CN114249833B - 一种基因编码的无机镉荧光探针及其制备方法和应用 - Google Patents

一种基因编码的无机镉荧光探针及其制备方法和应用 Download PDF

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CN114249833B
CN114249833B CN202111568781.0A CN202111568781A CN114249833B CN 114249833 B CN114249833 B CN 114249833B CN 202111568781 A CN202111568781 A CN 202111568781A CN 114249833 B CN114249833 B CN 114249833B
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胡树林
梁书利
林影
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Abstract

本发明公开了一种基因编码的无机镉荧光探针及其制备方法和应用，所述荧光探针包括对具有对镉离子结合能力的蛋白CadR和荧光蛋白cpYFP；所述的荧光蛋白cpYFP插入到结合蛋白CadR中，将CadR分为CadR1和CadR2两个部分；本发明提供的镉离子荧光探针，荧光动态变化大，特异性较好，并且能在大肠杆菌中表达，能定量检测镉离子。实验效果表明，本申请所提供的镉离子荧光探针对镉离子的检测限可达10nM，动态范围可达220％。

Description

一种基因编码的无机镉荧光探针及其制备方法和应用
技术领域
本发明属于荧光探针制备领域，具体涉及一种基因编码的无机镉荧光探针及其制备方法和应用。
背景技术
镉是一种有害的重金属元素。它广泛分布于水、土壤、农产品中，通过食物链传播，在人体内蓄积。对人体造成极大危害，引起广泛关注。镉的摄入会对人体的肾脏、肺、骨骼、神经系统产生不利影响，在人体内的生物半衰期为17-30年。镉的长期存在会引起肾功能障碍、钙代谢紊乱，增加各种疾病的发病率。由于这些原因，检测痕量镉至关重要。因此，检测和量化环境和食品样品中痕量镉至关重要。
镉的传统检测主要是通过电感耦合等离子体质谱(IPC-MS)和原子吸收光谱(AAS)等技术进行灵敏检测。但这些方法存在成本高、技术难度大、样品前处理耗时、无法进行实时检测、需要精密仪器参与等缺点。
发明内容
本发明第一方面的目的，在于提供一种荧光探针。本发明第二方面的目的，在于提供本发明第一个方面的荧光探针相关的生物材料。本发明第三方面的目的，在于提供本发明第一方面的探针或本发明第二方面的生物材料在检测样品中的镉离子中的应用。本发明第四方面的目的，在于提供本发明第一方面的探针或本发明第二方面的生物材料在制备镉离子检测产品中的应用。本发明第五方面的目的，在于提供本发明第一方面的探针的制备方法。本发明第六方面的目的，在于提供一种产品。
本发明所采取的技术方案是：
本发明的第一方面，提供一种荧光探针，包含镉离子结合蛋白和荧光蛋白，荧光蛋白位于镉离子结合蛋白的序列内。优选地，所述镉离子结合蛋白具有SEQ ID NO:10所示的序列或与其至少有80％序列相同性的序列，所述荧光蛋白具有SEQ ID NO:11所示的序列或与其至少有80％序列相同的序列。优选地，所述荧光蛋白位于镉离子结合蛋白的残基112-119中。优选地，所述荧光蛋白位于镉离子结合蛋白的选自以下的一个或多个位点：112/113,112/114,112/115,112/116,112/117,112/118,112/119,113/114,113/115,113/116,113/117,113/118,113/119,114/115,114/116,114/117,114/118,114/119,115/116,115/117,115/118,115/119,116/117,116/118,116/119,117/118,117/119,118/119。优选地，所述荧光蛋白位于镉离子结合蛋白的选自以下的一个或多个位点：112/116,112/117,114/117,114/118，116/117。优选地，所述荧光蛋白位于镉离子结合蛋白的114/117位点。优选地，所述荧光探针还包含柔性连接子，所述柔性连接子位于荧光蛋白的C端和N端。优选地，所述荧光蛋白的C端和N端柔性连接子选自以下的0～3个连接子：SAG/GTG，SAG/GT，SAG/G，SAG/0，AG/GTG，AG/GT，AG/G，AG/0，G/GTG，G/GT，G/G，G/0，0/GTG，0/GT，0/G，0/0。优选地，所述柔性连接子为SAG/GTG或AG/GT。优选地，所述荧光探针的序列如SEQ ID NO:12～SEQ IDNO:18任一项所示。
本发明的第二方面，提供本发明第一方面所述荧光探针相关的生物材料：所述相关生物材料为下述(A1)～(A8)中的任一种：(A1)编码所述荧光探针的核酸分子；(A2)含有(A1)所述核酸分子的表达盒；(A3)含有(A1)所述核酸分子的重组载体；(A4)含有(A2)所述表达盒的重组载体；(A5)含有(A1)所述核酸分子的重组微生物；(A6)含有(A2)所述表达盒的重组微生物；(A7)含有(A3)所述重组载体的重组微生物；(A8)含有(A4)所述重组载体的重组微生物。优选地，所述核酸分子的序列如SEQ ID NO:3～SEQ ID NO:9任一项所示。
本发明的第三方面，提供本发明第一方面所述探针或本发明第二方面所述生物材料在检测样品中的镉离子中的应用。优选地，所述样品为食品、细胞、水样等。
本发明第四方面的目的，在于提供本发明第一方面的探针或本发明第二方面的生物材料在制备镉离子检测产品中的应用。优选地，所述检测产品为试剂盒、试纸或芯片。在本发明的一些实施方式中，还可以提供一种检测系统，包括本发明第一方面的探针、本发明第二方面的生物材料或前述的试剂盒。
本发明的第五方面，提供一种制备荧光探针的方法，通过培养本发明第二方面所述的重组微生物获得荧光探针。
本发明的第六方面，提供一种产品，所述产品包含本发明第一方面所述荧光探针或本发明第二方面所述生物材料。优选地，所述产品中还可以包含检测镉离子所需的其他物质。优选地，所述物质为仪器或试剂。
本发明的有益效果是：
本发明提供镉离子荧光探针，包括对具有对镉离子结合能力的蛋白CadR和荧光蛋白cpYFP；所述的荧光蛋白cpYFP插入到结合蛋白CadR中，将CadR分为CadR1和CadR2两个部分；本发明提供的镉离子荧光探针，荧光动态变化大，特异性较好，并且能在大肠杆菌中表达，能定量检测镉离子。实验效果表明，本申请所提供的镉离子荧光探针对镉离子的检测限可达10nM，动态范围可达220％。
附图说明
图1为镉离子荧光探针SAG/GTG的SDS-PAGE分析图。
图2为黄色荧光蛋白cpYFP在镉离子结合蛋白不同插入位点形成的镉离子荧光探针对镉离子响应变化图。
图3为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行的连接子删减对镉离子响应变化图。
图4为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行删减的SAG/GTG和AG/GT纯化蛋白对不同浓度镉离子的滴定曲线。
图5为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行删减的SAG/GTG和AG/GT纯化蛋白对不同金属离子的特异性检测。
图6为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行删减的SAG/GTG和AG/GT纯化蛋白对过氧化氢下对镉离子的响应图。
图7为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行删减的SAG/GTG和AG/GT纯化蛋白的荧光光谱性质图。
图8为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行删减的SAG/GTG和AG/GT纯化蛋白对吸收光谱性质图。
图9为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行删减的SAG/GTG和AG/GT纯化蛋白对不同浓度pH的响应图。
图10为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行删减的SAG/GTG和AG/GT纯化蛋白对不同温度对镉离子的响应图。
图11为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行删减的SAG/GTG和AG/GT纯化蛋白的实时监控图。
图12为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行删减的SAG/GTG和AG/GT的大肠杆菌细胞的滴定曲线。
图13为黄色荧光蛋白cpYFP在镉离子结合蛋白114/117插入位点形成的镉离子荧光探针基础上进行删减的SAG/GTG和AG/GT的大肠杆菌细胞的实时监控图。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。
本发明提供了一种镉离子荧光探针，包括对镉离子敏感的结合蛋白CadR和对镉离子进行表现的荧光蛋白cpYFP；所述的荧光蛋白cpYFP插入到结合蛋白CadR中，将CadR分为CadR1和CadR2两个部分，形成CadR1-cpYFP-CadR2式的探针结构；所述结合蛋白CadR和镉离子结合导致荧光蛋白cpYFP荧光信号改变；在本发明中所述CadR蛋白来源于恶臭假单胞菌(Pseudomonas putida)。
下面结合具体实施例，进一步阐述本发明。
(1)实验材料和试剂
实施例中所用的基于pRSETB-CadR质粒由华南理工大学微生物酶学实验室构建，pRSETB质粒载体购自Invitrogen公司，CadR序列来自NCBI(NCBI序列号：AF333961)。所有用于PCR的引物均由北京擎科生物有限公司合成、纯化和经质谱法鉴定正确。实施例中构建的表达质粒都经过序列测定，序列测定由生工生物工程(上海)股份有限公司和北京擎科生物有限公司完成。各实施例所用的KOD高保真聚合酶购自日本东洋纺公司，PCR MIX购自北京庄盟国际生物公司。同源重组酶购自clones marter公司。除非特别声明，无机盐类等化学试剂均为国产分析纯试剂。HEPES盐购自上海麦克林生化科技有限公司，氨苄青霉素(Amp)购自上海麦克林生化科技有限公司。实施例中所用的DNA纯化试剂盒购自Magen公司，普通质粒小抽提试剂盒购自天根生化科技(北京)有限公司。克隆菌株Top10由微生物酶学实验室保存。镍柱亲和层析柱和脱盐柱均来自思拓凡公司。实施例中用到的主要仪器：TECANinfinite M200多功能酶标仪，5804R高速冷冻离心机(德国Eppendorf公司)，PCR扩增仪(上海桑晒生物科技有限公司)，超声破碎仪(宁波新芝公司)，核酸电泳仪(上海天能科技有限公司)，荧光分光光度计(Shimadzu RF-6000)。
(2)实施例中用到的常规分子生物学方法和细胞实验方法
1、聚合酶链式反应(PCR)：目的片段扩增PCR和鉴定，该方法主要用于基因片段扩增和菌落PCR鉴定阳性克隆；PCR反应体系见表1。
表1 PCR反应体系
2、DNA片段5’端磷酸化反应
PCR产物是不含磷酸基团的，故需对PCR产物的5’端碱基进行磷酸基团加成反应，只有末端含有磷酸基团DNA分子才能发生连接反应，磷酸化反应体系见表2。
表2磷酸化反应体系
3、目的片段和载体的连接反应
本实施例使用同源重组和T4连接酶的方法进行连接：通过同源重组酶将目的片段和载体的同源臂部分进行连接；连接反应体系见表3。
表3连接反应体系
注：载体和目的基因的摩尔比大致在1:1～1:3之间。
4、反向PCR引入定点突变后5’端磷酸化的DNA片段产物自身环化：将5’端磷酸化的DNA片段通过自身环化连接反应将载体的3’和5’端连接从而得到重组质粒；自身环化连接反应体系见表4。
表4自身环化连接反应体系
5、感受态细胞的制备
1)TOP10菌液在LB平板上划线分离出单菌落，在37℃培养箱中生长14h；
2)从平板上挑取单菌落接种到10mL的LB液体培养基中，3℃，200rpm培养约14h；
3)取过夜培养的菌液接种到50mL新鲜的LB液体培养基中，将起始OD600控制在0.1，37℃，220rpm培养至OD600＝0.4-0.6；
4)将50mL培养液装到50mL的离心管中，冰浴10min，4℃，6000rpm离心5min；
5)弃上清，加入10mL预冷的0.1M氯化钙溶液洗涤菌体2次，6000rpm离心5min；
6)弃上清，加入10mL预冷的0.1M氯化钙溶液洗涤菌体冰浴20min，4℃，6000rpm离心5min；
7)弃上清，加入1mL预冷的0.1M氯化钙溶液和1mL预冷30％甘油重悬菌体，分装至1.5mL离心管，每管分装80μL，立即使用或者-80℃保存。
6、感受态细胞的转化
1)取1管感受态细胞于冰浴上融化；
2)加入连接产物，轻轻吹打混匀，冰浴30min。通常加入的连接产物的体积少于感受态细胞体积的1/10；
3)将菌液放入42℃水浴中热激90s，迅速转移至冰浴中放置5min；
4)加入900μL LB培养基，于37℃恒温摇床上220rpm培养45min；
5)将菌液6000rpm离心5min，留100μL上清液将菌体吹打混匀，均匀涂布于含适当抗生素的琼脂平板表面，将平板放置在37℃恒温培养箱内倒置过夜。
7、蛋白质的表达，纯化和荧光检测
1)将pRSETB为基础的镉离子探针质粒转化到感受态BL21(DE3)中，倒置培养过夜，从平板上挑取单克隆到100ml LB锥形瓶中，加入AMP，置于37℃，220rpm培养至OD600＝0.4-0.6，加入0.1M的IPTG，18℃诱导表达20-24h；
2)诱导表达完成后，通过冷冻离心机6000rpm，3min离心收集发酵菌体，用去离子水和HEPEs Buffer清洗菌体表面的培养液，重复一次，保留菌体，向离心管中加入10mlHEPEs Buffer溶液，振荡重悬菌体；对发酵菌体进行超声破碎，功率设置为35％，频率设置为开3s，关3s，持续15min。破碎过程中样品须放置在冰上以防止温度过高破坏蛋白质样品；超声破碎结束后，将破碎产物4℃，4000×g，离心30min，保留上清液；将离心得到的样品通过水系滤膜过滤，以备纯化使用；
3)上清液通过镍柱亲和层析柱纯化获得蛋白，并加入100mM的EDTA处理在通过脱盐柱获得溶解在100mM HEPEs Buffer中；
4)纯化的蛋白经过SDS-PAGE鉴定后，使用HEPES Buffer稀释探针终浓度为2-20μM的蛋白溶液；
取100μL稀释的荧光探针溶液，加入0-25μM的不同浓度的镉离子进行滴定，用多功能酶标仪测量蛋白的485nm和420nm处激发，528nm处发射的荧光强度。
取100μL稀释的荧光探针溶液，加入或者不加入相应浓度的镉离子，等待5min，用分光光度计测定探针蛋白的吸光光谱，用荧光分光光度计测定探针蛋白的荧光光谱。
8、大肠杆菌荧光检测
1)将pRSETB为基础的镉离子探针质粒转化到感受态BL21(DE3)中，导致培养过夜，从平板上挑取单克隆到100ml LB锥形瓶中，置于37℃，220rpm培养至OD600＝0.4～0.6，加入0.1M的IPTG，18℃诱导表达20～24h。
2)诱导表达完成后，通过冷冻离心机6000rpm，3min离心收集发酵菌体，用去离子水和HEPEs Buffer清洗菌体表面的培养液，重复一次，保留菌体，向离心管中加入10mlHEPES Buffer溶液，振荡重悬菌体；将样品稀释至终浓度为OD600＝1。
3)将稀释的发酵菌加入到96孔板中，依次加入相应浓度的金属离子和螯合剂DMSA，用酶标仪监控荧光的变化。
实施例1不同插入位点的cpYFP探针的表达和检测
本实施例中，pRSETB-CadR质粒是由苏州金唯智生物科技有限公司合成，并以该质粒为基础根据CadR结合蛋白晶体结构选择了下述位点插入cpYFP，得到相应质粒：112/113，112/114，112/115，112/116，112/117，112/118，112/119，113/114，113/115，113/116，113/117，113/118，113/119，114/115，114/116，114/117，114/118，114/119，115/116，115/117，115/118，115/119，116/117，116/118，116/119，117/118，117/119，118/119。
利用PCR产生cpYFP并且带有pRSETB-CadR同源臂的DNA片段，对该DNA片段进行DpnI酶消化纯化，同时反向PCR扩增产生含有不同断裂位点的pRSETB-CadR线性化载体，将线性化载体的pRSETB-CadR和cpYFP片段在同源重组酶的作用下连接产生重组质粒，将重组质粒转化入Top10大肠杆菌感受态细胞，并设计引物，挑选单克隆抗体进行鉴定，将阳性菌落培养并提质粒送往北京擎科生物有限公司完成测序。
所用引物：
表达盒测序-F：GAGTCAGTGAGCGAGGAAGC(SEQ ID NO:19)；
表达盒测序-R：CCTCTTCGCTATTACGCCAG(SEQ ID NO:20)；
经过测序正确后，将重组质粒转化到BL21(DE3)中诱导表达，并纯化蛋白质，通过SDS-PAGE电泳大小在48Kda附近，该大小符合pRSETB-CadR-cpYFP表达出的含His纯化标签的融合蛋白质的大小。结果如图1所示。
将纯化的融合蛋白质进行镉离子响应筛选，将含有不同浓度的镉离子荧光检测信号除以无镉离子荧光检测信号，并做归一化处理，结果如图2所示，检测结果显示对镉离子明显响应的有112/116，112/117，114/117，114/118，116/117；其中114/117提高200％，112/116提高145％，112/117提高159％，114/118提高158％，116/117提高142％；其中114/117的核酸序列如SEQ ID NO:3所示，氨基酸序列如SEQ ID NO:12所示；112/116的核酸序列如SEQID NO:4所示，氨基酸序列如SEQ ID NO:13所示；112/117的核酸序列如SEQ ID NO:5所示，氨基酸序列如SEQ ID NO:14所示；114/118的核酸序列如SEQ ID NO:6所示，氨基酸序列如SEQ ID NO:15所示；116/117的核酸序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:16所示。
实施例2氨基酸删减的镉离子荧光探针的表达和检测
通过反向PCR线性化质粒pRSETB-114/117，获得线性化片段，同时对cpYFP进行扩增，并带有pRSETB-114/117同源臂，PCR删减cpYFP的第一个氨基酸甲硫氨酸位点的碱基序列，将两个片段经过同源重组酶进行连接，并由北京擎科生物有限公司完成测序。按照实施例1中的方法表达和检测，示例的荧光探针(SAG/GTG)的核酸序列如SEQ ID NO:8所示，氨基酸序列如SEQ ID NO:17所示，所述荧光蛋白SAG/GTG位于镉离子结合蛋白的114/117位点。删减掉的甲硫氨酸相比较于之前的114/117融合蛋白对0.5μM的镉离子具有220％的响应值，对痕量镉离子更具有响应能力。
将SAG/GTG序列进行连接的删减，依次通过同源重组的方法，删减掉cpYFP的N端SAG和C端GTG的连接子氨基酸，得到16个删减子。按照实施例1的方法表达和检测。结果如图3所示，荧光检测结果显示对镉离子具有最大增高和降低为SAG/GTG和AG/GT删减子，其中AG/GT删减子的荧光探针的核酸序列如SEQ ID NO:9所示，氨基酸序列如SEQ ID NO:18所示。SAG/GTG对镉离子的最大正响应为220％在0.5μM下，AG/GT对镉离子的最大负响应为167％，通过优化，提高了对镉离子的检测能力，得到了对镉离子具有正向响应和负向响应的的融合蛋白，对实现对痕量镉离子浓度检测具有重要意义。
实施例3荧光探针的滴定曲线和特异性
选择SAG/GTG和AG/GT融合蛋白进行浓度梯度的镉离子检测，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。SAG/GTG和AG/GT的Kd(结合常数)分别为0.12μM和0.10μM，变化幅度分别为2.36倍和1.51倍，结果如图4所示。
对SAG/GTG和AG/GT进行特异性检测，使用100μL含有不同离子成分的缓冲液和100μL纯化蛋白进行反应，含或不含0.5/5μM CdCl2。离子浓度如下所列：300mM Na+或K+，而其他离子为100μM(Fe3+、Mg2+、Ca2+、Ni2+、Mn2+、Fe2+、Li+、Cs+、Ba2+、Co2+)或0.5μM(Ag+、Zn2+、Cu2+、Pb2 +、Hg+、Cu+)。结果表明探针在大部分金属离子存在下对镉离子具有很好的专一性。如图5和图6所示。
实施例4探针的光谱性能
将纯化的SAG/GTG和AG/GT荧光探针与0μM、0.5μM、5μM镉离子反应5min，使用紫外分光光度计和荧光分光光度计进行吸收光谱和荧光光谱的检测。对吸收光谱的测定：扫描300nm-600nm的紫外吸收；对发射光谱的测定：固定激发波长为420nm和485nm，记录420-600nm的发射光谱，每1nm读取一次；对激发光谱的测定：固定发射光谱为530nm，扫描400nm-530nm的激发光谱，每1nm读取一次。镉离子荧光探针SAG/GTG和AG/GT的光谱曲线如图7和图8所示。在添加了0.5μM的镉离子后，在485nm与420nm下的比值的荧光强度提升为0μM镉离子的2.3倍；同理，AG/GT在添加了5μM的镉离子后，在485nm与420nm下的比值的荧光强度降低为0μM镉离子的1.5倍。
实施例5探针的物理性质
将纯化的SAG/GTG和AG/GT荧光探针与0μM、0.5μM、5μM镉离子反应5min，检测420nm激发528nm发射处荧光强度和485nm激发528nm发射处荧光强度在25～40℃及pH为6.8～8.4变化范围内的响应情况。如图9和10所示，SAG/GTG和AG/GT在25-40℃的范围内对其荧光性质基本无影响，在pH变化的条件下，SAG/GTG和AG/GT的荧光性质是随pH增大而增大的且具有线性关系。量子产率的测定，以通过测定蛋白的消光系数，在通过荧光分光光度计测得。
实施例6镉离子的动态监控
将纯化的SAG/GTG和AG/GT蛋白荧光探针在37℃孵育5min之后，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，之后加入0μM、0.5μM、5μM镉离子监测100s-400s，在之后加入DMSA监测200s，每20s监测1次。如图11所示，在添加了镉离子之后，蛋白SAG/GTG和AG/GT均作出变化，SAG/GTG的荧光响应逐渐增加，最高可达2.3倍，AG/GT的荧光响应逐渐降低，最低降至0.6附近。在加入DMSA螯合镉离子之后，荧光迅速回归。
检测限的计算方法用以下公式计算：
3：3倍信噪比作为检出限计算标准；Sb：空白的标准偏差；K：线性拟合之后的斜率；通过计算得出SAG/GTG具有最低检测限为10nM。
实施例7大肠杆菌的镉离子监控和滴定
将含有SAG/GTG和AG/GT蛋白荧光探针的大肠杆菌，在37℃孵育5min之后，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，之后加入0μM、0.5μM、5μM镉离子监测20min，在之后加入DMSA监测10min，每30s监测1次。如图13所示，在添加了镉离子之后，AG/GT大肠杆菌作出变化，AG/GT的荧光响应逐渐降低，最低降至0.65附近。选择有SAG/GTG和AG/GT融合蛋白的大肠杆菌进行浓度梯度的镉离子检测，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。结果如果图12所示，大肠杆菌AG/GT也具有跟纯化蛋白相似的性质。
由以上实施例可知，本发明提供的镉离子荧光探针，蛋白分子相对小且易成熟，荧光的动态范围大，对镉离子检测限低，特异性较好，并且在细胞内表达也具有检测镉离子的能力，具有快速，实时检测镉离子的能力。
上述具体实施方式对本发明作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。
SEQUENCE LISTING
<110> 华南理工大学
<120> 一种基因编码的无机镉荧光探针及其制备方法和应用
<130>
<160> 20
<170> PatentIn version 3.5
<210> 1
<211> 444
<212> DNA
<213> CadR
<400> 1
atgaagatcg gagaactggc caaagccacc gactgcgccg tggaaaccat ccgctactac 60
gagcgtgaac agctgctgcc ggagccggca cgcagcgacg gcaactaccg gctgtacacc 120
caggcccacg tcgagcggct taccttcatc cgcaactgcc gcaccctgga catgaccctg 180
gatgaaatcc gcagcctgct acgcctgcgc gacagccccg atgattcgtg cggcagcgtc 240
aatgcgctga tcgacgagca tatcgagcat gtgcaggcac ggatcgatgg tctggtggcg 300
ttgcaggaac agctggtgga gctgcggcgg cgctgcaatg cacaaggggc ggagtgtgcg 360
atcttgcagc aactggagac gaacggggcg gtatcggtgc cggaaaccga gcattcgcat 420
gtagggcgaa gccacgggca ttaa 444
<210> 2
<211> 744
<212> DNA
<213> cpYFP
<400> 2
atgtacaaca gcgacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggcc 60
aacttcaaga tccgccacaa cgtcgaggac ggcagcgtgc agctcgccga ccactaccag 120
cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcttc 180
cagtccgtcc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 240
gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaacgtgga tggcggtagc 300
ggtggcaccg gcagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 360
ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 420
acctacggca agctgaccct gaagctgatc tgcaccaccg gcaagctgcc cgtgccctgg 480
cccaccctcg tgaccaccct cggctacggc ctgaagtgct tcgcccgcta ccccgaccac 540
atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 600
atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 660
accctggtga accgcatcga gctgaagggc atcggcttca aggaggacgg caacatcctg 720
gggcacaagc tggagtacaa ctag 744
<210> 3
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<213> 人工序列
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atgaagatcg gagaactggc caaagccacc gactgcgccg tggaaaccat ccgctactac 60
gagcgtgaac agctgctgcc ggagccggca cgcagcgacg gcaactaccg gctgtacacc 120
caggcccacg tcgagcggct taccttcatc cgcaactgcc gcaccctgga catgaccctg 180
gatgaaatcc gcagcctgct acgcctgcgc gacagccccg atgattcgtg cggcagcgtc 240
aatgcgctga tcgacgagca tatcgagcat gtgcaggcac ggatcgatgg tctggtggcg 300
ttgcaggaac agctggtgga gctgcggcgg cgctgcaatg catctgcagg catgtacaac 360
agcgacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc caacttcaag 420
atccgccaca acgtcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 480
cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagctt ccagtccgtc 540
ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 600
gccgggatca ctctcggcat ggacgagctg tacaacgtgg atggcggtag cggtggcacc 660
ggcagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 720
gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 780
aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 840
gtgaccaccc tcggctacgg cctgaagtgc ttcgcccgct accccgacca catgaagcag 900
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 960
aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 1020
aaccgcatcg agctgaaggg catcggcttc aaggaggacg gcaacatcct ggggcacaag 1080
ctggagtaca acggtaccgg cgcggagtgt gcgatcttgc agcaactgga gacgaacggg 1140
gcggtatcgg tgccggaaac cgagcattcg catgtagggc gaagccacgg gcattaa 1197
<210> 4
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<212> DNA
<213> 人工序列
<400> 4
atgaagatcg gagaactggc caaagccacc gactgcgccg tggaaaccat ccgctactac 60
gagcgtgaac agctgctgcc ggagccggca cgcagcgacg gcaactaccg gctgtacacc 120
caggcccacg tcgagcggct taccttcatc cgcaactgcc gcaccctgga catgaccctg 180
gatgaaatcc gcagcctgct acgcctgcgc gacagccccg atgattcgtg cggcagcgtc 240
aatgcgctga tcgacgagca tatcgagcat gtgcaggcac ggatcgatgg tctggtggcg 300
ttgcaggaac agctggtgga gctgcggcgg cgctgctctg caggcatgta caacagcgac 360
aacgtctata tcatggccga caagcagaag aacggcatca aggccaactt caagatccgc 420
cacaacgtcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 480
ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcttccagtc cgtcctgagc 540
aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 600
atcactctcg gcatggacga gctgtacaac gtggatggcg gtagcggtgg caccggcagc 660
aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 720
aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 780
accctgaagc tgatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 840
accctcggct acggcctgaa gtgcttcgcc cgctaccccg accacatgaa gcagcacgac 900
ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 960
gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 1020
atcgagctga agggcatcgg cttcaaggag gacggcaaca tcctggggca caagctggag 1080
tacaacggta ccggcggggc ggagtgtgcg atcttgcagc aactggagac gaacggggcg 1140
gtatcggtgc cggaaaccga gcattcgcat gtagggcgaa gccacgggca ttaa 1194
<210> 5
<211> 1191
<212> DNA
<213> 人工序列
<400> 5
atgaagatcg gagaactggc caaagccacc gactgcgccg tggaaaccat ccgctactac 60
gagcgtgaac agctgctgcc ggagccggca cgcagcgacg gcaactaccg gctgtacacc 120
caggcccacg tcgagcggct taccttcatc cgcaactgcc gcaccctgga catgaccctg 180
gatgaaatcc gcagcctgct acgcctgcgc gacagccccg atgattcgtg cggcagcgtc 240
aatgcgctga tcgacgagca tatcgagcat gtgcaggcac ggatcgatgg tctggtggcg 300
ttgcaggaac agctggtgga gctgcggcgg cgctgctctg caggcatgta caacagcgac 360
aacgtctata tcatggccga caagcagaag aacggcatca aggccaactt caagatccgc 420
cacaacgtcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 480
ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcttccagtc cgtcctgagc 540
aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 600
atcactctcg gcatggacga gctgtacaac gtggatggcg gtagcggtgg caccggcagc 660
aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 720
aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 780
accctgaagc tgatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 840
accctcggct acggcctgaa gtgcttcgcc cgctaccccg accacatgaa gcagcacgac 900
ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 960
gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 1020
atcgagctga agggcatcgg cttcaaggag gacggcaaca tcctggggca caagctggag 1080
tacaacggta ccggcgcgga gtgtgcgatc ttgcagcaac tggagacgaa cggggcggta 1140
tcggtgccgg aaaccgagca ttcgcatgta gggcgaagcc acgggcatta a 1191
<210> 6
<211> 1194
<212> DNA
<213> 人工序列
<400> 6
atgaagatcg gagaactggc caaagccacc gactgcgccg tggaaaccat ccgctactac 60
gagcgtgaac agctgctgcc ggagccggca cgcagcgacg gcaactaccg gctgtacacc 120
caggcccacg tcgagcggct taccttcatc cgcaactgcc gcaccctgga catgaccctg 180
gatgaaatcc gcagcctgct acgcctgcgc gacagccccg atgattcgtg cggcagcgtc 240
aatgcgctga tcgacgagca tatcgagcat gtgcaggcac ggatcgatgg tctggtggcg 300
ttgcaggaac agctggtgga gctgcggcgg cgctgcaatg catctgcagg catgtacaac 360
agcgacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc caacttcaag 420
atccgccaca acgtcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 480
cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagctt ccagtccgtc 540
ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 600
gccgggatca ctctcggcat ggacgagctg tacaacgtgg atggcggtag cggtggcacc 660
ggcagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 720
gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 780
aagctgaccc tgaagctgat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 840
gtgaccaccc tcggctacgg cctgaagtgc ttcgcccgct accccgacca catgaagcag 900
cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 960
aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 1020
aaccgcatcg agctgaaggg catcggcttc aaggaggacg gcaacatcct ggggcacaag 1080
ctggagtaca acggtaccgg cgagtgtgcg atcttgcagc aactggagac gaacggggcg 1140
gtatcggtgc cggaaaccga gcattcgcat gtagggcgaa gccacgggca ttaa 1194
<210> 7
<211> 1203
<212> DNA
<213> 人工序列
<400> 7
atgaagatcg gagaactggc caaagccacc gactgcgccg tggaaaccat ccgctactac 60
gagcgtgaac agctgctgcc ggagccggca cgcagcgacg gcaactaccg gctgtacacc 120
caggcccacg tcgagcggct taccttcatc cgcaactgcc gcaccctgga catgaccctg 180
gatgaaatcc gcagcctgct acgcctgcgc gacagccccg atgattcgtg cggcagcgtc 240
aatgcgctga tcgacgagca tatcgagcat gtgcaggcac ggatcgatgg tctggtggcg 300
ttgcaggaac agctggtgga gctgcggcgg cgctgcaatg cacaagggtc tgcaggcatg 360
tacaacagcg acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggccaac 420
ttcaagatcc gccacaacgt cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 480
aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcttccag 540
tccgtcctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 600
accgccgccg ggatcactct cggcatggac gagctgtaca acgtggatgg cggtagcggt 660
ggcaccggca gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg 720
gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc 780
tacggcaagc tgaccctgaa gctgatctgc accaccggca agctgcccgt gccctggccc 840
accctcgtga ccaccctcgg ctacggcctg aagtgcttcg cccgctaccc cgaccacatg 900
aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc 960
ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc 1020
ctggtgaacc gcatcgagct gaagggcatc ggcttcaagg aggacggcaa catcctgggg 1080
cacaagctgg agtacaacgg taccggcgcg gagtgtgcga tcttgcagca actggagacg 1140
aacggggcgg tatcggtgcc ggaaaccgag cattcgcatg tagggcgaag ccacgggcat 1200
taa 1203
<210> 8
<211> 1194
<212> DNA
<213> 人工序列
<400> 8
atgaagatcg gagaactggc caaagccacc gactgcgccg tggaaaccat ccgctactac 60
gagcgtgaac agctgctgcc ggagccggca cgcagcgacg gcaactaccg gctgtacacc 120
caggcccacg tcgagcggct taccttcatc cgcaactgcc gcaccctgga catgaccctg 180
gatgaaatcc gcagcctgct acgcctgcgc gacagccccg atgattcgtg cggcagcgtc 240
aatgcgctga tcgacgagca tatcgagcat gtgcaggcac ggatcgatgg tctggtggcg 300
ttgcaggaac agctggtgga gctgcggcgg cgctgcaatg catctgcagg ctacaacagc 360
gacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggccaa cttcaagatc 420
cgccacaacg tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 480
atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcttcca gtccgtcctg 540
agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 600
gggatcactc tcggcatgga cgagctgtac aacgtggatg gcggtagcgg tggcaccggc 660
agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac 720
gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag 780
ctgaccctga agctgatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg 840
accaccctcg gctacggcct gaagtgcttc gcccgctacc ccgaccacat gaagcagcac 900
gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag 960
gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac 1020
cgcatcgagc tgaagggcat cggcttcaag gaggacggca acatcctggg gcacaagctg 1080
gagtacaacg gtaccggcgc ggagtgtgcg atcttgcagc aactggagac gaacggggcg 1140
gtatcggtgc cggaaaccga gcattcgcat gtagggcgaa gccacgggca ttaa 1194
<210> 9
<211> 1188
<212> DNA
<213> 人工序列
<400> 9
atgaagatcg gagaactggc caaagccacc gactgcgccg tggaaaccat ccgctactac 60
gagcgtgaac agctgctgcc ggagccggca cgcagcgacg gcaactaccg gctgtacacc 120
caggcccacg tcgagcggct taccttcatc cgcaactgcc gcaccctgga catgaccctg 180
gatgaaatcc gcagcctgct acgcctgcgc gacagccccg atgattcgtg cggcagcgtc 240
aatgcgctga tcgacgagca tatcgagcat gtgcaggcac ggatcgatgg tctggtggcg 300
ttgcaggaac agctggtgga gctgcggcgg cgctgcaatg cagcaggcta caacagcgac 360
aacgtctata tcatggccga caagcagaag aacggcatca aggccaactt caagatccgc 420
cacaacgtcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc 480
ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcttccagtc cgtcctgagc 540
aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 600
atcactctcg gcatggacga gctgtacaac gtggatggcg gtagcggtgg caccggcagc 660
aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 720
aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 780
accctgaagc tgatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 840
accctcggct acggcctgaa gtgcttcgcc cgctaccccg accacatgaa gcagcacgac 900
ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 960
gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 1020
atcgagctga agggcatcgg cttcaaggag gacggcaaca tcctggggca caagctggag 1080
tacaacggta ccgcggagtg tgcgatcttg cagcaactgg agacgaacgg ggcggtatcg 1140
gtgccggaaa ccgagcattc gcatgtaggg cgaagccacg ggcattaa 1188
<210> 10
<211> 147
<212> PRT
<213> CadR
<400> 10
Met Lys Ile Gly Glu Leu Ala Lys Ala Thr Asp Cys Ala Val Glu Thr
1 5 10 15
Ile Arg Tyr Tyr Glu Arg Glu Gln Leu Leu Pro Glu Pro Ala Arg Ser
20 25 30
Asp Gly Asn Tyr Arg Leu Tyr Thr Gln Ala His Val Glu Arg Leu Thr
35 40 45
Phe Ile Arg Asn Cys Arg Thr Leu Asp Met Thr Leu Asp Glu Ile Arg
50 55 60
Ser Leu Leu Arg Leu Arg Asp Ser Pro Asp Asp Ser Cys Gly Ser Val
65 70 75 80
Asn Ala Leu Ile Asp Glu His Ile Glu His Val Gln Ala Arg Ile Asp
85 90 95
Gly Leu Val Ala Leu Gln Glu Gln Leu Val Glu Leu Arg Arg Arg Cys
100 105 110
Asn Ala Gln Gly Ala Glu Cys Ala Ile Leu Gln Gln Leu Glu Thr Asn
115 120 125
Gly Ala Val Ser Val Pro Glu Thr Glu His Ser His Val Gly Arg Ser
130 135 140
His Gly His
145
<210> 11
<211> 247
<212> PRT
<213> cpYFP
<400> 11
Met Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
1 5 10 15
Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser
20 25 30
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
35 40 45
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln Ser Val Leu
50 55 60
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
65 70 75 80
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Asn Val
85 90 95
Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu Leu Phe Thr
100 105 110
Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
115 120 125
Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys
130 135 140
Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
145 150 155 160
Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys Phe Ala Arg
165 170 175
Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro
180 185 190
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn
195 200 205
Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
210 215 220
Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly Asn Ile Leu
225 230 235 240
Gly His Lys Leu Glu Tyr Asn
245
<210> 12
<211> 398
<212> PRT
<213> 人工序列
<400> 12
Met Lys Ile Gly Glu Leu Ala Lys Ala Thr Asp Cys Ala Val Glu Thr
1 5 10 15
Ile Arg Tyr Tyr Glu Arg Glu Gln Leu Leu Pro Glu Pro Ala Arg Ser
20 25 30
Asp Gly Asn Tyr Arg Leu Tyr Thr Gln Ala His Val Glu Arg Leu Thr
35 40 45
Phe Ile Arg Asn Cys Arg Thr Leu Asp Met Thr Leu Asp Glu Ile Arg
50 55 60
Ser Leu Leu Arg Leu Arg Asp Ser Pro Asp Asp Ser Cys Gly Ser Val
65 70 75 80
Asn Ala Leu Ile Asp Glu His Ile Glu His Val Gln Ala Arg Ile Asp
85 90 95
Gly Leu Val Ala Leu Gln Glu Gln Leu Val Glu Leu Arg Arg Arg Cys
100 105 110
Asn Ala Ser Ala Gly Met Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala
115 120 125
Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn
130 135 140
Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
145 150 155 160
Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
165 170 175
Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met
180 185 190
Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
195 200 205
Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly
210 215 220
Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
225 230 235 240
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp
245 250 255
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys
260 265 270
Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu
275 280 285
Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe
290 295 300
Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe
305 310 315 320
Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly
325 330 335
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu
340 345 350
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Thr Gly Ala
355 360 365
Glu Cys Ala Ile Leu Gln Gln Leu Glu Thr Asn Gly Ala Val Ser Val
370 375 380
Pro Glu Thr Glu His Ser His Val Gly Arg Ser His Gly His
385 390 395
<210> 13
<211> 397
<212> PRT
<213> 人工序列
<400> 13
Met Lys Ile Gly Glu Leu Ala Lys Ala Thr Asp Cys Ala Val Glu Thr
1 5 10 15
Ile Arg Tyr Tyr Glu Arg Glu Gln Leu Leu Pro Glu Pro Ala Arg Ser
20 25 30
Asp Gly Asn Tyr Arg Leu Tyr Thr Gln Ala His Val Glu Arg Leu Thr
35 40 45
Phe Ile Arg Asn Cys Arg Thr Leu Asp Met Thr Leu Asp Glu Ile Arg
50 55 60
Ser Leu Leu Arg Leu Arg Asp Ser Pro Asp Asp Ser Cys Gly Ser Val
65 70 75 80
Asn Ala Leu Ile Asp Glu His Ile Glu His Val Gln Ala Arg Ile Asp
85 90 95
Gly Leu Val Ala Leu Gln Glu Gln Leu Val Glu Leu Arg Arg Arg Cys
100 105 110
Ser Ala Gly Met Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys
115 120 125
Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu
130 135 140
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
145 150 155 160
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln
165 170 175
Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
180 185 190
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
195 200 205
Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu
210 215 220
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
225 230 235 240
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
245 250 255
Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro
260 265 270
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys
275 280 285
Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
290 295 300
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
305 310 315 320
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
325 330 335
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly
340 345 350
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Thr Gly Gly Ala Glu
355 360 365
Cys Ala Ile Leu Gln Gln Leu Glu Thr Asn Gly Ala Val Ser Val Pro
370 375 380
Glu Thr Glu His Ser His Val Gly Arg Ser His Gly His
385 390 395
<210> 14
<211> 396
<212> PRT
<213> 人工序列
<400> 14
Met Lys Ile Gly Glu Leu Ala Lys Ala Thr Asp Cys Ala Val Glu Thr
1 5 10 15
Ile Arg Tyr Tyr Glu Arg Glu Gln Leu Leu Pro Glu Pro Ala Arg Ser
20 25 30
Asp Gly Asn Tyr Arg Leu Tyr Thr Gln Ala His Val Glu Arg Leu Thr
35 40 45
Phe Ile Arg Asn Cys Arg Thr Leu Asp Met Thr Leu Asp Glu Ile Arg
50 55 60
Ser Leu Leu Arg Leu Arg Asp Ser Pro Asp Asp Ser Cys Gly Ser Val
65 70 75 80
Asn Ala Leu Ile Asp Glu His Ile Glu His Val Gln Ala Arg Ile Asp
85 90 95
Gly Leu Val Ala Leu Gln Glu Gln Leu Val Glu Leu Arg Arg Arg Cys
100 105 110
Ser Ala Gly Met Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys
115 120 125
Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu
130 135 140
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
145 150 155 160
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln
165 170 175
Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
180 185 190
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
195 200 205
Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu
210 215 220
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
225 230 235 240
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
245 250 255
Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro
260 265 270
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys
275 280 285
Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
290 295 300
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
305 310 315 320
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
325 330 335
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly
340 345 350
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Thr Gly Ala Glu Cys
355 360 365
Ala Ile Leu Gln Gln Leu Glu Thr Asn Gly Ala Val Ser Val Pro Glu
370 375 380
Thr Glu His Ser His Val Gly Arg Ser His Gly His
385 390 395
<210> 15
<211> 397
<212> PRT
<213> 人工序列
<400> 15
Met Lys Ile Gly Glu Leu Ala Lys Ala Thr Asp Cys Ala Val Glu Thr
1 5 10 15
Ile Arg Tyr Tyr Glu Arg Glu Gln Leu Leu Pro Glu Pro Ala Arg Ser
20 25 30
Asp Gly Asn Tyr Arg Leu Tyr Thr Gln Ala His Val Glu Arg Leu Thr
35 40 45
Phe Ile Arg Asn Cys Arg Thr Leu Asp Met Thr Leu Asp Glu Ile Arg
50 55 60
Ser Leu Leu Arg Leu Arg Asp Ser Pro Asp Asp Ser Cys Gly Ser Val
65 70 75 80
Asn Ala Leu Ile Asp Glu His Ile Glu His Val Gln Ala Arg Ile Asp
85 90 95
Gly Leu Val Ala Leu Gln Glu Gln Leu Val Glu Leu Arg Arg Arg Cys
100 105 110
Asn Ala Ser Ala Gly Met Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala
115 120 125
Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn
130 135 140
Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr
145 150 155 160
Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
165 170 175
Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met
180 185 190
Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp
195 200 205
Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly
210 215 220
Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly
225 230 235 240
Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp
245 250 255
Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys
260 265 270
Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu
275 280 285
Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe
290 295 300
Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe
305 310 315 320
Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly
325 330 335
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu
340 345 350
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Thr Gly Glu
355 360 365
Cys Ala Ile Leu Gln Gln Leu Glu Thr Asn Gly Ala Val Ser Val Pro
370 375 380
Glu Thr Glu His Ser His Val Gly Arg Ser His Gly His
385 390 395
<210> 16
<211> 400
<212> PRT
<213> 人工序列
<400> 16
Met Lys Ile Gly Glu Leu Ala Lys Ala Thr Asp Cys Ala Val Glu Thr
1 5 10 15
Ile Arg Tyr Tyr Glu Arg Glu Gln Leu Leu Pro Glu Pro Ala Arg Ser
20 25 30
Asp Gly Asn Tyr Arg Leu Tyr Thr Gln Ala His Val Glu Arg Leu Thr
35 40 45
Phe Ile Arg Asn Cys Arg Thr Leu Asp Met Thr Leu Asp Glu Ile Arg
50 55 60
Ser Leu Leu Arg Leu Arg Asp Ser Pro Asp Asp Ser Cys Gly Ser Val
65 70 75 80
Asn Ala Leu Ile Asp Glu His Ile Glu His Val Gln Ala Arg Ile Asp
85 90 95
Gly Leu Val Ala Leu Gln Glu Gln Leu Val Glu Leu Arg Arg Arg Cys
100 105 110
Asn Ala Gln Gly Ser Ala Gly Met Tyr Asn Ser Asp Asn Val Tyr Ile
115 120 125
Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg
130 135 140
His Asn Val Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln
145 150 155 160
Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr
165 170 175
Leu Ser Phe Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp
180 185 190
His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly
195 200 205
Met Asp Glu Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser
210 215 220
Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu
225 230 235 240
Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu
245 250 255
Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr
260 265 270
Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr
275 280 285
Gly Leu Lys Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp
290 295 300
Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile
305 310 315 320
Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe
325 330 335
Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe
340 345 350
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Thr
355 360 365
Gly Ala Glu Cys Ala Ile Leu Gln Gln Leu Glu Thr Asn Gly Ala Val
370 375 380
Ser Val Pro Glu Thr Glu His Ser His Val Gly Arg Ser His Gly His
385 390 395 400
<210> 17
<211> 397
<212> PRT
<213> 人工序列
<400> 17
Met Lys Ile Gly Glu Leu Ala Lys Ala Thr Asp Cys Ala Val Glu Thr
1 5 10 15
Ile Arg Tyr Tyr Glu Arg Glu Gln Leu Leu Pro Glu Pro Ala Arg Ser
20 25 30
Asp Gly Asn Tyr Arg Leu Tyr Thr Gln Ala His Val Glu Arg Leu Thr
35 40 45
Phe Ile Arg Asn Cys Arg Thr Leu Asp Met Thr Leu Asp Glu Ile Arg
50 55 60
Ser Leu Leu Arg Leu Arg Asp Ser Pro Asp Asp Ser Cys Gly Ser Val
65 70 75 80
Asn Ala Leu Ile Asp Glu His Ile Glu His Val Gln Ala Arg Ile Asp
85 90 95
Gly Leu Val Ala Leu Gln Glu Gln Leu Val Glu Leu Arg Arg Arg Cys
100 105 110
Asn Ala Ser Ala Gly Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp
115 120 125
Lys Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val
130 135 140
Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro
145 150 155 160
Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe
165 170 175
Gln Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val
180 185 190
Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu
195 200 205
Leu Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu
210 215 220
Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp
225 230 235 240
Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala
245 250 255
Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu
260 265 270
Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys
275 280 285
Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys
290 295 300
Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys
305 310 315 320
Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp
325 330 335
Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp
340 345 350
Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Thr Gly Ala Glu
355 360 365
Cys Ala Ile Leu Gln Gln Leu Glu Thr Asn Gly Ala Val Ser Val Pro
370 375 380
Glu Thr Glu His Ser His Val Gly Arg Ser His Gly His
385 390 395
<210> 18
<211> 395
<212> PRT
<213> 人工序列
<400> 18
Met Lys Ile Gly Glu Leu Ala Lys Ala Thr Asp Cys Ala Val Glu Thr
1 5 10 15
Ile Arg Tyr Tyr Glu Arg Glu Gln Leu Leu Pro Glu Pro Ala Arg Ser
20 25 30
Asp Gly Asn Tyr Arg Leu Tyr Thr Gln Ala His Val Glu Arg Leu Thr
35 40 45
Phe Ile Arg Asn Cys Arg Thr Leu Asp Met Thr Leu Asp Glu Ile Arg
50 55 60
Ser Leu Leu Arg Leu Arg Asp Ser Pro Asp Asp Ser Cys Gly Ser Val
65 70 75 80
Asn Ala Leu Ile Asp Glu His Ile Glu His Val Gln Ala Arg Ile Asp
85 90 95
Gly Leu Val Ala Leu Gln Glu Gln Leu Val Glu Leu Arg Arg Arg Cys
100 105 110
Asn Ala Ala Gly Tyr Asn Ser Asp Asn Val Tyr Ile Met Ala Asp Lys
115 120 125
Gln Lys Asn Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu
130 135 140
Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
145 150 155 160
Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Phe Gln
165 170 175
Ser Val Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu
180 185 190
Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu
195 200 205
Tyr Asn Val Asp Gly Gly Ser Gly Gly Thr Gly Ser Lys Gly Glu Glu
210 215 220
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
225 230 235 240
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
245 250 255
Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr Thr Gly Lys Leu Pro
260 265 270
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly Tyr Gly Leu Lys Cys
275 280 285
Phe Ala Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
290 295 300
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
305 310 315 320
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
325 330 335
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Gly Phe Lys Glu Asp Gly
340 345 350
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Gly Thr Ala Glu Cys Ala
355 360 365
Ile Leu Gln Gln Leu Glu Thr Asn Gly Ala Val Ser Val Pro Glu Thr
370 375 380
Glu His Ser His Val Gly Arg Ser His Gly His
385 390 395
<210> 19
<211> 20
<212> DNA
<213> 人工序列
<400> 19
gagtcagtga gcgaggaagc 20
<210> 20
<211> 20
<212> DNA
<213> 人工序列
<400> 20
cctcttcgct attacgccag 20

Claims (7)

1.一种荧光探针，包含镉离子结合蛋白和荧光蛋白，荧光蛋白位于镉离子结合蛋白的序列内；所述镉离子结合蛋白具有如SEQ ID NO:10所示的序列，所述荧光蛋白具有如SEQID NO:11所示的序列；所述荧光探针的氨基酸序列如SEQ ID NO:17或SEQ ID NO:18所示。
2.权利要求1所述荧光探针相关的生物材料：所述相关生物材料为下述(A1)～(A8)中的任一种：
(A1)编码所述荧光探针的核酸分子；
(A2)含有(A1)所述核酸分子的表达盒；
(A3)含有(A1)所述核酸分子的重组载体；
(A4)含有(A2)所述表达盒的重组载体；
(A5)含有(A1)所述核酸分子的重组微生物；
(A6)含有(A2)所述表达盒的重组微生物；
(A7)含有(A3)所述重组载体的重组微生物；
(A8)含有(A4)所述重组载体的重组微生物。
3.根据权利要求2所述的生物材料，其特征在于，所述核酸分子的核苷酸序列如SEQ IDNO.8或SEQ ID NO.9所示。
4.权利要求1所述荧光探针或权利要求2或3所述生物材料在检测样品中的镉离子中的应用。
5.权利要求1所述荧光探针或权利要求2或3所述生物材料在制备镉离子检测产品中的应用。
6.一种制备荧光探针的方法，通过培养权利要求2或3中所述的重组微生物获得荧光探针。
7.一种产品，所述产品包含权利要求1所述荧光探针或权利要求2或3所述生物材料。
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