CN115029333B - 一种核酸内切酶及其纯化方法和应用 - Google Patents
一种核酸内切酶及其纯化方法和应用 Download PDFInfo
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Abstract
本发明公开一种核酸内切酶及其纯化方法和应用。所述的核酸内切酶包含GajA蛋白和识别标签。编码GajA蛋白的核苷酸序列见序列表SEQ ID NO.1，识别标签为组氨酸标签、FLAG标签、HA标签、SBP标签、Avi标签、Nus标签、V5标签中的任意一种，GajA蛋白的氨基端与所述识别标签连接。本发明提供的核酸内切酶是一个全新的具有序列识别特异性的缺刻内切酶，而且根据识别序列的不同，可同时作为限制性内切酶和缺刻内切酶发挥作用。因此，该酶可广泛应用于DNA加工、基因编辑、基因工程等领域。另外，核酸内切酶的活性受核苷酸的负调控，拓展了该酶的应用。
Description
技术领域
本发明属于核酸内切酶技术领域，特别涉及核酸内切酶及其纯化方法和应用。
背景技术
核酸内切酶(endonuclease)是一类广泛存在于生物体内的核酸水解酶，可水解分子链内部磷酸二酯键生成寡核苷酸。从对底物的特异性来看，核酸内切酶可分为DNaseⅠ、DNaseⅡ等分解DNA的酶，以及RNase、RNaseT1等分解RNA的酶。一般来说，大都不具碱基特异性，但也有能够识别并切断特定的碱基或碱基序列的酶。所以，把用来识别特定的脱氧核苷酸序列，并对每条链中特定部位的两个脱氧核糖核苷酸之间的磷酸二酯键进行切割的一类核酸内切酶，称为限制性核酸内切酶，简称限制酶。根据限制酶的结构、辅因子的需求、切割位点与作用方式，可将限制酶分为三种类型，分别是第一型(Type I)、第二型(Type II)、第三型(Type III)及第四型(Type IV)。Ⅰ型限制性内切酶既能催化宿主DNA的甲基化，又催化非甲基化的DNA的水解，需要ATP，切割位点与识别位点间的间距不定；而Ⅱ型限制性内切酶只催化非甲基化的DNA的水解，具有特异性的识别序列，切割位点位于识别序列内或邻近识别序列。Ⅲ型限制性内切酶同时具有修饰及认知切割的作用，需要ATP。IV型限制性内切酶仅切割甲基化的DNA，切割位点大约距离识别位点30bp。其中，Type II型限制性内切酶被广泛应用于生物科学的研究，使基因重组技术成为可能，是基因工程的基础。限制性内切酶可用于DNA基因组物理图谱的组建、基因的定位和基因分离、DNA分子碱基序列分析、比较相关的DNA分子和遗传工程、DNA加工和基因工程等领域。限制性核酸内切酶是由细菌产生的，限制酶一般不切割自身的DNA分子，只切割外源DNA，其生理意义是提高自身的防御能力。
限制性内切酶结合到它们的识别序列上，通常同时切割DNA的两条链，两个独立的水解反应同时进行，通常是由限制酶内存在的两个催化位点驱动的，每一个用于水解一条链。只能水解双链结构中的一条链，从而产生“切口”而不是切割的DNA分子，这一类酶称为缺刻内切酶。缺刻内切酶包括天然的缺刻内切酶，如：Nt.BstNBI、Nb.BtsI、Nb.BsrDI；也可以通过人工改造限制酶得到缺刻内切酶，如：Nt.BspQI、Nt.BbvCI、Nb.BsmI。缺刻内切酶产生的缺口可以作为各种进一步酶反应的起始点，如替换DNA合成、链位移放大(Walker etal.,1992)、核酸外切降解或小缺口的产生(Wang et al.,2000)，还可用于基因组制图。随着科学技术的发展，缺刻内切酶将发展出越来越多的用途。
发明内容
针对以上现有技术的不足，本发明发现了一种全新的核酸内切酶，成功克隆并建立了其表达纯化方法，进行了功能鉴定和应用探索，为DNA加工、基因工程和基因编辑等领域的研究与应用提供一种有效的候选工具酶。具体通过以下技术实现。
一种核酸内切酶，包括GajA蛋白和识别标签；
所述GajA蛋白的氨基酸序列如SEQ ID NO.1所示；
所述识别标签为组氨酸标签、FLAG标签、HA标签、SBP标签、Avi标签、Nus标签、V5标签中的任意一种；所述组氨酸标签的编码核苷酸序列如SEQ ID NO.2所示；
所述GajA蛋白的氨基端与所述识别标签连接。
虽然现有的技术文献中提到了Gabija细菌防御系统包含GajA蛋白和GajB蛋白两个组分，主要作用抵抗噬菌体队细菌的入侵。然而，GajA蛋白的功能还没有人进行相关研究。本发明的技术人员参考了现有的技术文献，预测GajA蛋白是ATP依赖的核酸内切酶。并经过大量的实验数据得以证实这种推论。GajA蛋白包含一个ATPase结构域和一个TOPRIM结构域，其在细菌防御系统中具有特异性的核酸切割加工能力，是Gabija细菌防御系统的核心组分。
本发明提供的核酸内切酶以GajA蛋白为主体，在GajA蛋白的氨基端还连接有识别标签。在GajA蛋白上接入这种识别标签有利于后续进行亲和层析纯化，得到不含RNA酶污染的应用级蛋白质。本发明所得到的GajA蛋白来自于Bacillus cereus VD045(蜡样芽孢杆菌VD045)，或者来自与所述GajA的蛋白质序列同源性高于20％并含有一个ATPase结构域和一个TOPRIM结构域的其他同源蛋白。本发明提供的核算内切酶的识别标签主要为组氨酸标签、FLAG标签、HA标签、SBP标签、Avi标签、Nus标签、V5标签中的任意一种。上述识别标签中，FLAG标签、HA标签、SBP标签、Avi标签、Nus标签、V5标签是目前行业内常用的氨基酸序列已知得标签。
本发明对GajA蛋白的功能的解析和发现，为生物学研究提供新的分子生物学工具酶乃至创造出新的核酸生物技术。
优选地，所述组氨酸标签的编码核苷酸序列如SEQ ID NO.2所示，由6个组氨酸串联而成。
更优选地，所述核酸内切酶的氨基端与所述识别标签之间还连接有柔性肽段，所述柔性肽段由1-10个氨基酸组成。在GajA蛋白和识别标签之间设置柔性肽段的作用是使所关联的标签充分展示，同时不影响目的蛋白的正确折叠。本发明所述的柔性肽段可采用现有富含甘氨酸或丝氨酸的串联组合，例如Gly-Gly-Ser-Gly-Gly-Ser(GGSGGS，6个氨基酸)、Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(GGGGSGGGGS，10个氨基酸)等。这些串联组合中也可以包含蛋白酶切位点，例如Leu-Val-Pro-Arg-Gly-Ser(LVPAGS)。
进一步优选地，所述柔性肽段为若干甘氨酸和若干丝氨酸的氨基酸串联组合，或含有蛋白酶切位点的氨基酸串联组合。
更进一步优选地，所述柔性肽段为SSGLVPAGSH。该柔性肽段即包含了序列为SSG的甘氨酸和丝氨酸的串联组合，也包含了蛋白酶切位点LVPAGS，还含有1个组氨酸(H)。
更优选地，所述核酸内切酶的缺刻位点的核苷酸识别序列为：
5’-TNNNSXRGGNNA-3’；其中，S为G或C，R为A或G，N为A、G、C或T，X为缺刻位点。上述核苷酸识别序列即本发明的核酸内切酶的缺刻内切识别序列，序列中其中，S可以为鸟嘌呤(G)或胞嘧啶(C)，R可以为腺嘌呤(A)或胸腺嘧啶(T)，N则可以是A、G、C、T中任意一种碱基。
本发明还提供了上述核酸内切酶的纯化方法，包括以下步骤，
S1、蛋白表达：
S11、合成所述核酸内切酶的编码基因，将所述核酸内切酶的基因克隆到原核表达载体pET28a中，获得重组质粒；
S12、将重组质粒转化BL21(DE3)感受态细胞，涂布于含有50μg/ml卡那霉素的LB培养基平板上，置于37℃培养箱中静置培养过夜，挑取单克隆鉴定获得所述核酸内切酶表达菌种并于-80℃环境中保种；
S13、所述核酸内切酶表达菌种转接LB培养基中37℃过夜活化，按1％比例稀释后在37℃下放大培养；待OD600为0.6-0.8时，加入终浓度为0.05-0.4mM的IPTG诱导所述核酸内切酶表达，在10℃-15℃的温度下诱导表达16-20h后收集菌体，再加入细菌裂解液裂解后离心得上清液；
S2、蛋白纯化：
S21、将所述步骤S13中收集的菌体裂解上清液通过镍柱，按照浓度由低到高的顺序加入咪唑溶液与镍柱竞争结合洗脱所述核酸内切酶，所述核酸内切酶在加入高浓度的咪唑溶液时被洗脱，收集目的蛋白；
S22、将镍柱亲和层析收集的所述核酸内切酶溶液进行超滤浓缩，然后用凝胶过滤层析法进一步纯化，收集洗脱峰溶液；
S23、将洗脱峰溶液超滤浓缩后用透析液多次透析，收集透析后蛋白置于-20℃保存。
发明人通过上述基因克隆的方法，得到了来源于Bacillus cereus VD045的GajA基因，将其插入到原核表达载体pET-28a中，然后转到大肠杆菌中进行诱导表达。为了得到高纯度及活性的GajA蛋白，申请人进一步利用镍柱层析、凝胶过滤层析、离子交换层析、蛋白透析等多种方法对表达的蛋白进行纯化，并对纯化方法进行优化，最终得到了高纯度的GajA蛋白。为了检验得到的GajA蛋白的活性，申请人进行了体外功能鉴定，发现了上述的切割特异性和缺刻位点，且具有较高活性。
优选的，上述纯化方法的步骤S13中，细菌裂解上清液的制备方法为：将步骤S13诱导表达获得的菌体混合物在5000rpm、4℃离心15min，收集菌体沉淀，将菌体重悬于裂解液中；立即于-80℃冷冻，凝固后取出置于冰上融化1h，重复冻融两次；再在35000rpm、4℃离心1h，离心后取上清液通过0.45μm孔径的滤膜过滤，即得细胞裂解上清液；所述裂解液中含有20mM Tris-HCl(pH值7.5)、300mM NaCl、0.5mg/ml溶菌酶、0.5mM DTT；
步骤S21中，所述咪唑溶液的浓度由低到高分别为20mM、50mM、100mM三种浓度，由pH值7.5、50mM Tris-HCl和300mM NaCl的缓冲液对咪唑进行稀释得到；
步骤S23具体为：将超滤浓缩后的洗脱峰溶液加入透析袋中密封，置于1L透析液中进行透析，间隔6h更换干净透析液，透析三次；透析液的成分为pH值7.5、50mM Tris-HCl，100mM NaCL，1mM DTT，0.5mM EDTA，1％Triton X-100，50％甘油。
本发明提供的核酸内切酶利用其特殊得缺刻内切功能、缺刻识别序列和缺刻位点，将能在DNA加工、基因工程、基因编辑以及监测体内NTP含量中的应用。
在利用本发明所述核酸内切酶应用在上述技术领域时，能够使核酸内切酶活性最佳的最佳反应缓冲液为pH值9，20mM Tris-HCl，1mM MgCl2，1mM DTT，反应温度为37℃。
与现有技术相比，本发明的有益之处在于：本发明提供了一种全新的具有缺刻内切功能的核酸内切酶，采用本发明的纯化方法能够获得纯度更高的这种核酸内切酶，其特殊得缺刻内切和识别位点，为DNA加工、基因编辑和基因工程等领域的研究与应用提供一种有效的候选工具酶。
附图说明
图1为实施例1提供的核酸内切酶的结构域组成；
图2为实施例1中SDS-PAGE电泳检测核酸内切酶的纯化效果图；
图3为实施例2中利用实施例1纯化的核酸内切酶进行体外切割的结果图；
图4为实施例2中实施例1纯化的核酸内切酶所需的最适Mg2+浓度的试验结果；
图5为图4的量化统计结果；
图6为实施例2中，在生理浓度Mg2+和Mn2+条件下，核酸内切酶表现出特异性切割活性，CK表示不加GajA蛋白的对照组反应；
图7为实施例2中实施例1纯化的核酸内切酶切割效率的检测结果；
图8为实施例2中图7的量化统计结果；
图9为实施例2中核酸内切酶对双链DNA(dsDNA)和单链DNA(ssDNA)的切割结果；
图10为实施例2中核酸内切酶对双链RNA(dsRNA)和单链RNA的切割结果，用相应的转录模板作为对照；
图11为实施例2中通过DNA测序总结出的实施例1的核酸内切酶的缺刻位点的识别序列，图中箭头表示缺刻位点；
图12为实施例2中ATP对核酸内切酶活性的抑制效果；
图13为实施例2中NDP、NMP、dNMP和核苷对核酸内切酶活性的影响。
具体实施方式
本发明所得到的GajA蛋白来自于Bacillus cereus VD045(蜡样芽孢杆菌VD045)，或者来自与所述GajA的蛋白质序列同源性高于20％并含有一个ATPase结构域和一个TOPRIM结构域的其他同源蛋白。经标签修饰得到的核酸内切酶均可以达到本发明所述的有益效果。由于实验原理大体相同，以下实施例仅以来自Bacillus cereus VD045的GajA蛋白为例。本发明包含的其他GajA蛋白均可以参照实施例中的方法获得。所有本发明所述的新颖核酸内切酶GajA的氨基酸序列均可在基因序列查询网站(https://www.ncbi.nlm.nih.gov/genbank/)上查询。
下面将对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，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条件下所获得的所有其它实施例，都属于本发明保护的范围。
实施例1：GajA蛋白基因扩增以及核酸内切酶的纯化
S1、GajA蛋白的基因扩增及蛋白表达
利用PCR的方法扩增GajA基因，将其克隆到原核表达载体pET-28a的酶切位点NdeI和Not I之间，使其基因5’末端融合60个碱基长度的DNA片段，该DNA片段编码所述如序列表SEQ ID NO.2所示的组氨酸标签(识别标签)，以及组氨酸标签与GajA蛋白之间的柔性肽段，连接肽段序列为Ser-Ser-Gly-Leu-Val-Pro-Arg-Gly-Ser-His(SSGLVPAGSH)，将得到的重组载体转化E.coli BL21(DE3)，即重组质粒转化的感受态细胞。
将重组质粒转化的感受态细胞置于37℃含有50μg/ml卡那霉素的LB培养基中，摇床培养至OD600值为0.6-0.8，然后加入终浓度为0.1mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)于12℃摇床诱导表达20h。
S2、GajA蛋白的纯化
细菌裂解：将步骤S1获得的混合物在5000rpm、4℃离心15min，收集菌体沉淀，将菌体重悬于含有20mM Tris-HCl(pH 7.5)、300mM NaCl、0.5mg/ml溶菌酶、0.5mM DTT的裂解液中，立即于-80℃冷冻，凝固后取出置于冰上融化1h，接着再反复冻融两次；置于冷冻高速离心机中15000rpm、4℃离心1h，离心后将上清转移至另一干净的离心管中，然后将分离出的上清液通过0.45μm孔径的滤膜过滤进一步去除杂质，过滤后的细胞裂解液可直接用于镍柱纯化或4℃短暂保存。
镍柱纯化：配制含有20mM Tris-HCl(pH 7.5)、300mM NaCl的洗脱缓冲液，利用洗脱缓冲液对咪唑进行稀释，得到20mM、50mM、100mM三种浓度的咪唑溶液备用；使用10倍镍柱填料体积的洗脱缓冲液平衡镍柱，然后将所述过滤后的细胞裂解液缓慢流过镍柱，下一步按照由低浓度到高浓度的顺序分批加入20mM、50mM、100mM的咪唑溶液过柱，洗脱非特异性结合的杂蛋白并最终将与镍柱结合的蛋白竞争性的洗脱下来；在加入上述不同浓度梯度的咪唑溶液洗脱时，每一浓度洗脱下来的蛋白液需用若干干净的冻存管保存，并按洗脱的先后顺序及咪唑浓度作好标记，最后将所有洗脱下来的蛋白液通过SDS-PAGE电泳进行检测，选择较高纯度的GajA蛋白于4℃保存。
凝胶过滤层析：将镍柱亲和层析收集的蛋白溶液通过超滤浓缩后用凝胶过滤层析法进一步纯化，收集洗脱峰溶液。本实施例所用的凝胶过滤层析洗脱缓冲液成分：pH7.5、20mM Tris-HCl，300mM NaCl，0.5mM DTT。
蛋白透析：剪取一段～10cm透析袋，底部用重力夹夹紧，将蛋白加入透析袋中然后塑料夹封口，将加有蛋白的透析袋置于1L含有pH 7.5、50mM Tris-HCl，100mM NaCL，1mMDTT，0.5mM EDTA，1％Triton X-100，50％甘油的透析液中，置于磁力搅拌器上搅拌促进溶液交换，间隔6h更换干净透析液透析三次，收集透析后蛋白置于-20℃保存。
SDS-PAGE电泳检测GajA蛋白的纯化效果：
透析后的蛋白进行SDS-PAGE电泳，然后利用考马斯亮蓝染色对透析后的GajA蛋白的纯度进行检测，结果如图2所示，可以看出透析后蛋白条带单一，表明GajA蛋白经过以上纯化步骤后得到的纯度较高。
实施例2：实施例1纯化的核酸内切酶的体外功能鉴定
(1)实施例1纯化的核酸内切酶的特异性活性检测
为了探究实施例1纯化的核酸内切酶的核酸酶活性及其作用底物，我们分别采用了环状质粒DNA、线性双链DNA、单链DNA、RNA等不同的核酸底物进行活性检测，结果发现实施例1纯化的核酸内切酶对λDNA具有特异性切割活性，用从λDNA中扩增得到的一段955bp(λ955)的DNA为反应底物，结果表实施例1纯化的核酸内切酶能切割为两个分别为583bp和372bp的特异片段(如图3所示)。酶促反应一般是需要金属离子的，所以我们检测了核酸内切酶反应所需的最适浓度Mg2+，结果表明核酸内切酶在1-5mM Mg2+浓度下的活性是最高的(如图4、5所示)。在细菌体内，Mg2+的生理浓度是4-5mM，Mn2+的生理浓度约为15μM，我们进一步检测了生理浓度的Mg2+和Mn2+下，核酸内切酶的活性结果表明在生理浓度的Mg2+和Mn2+下表现出特异性的切割活性(如图6所示)。另外，还建立了核酸内切酶的最适反应条件，最适反应缓冲液为20mM Tris-HCl，pH 9，1mM MgCl2，1mM DTT，最适反应温度为37℃。
(2)核酸内切酶的切割效率检测和底物专一性
以λ955DNA为反应底物，反应体系成分如下：125ngλ955DNA，0.2μM实施例1的核酸内切酶，选用最适反应缓冲液，补水至10μl。将反应体系混合好后置于37℃，分别反应不同时间，从而检测核酸内切酶的切割效率。结果如图7、8所示，反应30s时，超过60％的DNA底物已被消化；反应120s时，超过96％的DNA底物已被消化，表明核酸内切酶表现出快速的DNA切割活性。为检测核酸内切酶对不同底物的作用，我们分别检测了核酸内切酶对dsDNA、ssDNA、dsRNA和ssRNA的切割活性，这些底物均含有本发明的核酸内切酶的最适切割位点。结果如图9所示，核酸内切酶对dsDNA表现出切割活性，对ssDNA无切割活性；如图10所示，核酸内切酶对dsRNA和ssRNA均不表现出切割活性。这些结果表明核酸内切酶只对dsDNA具有切割活性，对ssDNA、dsRNA和ssRNA均无切割活性。
(3)核酸内切酶的缺刻位点的确定
为了探究核酸内切酶的缺刻位点，以T7 DNA为反应底物，通过DNA测序得出缺刻位点，再总结出识别序列的规律，从而得出核酸内切酶的缺刻位点。由于T7 DNA片段过大，无法直接测序，所以把T7 DNA分为6段，分别扩增出来，作为反应底物，反应体系成分如下：20ng/μl DNA，0.2μM核酸内切酶，选用最适反应缓冲液，补水至200μl，将反应体系混合好后置于37℃反应5min。经过核酸内切酶处理后，用乙醇沉淀法进行DNA纯化，再进行DNA测序，共得到110个缺刻位点。通过WebLogo软件进行作图，总结出核酸内切酶缺刻位点的识别序列为5’-TNNNSXRGGNNA-3’；其中，S为G或C，R为A或G，N为A、G、C或T，X为缺刻位点，如图11中的箭头Nicking即为缺刻位点。
(4)核苷酸对核酸内切酶的活性的影响
在反应体系中加入ATP，反应体系成分如下：125ngλ955DNA，0.2μM核酸内切酶，采用最适反应缓冲液，-/+0.5mM ATP，补水至10μl，将反应体系混合好后置于37℃反应5min，与不加ATP的反应结果进行比较，发现核酸内切酶的活性受到明显抑制(如图12所示)；进而发现其他NTP、dNTP对核酸内切酶的活性也具有明显的抑制作用。进一步用同样的方法检测了其他NDP、NMP、dNMP和核苷对核酸内切酶的活性的影响，结果发现NDP对核酸内切酶的活性具有明显的抑制作用，但NMP、dNMP和核苷不影响核酸内切酶的活性(如图13所示)。这些结果表明核酸内切酶的活性受NTP、dNTP、NDP和dNDP的调控，但不受NMP、dNMP和核苷的影响。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
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Claims (2)
1.一种GajA蛋白在DNA加工中的应用，其特征在于，将GajA蛋白的氨基端连接识别标签后制得核酸内切酶，所述应用中该核酸内切酶的活性受ATP、CTP、GTP、UTP、dNTP、ADP、CDP或GDP的负调控，对dsDNA具有切割活性。
2.根据权利要求1所述的应用，其特征在于，所述核酸内切酶的最佳反应缓冲液为pH值9，20mM Tris-HCl，1mM MgCl2，1mM DTT，反应温度为37℃。
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| Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (4)
| Title |
|---|
| A nucleotide-sensing endonuclease from the Gabija bacterial defense system;Cheng R等;《Nucleic Acids Research》;第49卷(第9期);第5216-5229页 * |
| AAA family ATPase [Bacillus cereus];NCBI Author;GenBank DataBase;Accession No. WP_000672733 * |
| NCBI Author.AAA family ATPase [Bacillus cereus].GenBank DataBase.2020,Accession No. WP_000672733. * |
| 基因编辑技术研究进展;何景进;《发育医学电子杂志》;第7卷(第3期);第253-240页 * |
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