的潜力的cDNA插入片段的重组噬菌体。
为此，将由大肠杆菌XL1-Blue得到的菌苔铺平铺到90毫米的培养皿上，并且在37℃下培养10小时。然后每个培养皿用2500个噬菌体感染，然后在37℃下培养8小时，以便形成噬菌斑。然后将培养皿在4℃下保存1小时，以便使软的琼脂糖变硬。
为了进行复制铺板，用大肠杆菌XL1-Blue悬浮液饱和硝酸纤维素膜并且干燥。将干燥的膜在噬菌斑上放置60秒，然后铺放在新的琼脂糖平板上。然后在37℃下培养所述琼脂糖平板2小时，并且添加4毫升SOB培养基(+10mM MgSO4，0.2％麦芽糖)。分离菌苔，重新悬浮在LB培养基中(+20mM IPTG)，并且在37℃下培养1小时。通过离心收获细菌，并且通过超声波破碎，在添加Coelenterazin之后，在发光计中测定生物发光活性。
将能产生阳性生物发光信号的培养皿划分成多个扇区，并进行新的复制铺板。连续进行复制铺板，直到鉴定了活性个体噬菌斑。为了亚克隆阳性噬菌斑的噬菌体cDNA插入片段，按照SMART cDNA库构建试剂盒的生产商的方法在大肠杆菌BM25.8中的pTriplEx2载体中进行。在含有浓度为100微克/毫升的氨苄青霉素的LB培养基中在37℃下培养pTriplEx2-cDNA转染过的大肠杆菌过夜。为了获得超量表达，用LB培养基将过夜培养物按1∶150倍稀释，并且在37℃下培养1小时。然后通过添加IPTG(异丙基硫代半乳糖苷)至20mM的浓度进行培养。将诱导的培养基在37℃下培养1小时，并且通过离心收获细菌。通过在0.5毫升SM缓冲液中超声处理破碎所述细胞。在添加10微升Coelenterazin(10-4M，溶解在甲醇中)之后在发光计中测定化学发光。
鉴定了具有Coelenterazine依赖性荧光素酶活性的三种荧光素酶。所述荧光素酶被命名为Lu164、LuAL和Lu22。下面将详细披露所述荧光素酶。
本发明还涉及这三种荧光素酶的功能性等同物。功能性等同物是具有可比较的底物范围的荧光素酶，这种荧光素酶是分泌的并且具有至少70％的同源性。优选80％或90％的同源性。特别优选95％的同源性。
荧光素酶适合用作细胞系统的报导基因，特别是用作受体、离子通道、转载体、转录因子或诱导系统的报导基因。
可以将荧光素酶用于细菌系统中，例如，用于效价测定或用作生物化学系统，特别是蛋白酶的底物。
荧光素酶还可以用作与抗体或其他蛋白连接的报导酶，例如，用于ELISA，用于免疫组织化学或用于Western印迹。
可以将荧光素酶用于BRET(生物发光共振能量转移)系统。
荧光素酶还适合用作融合蛋白，用于进行共聚焦显微分析，或用于分析蛋白-蛋白相互作用。
荧光素酶还可用作与生物素、NHS，CN-Br或其他结合介体连接的报导酶，例如，用于ELISA或用于固定化。
另外，可以将荧光素酶用作与DNA或RNA寡核苷酸连接的报导酶，例如，用于Northern和Southern印迹或用于实时PCR。
本发明还涉及作为野生型或标记蛋白纯化荧光素酶，并且涉及荧光素酶在体外翻译系统中的应用。核苷酸和氨基酸序列LuAL
荧光素酶LuAL是分子量为23.7kDa，等电点为8.32的蛋白。其编码核苷酸序列为：5`atggatatgagggttatctttgctcttgttttctcatcattggttcaggccaaatcaactgaattcgatcctaacattaacattgttggtttagaaggaaaatttggtataacaaaccttgagacggatttattcacaatatgggagacaatggatgtcatcaaatcagatattacagatactgatagagtcagcaactttgttgcaactgaaaccgatgctaaccgtgggaaaatgcctggcaaaaaactgccactggcagttatcatggaaatggaagccaatgctttcaaagctggctgcaccaggggatgccttatctgtctttcaaaaataaagtgtacagccaaaatgaaggtgtacattccaggaagatgtcatgattatggtggtgacaagaaaactggacaggcaggaatagttggtgcaattgttgacattcccgaaatctctggatttaaggagatggcacccatggaacagttcattgctcaagttgatctttgcgctacctgcactactggatgtctcaaaggtcttgccaatgttaagtgctctgaactcctgaagaaatggctgcctggcagatgtgcaagttttgctgacaagattcaaaaagaagttcacaatatcaaaggcatggctggagatcgttga 3`
它能产生以下氨基酸序列：MDMRVIFALVFSSLVQAKSTEFDPNINIVGLEGKFGITNLETDLFTIWETMDVIKSDITDTDRVSNFVATETDANRGKMPGKKLPLAVIMEMEANAFKAGCTRGCLICLSKIKCTAKMKVYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMAPMEQFIAQVDLCATCTTGCLKGLANVKCSELLKKWLPGRCASFADKIQKEVHNIKGMAGDR
和以下氨基酸组成：Ala：18(8.3％) Cys：10(4.6％) Asp：14(6.4％) Glu：12(5.5％)Phe：10(4.6％) Gly：19(8.7％) His： 2(0.9％) Ile：18(8.3％)Lys：21(9.6％) Leu：15(6.9％) Met：10(4.6％) Asn： 8(3.7％)Pro： 7(3.2％) Gln： 5(2.3％) Arg： 7(3.2％) Ser： 9(4.1％)Thr：15(6.9％) Val：14(6.4％) Trp： 2(0.9％) Tyr： 2(0.9％)Lu164
荧光素酶Lu164是分子量为23.8kDa，等电点为7.81的蛋白。其编码核苷酸序列为：5`atggatataaaggttgtctttactcttgttttctcagcattggttcaggcaaaatcaactgaattcgatcctaacattgacattgttggtttagaaggaaaatttggtataacaaaccttgagacggatttattcacaatatgggagacaatggaggtcatgatcaaagcagatattgcagatactgatagagccagcaactttgttgcaactgaaaccgatgctaaccgtggaaaaatgcctggcaaaaaactgccactggcagttatcatggaaatggaagccaatgctttcaaagctggctgcaccaggggatgccttatctgtctttcaaaaataaagtgtacagccaaaatgaaggtgtacattccaggaagatgtcatgattatggtggtgacaagaaaactggacaggcaggaatagttggtgcaattgttgacattcccgaaatctctggatttaaggagatggcacccatggaacagttcattgctcaagttgaacgttgcgcttcctgcactactggatgtctcaaaggtcttgccaatgttaagtgctctgaactcctgaagaaatggctgcctgacagatgtgcaagttttgctgacaagattcaaaaagaagttcacaatatcaaaggcatggctggagatcgttga 3`
它能产生以下氨基酸序列：MDIKVVFTLVFSALVQAKSTEFDPNIDIVGLEGKFGITNLETDLFTIWETMEVMIKADIADTDRASNFVATETDANRGKMPGKKLPLAVIMEMEANAFKAGCTRGCLICLSKIKCTAKMKVYIPGRCHDYGGDKKTGQAGIVGAIVDIPEISGFKEMAPMEQFIAQVDRCASCTTGCLKGLANVKCSELLKKWLPDRCASFADKIQKEVHNIKGMAGDR
并且具有以下氨基酸组成：Ala：21(9.6％) Cys：10(4.6％) Asp：15(6.8％) Glu：13(5.9％)Phe：10(4.6％) Gly：18(8.2％) His： 2(0.9％) Ile：18(8.2％)Lys：22(10.0％) Leu：14(6.4％) Met：10(4.6％) Asn： 7(3.2％)Pro： 7(3.2％) Gln： 5(2.3％) Arg： 7(3.2％) Ser： 8(3.7％)Thr：14(6.4％) Val：14(6.4％) Trp： 2(0.9％) Tyr： 2(0.9％)Lu22
荧光素酶Lu22是分子量为20.2kDa，等电点为7.89的蛋白。其编码核苷酸序列为：5`atgggagtcaaacttatttttgctgttgtttgtgtcgcagttgcccaggctgccacaattcaggaaaattttgaagacattgatcttgtagccataggtggcagctttgcatcagatgttgatgctaacagaggtggacatggtggacatcctggcaaaaagatgccaaaagaagtacttatggaaatggaagccaatgctaaacgagctggctgccacaggggttgtctggtttgtctgtcacacatcaagtgcacagcacaaatgcagaagtttatcccaggaagatgccatagttatgcaggagacaaggattctgctcagggaggaattgccggtggtgccattgttgatatacctgaaattgccggatttaaagaaatgaagcccatggaacagttcattgctcaagttgatctctgtgaagattgcacaactggatgcctcaaaggtcttgccaatgttcattgctctgatctcctgaagaagtggctgccatcaagatgtaagacatttgcttccaaaattcaatctcaagtggataccatcaaaggtttggctggagatcgttga 3`
它能产生以下氨基酸序列：MGVKLIFAVVCVAVAQAATIQENFEDIDLVAIGGSFASDVDANRGGHGGHPGKKMPKEVLMEMEANAKRAGCHRGCLVCLSHIKCTAQMQKFIPGRCHSYAGDKDSAQGGIAGGAIVDIPEIAGFKEMKPMEQFIAQVDLCEDCTTGCLKGLANVHCSDLLKKWLPSRCKTFASKIQSQVDTIKGLAGDR
并且具有以下氨基酸组成：Ala：21(11.1％) Cys：11(5.8％) Asp：12(6.3％) Glu： 9(4.7％)Phe： 7(3.7％) Gly：21(11.1％) His： 6(3.2％) Ile：13(6.8％)Lys：16(8.4％) Leu：12(6.3％) Met： 7(3.7％) Asn： 4(2.1％)Pro： 6(3.2％) Gln： 9(4.7％) Arg： 6(3.2％) Ser： 9(4.7％)Thr： 6(3.2％) Val：13(6.8％) Trp： 1(0.5％) Tyr： 1(0.5％)
在序列表中也示出了上述序列。荧光素酶的酶促活性和生物化学表征
蛋白LuAL、Lu164和Lu22是在转化Coelenterazin时能发出光线的酶。因此，它们属于荧光素酶。荧光素酶可以在细菌和真核细胞中有效表达。在真核细胞中表达的荧光素酶LuAl、Lu164和Lu22是分泌型的，不会发生与细菌表达相关的分泌。
荧光素酶的活性是温度依赖型的。测定荧光素酶LuAL和Lu164的最佳温度分别为22℃(LuAL)和27℃(Lu164)。荧光素酶Lu22活性的最佳温度为4℃或更低。
实施例质粒/结构
用于制备下文所披露的结构的载体是购自Clontech公司的载体pcDNA3.1(+)和pTriplEx2，以及载体pASMplr(具有cAMP-敏感型启动子元件的固有结构；cre)。所述载体的衍生物被命名为pcDNA3-x、pTriplEx2-x和pASM-x。LuAL
图1表示载体pTriplEX2-LuAL、pcDNA3-LuAL和pASM-LuAL的质粒图谱。
图2表示载体pTriplEX2-Lu164、pcDNA3-Lu164和pASM-Lu164的质粒图谱。
图3表示载体pTriplEX2-Lu22、pcDNA3-Lu22和pASM-Lu22的质粒图谱。作为Lu164底物的Coelenterazin衍生物
为了鉴定Lu164的底物，在每一种情况下将10微升不同的Coelenterazin衍生物(10-4M)的溶液与来自CHO-pcDNA3-Lu164细胞系的10微升上清液一起培养，并且测定发光。Coelenterazine获自Molecular Probes(USA)。
荧光素酶LuAL和Lu22与荧光素酶Lu164相比没有差别。确定未修饰过的Colenterazin(图B，Coelenterazin a)是Lu164、LuAl和Lu22的最佳底物。
图4表示作为Lu164的潜在底物的Coelenterazin衍生物和在发光计(RLU，相对光学单位)中以8.7kV的电压测定发光30秒所获得的曲线；以及Coelenterazin衍生物的分子结构示意图。荧光素酶Lu164、LuAL和Lu22依赖于Coelenterazin的酶促活性细菌表达
细菌表达是通过用表达质粒pTriplEX2-Lu164、pTriplEX2-LuAL和pTriplEX2-Lu22转化细菌在大肠杆菌菌株BL21(DE3)中进行的。在37℃下，在LB培养基中培养转化过的细菌3小时，通过添加IPTG至1mM的最终浓度诱导表达4小时。通过离心收获诱导过的细菌，重新悬浮在PBS中，并且通过超声波破碎。将Coelenterazin(10-4M，溶解在甲醇中)或荧光素(萤火虫荧光素)添加到5微升裂解物(5mg/ml)中，并且测定化学发光。
所述测定，用RLU(相对发光单位)表示，是在9.5kV的电压下进行30秒。所测定的Lu164、LuAL和Lu22的值分别为230000、320000和260000 RLU。所述表达是使用载体pTriplEx2-Lu164、pTriplEx2-LuAL和pTriplEx2-Lu22在大肠杆菌BL21(DE3)中进行的。真核表达
在瞬时实验中，通过用表达质粒pcDNA3-Lu164、pcDNA3-LuAL和pcDNA3-Lu22转染细胞影响在CHO细胞中的组成型真核表达。为此，在96孔微量滴定板的每个孔中接种10000个悬浮在DMEM-F12培养基中的细胞，并且在37℃下培养过夜。转染是用Fugene 6试剂盒(Roche)按照生产商的说明进行的。转染过的细胞在37℃下，在DMEM-F 12培养基中培养过夜。在添加Coelenterazin(10-4M，溶解在甲醇中)之后，在室温下，用发光计以9.5kV的电压测定培养基(5微升)和细胞裂解物(5微升)中的化学发光30秒。
所测定的Lu164、LuAL和Lu22的值分别为680000、670000和510000 RLU(相对发光单位)。所述表达是使用载体pcDNA3-Lu164、pcDNA3-LuAL和pcDNA3-Lu22在CHO细胞中进行的。荧光素酶Lu164、LuAL和Lu22的发光光谱
为了测定发光光谱，用质粒pTriplEx2-Lu164、pTriplEx2-LuAL和pTriplEx2-Lu22转化大肠杆菌BL21(DE3)细胞，并且按照下面的3.1部分所披露的方法进行超量表达。将50微升Coelenterazin(10-4M)添加到100微升体积的细菌裂解物中，并且测定发光光谱。荧光素酶的发光光谱曲线如下文所示。
对于荧光素酶LuAL、Lu164和Lu22来说，通过所述的底物转化所产生的最大发光发生在大约490纳米的波长下。
图5表示在细菌表达之后荧光素酶Lu164(A)、LuAL(B)和Lu22(C)的发光光谱(RLU，相对发光单位)。荧光素酶Lu164、LuAL和Lu22从CHO细胞中的分泌，以Lu164和LuAL为例
为了表征荧光素酶LuAL、Lu164和Lu22在真核细胞中的表达，用质粒pcDNA3-LuAl、pcDNA3-Lu164、pcDNA3-Fireluc和pcDNA3.1(+)稳定转染CHO细胞。所得到的克隆在DMEM-F12培养基中培养。将萤火虫荧光素酶用作非分泌型荧光素酶的阳性对照。将质粒pcDNA3.1(+)用作对照质粒，检测CHO亲本细胞中的潜在内源活性。
为了检测荧光素酶的分泌，将2000个细胞平铺到384孔微量滴定板上。在24小时之后，取出培养基，并且用Tyrode溶液洗涤细胞，并且添加30微升新的培养基。对于萤火虫荧光素酶来说，在添加5微升Coelenterazin(10-4M)或荧光素之后，用发光计以9.5kV的电压首次测定(0小时)30秒。在经过1-5小时之后进行1-5小时的测定。
图6表示培养基中荧光素酶活性随着时间的提高。没有分泌萤火虫荧光素酶。荧光素酶LuAL、Lu164和Lu22是分泌型荧光素酶。
图6表示在用pcDNA3-LuAL、pcDNA3-Firefly、pcDNA3-Lu164和pcDNA3作为对照载体的无cDNA插入片段的转染之后CHO细胞培养基(5微升)中的荧光素酶活性(RLU，相对发光单位；h是小时；Firefly：萤火虫荧光素酶)。荧光素酶活性对温度的依赖性
为了测定荧光素酶Lu22、Lu164和LuAL对温度的依赖性，用载体pcDNA3-Lu22、pcDNA3-Lu164和pcDNA3-LuAL瞬时转染CHO细胞，并且在0-47℃下测定上清液中的荧光素酶活性。为此，让细胞上清液和Coelenterazin溶液适应测定温度5分钟。测定是在发光计中以9.5kV的电压进行30秒。
图7表示所测定的荧光素酶LuAl、Lu164和Lu22依赖于温度的发光情况。LuAL的荧光素酶活性的最佳温度为27℃。对于Lu164最佳温度为22℃和对于Lu22所测定的最佳温度为4℃或更低。
图7表示在用pcDNA3-LuAL、pcDNA3-Firefly和pcDNA3-Lu164转染之后CHO细胞培养基(5微升)中的温度依赖型荧光素酶活性(RLU：相对发光单位；培养基：DMEM-F12+10％FCS)在CHO细胞中诱导荧光素酶Lu164、LuAL和Lu22表达，以LuAL为例
在瞬时实验中，通过用表达质粒pASM-LuAL转染细胞在CHO细胞中诱导真核表达。为此，在96孔微量滴定板的每个孔中接种10000个悬浮在DMEM-F12培养基中的细胞，并且在37℃下培养过夜。用Fugene6试剂盒(Roche)按照生产商的说明进行转染。转染过的细胞在37℃下，在DMEM-F12培养基中培养过夜。然后用毛喉萜(Forskolin)(10-5M)诱导所述细胞5小时。在添加Coelenterazin(10-4M，溶解在甲醇中)之后，用发光计以9.5kV的电压测定培养基和细胞裂解物的化学发光30秒。
图8表示LuAL在CHO细胞中的诱导表达。在37℃下用毛喉萜(10-5M)诱导表达5小时。测定10微升细胞上清液中的活性(RLU：相对发光单位；诱导因子：诱导过的RLU与未诱导过的RLU的比例)在细胞系统中用荧光素酶Lu164、LuAL和Lu22作为报导基因，以报导体NPY2和A2A为例，并且使用LuAL作为报导基因
为了能够分析受体专一性配体在基于细胞的系统中对G蛋白结合受体的激活作用，将荧光素酶LuAL的cDNA序列克隆到表达载体pASMplr中。表达载体pASMplr含有cAMP-敏感型启动子元件(CRE)，它能够间接测定cAMP的细胞内浓度。在该系统中，荧光素酶被用作报导基因。
以G蛋白结合受体NPY2(神经肽受体2)和A2A(腺苷受体2a)为例说明了在细胞系统中将荧光素酶Lu22、Lu164和Lu22用作报导基因的用途。为此，用载体pcDNA3-NPY2或pcDNA3-A2A对稳定克隆CHO-pASM-LuAL进行瞬时转染。受体NPY2是Gi结合受体，而A2A受体是Gs结合受体。
通过添加1μM NECA活化A2A受体4小时。在存在10-5M毛喉萜的条件下，通过添加10μM NPY2肽活化NPY2受体。在添加Coelenterazin(10-4M)之后用发光计以9.5kV的电压测定培养基(30微升)中的荧光素酶活性30秒。
图9表示将荧光素酶用作细胞系统的报导基因的用途，以G蛋白结合受体A2A和NPY2为例(RLU：相对发光单位)。参考文献/专利文献
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序列表<110>拜尔公司<120>分离的荧光素酶及其用途<130>LeA34790<150>10058091.2<151>2000-11-23<160>10<170>PatentIn version 3.1<210>1<211>660<212>DNA<213>Metridia longa<400>1atggatataa aggttgtctt tactcttgtt ttctcagcat tggttcaggc aaaatcaact 60gaattcgatc ctaacattga cattgttggt ttagaaggaa aatttggtat aacaaacctt 120gagacggatt tattcacaat atgggagaca atggaggtca tgatcaaagc agatattgca 180gatactgata gagccagcaa ctttgttgca actgaaaccg atgctaaccg tggaaaaatg 240cctggcaaaa aactgccact ggcagttatc atggaaatgg aagccaatgc tttcaaagct 300ggctgcacca ggggatgcct tatctgtctt tcaaaaataa agtgtacagc caaaatgaag 360gtgtacattc caggaagatg tcatgattat ggtggtgaca agaaaactgg acaggcagga 420atagttggtg caattgttga cattcccgaa atctctggat ttaaggagat ggcacccatg 480gaacagttca ttgctcaagt tgaacgttgc gcttcctgca ctactggatg tctcaaaggt 540cttgccaatg ttaagtgctc tgaactcctg aagaaatggc tgcctgacag atgtgcaagt 600tttgctgaca agattcaaaa agaagttcac aatatcaaag gcatggctgg agatcgttga 660<210>2<211>219<212>PRT<213>Metridia longa<400>2Met Asp Ile Lys Val Val Phe Thr Leu Val Phe Ser Ala Leu Val Gln1 5 10 15Ala Lys Ser Thr Glu Phe Asp Pro Asn Ile Asp Ile Val Gly Leu Glu
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130 135 140Thr Thr Gly Cys Leu Lys Gly Leu Ala Asn Val His Cys Ser Asp Leu145 150 155 160Leu Lys Lys Trp Leu Pro Ser Arg Cys Lys Thr Phe Ala Ser Lys Ile
165 170 175Gln Ser Gln Val Asp Thr Ile Lys Gly Leu Ala Gly Asp Arg
180 185 190<210>7<211>657<212>DNA<213>Metridia longa<400>7atggatatga aggttatctt tgctcttatt ttctcagcat tggttcaggc caaatcaact 60gaattcgatc ctaacattga cattgttggt ttagaaggaa aatttggtat aacaaacctt 120gagacggatt tattcacaat atgggagaca atggaggtca tcaaatcaga tattgcagat 180actgatagag tcagcaactt tgttgcaact gaaaccgatg ctaaccgtgg gaaaatgcct 240ggcaaaaaac tgccactggc agttatcatg gaaatggaag ccaatgcttt caaagctggc 300tgcaccaggg gatgccttat ctgtctttca aaaataaagt gtacagccaa aatgaaggtg 360tacattccag gaagatgtca tgattatggt ggtgacaaga aaactggaca ggcaggaata 420gttggtgcaa ttgttgacat tcccgaaatc tctggattta aggagatggc acccatggaa 480cagttcattg ctcaagttga tcgttgcact tcctgcacta ctggatgtct caaaggtctt 540gccaatgtta agtgctctga actcctgaag aaatggctgc ctgacagatg tgcaagtttt 600gctgacaaga ttcaaaaaga agttcacaat atcaaaggca tggctggaga tcgttga 657<210>8<211>630<212>DNA<213>Metridia longa<400>8atggatatca aagttctttt tgctcttatt tgcattgcat tggtccaggc caatccaact 60gaaaacaatg atcacattaa tattgttggt atagaaggga aatttggtat aacagatctt 120gaaacggatt tattcaccat atgggagaca aaccgtatga tcagtacaga taatgaacaa 180gccaacacag attctaaccg tggtaaaatg cctgggaaaa aattaccact ggcagtactc 240atagaaatgg aagccaatgc ttttaaagct ggctgcacca ggggatgtct tatttgtctt 300tctaaaatca agtgtacagc caaaatgaag aagtacattc caggaagatg tcatgattac 360ggaggagaca agaaaactgg acaggcaggc atagttggag ctattgttga cattcctgat 420atctctggat ttaaagagat gggacccatg gagcagttca ttgctcaagt tgatcgctgc 480accgactgca ctactggctg cctcaaaggt cttgccaatg tcaagtgctc tgaactcctc 540aaaaaatggc tgccagacag atgtgcaagt tttgctgaca aaattcaaag tgaagtgcac 600aacattaagg gccttgctgg agatcgttga 630<210>9<211>657<212>DNA<213>Metridia longa<400>9atggatataa aggttgtctt tgctcttgtt ttctctgcat tggttcaggc caaatcaact 60gaattcgatc ctaacattga cattgttggt ttagaaggaa aatttggtat aacaaacctt 120gagacggatt tattcacaat atgggagaca atggaggtca tcaaaacaga tattgcagat 180actgatagag ccagaagctt tgttgcaact gaaaccgatg ctaaccgtgg gaaaatgcct 240ggcaaaaaac tgccactggc agttatcatg gaaatggaag ccaatgcttt caaagctggc 300tgcaccaggg gatgccttat ctgtctttca aaaataaagt gtacagccaa aatgaaggtg 360tacattccag gaagatgcca tgattatggt ggtgacaaga aaactggaca ggcaggaata 420gtaggtgcaa ttgttgacat tcccgaaatc tctggattta aggagatgga acccatggaa 480cagttcattg ctcaagttga tcgttgcgct tcctgcacta ctggatgtct caaaggtctt 540gccaatgtta agtgctctga actcctgaag aaatggctgc ctgacagatg tgcaagtttt 600gctgacaaga ttcaaaaaga agttcacaat atcaaaggca tggctggaga tcgttga 657<210>10<211>657<212>DNA<213>Metridia longa<400>10atggatataa aggttgtctt tgctcttgtt ttctctgcat tggttcaggc caaatcaact 60gaattcgatc ctaacattga cgttgttggt ttagaaggaa aatttggtat aacaaacctt 120gagacggatt tattcacaat atgggagaca atggaggtca tcaaaacaga tattgcagat 180actgatagag ccagaaactt tgttgcaact gaaaccgatg ctaaccgtgg gaaaatgcct 240ggcaaaaaac tgccactggc agttatcatg gaaatggaag ccaatgcttt caaagctggc 300tgcaccaggg gatgccttat ctgtctttca aaaataaagt gtacagccaa aatgaaggtg 360tacattccag gaagatgcca tgattatggt ggtgacaaga aaactggaca ggcaggaata 420gtaggtgcaa ttgttgacat tcccgaaatc tctggattta aggagatgga acccatggaa 480cagttcattg ctcaagttga tcgttgcgct tcctgcaata ctggatgtct caaaggtctt 540gccaatgtta agtgctctga actcctgaag aaatggctgc ctgacagatg tgcaagtttt 600gctgacaaga ttcaaaaaga agttcacaat atcaaaggca tggctggaga tcgttga 657
Claims (8)
1.一种DNA或RNA分子，它们具有荧光素酶LuAL、Lu164、Lu16、Lu39、Lu45，Lu52或Lu22的序列或其功能性等同物。
2.如权利要求1的分子，其中所述序列包括位于该序列5’方向的功能性启动子。
3.如权利要求2的分子，它是重组DNA或RNA载体的一部分。
4.一种包含权利要求3所述载体的生物。
5.一种寡核苷酸，它包括与所述DNA或RNA序列相同或互补的10个以上的连续核苷酸。
6.一种肽，它是由权利要求1的核苷酸序列编码的。
7.一种肽，它包括被抗Lu164、LuAL或Lu22的抗体免疫识别的5个以上连续的氨基酸。
8.如权利要求1-6的荧光素酶作为细胞系统报导基因的用途。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE10058091A DE10058091A1 (de) | 2000-11-23 | 2000-11-23 | Isolierte Luziferasen Lu164, LuAL und Lu22, sowie deren Verwendung |
| DE10058091.2 | 2000-11-23 |
Related Child Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200510118849XA Division CN1847399B (zh) | 2000-11-23 | 2001-11-22 | 分离的荧光素酶及其用途 |
| CN2012102094057A Division CN102732537A (zh) | 2000-11-23 | 2001-11-22 | 分离的荧光素酶及其用途 |
| CN2010105056541A Division CN101979586B (zh) | 2000-11-23 | 2001-11-22 | 分离的荧光素酶及其用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1476481A true CN1476481A (zh) | 2004-02-18 |
| CN1303214C CN1303214C (zh) | 2007-03-07 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200510118849XA Expired - Lifetime CN1847399B (zh) | 2000-11-23 | 2001-11-22 | 分离的荧光素酶及其用途 |
| CN2010105056541A Expired - Lifetime CN101979586B (zh) | 2000-11-23 | 2001-11-22 | 分离的荧光素酶及其用途 |
| CN2012102094057A Pending CN102732537A (zh) | 2000-11-23 | 2001-11-22 | 分离的荧光素酶及其用途 |
| CNB018194338A Expired - Lifetime CN1303214C (zh) | 2000-11-23 | 2001-11-22 | 分离的荧光素酶及其用途 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200510118849XA Expired - Lifetime CN1847399B (zh) | 2000-11-23 | 2001-11-22 | 分离的荧光素酶及其用途 |
| CN2010105056541A Expired - Lifetime CN101979586B (zh) | 2000-11-23 | 2001-11-22 | 分离的荧光素酶及其用途 |
| CN2012102094057A Pending CN102732537A (zh) | 2000-11-23 | 2001-11-22 | 分离的荧光素酶及其用途 |
Country Status (19)
| Country | Link |
|---|---|
| US (4) | US7297483B2 (zh) |
| EP (1) | EP1339853B1 (zh) |
| JP (2) | JP4179877B2 (zh) |
| KR (1) | KR100841808B1 (zh) |
| CN (4) | CN1847399B (zh) |
| AT (1) | ATE335824T1 (zh) |
| AU (3) | AU2002220697A1 (zh) |
| BR (2) | BRPI0115534B1 (zh) |
| CA (1) | CA2429451C (zh) |
| DE (2) | DE10058091A1 (zh) |
| DK (1) | DK1339853T3 (zh) |
| ES (1) | ES2271111T3 (zh) |
| HK (3) | HK1096423A1 (zh) |
| IL (2) | IL155878A0 (zh) |
| MX (1) | MXPA03004539A (zh) |
| PL (1) | PL206385B1 (zh) |
| PT (1) | PT1339853E (zh) |
| WO (2) | WO2002042469A1 (zh) |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103582701A (zh) * | 2010-12-03 | 2014-02-12 | 基因流股份有限公司 | 改善的光-发射分子 |
| CN109612981A (zh) * | 2012-12-12 | 2019-04-12 | 普洛麦格公司 | 使用细胞内生物发光共振能量转移识别生物活性剂的细胞靶标结合 |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10058091A1 (de) | 2000-11-23 | 2002-06-06 | Bayer Ag | Isolierte Luziferasen Lu164, LuAL und Lu22, sowie deren Verwendung |
| DE102007005804A1 (de) * | 2007-02-06 | 2008-08-07 | Bayer Healthcare Ag | Sekretierte Luziferase Lu164M3 und deren Verwendung |
| DE102007005803A1 (de) | 2007-02-06 | 2008-08-07 | Bayer Healthcare Ag | Sekretierte Luziferase MLuc7 und deren Verwendung |
| JP5605727B2 (ja) | 2010-04-06 | 2014-10-15 | Ｊｎｃ株式会社 | セレンテラジン類縁体及びセレンテラミド類縁体 |
| US8435777B1 (en) | 2012-01-20 | 2013-05-07 | Cayla | CPG-free gene for a new secreted reporter protein |
| US10533231B2 (en) | 2012-10-26 | 2020-01-14 | National Institute Of Advanced Industrial Science And Technology | Artificial bioluminescent enzyme |
| JP6132350B2 (ja) * | 2012-10-26 | 2017-05-24 | 国立研究開発法人産業技術総合研究所 | 人工生物発光酵素 |
| WO2015056762A1 (ja) * | 2013-10-18 | 2015-04-23 | 独立行政法人産業技術総合研究所 | 人工生物発光酵素に用いるための発光基質 |
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| US5604123A (en) * | 1988-08-09 | 1997-02-18 | Toray Industries, Inc. | Luciferase, gene encoding the same and production process of the same |
| US5838313A (en) * | 1995-11-20 | 1998-11-17 | Siemens Corporate Research, Inc. | Multimedia-based reporting system with recording and playback of dynamic annotation |
| CA2324648C (en) * | 1998-03-27 | 2013-02-26 | Prolume, Ltd. | Luciferases, fluorescent proteins, nucleic acids encoding the luciferases and fluorescent proteins and the use thereof in diagnostics, high throughput screening and novelty items |
| US6853375B2 (en) * | 2000-05-10 | 2005-02-08 | Cognos Incorporated | Method for preemptive screen rendering |
| DE10058091A1 (de) * | 2000-11-23 | 2002-06-06 | Bayer Ag | Isolierte Luziferasen Lu164, LuAL und Lu22, sowie deren Verwendung |
| US6643635B2 (en) * | 2001-03-15 | 2003-11-04 | Sagemetrics Corporation | Methods for dynamically accessing, processing, and presenting data acquired from disparate data sources |
| US8036939B2 (en) * | 2001-06-08 | 2011-10-11 | Servigistics, Inc. | Reporting in a supply chain |
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| US20060230234A1 (en) * | 2005-03-30 | 2006-10-12 | Sap Ag. | Browser cache management |
| US7779347B2 (en) * | 2005-09-02 | 2010-08-17 | Fourteen40, Inc. | Systems and methods for collaboratively annotating electronic documents |
| US7831464B1 (en) * | 2006-04-06 | 2010-11-09 | ClearPoint Metrics, Inc. | Method and system for dynamically representing distributed information |
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2000
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- 2001-11-12 WO PCT/EP2001/013057 patent/WO2002042469A1/de unknown
- 2001-11-22 CN CN200510118849XA patent/CN1847399B/zh not_active Expired - Lifetime
- 2001-11-22 WO PCT/EP2001/013597 patent/WO2002042470A1/de active IP Right Grant
- 2001-11-22 KR KR1020037006911A patent/KR100841808B1/ko active IP Right Grant
- 2001-11-22 PL PL362774A patent/PL206385B1/pl unknown
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- 2001-11-22 CN CN2012102094057A patent/CN102732537A/zh active Pending
- 2001-11-22 IL IL15587801A patent/IL155878A0/xx active IP Right Grant
- 2001-11-22 DK DK01994705T patent/DK1339853T3/da active
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- 2001-11-22 BR BR0115534-2A patent/BR0115534A/pt active IP Right Grant
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103582701A (zh) * | 2010-12-03 | 2014-02-12 | 基因流股份有限公司 | 改善的光-发射分子 |
| CN103582701B (zh) * | 2010-12-03 | 2018-08-14 | 基因流股份有限公司 | 改善的光-发射分子 |
| US10428317B2 (en) | 2010-12-03 | 2019-10-01 | Gene Stream Pty Ltd | Light-emitting molecules |
| US11661587B2 (en) | 2010-12-03 | 2023-05-30 | Promega Corporation | Light-emitting molecules |
| CN109612981A (zh) * | 2012-12-12 | 2019-04-12 | 普洛麦格公司 | 使用细胞内生物发光共振能量转移识别生物活性剂的细胞靶标结合 |
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