CN113512541A - 一种新型磷酸化腺苷酰化酶及其制备方法与应用 - Google Patents

一种新型磷酸化腺苷酰化酶及其制备方法与应用 Download PDF

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CN113512541A
CN113512541A CN202110470660.6A CN202110470660A CN113512541A CN 113512541 A CN113512541 A CN 113512541A CN 202110470660 A CN202110470660 A CN 202110470660A CN 113512541 A CN113512541 A CN 113512541A
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杨政权
丁春明
张成良
金胜男
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Abstract

一种新型磷酸化腺苷酰化酶及其制备方法与应用，构建了Adenylase（PF0353）与T4 PNK（T4 Polynucleotide Kinase）融合酶的原核诱导表达质粒，并通过表达和纯化得到了目的融合酶，该酶水溶性强，表达纯化简单，得到的融合酶同时拥有T4多核苷酸激酶和腺苷酰化酶的活性，可以一步法将5’‑羟基DNA在ATP存在的条件下直接催化反应得到5’腺苷酰化DNA。使用此融合酶制备腺苷酰化DNA的方法成本低、反应效率高、简便快速，可供分子生物学技术灵活使用。

Description

一种新型磷酸化腺苷酰化酶及其制备方法与应用
技术领域
本发明具体涉及核酸的腺苷酰化修饰技术领域，具体涉及一种新型磷酸化腺苷酰化酶及其制备方法与应用。
背景技术
核酸的腺苷酰化（Adenylation）是指一磷酸腺苷（AMP）分子共价连接到核酸末端的过程。腺苷酰化核酸是含有5’5’-腺苷焦磷酸基帽的核酸，是核酸连接酶催化的核酸连接反应的中间产物，具有高能焦磷酸键，是一种活化形式的核酸分子，在分子生物学中具有较多的应用。腺苷酰化的核酸包括腺苷酰化的DNA和腺苷酰化的RNA两种，目前以腺苷酰化DNA（AppDNA）应用较多。
AppDNA广泛用于RNA测序文库（尤其是miRNA测序文库）接头制备、单链DNA连接、特定连接酶催化的连接反应等分子生物学应用。以miRNA建库过程为例，Illumina测序平台miRNA测序文库的构建需要在RNA的3’端连接腺苷酰化的通用接头，该连接反应在截短型T4RNA连接酶2催化下将5'端有腺苷酰化修饰的末端与RNA的3'端的羟基连接，形成3, 5-磷酸二酯键完成连接反应。由于截短型T4RNA连接酶2仅能识别核酸的腺苷酰化末端，因此避免了5’末端磷酸化RNA自身环化副产物和RNA分子间连接副产物的形成，有效提升建库效率。在单链DNA连接中，5’腺苷酰化DNA底物由于含有高能焦磷酸键处于活化状态，比5’磷酸化DNA底物更易于进行连接反应。有些DNA连接酶，如K159S位点突变的T4 DNA连接酶，仅能催化5’腺苷酰化DNA底物与3’羟基末端连接。
DNA或RNA的腺苷酰化主要通过化学合成或酶法合成。化学合成法合成难度大、效率低、成本高，目前中国境内尚无公司可以完成，国外也仅有少数引物合成公司如Integrated DNA Technologies（IDT）能够完成。这些因素导致此类化学修饰合成价格昂贵，单条引物合成价格超过万元，且到货周期长，无法满足飞速进展的分子生物学研究和应用。酶法合成腺苷酰化具有操作简单、反应迅速、催化效率高等优势，非常适合各种实验室研究和技术研发使用。目前，酶法催化腺苷酰化反应以NEB公司的5’DNA Adenylation Kit为代表。该试剂盒利用Mth RNA连接酶在高浓度ATP作用下连接效率较低的特性，催化5’磷酸化DNA或RNA末端在ATP的作用下形成5’腺苷酰化产物。由于Mth RNA连接酶催化连接反应活性的存在，该酶无法完全避免5’磷酸化底物连接副产物的产生。同时，该酶以5’磷酸化核酸为底物，因此仍然需要合成5’磷酸化核酸后再进行酶法腺苷酰化合成。
与核酸的腺苷酰化相关的现有的技术：
现有技术方案一
通过对合成的5’羟基末端DNA或RNA底物依次单独进行磷酸化激酶处理磷酸化和腺苷酰化酶处理腺苷酰化
此技术的方案是：第一步，先通过引物合成公司化学合成5’羟基末端的DNA或RNA。第二步，使用商业化T4多核苷酸激酶（T4 Polynucleotide Kinase， T4 PNK）进行磷酸化反应，使核酸的5’端加上磷酸基团成为磷酸化产物。第三步，对磷酸化反应产物进行纯化处理。第四步，使用腺苷酰化酶对纯化后的5’磷酸末端产物进行腺苷酰化反应。第五步，将腺苷酰化的核酸从酶反应液中纯化出来，得到腺苷酰化修饰的DNA或RNA。
存在的的缺点：
（1）需要经过多步的反应和纯化，操作步骤较为繁琐；（2）商业化磷酸化试剂、腺苷酰化试剂、纯化试剂的使用都增加了腺苷酰化DNA或RNA合成的成本；（3）不同纯化方法的核酸回收率有差别，对于片段较短的核酸分子回收效率更差，多个步骤的纯化过程导致更多的核酸损失，影响腺苷酰化核酸的产量。此技术方案合成腺苷酰化核酸成本较高，耗时耗力，不适合用于大用量腺苷酰化DNA或RNA的制备。
现有技术方案二
其与技术一类似，不同之处在于通过公司化学合成磷酸化底物直接进行腺苷酰化。
此技术的方案是：第一步，通过引物合成公司先合成5’末端磷酸化修饰的DNA或RNA。第二步，使用腺苷酰化酶对磷酸化样品进行腺苷酰化。第三步，通过纯化回收腺苷酰化产物。
存在的的缺点：
（1）需要从公司化学合成5’磷酸化的DNA或RNA作为反应底物，如果需要防止底物自身环化副产物产生还需要在底物3’端进行特殊修饰以防止核酸自连，这些化学修饰耗时较长且增加了腺苷酰化DNA或RNA的合成成本（2）现有商品化腺苷酰化酶价格较为昂贵，反应成本较高。现有技术二合成腺苷酰化DNA或RNA需要订购磷酸化底物，周期较长且成本仍然较高。
现有技术方案三
现有技术方案三是通过引物合成公司使用化学合成法合成5’腺苷酰化的DNA或RNA。化学合成腺苷酰化DNA或RNA的关键步骤是焦磷酸键的形成，合成方法一般包括五步：第一步，以常规引物合成的方式采用β-乙腈亚磷酰胺合成一段DNA或RNA序列；第二步，在核酸的5’端偶联上磷酸基团；第三步，使用磷酰咪唑烷、磷酸N-甲基咪唑烷等化合物合成活化形式的腺苷酸衍生物；第四步，将腺苷5’-咪唑磷酸酯（adenosine 5’-phosphorimidazolidate）与固定化的DNA或RNA 5’磷酸基团偶联，生成腺苷酰化的DNA或RNA；第五步，通过PAGE电泳纯化相差一个碱基的腺苷酰化DNA或RNA产物。具体反应方案见图1。
存在的缺点：
（1）通过化学合成法直接合成腺苷酰化DNA或RNA的方案要经过多步反应，步骤繁琐，需要有较高的专业技术知识和操作能力的技术人员才能进行合成，不适合普通实验室独立制备；（2）合成原料价格高；（3）在合成大于11碱基的DNA或RNA时反应效率不高，产物需要通过PAGE电泳从相差1个碱基的底物和产物之间分离开来，纯化过程耗时费力，进一步增加了合成的成本。（4）目前国内还没有提供直接化学合成腺苷酰化DNA或RNA的公司，从国外合成公司订购成本高昂且货期较长。（5）合成过程中，5’腺苷酰化末端一与3’羟基末端连接，因此需要对3’末端进行修饰，以阻断产物自连。
发明内容
为了解决现有技术存在的缺陷，本发明提供了一种新型磷酸化腺苷酰化融合酶及其制备方法与应用，可以一步法从5’-羟基末端DNA或RNA合成腺苷酰化DNA或RNA，只需要进行一次反应和纯化，可以简便、快捷、低成本、高效地合成腺苷酰化DNA或RNA。
本发明采用的技术解决方案是：一种新型磷酸化腺苷酰化融合酶，包含以下项或由其组成：
（a）SEQ ID NO：3至6中所示的氨基酸序列；或；
（b）SEQ ID NO：3至6中所示的序列具有至少80％的序列同一性的氨基酸序列；或；
（c）包含表达Adenylase（由PF0353基因编码，来源于Pyrococcus furiosus,UniProtKB数据库名称为Q8U3V2_PYRFU，我们将其命名为Adenylase）蛋白的氨基酸序列见SEQ ID NO：1和表达T4 PNK蛋白的氨基酸序列见SEQ ID NO：2，中间接或不接linker肽段，linker肽段可为长度1至任意长度的氨基酸序列。
所述的包含表达Adenylase蛋白的氨基酸序列和/或表达T4 PNK蛋白的氨基酸序列的任意端具有亲和纯化氨基酸标签。
所述的包含表达Adenylase蛋白的氨基酸序列的N端带有8个组氨酸纯化标签和/或所述的表达T4 PNK蛋白的氨基酸序列的C端带有6个组氨酸纯化标签。
所述的包含表达Adenylase蛋白的氨基酸序列和/或表达T4 PNK蛋白的氨基酸序列的任意端的亲和纯化氨基酸标签，可以在蛋白制备和纯化过程中通过酶切方法去除亲和纯化标签。
所述的包含表达Adenylase蛋白的氨基酸序列和/或表达T4 PNK蛋白的氨基酸序列不含亲和纯化标签，可以利用本申请目标蛋白的热稳定性，通过加热变性去除非目标蛋白。
一种新型磷酸化腺苷酰化酶的制备方法，包括以下步骤：
（1）将腺苷酰化酶Adenylase和磷酸化激酶T4 PNK两种酶的基因编码序列通过一段连接肽（Linker）顺序连接在一起，分别设计Adenylase在T4 PNK编码序列的N端或C端两种不同的融合顺序，形成Adenylase-T4PNK或T4PNK-Adenylase融合酶的编码序列；
（2）在融合酶的编码序列N端和/或C端分别加上纯化氨基酸标签用于蛋白纯化，构建到pET28a原核表达载体中，转化到大肠杆菌感受态细胞中；
（3）诱导表达Adenylase-T4PNK和T4PNK-Adenylase融合蛋白并进行纯化，得到的融合酶用于一步法腺苷酰化DNA或RNA的合成；
（4）使用5’-羟基DNA对融合酶的腺苷酰化活性进行测试，经质谱和PAGE电泳验证融合酶反应产物确定为腺苷酰化修饰的DNA。
（5）使用5’-磷酸化DNA对融合酶的腺苷酰化活性进行测试，经PAGE电泳验证融合酶反应产物确定为腺苷酰化修饰的DNA。所述的大肠杆菌感受态细胞为BL21（DE3）大肠杆菌感受态细胞。
一种新型磷酸化腺苷酰化酶在作为制备催化核酸的腺苷酰化试剂上的应用。
所述的核酸的腺苷酰化包括DNA的腺苷酰化和RNA的腺苷酰化。
所述的大肠杆菌感受态细胞为BL21（DE3）大肠杆菌感受态细胞。
本发明的有益效果是：本发明提供了一种新型磷酸化腺苷酰化酶及其制备方法与应用，构建了Adenylase-T4PNK和T4PNK-Adenylase融合酶的原核诱导表达质粒，并通过表达和纯化得到了目的融合酶，融合酶可溶性强，表达量高，纯化简单，得到的融合酶同时拥有T4多核苷酸激酶和腺苷酰化酶的活性，可以一步法将5’-羟基DNA或RNA在ATP存在的条件下直接催化反应得到5’腺苷酰化DNA或RNA。也可以将5’磷酸化DNA或RNA在ATP存在的条件下催化反应得到5’腺苷酰化DNA或RNA。使用此融合酶制备腺苷酰化DNA或RNA的方法成本低、反应效率高、简便快速，可供分子生物学技术灵活使用。
附图说明
图1为化学合成法制备腺苷酰化DNA方案示意图；其中，A：5-DNA-CPG；C：5-pDNA-CPG；1为亚磷酰胺单体；2为中间产物；3为5’-亚磷酸酯；4为腺苷5’-咪唑磷酸酯。
图2为不同连接方式产生的融合酶结构示意图及命名；其中，左边为融合酶的简称代号，右边为对应质粒编码结构示意图；Linker使用柔性连接肽SGGSGGSGGSAG，分别将Adenylase序列连接在T4PNK的N端或C端，分别将His组氨酸标签加在融合酶的N端或C端。
图3为融合酶在不同反应条件下的腺苷酰化活性鉴定；其中，图A：尿素PAGE电泳图，oligo为17nt，OH为5’羟基底物，p为磷酸化修饰底物，App为腺苷酰化修饰DNA对照，腺苷酰化DNA电泳比磷酸化DNA电泳速度约慢1个碱基；P+A为联合使用T4 PNK和Adenylase反应产物。其他条带为所注条件的反应产物。图B：相对腺苷酰化效率比较，通过融合酶反应产物量与T4 PNK加Adenylase联合使用反应产物量之比得到；三种融合酶都成功将5’-羟基底物反应为腺苷酰化产物，8H-A-L-P和8H-P-L-A在80℃ 1h的条件下反应效率较高。
图4为腺苷酰化反应的底物与产物质谱鉴定图；其中，OH为5’羟基底物；P为5’磷酸化DNA；MthRnl为商品化腺苷酰化试剂盒反应产物，底物为5’磷酸化DNA；Ade为Adenylase反应产物；8H-A-L-P和8H-P-L-A反应产物峰图如图所示。
图5为融合酶对RNA样品的腺苷酰化活性测试。Input为19碱基RNA，8H-A-L-P为5’羟基RNA为底物时，经8H-A-L-P融合酶催化产物。
图6为融合酶对5’磷酸化底物腺苷酰化的催化效率。P为磷酸化55nt底物对照，余2条带为2种融合酶催化产物的电泳结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整的描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获的的所有其他实施例，都属于本发明保护的范围。
构建表达T4 PNK和Adenylase融合酶的重组质粒
表达Adenylase（PF0353）蛋白的氨基酸序列见SEQIDNO：1，在N端带有8个组氨酸的纯化标签。表达T4 PNK蛋白的氨基酸序列见SEQIDNO：2，在C端添加了6个组氨酸纯化标签。将上述氨基酸序列进行密码子优化后以现有的任意方法合成DNA序列，在两蛋白之间加入linker序列（本具体实例中linker序列为SGGSGGSGGSAG），在融合酶的任意端加入亲和纯化标签（本实例中为连续组氨酸标签）。不同方式连接的融合酶及简易命名如图2所示。不同融合酶氨基酸序列见SEQIDNO:3-SEQIDNO:6。
质粒构建过程如下：
（1）分别设计构建编码融合酶Adenylase和T4 PNK的特异性引物用于同源重组反应，引物信息见表1。使用特异性引物分别对带有Adenylase和T4 PNK编码基因的质粒进行PCR扩增，按照Phanta Max Super-Fidelity DNA Polymerase试剂盒说明书配制25μL PCR反应体系，设置PCR扩增条件95℃30s，1个循环，95℃ 15s，60℃15s，72℃3min，35个循环，72℃ 5min，1个循环。获得的PCR产物用1%琼脂糖凝胶电泳进行鉴定，对条带单一的PCR产物进行柱纯化，纯化方法按DNA Clean and Concentrator试剂盒说明书进行。
Figure 250134DEST_PATH_IMAGE001
（2）获得的PCR基因片段与pET28a载体按照ClonExpress II One Step CloningKit试剂盒进行同源重组克隆。冰上配制反应体系
Figure 798927DEST_PATH_IMAGE002
在PCR仪上37℃孵育30min，反应结束产物立即置于冰上。
（3）立即取10μL重组反应产物转化入BL21（DE3）化学感受态细胞中，涂布在含卡那霉素的LB固体平板上，37℃恒温培养16h过夜后挑取单克隆菌落培养。
（4）通过Champagne Taq DNA Polymerase试剂盒进行菌液PCR验证克隆结果，将菌液PCR片段条带位置正确的重组菌提取质粒进行一代测序鉴定DNA序列正确。
融合酶的诱导表达和纯化
融合酶的诱导表达
按照上述方法成功构建四种不同连接顺序的融合酶表达重组质粒及其表达菌株，将表达融合酶的重组大肠杆菌在37℃，250rpm条件LB培养8h 后按1:50比例转入1L体积LB培养基中，扩大培养至OD600=0.6时，加入终浓度为1mM的IPTG，在18℃，250rpm诱导表达16h。
融合酶的纯化
诱导表达结束后将菌液离心，用LysisBuffer（20mM NaH2PO4、300mMNaCl、20mM咪唑）洗涤两次后再重新悬浮菌体沉淀，超声破碎后离心取上清液在AKTA蛋白纯化系统上进行Ni-NTA柱亲和纯化和Heparin柱纯化，纯化过程如下：
（1）充分平衡Ni-NTA柱柱后，将样品通过进样泵输送到Ni-NTA柱柱中，用平衡液充分洗涤Ni-NTA柱柱10个柱体积，分别使用含有50mM、100mM、250mM、500mM浓度咪唑的洗脱液对Ni-NTA柱进行洗脱，在250mM咪唑洗脱液条件下得到了最高的峰收集产物。
（2）将Ni-NTA柱峰收集产物稀释5倍后过Heparin柱纯化，经700mM的NaCl浓度洗脱液从Heparin柱洗脱得到产物峰，洗脱产物加入等体积甘油，混匀后保存于-20℃。纯化得到的蛋白量见表3，除P-L-A-6H表达量过低外，剩余三个融合酶均纯化得到酶蛋白。
Figure 621389DEST_PATH_IMAGE003
融合酶可以成功将5’-OH底物催化为腺苷酰化DNA产物
Oligo信息
Figure 59062DEST_PATH_IMAGE004
融合酶的腺苷酰化反应活性鉴定
为鉴定融合酶的腺苷酰化催化活性，对融合酶进行腺苷酰化测试：
（1）按表5冰上配制反应体系
Figure 992383DEST_PATH_IMAGE005
80℃反应30min，产物加入等体积的2×甲酰胺Loading（95%甲酰胺、18mM EDTA、0.025% SDS、0.025% Xylene Cyanol、0.025% Bromophenol Blue），20%变性Urea-PAGE电泳鉴定产物。
（2）对纯化得到的三种融合酶使用与Adenylase相同的反应体系，ATP终浓度增加到1mM，配制如表6所示反应体系
Figure 457999DEST_PATH_IMAGE006
分别按37℃反应30min后80℃反应30min条件或80℃反应1h条件进行反应，反应产物使用20%变性Urea-PAGE鉴定。电泳结果如图3所示，结果表明融合酶具有T4 PNK和Adenylase两种酶活性，成功催化5’羟基末端DNA底物为腺苷酰化产物，且反应直接在80℃进行时催化效果更好。不同融合酶催化产物对比显示，融合酶A-L-P-6H的产物亮度相较于另外两个融合酶更低，N端8×His标签对应的两个融合蛋白的反应活性更高。值得注意的是，直接进行80℃的催化反应将羟基底物催化为腺苷酰化产物，表明融合酶的T4 PNK酶活性能够在高温下进行，提示两酶融合提高了融合酶中T4 PNK酶的热稳定性，使原来热敏感的T4 PNK蛋白拥有了良好的热稳定性。
同上述反应条件，同时进行了对RNA样品腺苷酰化的活性测试。如图4所示，融合酶成功催化5’羟基的RNA底物为腺苷酰化产物。
同上述反应条件，融合酶仍然具有催化5’磷酸化底物为腺苷酰化产物的能力（图5）。
质谱鉴定融合酶反应产物
将5’羟基底物、5’磷酸化底物、5’腺苷酰化DNA产物作为标准品对照，将8H-AL-P、8H-P-L-A融合酶反应产物在Agena MassArray核酸飞行质谱平台进行分子量鉴定。质谱鉴定结果如图6所示。根据质谱验证结果，融合酶成功将5’OH底物转化为了5’腺苷酰化产物，融合酶催化产物分子量与标准品对照完全相同表明融合酶催化的产物确为腺苷酰化DNA产物。根据核酸飞行质谱定量分析显示， H-A-L-P融合酶腺苷酰化效率达90%以上。
各位技术人员须知：虽然本发明已按照上述具体实施方式做了描述，但是本发明的发明思想并不仅限于此发明，任何运用本发明思想的改装，都将纳入本专利专利权保护范围内。
以上所述仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
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Asn Pro Glu Arg Arg Leu Ala Trp Glu Thr Phe Ala Lys Glu Tyr Gly
100 105 110
Trp Lys Val Glu His Lys Val Phe Asp Val Pro Trp Thr Glu Leu Val
115 120 125
Lys Arg Asn Ser Lys Arg Gly Thr Lys Ala Val Pro Ile Asp Val Leu
130 135 140
Arg Ser Met Tyr Lys Ser Met Arg Glu Tyr Leu Gly Leu Pro Val Tyr
145 150 155 160
Asn Gly Thr Pro Gly Lys Pro Lys Ala Val Ile Phe Asp Val Asp Gly
165 170 175
Thr Leu Ala Lys Met Asn Gly Arg Gly Pro Tyr Asp Leu Glu Lys Cys
180 185 190
Asp Thr Asp Val Ile Asn Pro Met Val Val Glu Leu Ser Lys Met Tyr
195 200 205
Ala Leu Met Gly Tyr Gln Ile Val Val Val Ser Gly Arg Glu Ser Gly
210 215 220
Thr Lys Glu Asp Pro Thr Lys Tyr Tyr Arg Met Thr Arg Lys Trp Val
225 230 235 240
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245 250 255
Gly Asp Thr Arg Lys Asp Asp Val Val Lys Glu Glu Ile Phe Trp Lys
260 265 270
His Ile Ala Pro His Phe Asp Val Lys Leu Ala Ile Asp Asp Arg Thr
275 280 285
Gln Val Val Glu Met Trp Arg Arg Ile Gly Val Glu Cys Trp Gln Val
290 295 300
Ala Ser Gly Asp Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ala
305 310 315 320
Gly Glu Asn Met Val Ser Ser Lys Phe Lys Glu Leu Leu Tyr Thr Leu
325 330 335
Gly Ile Pro Glu Asp Lys Val Glu Ile Leu Glu Ala Arg Gly Gly Ile
340 345 350
Met Glu Asp Glu Phe Glu Gly Ile Arg Tyr Leu Arg Phe Lys Asn Ser
355 360 365
Val Gly Lys Leu Arg Arg Gly Thr Val Leu Phe Glu Asp Gly Thr Thr
370 375 380
Val Phe Gly Phe Pro His Ile Lys Arg Ile Val Asn Leu Ser Ala Gly
385 390 395 400
Val Arg Lys Ile Phe Lys Ser Ser Glu Phe Tyr Val Glu Glu Lys Val
405 410 415
Asp Gly Tyr Asn Val Arg Val Val Lys Phe Lys Asp Arg Ile Leu Gly
420 425 430
Ile Thr Arg Gly Gly Phe Ile Cys Pro Tyr Thr Thr Glu Arg Ile Ala
435 440 445
Glu Phe Val Pro Glu Glu Phe Phe Lys Asp His Lys Asp Leu Val Leu
450 455 460
Val Gly Glu Met Ala Gly Pro Glu Ser Pro Tyr Leu Val Glu Gly Pro
465 470 475 480
Pro Tyr Val Lys Glu Asp Ile Gln Phe Phe Leu Phe Asp Ile Gln Asp
485 490 495
Ile Lys Thr Gly Ser Ser Leu Pro Val Glu Glu Arg Leu Lys Leu Ala
500 505 510
Glu Glu Tyr Gly Ile Asn His Val Glu Val Phe Gly Arg Tyr Ser Tyr
515 520 525
Lys Asp Ile Asp Asp Leu Tyr Glu Leu Ile Glu Arg Leu Ser Arg Glu
530 535 540
Gly Arg Glu Gly Ile Val Met Lys Ser Pro Asp Met Lys Lys Ile Val
545 550 555 560
Lys Tyr Val Thr Pro Tyr Ala Asn Ile Asn Asp Ile Lys Ile Gly Ala
565 570 575
Arg Val Phe Tyr Glu Leu Pro Gly Gly Tyr Phe Thr Ser Arg Ile Ser
580 585 590
Arg Leu Ala Phe Tyr Ile Ala Glu Lys Lys Ile Arg Gly Glu Glu Leu
595 600 605
His Asn Leu Ala Leu Gln Leu Gly Lys Ala Leu Leu Gln Pro Leu Val
610 615 620
Glu Ala Ile His Asp Val Thr Gln Gly Asp Val Ile Ala Glu Arg Phe
625 630 635 640
Arg Val Arg Val Arg Lys Ile Glu Thr Ala Tyr Lys Met Val Thr His
645 650 655
Phe Glu Lys Leu Gly Leu Glu Ile Glu Ile Glu Asp Ile Glu Glu Ile
660 665 670
Glu Gly Gly Trp Arg Val Thr Phe Lys Arg Val Tyr Pro Glu Ala Thr
675 680 685
Arg Glu Ile Arg Asp Leu Ile Gly Gly Lys Ala Phe Val Asp
690 695 700
<210> 6
<211> 700
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Lys Lys Ile Ile Leu Thr Ile Gly Cys Pro Gly Ser Gly Lys Ser
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Thr Trp Ala Arg Glu Phe Ile Ala Lys Asn Pro Gly Phe Tyr Asn Ile
20 25 30
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35 40 45
Glu Tyr Lys Tyr Thr Lys Lys Lys Glu Gly Ile Val Thr Gly Met Gln
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Phe Asp Thr Ala Lys Ser Ile Leu Tyr Gly Gly Asp Ser Val Lys Gly
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Val Ile Ile Ser Asp Thr Asn Leu Asn Pro Glu Arg Arg Leu Ala Trp
85 90 95
Glu Thr Phe Ala Lys Glu Tyr Gly Trp Lys Val Glu His Lys Val Phe
100 105 110
Asp Val Pro Trp Thr Glu Leu Val Lys Arg Asn Ser Lys Arg Gly Thr
115 120 125
Lys Ala Val Pro Ile Asp Val Leu Arg Ser Met Tyr Lys Ser Met Arg
130 135 140
Glu Tyr Leu Gly Leu Pro Val Tyr Asn Gly Thr Pro Gly Lys Pro Lys
145 150 155 160
Ala Val Ile Phe Asp Val Asp Gly Thr Leu Ala Lys Met Asn Gly Arg
165 170 175
Gly Pro Tyr Asp Leu Glu Lys Cys Asp Thr Asp Val Ile Asn Pro Met
180 185 190
Val Val Glu Leu Ser Lys Met Tyr Ala Leu Met Gly Tyr Gln Ile Val
195 200 205
Val Val Ser Gly Arg Glu Ser Gly Thr Lys Glu Asp Pro Thr Lys Tyr
210 215 220
Tyr Arg Met Thr Arg Lys Trp Val Glu Asp Ile Ala Gly Val Pro Leu
225 230 235 240
Val Met Gln Cys Gln Arg Glu Gln Gly Asp Thr Arg Lys Asp Asp Val
245 250 255
Val Lys Glu Glu Ile Phe Trp Lys His Ile Ala Pro His Phe Asp Val
260 265 270
Lys Leu Ala Ile Asp Asp Arg Thr Gln Val Val Glu Met Trp Arg Arg
275 280 285
Ile Gly Val Glu Cys Trp Gln Val Ala Ser Gly Asp Ser Ser Gly Gly
290 295 300
Ser Gly Gly Ser Gly Gly Ser Ala Gly Glu Asn Met Val Ser Ser Lys
305 310 315 320
Phe Lys Glu Leu Leu Tyr Thr Leu Gly Ile Pro Glu Asp Lys Val Glu
325 330 335
Ile Leu Glu Ala Arg Gly Gly Ile Met Glu Asp Glu Phe Glu Gly Ile
340 345 350
Arg Tyr Leu Arg Phe Lys Asn Ser Val Gly Lys Leu Arg Arg Gly Thr
355 360 365
Val Leu Phe Glu Asp Gly Thr Thr Val Phe Gly Phe Pro His Ile Lys
370 375 380
Arg Ile Val Asn Leu Ser Ala Gly Val Arg Lys Ile Phe Lys Ser Ser
385 390 395 400
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405 410 415
Lys Phe Lys Asp Arg Ile Leu Gly Ile Thr Arg Gly Gly Phe Ile Cys
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Pro Tyr Thr Thr Glu Arg Ile Ala Glu Phe Val Pro Glu Glu Phe Phe
435 440 445
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450 455 460
Ser Pro Tyr Leu Val Glu Gly Pro Pro Tyr Val Lys Glu Asp Ile Gln
465 470 475 480
Phe Phe Leu Phe Asp Ile Gln Asp Ile Lys Thr Gly Ser Ser Leu Pro
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Val Glu Glu Arg Leu Lys Leu Ala Glu Glu Tyr Gly Ile Asn His Val
500 505 510
Glu Val Phe Gly Arg Tyr Ser Tyr Lys Asp Ile Asp Asp Leu Tyr Glu
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Leu Ile Glu Arg Leu Ser Arg Glu Gly Arg Glu Gly Ile Val Met Lys
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Ser Pro Asp Met Lys Lys Ile Val Lys Tyr Val Thr Pro Tyr Ala Asn
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Ile Asn Asp Ile Lys Ile Gly Ala Arg Val Phe Tyr Glu Leu Pro Gly
565 570 575
Gly Tyr Phe Thr Ser Arg Ile Ser Arg Leu Ala Phe Tyr Ile Ala Glu
580 585 590
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595 600 605
Lys Ala Leu Leu Gln Pro Leu Val Glu Ala Ile His Asp Val Thr Gln
610 615 620
Gly Asp Val Ile Ala Glu Arg Phe Arg Val Arg Val Arg Lys Ile Glu
625 630 635 640
Thr Ala Tyr Lys Met Val Thr His Phe Glu Lys Leu Gly Leu Glu Ile
645 650 655
Glu Ile Glu Asp Ile Glu Glu Ile Glu Gly Gly Trp Arg Val Thr Phe
660 665 670
Lys Arg Val Tyr Pro Glu Ala Thr Arg Glu Ile Arg Asp Leu Ile Gly
675 680 685
Gly Lys Ala Phe Val Asp His His His His His His
690 695 700

Claims (10)

1.一种新型磷酸化腺苷酰化酶，其特征在于，包含以下项或由其组成：
（a）SEQ ID NO：3至6中所示的氨基酸序列；或；
（b）SEQ ID NO：3至6中所示的序列具有至少80％的序列同一性的氨基酸序列；或；
（c）包含表达Adenylase蛋白的氨基酸序列见SEQ ID NO：1和表达T4 PNK蛋白的氨基酸序列见SEQ ID NO：2，中间接或不接linker肽段，linker肽段可为长度1至任意长度的氨基酸序列。
2.根据权利要求1所述的一种新型磷酸化腺苷酰化酶，其特征在于，所述的包含表达Adenylase蛋白的氨基酸序列和/或表达T4 PNK蛋白的氨基酸序列的任意端具有亲和纯化氨基酸标签。
3.根据权利要求2所述的一种新型磷酸化腺苷酰化酶，其特征在于，所述的包含表达Adenylase蛋白的氨基酸序列的N端带有8个组氨酸的纯化氨基酸标签和/或所述的表达T4PNK蛋白的氨基酸序列的C端带有6个组氨酸纯化标签。
4.根据权利要求3所述的一种新型磷酸化腺苷酰化酶，其特征在于，所述的包含表达Adenylase蛋白的氨基酸序列和/或表达T4 PNK蛋白的氨基酸序列的任意端的亲和纯化氨基酸标签，可以在蛋白制备和纯化过程中通过酶切方法去除亲和纯化标签。
5.一种权利要求1所述的新型磷酸化腺苷酰化酶的制备方法，其特征在于，包括以下步骤：
（1）将腺苷酰化酶Adenylase和T4 PNK两种酶的基因编码序列通过一段编码连接肽（Linker）的序列连接在一起，分别设计Adenylase在T4 PNK编码序列的N端或C端两种不同的融合顺序，形成Adenylase-T4 PNK融合酶的编码序列；
（2）在融合酶的编码序列N端和/或C端分别加上纯化氨基酸标签用于蛋白纯化，构建到pET28a原核表达载体中，转化到大肠杆菌感受态细胞中；
（3）诱导表达Adenylase-T4 PNK融合蛋白并进行纯化，得到的融合酶用于一步法DNA腺苷酰化反应；
（4）使用5’-羟基DNA对融合酶的磷酸化腺苷酰化活性进行测试，经质谱或PAGE电泳验证融合酶反应产物确定为腺苷酰化修饰的DNA。
6.根据权利要求5所述的制备方法，其特征在于，所述的大肠杆菌感受态细胞为BL21（DE3）大肠杆菌感受态细胞。
7.一种权利要求1所述的新型磷酸化腺苷酰化酶在作为制备催化核酸腺苷酰化试剂上的应用。
8.根据权利要求7所述的应用，其特征在于，所述的核酸的腺苷酰化包括DNA的腺苷酰化和RNA的腺苷酰化。
9.根据权利要求7所述的应用, 所述的核酸为5’末端为羟基末端。
10.根据权利要求7所述的应用, 所述的核酸为5’末端为磷酸化末端。
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