CN110438103B - 一种新型高效的常温ii型限制性内切酶 - Google Patents
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Abstract
本发明提供了一种新型高效的常温II型限制性内切酶DFc，该酶的氨基酸序列如SEQ NO：2所示，其编码基因序列如SEQ NO：1所示；DFc酶可识别5’…AGCT…3’核酸片段中的AGCT回文结构区并在G和C之间进行切割形成平末端。酶DFc具有良好的酶活，1.0μg限制酶DFc在1×CutSmart Buffer缓冲体系和37℃或40℃温度下，在1h内可以将1nmol的含AGCT回文结构区的双链DNA片段在G和C之间完全酶切形成平末端。此外，限制酶DFc具有良好的热稳定性，该酶置于室温下30min，酶活力仍保留99%以上。本发明提供的特定全新II型常温限制酶DFc，可以作为基因工程和基因检测的工具酶。
Description
技术领域
本发明涉及分子生物学领域，具体涉及一种靶向AGCT位点的新型高效的常温II型限制性内切酶及其应用。
背景技术
限制性核酸内切酶是可以识别双链DNA上特定的核苷酸序列（通常为4到8个核苷酸），并对每条链中特定部位的两个核苷酸之间的磷酸二酯键进行切割的一类酶，简称限制酶。按照亚基组成、酶切位置、识别位点和辅助因子等因素，可将限制酶分为I、Ⅱ和Ⅲ三种类型。Ⅱ型限制性内切酶（以下简称限制酶）是基因工程中重要的工具酶，其所识别的位置多为短的回文序列，所剪切的碱基序列通常即为所识别的序列。特定的II型限制酶在特定的识别序列切割DNA分子而不会在其它地方切割，精准的切割性能使其在基因重组、基因克隆、质粒构建、DNA片段分析和突变检测等方面应用广泛。然而目前发现和商品化的限制酶基本上均为国外专利产品。
申请人取福建省境内普通农田中的牛粪秸秆类高温堆肥开展研究，在其微生态体系蛋白提取液中筛选限制酶。首先通过含4核苷酸回文结构区的DNA片段即酶切底物筛选体系获知了堆肥蛋白提取液中含有靶向AGCT位点的常温限制酶，接着利用转录组测序和目标基因捕获测序并结合生物信息分析，初步锁定了限制酶的候选编码基因，进行基因克隆和蛋白表达纯化并探究酶切特性，结果发现1种含有396个氨基酸的常温限制酶DFc，可以识别5’...AGCT...3’核酸片段的AGCT回文结构区并在G和C之间进行切割形成平末端，酶DFc常温下具有良好的酶活和热稳定性。本发明提供的特定全新II型常温限制酶DFc，可以作为基因工程和基因检测的工具酶。
发明内容
本发明提供了新型的可识别5’...AGCT...3’核酸片段中的AGCT回文结构区并在G和C之间进行切割形成平末端的常温Ⅱ型限制性内切酶，并探究了其酶切特性。
本发明解决其技术问题所采用的技术方案如下：
本发明的目的在于提供一种新型的常温Ⅱ型限制性内切酶DFc，该酶的氨基酸序列如SEQ NO：2所示，其编码基因序列如SEQ NO：1所示；DFc酶可识别5’...AGCT...3’核酸片段中的AGCT回文结构区并在G和C之间进行切割形成平末端。
酶DFc具有良好的酶活，1μg限制酶DFc在1×CutSmart Buffer缓冲体系和37℃温度下，在1h内可以将1nmol的含AGCT回文结构区的双链DNA片段（5’-FAM-F15-AGCT-R15-TAMRA-3’）在G和C之间完全酶切形成平末端。此外，DFc酶置于室温下30min，酶活力仍保留95%以上，具有良好的热稳定性。
附图说明
图1是5’-FAM-F15-NNNN-R15-TAMRA-3’即含有4核苷酸回文结构区的DNA片段体系的结构示意图，未免大量重复图中含义详见文中表述。
图2是目的基因扩增片段电泳图谱，左起第一泳道的DNA Marker从上到下长度分别为2000bp、1000bp、500bp和250bp，第二泳道为反转录扩增体系产物，第二泳道为基因组扩增体系产物。
图3是目标基因蛋白表达产物的SDS-PAGE图谱，左起第一泳道的蛋白Marker从上到下分子量分别为50kDa、33kDa、28kDa和19kDa，第二泳道为目标蛋白。
具体实施方式
以下结合具体实施例，进一步阐述本发明。
实施例1 堆肥蛋白提取液含有耐高温限制酶
高温堆肥就是将人粪尿、禽畜粪尿和秸秆等堆积起来，使样本和环境中的细菌和真菌等微生物大量繁殖，细菌和真菌等可以将有机物分解，并且释放出能量形成高温。高温堆肥是生产农家肥料的重要方式，也是分离耐高温微生物和耐高温蛋白的良好来源。取福建省境内普通农田中的牛粪秸秆类高温堆肥样本10份（5g/份），混合均匀后从中取10g，采用德国Qiagen公司的土壤蛋白提取试剂盒（NoviPure Soil Protein Extraction Kit），按照说明书中方法进行总蛋白（包含细胞内和细胞外的蛋白）提取。对提取的总蛋白，经美国Thermo Fisher公司的的Qubit3.0和配套的Protein Quantitation Assay试剂盒进行浓度测定，结果为9.76mg/mL。
接着探究堆肥样品总蛋白中的耐高温限制性内切酶特征谱，首先要制备特定酶切底物即含有拟探究的识别序列的DNA片段筛选体系。由于限制性内切酶的识别序列有一大类是回文结构，因此选择含有回文结构区的DNA片段体系先行进行筛选。合成并制备含有4核苷酸回文结构区的DNA双链片段体系“5’-FAM-F15-NNNN-R15-TAMRA-3’”，其结构如图1所示，类似于TaqMan探针的双链形式。该体系包含8种短DNA片段，每条片段5’端序列都一样，都是15个核苷酸（序列为5’-AAAAAAAAAAAAAAA-3’即5’-（A）15-3’，其中5’端的第一个核苷酸用荧光报告基团FAM进行标记），记为F15；每条片段3’端序列都一样，都是15个核苷酸（序列为5’-CCCCCCCCCCCCCCC-3’即5’-（C）15-3’， 其中3’端的最后一个核苷酸用荧光淬灭基团TAMRA进行标记），记为R15；8条片段唯一不同的是中间的4个核苷酸，是回文结构区（NNNN代表4核苷酸的所有8种回文结构可能，序列都有AATT、ATAT、ACGT、AGCT、CATG、CTAG、CCGG和CGCG）。首先分别单独合成8种DNA单链片段（带有所述荧光报告基团FAM和荧光淬灭基团TAMRA的那条DNA单链，简称荧光链），并同时对应单独合成其互补链（互补链无荧光基团修饰），接着将每种荧光链和其对应的互补链等量混合，在表1所示的体系中进行退火反应（在PCR仪中孵育，先95℃下2min，再缓慢降温1h至25℃，并在25℃下维持15min），最终获得含有4核苷酸回文结构区的DNA双链片段体系，对获得的DNA双链片段进行纯化并测定浓度。所述DNA片段的合成、双链的制备和纯化均委托上海生工生物工程股份有限公司进行。
表1 双链制备体系（20μl）
| 组份 | 添加量/μl |
| 荧光链（0.25nmol/μl） | 5.0 |
| 互补链（0.25nmol/μl） | 5.0 |
| 10×PCR Buffer | 2.0 |
| 去离子水 | 8.0 |
如图1所示的DNA双链片段，其5’端的荧光报告基团FAM和3’端的荧光淬灭基团距离非常近，因此FAM发出的荧光将被TAMRA所直接吸收，而检测不到荧光的产生（参照TaqMan探针原理）。若堆肥样品总蛋白提取液中含有能够识别并切割图1所示的DNA双链底物片段的回文结构区的限制酶，则5’端的荧光报告基团FAM和3’端的荧光淬灭基团TAMRA会随切割片段而分离，因此会检测得到有荧光的产生，并且荧光强度能够表征限制酶的活力。酶切体系如表2所示，其中的10×CutSmart Buffer购买自美国NEB公司，是通用型限制酶缓冲液体系（1X CutSmart Buffer Components：50 mM Potassium Acetate、20 mM Tris-acetate、10 mM Magnesium Acetate和100 µg/ml BSA；pH 7.9，25°C），也适用于耐高温的TaqαI等限制性内切酶，前期筛选工作表明该缓冲液体系适合于所探究的酶系；为了更好筛选和探究酶切性能，添加十分足量的1nmol DNA双链底物片段。酶切反应在荧光定量PCR仪（Thermo公司的Steponeplus）上进行，初步探究时共进行了30℃、40℃、50℃、60℃、70℃和80℃六个温度下的酶切反应，反应时间为30min，在反应前和反应结束时分别收集荧光信号，其差值即为反应的相对荧光强度。以（1）仅含等量的5’-FAM-F15-NNNN-R15-3’即不含荧光淬灭基团TAMRA仅含荧光报告基团FAM的单链（阳性参照，不加堆肥蛋白提取液，其平均荧光强度为15.21，dRn）和（2）含制备的正常双链底物片段5’-FAM-F15-NNNN-R15-TAMRA-3’但不加堆肥蛋白提取液的空白反应体系（阴性参照，其相对荧光强度为0.09）为参照，计算酶切反应体系收集到的相对荧光强度，数据如表3所示。表3中结果显示，除含AGCT回文结构区的DNA片段体系外，其余7种片段体系在各个温度下其相对荧光强度均在0.08-0.15之间，处于空白背景荧光范围内，表明堆肥蛋白中不存在靶向AATT、ATAT、ACGT、CATG、CTAG、CCGG和CGCG这7种回文结构区的限制酶。而对于含AGCT回文结构区的DNA底物片段，其酶切体系在40℃和70℃反应时均检测到了强烈的荧光信号，分别达到了全部潜在荧光强度的50.76%（7.72/15.21）和62.20%（9.46/15.21），初步表明所提取的堆肥蛋白液中存在可识别和切割含AGCT回文结构区的DNA片段的常温和耐高温限制酶，接着需要进一步确定其编码序列。
表2 堆肥样品总蛋白提取液酶切体系 （10μl）
| 组份 | 添加量/μl |
| DNA双链底物片段（0.2nmol/μl） | 5.0 |
| 10×CutSmart Buffer | 1.0 |
| 堆肥总蛋白提取液（9.76mg/mL） | 2.0 |
| 去离子水 | 2.0 |
表3 荧光信号检测结果
实施例2 限制酶基因序列的筛选和鉴定
取实施例1中混匀的堆肥样本10g，将其装入盛有100mL无菌水并带有玻璃珠的1L锥形瓶中，剧烈震荡约5min制成悬液；接着用8层纱布快速过滤悬液得到滤液，滤液再次用8层纱布快速过滤得到新的滤液；再将滤液在4℃、5000r/pm条件下离心2min，弃上清得到沉淀。按照土壤总RNA和总DNA提取试剂盒（美国OMEGA公司）说明书中方法分别对堆肥样本沉淀物进行总RNA和总DNA提取。由于是对耐热酶的筛选，因此只需对mRNA进行测序。采用OMEGA的mRNA捕获试剂盒（Mag-Bind mRNA Enrichment Kit）对总RNA进行mRNA的富集。富集的mRNA经测定其获取量为10.2μg，OD260/OD280=1.93，RIN=8.12，总体评价良好。获取的mRNA送华大基因进行高通量测序分析，采用的是illumina公司的Truseq RNA sample prepKit构建cDNA文库，再利用Hiseq 2000进行测序（2×100bp），数据过滤后最终得到483Mbp的clean reads，Q20=91.2%，拼接后得到12538条转录本，平均长度为654.61bp。
将得到的转录本比对到Nr即非冗余数据库中，发现有5条相近序列（平均长度在561-744bp之间）比到了GeneBank数据库上登录号为HM569710.1的序列，覆盖度为45.7-60.6%，并且覆盖区具有很高的一致性（均大于80%）。需要特别指明的是，关注HM569710.1序列，是因为该序列是纤维化纤维微细菌（即Cellulosimicrobium cellulans）的Alu限制修饰酶基因簇，其中包含了AluI限制酶的编码基因（HM569710.1序列的第539-1765位点的DNA片段），而AluI限制酶恰好可以识别AGCT回文结构区并进行切割，这符合在实施例1中观察到的现象。提示了这5条候选序列其基因编码的可能是限制酶AluI的类似酶。
为了获取全长的mRNA序列（或其编码基因全长序列），选择mRNA测序结果中所有长度大于100bp，并且和纤维化纤维微细菌AluI限制酶的编码基因比对覆盖度不少于50bp且覆盖区域一致性大于60%的序列，委托华大基因公司设计成液相捕获探针（共124条），并以这些捕获探针对提取的mRNA进行捕获测序和拼接，平均测序深度大于1000×。经过捕获测序分析，得到了这5条mRNA的完整序列，并转换成对应的编码基因序列，比对发现可以分成2组，一组为2条相近的较长序列，序列长度分别是1242bp和1254bp；另外一组为3条相近的较短序列，序列长度分别是1191bp、1173bp和1048bp。
选择有2条相近长序列的这组中长度为1191bp的序列（编码基因序列如SEQ IDNO：1所示），根据序列设计引物对P1（5’-GTGGGATCAATCCCTGATGG-3’）和P2（5’-TCAGACACGTAGATCGATGCT-3’），以提取富集的mRNA（详见反转录扩增体系）和总DNA（详见基因组扩增体系）为模板进行扩增，扩增体系如表4所示。表4中的相关试剂均购买自天根生化科技（北京）有限公司。反转录体系扩增条件为：42°C逆转录30min；95°C预变性3min；94°C变性30s，53°C退火35s，72°C延伸30s，36个循环；72°C终延伸5 min。基因组体系扩增条件为：94°C预变性3min；94°C变性30s，53°C退火30s，72°C延伸45s，32个循环；72°C终延伸5min。扩增产物进行电泳，相关电泳图谱如图2所示，两种体系的扩增片段条带一致，并且条带清晰且单一，长度（约1200bp）符合预期。
表4两种扩增体系信息
PCR产物送上海生工进行Sanger测序，反转录扩增体系和基因组扩增体系的扩增产物测出的序列是一样的，如SEQ ID NO：1所示，这和捕获测序的结果是一致的。测定的序列SEQ ID NO：1在NCBI的核酸数据库进行比对，结果显示其序列和GeneBank数据库上登录号为HM569710.1的序列在99%的覆盖度下，一致性高达95.30%，因此提示SEQ ID NO：1为限制酶AluI的类似酶的基因编码序列，则其编码的蛋白的氨基酸序列如SEQ ID NO：2所示，其理论分子量为45.17kDa。对于获取的限制酶候选基因序列，进一步的需要在蛋白水平层面进行探究，首先对其进行基因克隆和蛋白表达纯化。
实施例3 限制酶基因克隆表达
选择Thermo公司的大肠杆菌蛋白表达体系pET303/CT-His，按照说明书中方法并参考相关文献进行SEQ ID NO：1所示基因编码序列的蛋白表达，一般性的步骤参照《精编分子生物学实验指南》（第五版）和《分子克隆实验指南》（第四版）进行。采用带有XbaⅠ和NsiⅠ双酶切位点的引物扩增目的基因（P3：5’-GATTCTAGAGTGGGATCAATCGTCGACCA-3’和P4：5’-TCAGACCGGACGACGTAGGATGCATAGC-3’，扩增体系和条件同表4中使用引物对P1和P2的基因组扩增体系），PCR产物经琼脂糖凝胶电泳检测无误后按照说明书中方法进行XbaⅠ和NsiⅠ双酶，酶切产物进行纯化回收。回收的目的基因扩增片段同经XbaⅠ和NsiⅠ双酶切的pET303/CT-His载体连接构建重组质粒。重组质粒转化感受态细胞BL21，筛选阳性重组子，最后进行阳性克隆的酶切、PCR扩增和扩增片段Sanger测序鉴定。挑取经测序鉴定正确（同SEQ IDNO：1序列）的阳性克隆子接种于100ml含50μg/mlAmp的LB培养基中，37℃摇动培养至对数生长期，按1：5接种至1000ml新鲜的LB培养基中，30℃摇动培养到D600=0.6-0.8，加IPTG至终浓度为0.25mmol/L，于16℃过夜震荡培养，诱导带组氨酸标签（His-tag）的目标蛋白表达。离心收集菌体，超声破碎，离心收集裂解液上清，用Ni-NTA亲和层析柱纯化目标蛋白，纯化产物以10.0%SDS-PAGE分析鉴定。图3的SDS-PAGE图谱显示获得了单一明亮的蛋白表达产物条带，并且分子量符合预期的目标蛋白。经Qubit3.0测定，纯化蛋白的浓度为1.11mg/ml。
由此借助商业化的大肠杆菌表达体系获得了大量纯净的目标蛋白产物，接着进行酶切底物和耐热性能的验证和分析。
实施例4 限制酶DFc相关酶学性质研究
以5’-FAM-F15-AGCT-R15-TAMRA-3’双链片段为底物，目标蛋白添加量为1μg，参照实施例1中的酶切体系和条件，分别在反应30min、1h和1.5h时测定目标蛋白的限制酶活性（表5）。初步探究可知，该酶只在30-40℃温度范围内显示出显著的酶活，表明这是一种常温限制酶，为行文方便将其命名为限制酶DFc。
表5 限制酶DFc的温度梯度酶活实验数据
综合来看酶DFc在37℃下的荧光强度最高，可知酶DFc的最适温度为37℃，且在此温度下反应1.5h即达到了饱和荧光强度，表明反应1.5h就可以将底物彻底酶解；40℃的酶活和37℃的基本一致，40℃下反应1.5h也可以将底物彻底酶解。将酶切产物纯化后送华大基因有限公司进行质谱测定，质谱形成了4个主峰，经分析分子量分别对应于FAM-（A）15-AG、CT-（C）15-TRMRA、（T15）-TC和GA-（G）15这4种片段，由酶切产物反推可知酶DFc识别AGCT回文结构区并在G和C之间进行切割形成平末端。由此可得，1μg限制酶DFc在1×CutSmartBuffer缓冲体系和37℃或40℃温度下，在1.5h内可以将含1nmol的AGCT回文结构区的双链DNA片段（5’-FAM-F15-NNNN-R15-TAMRA-3’）在G和C之间完全酶切形成平末端。
实施例5酶性能比较实验
对比本发明获得的耐高温限制酶DFc和市面上两款主流的商品化AluI酶（来源于不同的两大品牌公司）的酶活，按照表6中体系和条件进行酶切。这3种AluI酶的添加量均为1μg（均采用Qubit按照说明书方法定量分析），反应底物均是5’-FAM-F15-AGCT-R15-TAMRA-3’短双链DNA片段；购买的两款商品化AluI酶采用各自产品自带的缓冲液体系和添加量，反应温度是说明书推荐的37℃；酶DFc采用的是1×CutSmart Buffer，反应温度是拟实际应用的37℃；反应时间均为3h，设定实时荧光定量PCR信号采集时间点，分别在反应1h、2h和结束时测定相对荧光强度。结果显示，在反应1h、2h和3h时，酶DFc测得的相对荧光强度分别是13.28、15.21和15.21；AluI酶1测得的分别是10.28、15.05和15.21；AluI酶2测得的分别是7.14、10.85和15.21。由此可知在各自最适的反应体系和条件下，酶DFc最快达到彻底酶切，其AGCT限制性内切酶酶活高于市面上主流的商品化AluI酶。
表6 3种限制酶反应体系和条件
此外，将这三种酶至于室温（30℃左右）下1h，再按照表6中体系和条件进行酶切反应。结果显示酶切1h后，酶DFc、AluI酶1和AluI酶2测得的相对荧光强度分别是14.98、9.51和6.64，其酶活分别保留了98.49%（14.98/15.21）、92.96%（9.51/10.23）和94.05%（6.64/7.06）。由此可知置于室温下30min，酶DFc酶损失约1.5%，而AluI酶1和AluI酶2均损失了超过5%的酶活力，则表明酶DFc常温下更稳定，能够实现更加有效和简便地应用于实际。
综上可知，本发明提供的全新的常温限制酶DFc在酶活和热稳定方面均优于已商品化的同类酶，可以作为基因工程和基因检测的工具酶。
序列表
<110> 福建晨欣科生物科技有限公司
<120> 一种新型高效的常温II型限制性内切酶
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 1191
<212> DNA
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gtgggatcaa tccctgatgg gggcgtcgtt ccagacgtcg acacgacgct cagcgaaaag 60
gagaagaacg acctcctcct gtccgcgctg cccggcgcca cgacgtcgac gtacggtggc 120
gcccgcgtgg tcaggttcca cgaccagatc atcctcaagg tcgacgctgt tcaggtcaca 180
cacctcggac acccgtggcc cgcctacaag aagcgcatcc agatcccgaa gtcttggctg 240
gaggtcgagc gacgggccac gcgcgacgga ctggtgaccc gctttgtcgg catctatcgg 300
taccgcgccg tcaccgaccc gcggacgtac atccagcgcg ctgccaacaa ctctgcggcc 360
cacgtctcga ccaacgacct gcaccaggca cagacgctcg ggctgttcga gcgcgtcgat 420
cgcaacggga actgtctgac cagcgtccgg gctgaactgc tcgccgacta ccttgtcggt 480
gtcgcagaca ccactcaccc gagcgtcgat gtgttccggc ggttcaacca ggacttcttc 540
actggcagat ggctgcgcgc tctcgacgcg gttcaggaga tgcacgctgc cgcgtggccc 600
gacaggttcc agaacgagtg ggctggcttc tacttccgcg acaggttcta cgtgagagcg 660
gaggggcttg cggacgagct ccagaagaag aagcagcgcg ggggctacga ctacgacctg 720
gtcttccacg acgggccgcg tgtcgacttc tacggtgatc tgaaggcctc caacatcgcc 780
aagcaggagg ccatcggcaa cgaccgcgac gacctggtcc gctgcctcga ggagttcggc 840
cgcttctggt acgtgatcta cgagcacgac acggtccacg ggaaggccaa cggcgacgtc 900
gccacgatcg agtggaacga gtggcggcgg tccgtcgggc acgttcaggg caaggagtac 960
agcccgctct cctactctgg gcggttcaag gagtcggtgc ggttcgcccg gatgcaggtg 1020
ctcgaggtga acgaggcgaa cgctagcctc gttctcggcg accatcacca aggccggcaa 1080
cccagcggtg cctcgcgcga gcccaaggtc aagatcctga agaagcacat cgacaacttc 1140
ctgatcttct cccagctgcc cgaggtgatc agcatcgatc tacgtgtctg a 1191
<210> 2
<211> 396
<212> PRT
<213> 未知(Unknown)
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Val Gly Ser Ile Pro Asp Gly Gly Val Val Pro Asp Val Asp Thr Thr
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Leu Ser Glu Lys Glu Lys Asn Asp Leu Leu Leu Ser Ala Leu Pro Gly
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Ala Thr Thr Ser Thr Tyr Gly Gly Ala Arg Val Val Arg Phe His Asp
35 40 45
Gln Ile Ile Leu Lys Val Asp Ala Val Gln Val Thr His Leu Gly His
50 55 60
Pro Trp Pro Ala Tyr Lys Lys Arg Ile Gln Ile Pro Lys Ser Trp Leu
65 70 75 80
Glu Val Glu Arg Arg Ala Thr Arg Asp Gly Leu Val Thr Arg Phe Val
85 90 95
Gly Ile Tyr Arg Tyr Arg Ala Val Thr Asp Pro Arg Thr Tyr Ile Gln
100 105 110
Arg Ala Ala Asn Asn Ser Ala Ala His Val Ser Thr Asn Asp Leu His
115 120 125
Gln Ala Gln Thr Leu Gly Leu Phe Glu Arg Val Asp Arg Asn Gly Asn
130 135 140
Cys Leu Thr Ser Val Arg Ala Glu Leu Leu Ala Asp Tyr Leu Val Gly
145 150 155 160
Val Ala Asp Thr Thr His Pro Ser Val Asp Val Phe Arg Arg Phe Asn
165 170 175
Gln Asp Phe Phe Thr Gly Arg Trp Leu Arg Ala Leu Asp Ala Val Gln
180 185 190
Glu Met His Ala Ala Ala Trp Pro Asp Arg Phe Gln Asn Glu Trp Ala
195 200 205
Gly Phe Tyr Phe Arg Asp Arg Phe Tyr Val Arg Ala Glu Gly Leu Ala
210 215 220
Asp Glu Leu Gln Lys Lys Lys Gln Arg Gly Gly Tyr Asp Tyr Asp Leu
225 230 235 240
Val Phe His Asp Gly Pro Arg Val Asp Phe Tyr Gly Asp Leu Lys Ala
245 250 255
Ser Asn Ile Ala Lys Gln Glu Ala Ile Gly Asn Asp Arg Asp Asp Leu
260 265 270
Val Arg Cys Leu Glu Glu Phe Gly Arg Phe Trp Tyr Val Ile Tyr Glu
275 280 285
His Asp Thr Val His Gly Lys Ala Asn Gly Asp Val Ala Thr Ile Glu
290 295 300
Trp Asn Glu Trp Arg Arg Ser Val Gly His Val Gln Gly Lys Glu Tyr
305 310 315 320
Ser Pro Leu Ser Tyr Ser Gly Arg Phe Lys Glu Ser Val Arg Phe Ala
325 330 335
Arg Met Gln Val Leu Glu Val Asn Glu Ala Asn Ala Ser Leu Val Leu
340 345 350
Gly Asp His His Gln Gly Arg Gln Pro Ser Gly Ala Ser Arg Glu Pro
355 360 365
Lys Val Lys Ile Leu Lys Lys His Ile Asp Asn Phe Leu Ile Phe Ser
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Gln Leu Pro Glu Val Ile Ser Ile Asp Leu Arg Val
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Claims (2)
1.一种常温II型限制性内切酶DFc，其特征在于所述DFc酶的氨基酸序列如SEQ NO：2所示，其编码所述DFc酶的基因序列如SEQ NO：1所示；所述DFc酶可识别5’...AGCT...3’核酸片段中的AGCT回文结构区并在G和C之间进行切割形成平末端。
2.根据权利要求1所述的常温II型限制性内切酶DFc，其特征在于1.0μg限制酶DFc在1×CutSmart Buffer缓冲体系和37℃温度下，在1.5h内可以将1nmol如5’-FAM-F15-AGCT-R15-TAMRA-3’所示的含AGCT回文结构区的双链DNA片段在G和C之间完全酶切形成平末端；限制酶DFc置于室温下30min，酶活力仍保留95%以上。
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5334526A (en) * | 1993-05-28 | 1994-08-02 | Life Technologies, Inc. | Cloning and expression of AluI restriction endonuclease |
| US5405768A (en) * | 1992-07-07 | 1995-04-11 | New England Biolabs, Inc. | Method for cloning and producing the AATII and ALUI restriction endonuclease and methylase and related method for overexpressing restriction endonucleases |
| RU2340670C1 (ru) * | 2007-04-26 | 2008-12-10 | Общество с ограниченной ответственностью "СибЭнзайм" | ШТАММ БАКТЕРИЙ Arthrobacter luteus B-ПРОДУЦЕНТ САЙТ-СПЕЦИФИЧЕСКОЙ ЭНДОНУКЛЕАЗЫ Alu BI |
| CN103484556A (zh) * | 2013-10-14 | 2014-01-01 | 上海师范大学 | 检测水稻香味等位基因的分子标记的引物和方法 |
| CN105274204A (zh) * | 2015-09-24 | 2016-01-27 | 郑州大学 | 人PLIN1基因rs894160位点多态性检测方法及试剂盒 |
| CN105274212A (zh) * | 2015-09-24 | 2016-01-27 | 郑州大学 | 人POT1基因rs1034794位点多态性检测方法及试剂盒 |
-
2019
- 2019-08-20 CN CN201910770407.5A patent/CN110438103B/zh not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5405768A (en) * | 1992-07-07 | 1995-04-11 | New England Biolabs, Inc. | Method for cloning and producing the AATII and ALUI restriction endonuclease and methylase and related method for overexpressing restriction endonucleases |
| US5334526A (en) * | 1993-05-28 | 1994-08-02 | Life Technologies, Inc. | Cloning and expression of AluI restriction endonuclease |
| RU2340670C1 (ru) * | 2007-04-26 | 2008-12-10 | Общество с ограниченной ответственностью "СибЭнзайм" | ШТАММ БАКТЕРИЙ Arthrobacter luteus B-ПРОДУЦЕНТ САЙТ-СПЕЦИФИЧЕСКОЙ ЭНДОНУКЛЕАЗЫ Alu BI |
| CN103484556A (zh) * | 2013-10-14 | 2014-01-01 | 上海师范大学 | 检测水稻香味等位基因的分子标记的引物和方法 |
| CN105274204A (zh) * | 2015-09-24 | 2016-01-27 | 郑州大学 | 人PLIN1基因rs894160位点多态性检测方法及试剂盒 |
| CN105274212A (zh) * | 2015-09-24 | 2016-01-27 | 郑州大学 | 人POT1基因rs1034794位点多态性检测方法及试剂盒 |
Non-Patent Citations (1)
| Title |
|---|
| Cellulosimicrobium cellulans AluI restriction-modification gene cluster, complete sequence;Ware,J.等;《GenBank》;20100711;HM569710.1，参见全文 * |
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