CN112662647A - 重组NcoI限制性内切酶的制备方法 - Google Patents
重组NcoI限制性内切酶的制备方法 Download PDFInfo
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- CN112662647A CN112662647A CN202110021361.4A CN202110021361A CN112662647A CN 112662647 A CN112662647 A CN 112662647A CN 202110021361 A CN202110021361 A CN 202110021361A CN 112662647 A CN112662647 A CN 112662647A
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- ncoi
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Abstract
本发明提供了一种重组NcoI限制性内切酶的制备方法，包括以下步骤：一、合成NcoIM和NcoI的编码基因，分别构建至相应载体中，获得带有NcoIM基因的重组载体和融合了纯化标签的NcoI蛋白表达质粒；二、将带有NcoIM基因的重组载体转入大肠杆菌中，获得具备基底NcoIM表达的重组菌；三、制作成具备基底NcoIM表达的重组菌感受态细胞；四、将融合了纯化标签的NcoI蛋白表达质粒转入具备基底NcoIM表达的重组菌感受态细胞中，获得阳性单克隆，培养进行NcoI限制性内切酶的表达；五、NcoI限制性内切酶的纯化。通过本发明提供的方法可以获得大量的NcoI限制性内切酶，具有广阔的应用场景。
Description
技术领域
本发明属于分子生物学和细胞工程领域，具体涉及一种重组NcoI限制性内切酶的制备方法。
背景技术
NcoI是珊瑚红诺卡氏菌(Nocardia corallina)体内的一种二型限制性内切酶，其特点在于它特异性的识别双链DNA中的CCATGG，并分别在双链5’端之后第一个C残基进行切割，如图1所示。切割后的片段具有黏性末端，是遗传及基因工程上，实用性较高的限制酶种类。作为一种限制性核酸内切酶，其在大肠杆菌内的重组表达会严重抑制宿主菌基基因组复制，影响菌种增值，无法正常表达蛋白。国际上对于限性内切酶的表达常采用低背景表达菌株表达法，如使用BL2(DE3)plysS表达HindIII(Watanabe,N.等，2009，Actacrystallographica.Section D,Biological crystallography 65,1326-1333)，或者特殊菌株具有相应的甲基化酶表达，例如BamHI(Jack,W.E.等，1991，Nucleic acids research19,1825-1829)。目前尚无文献报道，NcoI的表达纯化方法，常规的方法难以拿到大量的NcoI蛋白。
发明内容
为了克服现有技术的难以获得大量NcoI蛋白的缺陷，本发明提供了一种重组NcoI限制性内切酶的制备方法，其特征在于，包括以下步骤：
步骤一、合成NcoIM编码基因，构建至原核表达载体，获得NcoIM表达质粒；合成NcoI编码基因，构建至带有纯化标签的原核表达载体，获得融合了纯化标签的NcoI蛋白表达质粒；NcoIM编码基因的DNA序列和NcoI编码基因的DNA序列分别根据NcoIM编码基因的氨基酸序列如SEQ ID NO:3所示和NcoI编码基因的氨基酸序列如SEQ ID NO:4所示优化且排除NcoI酶切位点获得；
步骤二、将NcoIM表达质粒转入不具备NcoIM天然表达特性的大肠杆菌感受态细胞中，获得具备基底NcoIM表达的重组菌；
步骤三、将步骤二获得的具备基底NcoIM表达的重组菌制作成具备基底NcoIM表达的重组菌感受态细胞；
步骤四、将步骤一得到的融合了纯化标签的NcoI蛋白表达质粒转入步骤三获得的具备基底NcoIM表达的重组菌感受态细胞中，获得阳性单克隆，培养进行NcoI限制性内切酶的表达；
步骤五、NcoI限制性内切酶的纯化。
进一步地，NcoIM编码基因的DNA及对应氨基酸序列如SEQ ID NO:1，SEQ ID NO:3所示；NcoI编码基因的DNA及对应氨基酸序列如SEQ ID NO:2，SEQ ID NO:4所示。或者NcoIM编码氨基酸序列与SEQ ID NO:3有95％及以上的相似度，NcoI编码氨基酸序列与SEQ IDNO:4有95％及以上的相似度。
进一步地，步骤一中的NcoIM的编码基因构建至原核表达载体pET16B；NcoI的编码基因构建至带有纯化标签的原核表达载体pET28A。
进一步地，步骤一具体为：使用高保真酶扩增NcoIM编码基因，同时以NcoI、BamHI限制性内切酶，双酶切pET16B载体后，通过同源重组方式插入至pET16B，确保读码框正确；使用高保真酶扩增NcoI编码基因，同时以NdeI、NotI限制性内切酶，双酶切pET29A载体后，通过同源重组方式插入至pET29A，确保读码框正确。
进一步地，步骤一中的纯化标签为MBP、GST、6*HIS中的一种或者多种。
进一步地，步骤二中的不具备NcoIM天然表达特性的大肠杆菌感受态细胞为BL21(DE3)感受态细胞。
进一步地，步骤五中采用亲和纯化层析、离子交换层析，或者亲和纯化层析、凝胶色谱层析，或者亲和纯化层析、离子交换层析、凝胶色谱层析进行NcoI限制性内切酶的纯化。
进一步地，还包括步骤六、NcoI限制性内切酶的酶活的测定。
进一步地，步骤六具体为：取不同稀释比的NcoI加入足量单位BamHI，在37℃下一小时，消化1μg鉴定用质粒，最终通过琼脂糖电泳确认酶的比活；鉴定所用质粒具有至少一个NcoI及BamHI识别位点。
进一步地，鉴定所用质粒的DNA序列如SEQ ID NO:9所示。
本发明通过利用表达NcoIM(NcoII甲基化酶)的特殊BL21(DE3)菌种，进行NcoI的表达。大肠杆菌中基底表达的NcoIM特异识别大肠杆菌基因组上的双链序列GAATTC，使两条链上A-3的特异性甲基化，保护基因组DNA不被NcoI内切酶切割，使大肠杆菌能够继续正常生长增殖。
传统方法将NcoI表达的构建质粒直接转入大肠杆菌中进行表达，NcoI限制菌种的正常增殖，无法拿到大量NcoI蛋白。本发明方法先将NcoIM表达基因构建至原核载体，并将其转入菌株，后以此菌株制备感受态细胞。后将NcoI内切酶构建至不同抗性的原核表达载体，转入表达NcoIM的感受态细胞中，进行表达纯化并最终得到高纯度的NcoI蛋白。
该方法利用常规易得的菌种BL21(DE3)，采取亲和纯化方案，操作简单易于实行，表达量高纯度高。
以下将结合附图对本发明作进一步说明，以充分说明本发明的目的、技术特征和技术效果。
附图说明
图1是NcoI限制性内切酶的酶切位点。
图2是本发明所涉及的重组NcoI限制性内切酶的制备方法的一个具体实施方式的操作流程图。
图3是实施例1中本发明构建好pET16b-NcoIM重组质粒的大肠杆菌感受态细胞与普通的BL21(DE3)(购自唯地生物)，转化融合了纯化标签的NcoI蛋白表达质粒涂布平板后的菌种生长效果。该平板为卡那和氨苄双抗性LB平板。左为普通的BL21(DE3)转化融合了纯化标签的NcoI蛋白表达质粒涂布平板后的菌种生长效果；右为pET16b-NcoIM重组质粒的大肠杆菌感受态细胞转化融合了纯化标签的NcoI蛋白表达质粒涂布平板后的菌种生长效果。
图4是金属离子螯合琼脂糖介质亲和纯化及离子交换层析后，经过凝胶色谱层析进一步纯化后的NcoI蛋白，SDS-PAGE胶图。
经凝胶色谱纯化后，蛋白峰不同取样管的跑胶确认。蛋白纯度在95％以上。
图5是构建有基因的商用pFastbac载体经过本发明得到的NcoI,及商用BamHI,NcoI双酶切割后琼脂糖凝胶电泳图。
反应条件为：20μl反应体系，10U NcoI，10U BamHI,反应缓冲体系：100mM NaCl,50mM Tris-HCl,10mM MgCl2,100μg/ml BSA(pH 7.9@25℃)，2.5μg质粒37℃反应10min，T代表相应酶购自ThermoFisher Scientific。
具体实施方式
本发明所涉及的试剂和耗材均可以通过商业途径购买，除特别标注的外，均购自上海百赛生物技术股份有限公司。
实施例1
在该实施例中，本发明所涉及的重组NcoI限制性内切酶的制备方法的操作流程如图2所示。
步骤一、使用原核表达载体分别插入构建表达NcoIM的质粒和NcoI的质粒。
基因合成NcoIM编码基因的DNA序列如SEQ ID NO:1所示，和NcoI编码基因的DNA序列如SEQ ID NO:2所示。DNA序列合成设计时排除NcoI酶切位点，基因序列根据氨基酸序列(UniProt ID：NcoIM O85488；NcoI O85489)优化得到。NcoIM编码基因的氨基酸序列如SEQID NO:3所示；NcoI编码基因的氨基酸序列如SEQ ID NO:4所示。
NcoIM编码基因(引物对如SEQ ID NO:5所示和如SEQ ID NO:6所示)和NcoI编码基因(引物对如SEQ ID NO:7所示和如SEQ ID NO:8所示)分别通过重组酶(Vazyme)介导的重组反应构建至原核表达载pET16B(NcoI/BamHI之间),pET28A(NdeI/HindIII之间)，并保证氨基酸翻译读码框正确，NcoIM不带有亲和纯化标签，NcoI带有亲和纯化标签，分别得到NcoIM表达质粒和融合了纯化标签的NcoI蛋白表达质粒。
步骤二、构建表达NcoIM蛋白的菌株。向不具备NcoIM天然表达特性的大肠杆菌转入NcoIM表达质粒，使其具备基底的NcoIM表达。
BL21感受态细胞-80℃取出后于冰上放置10min，后加入NcoIM表达质粒，缓慢吹打混匀，于冰上放置15min，后于42℃水浴锅中热激45s，取出在冰上放置10min，后加入1ml LB培养基于37℃摇床220rpm培养1hr。最后10000g离心去上清，菌体于氨苄抗性LB平板上涂布均匀。通过涂布氨苄抗性LB平板筛选出NcoIM成功转化进入的BL21大肠杆菌的克隆，即具备基底NcoIM表达的重组菌(pET16b-NcoIM重组质粒的大肠杆菌)。
步骤三、将步骤二所得到的具备基底NcoIM表达的重组菌通过常规化学处理制备为具有接收外来质粒能力的感受态细胞。
接菌：在已经转入NcoIM的BL21菌种平板上用镊子夹取枪头挑取BL21单克隆，接入装有5ml LB培养基的试管中。
小摇：将接完菌的试管放入摇床中，37℃，220rpm/min，培养8小时左右，待肉眼可见菌液完全浑浊后取出。
大摇：取1ml小摇后的BL21菌液接入装有250ml SOB的2L锥形瓶中，18℃，220rpm/min，培养过夜(此步骤应全程在超净台中进行操作SOB使用前需加入1.25ml 1M MgCl2与1.25ml 1M MgSO4，MgCl2与MgSO4溶液需提前过滤除菌)
测OD值：第二天测量OD值，当菌体OD值达到0.3-0.4左右时，将菌体倒入离心瓶中，冰浴30分钟，此时将重悬buffer放在冰上进行预冷，打开离心机进行预冷。
离心：4℃3000g离心10分钟，弃上清。
重悬：取50ml重悬buffer倒入离心瓶中，轻轻摇晃至菌体完全均匀，冰浴10分钟。
离心：4℃3000g离心10分钟，弃上清。
重悬及定量：取5-10ml重悬buffer倒入离心瓶中，轻轻摇晃至菌体完全均匀，将重悬后的菌液倒入一支带刻度的无菌50ml离心管中，取150μl菌液与2.85ml水加入比色皿中测OD值，OD值为0.5左右适宜，若OD值高于0.5可适量再加入重悬buffer，若OD值低于0.5待菌体沉淀后适量吸出重悬buffer。
在50ml离心管中加入体积为终体积7％的DMSO，盖上盖子缓慢颠倒混匀。快速的将菌体悬液快速分装进预冷灭菌的离心管中，然后置于于液氮浴中快速冷冻。将制作好的感受态细胞与-80℃冰箱保存。将1ng卡那抗性质粒通过热激转入制备好的感受态细胞中，涂布平板，次日通过长出的菌斑数量计算转化效率大于5*10^6。
步骤四、向步骤三得到的感受态细胞转入融合了纯化标签的NcoI蛋白表达质粒，并挑取平板上生长的单克隆(见图3中右边平板)进行培养并表达。右边平板上长出来的均是阳性单克隆；左边平板上没有长出单克隆是因为普通的BL21(DE3)不具有基底NcoIM表达的能力，其基因组对应位点没有被保护修饰，所以被NcoI切割使得大肠杆菌不能正常生长增殖，无法形成单克隆。
挑取pET16b-NcoIM重组质粒的大肠杆菌感受态细胞成功转入融合了纯化标签的NcoI蛋白表达质粒的单克隆接种至10ml氨苄(Ampicillin)，卡那(Kanamycin)双抗性的LB培养基中，于过夜摇床培养，次日接种于1L双抗的LB摇瓶中(即以1L表达体系为例)，220rpm37℃，摇床培养至摇至OD0.6-0.8，加入终浓度0.5mM的IPTG进行诱导，表达21小时。
步骤五、NcoI限制性内切酶的纯化
10000rcf离心力离心5min收集菌体，去除上清培养基，使用预冷buffer(100mMKCl,25mM Tris pH7.5)30ml悬浮菌体，后使用高压650bar进行破菌5min，破菌同时加入蛋白酶抑制剂，后14000rcf离心30min去除沉淀，上清加入相应的亲和纯化介质进行结合(Ni,Glutathione,Dextrin等)，以6*HIS标签为例，将50ml离心去沉淀的上清与2ml Ni-NTA(Qiagen)纯化介质混合，4-8℃结合2hr后，将beads滤出，并分别用0,10,15,25mM含咪唑缓冲液洗去杂蛋白，最后用含250mM咪唑缓冲液将目的蛋白洗下。跑SDS-PAGE胶确定纯度。之后将洗脱蛋白进行以AKTA(GE healthcare)介导的洗脱分离过程，进行离子交换层析或者凝胶色谱层析。离子交换层析，蛋白在约在330mM NaCl离子强度下洗出。凝胶色谱层析选用Hiload 16/600superdex 75pg(GE healthcare)型号柱，出峰洗脱柱体积位置约为65ml。最终SDS-PAGE确定纯度如图4所示，蛋白浓缩后通过A280吸光值结合消光系数确定最终蛋白质浓度。NcoIM虽然也过量表达，但由于其无亲和纯化标签，亲和纯化及后面的过柱纯化步骤，基本完全去除了NcoIM的残留。
步骤六、NcoI限制性内切酶的酶活的测定
一个单位被定义为在37℃下1小时内消化1μg线性化质粒DNA(BamHI消化物)，总反应体积为20μl所需的酶量。鉴定所用质粒的DNA序列如SEQ ID NO:9所示。
取不同稀释比的NcoI加入足量单位BamHI，在37℃下一小时，消化1μg质粒DNA，最终通过琼脂糖电泳确认酶的比活。
经过三次过柱(亲和纯化层析、离子交换层析、凝胶色谱层析)纯化去除了杂蛋白及NcoIM的污染，在最终纯化得到的产物中检测不到NcoIM活性。以1L表达体系为例，最终得到的NcoI内切酶总质量在约10mg/l,比活大6*10^6U/mg。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思做出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
序列表
<110> 上海咏科生物科技有限公司
<120> 重组NcoI限制性内切酶的制备方法
<130> CN100-20003PICN
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260 265 270
Thr Phe Gly Ser Ser Ser Asp Ala Ile Glu Phe Leu Asp Thr Phe Asn
275 280 285
<210> 5
<211> 40
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 5
ttaactttaa gaaggagata taatgtctca accactgctg 40
<210> 6
<211> 38
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 6
ttcgggcttt gttagcagcc ttagccttca atccacag 38
<210> 7
<211> 38
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 7
ctttaagaag gagatataca atggctactg ccccaggt 38
<210> 8
<211> 39
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 8
ggtgctcgag tgcggccgcg ttgaaagtgt ccaggaatt 39
<210> 9
<211> 7391
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 9
gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 60
gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 120
acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 180
agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg 240
ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 300
ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta 360
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 420
aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt tcggggaaat 480
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 540
agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 600
catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 660
ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 720
atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 780
ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 840
gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 900
ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 960
ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 1020
gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 1080
ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 1140
gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 1200
ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 1260
gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 1320
gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 1380
caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 1440
cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 1500
ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 1560
taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 1620
tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 1680
gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 1740
agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 1800
aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 1860
gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 1920
gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 1980
tacaccgaac tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg 2040
agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 2100
cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 2160
gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 2220
gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 2280
ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 2340
cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 2400
cggtattttc tccttacgca tctgtgcggt atttcacacc gcagaccagc cgcgtaacct 2460
ggcaaaatcg gttacggttg agtaataaat ggatgccctg cgtaagcggg tgtgggcgga 2520
caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt cttaaactag 2580
acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat actggacttt 2640
tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc gtattaaaga 2700
ggggcgtggc caagggcatg gtaaagacta tattcgcggc gttgtgacaa tttaccgaac 2760
aactccgcgg ccgggaagcc gatctcggct tgaacgaatt gttaggtggc ggtacttggg 2820
tcgatatcaa agtgcatcac ttcttcccgt atgcccaact ttgtatagag agccactgcg 2880
ggatcgtcac cgtaatctgc ttgcacgtag atcacataag caccaagcgc gttggcctca 2940
tgcttgagga gattgatgag cgcggtggca atgccctgcc tccggtgctc gccggagact 3000
gcgagatcat agatatagat ctcactacgc ggctgctcaa acctgggcag aacgtaagcc 3060
gcgagagcgc caacaaccgc ttcttggtcg aaggcagcaa gcgcgatgaa tgtcttacta 3120
cggagcaagt tcccgaggta atcggagtcc ggctgatgtt gggagtaggt ggctacgtct 3180
ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg gtcagggccg 3240
agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt agggcgactg 3300
ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata acatcaaaca 3360
tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact gtacaaaaaa 3420
acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc gttcggtcaa 3480
ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac gaaccgaaca 3540
ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc acccggcaac 3600
cttgggcagc agcgaagtcg aggcatttct gtcctggctg gcgaacgagc gcaaggtttc 3660
ggtctccacg catcgtcagg cattggcggc cttgctgttc ttctacggca aggtgctgtg 3720
cacggatctg ccctggcttc aggagatcgg aagacctcgg ccgtcgcggc gcttgccggt 3780
ggtgctgacc ccggatgaag tggttcgcat cctcggtttt ctggaaggcg agcatcgttt 3840
gttcgcccag gactctagct atagttctag tggttggcta cgtatactcc ggaatattaa 3900
tagatcatgg agataattaa aatgataacc atctcgcaaa taaataagta ttttactgtt 3960
ttcgtaacag ttttgtaata aaaaaaccta taaatattcc ggattattca taccgtccca 4020
ccatcgggcg caaggaggcc ttgaatggga ttctccaggg gttcagggtc atctactggg 4080
ccaacctcat agacggagag ctgggcgaga ttaagaacgt caccaccacg cagccttcct 4140
tggagttgga tggactagag aaatacacca actacagtat ccaggtcctg gccttcaccc 4200
gtgcagggga tggcgtccgg agcgagcaga tctttacccg taccaaggag gacgttccag 4260
gtcctcctgc cggtgtcaag gcggcggcag cctcggcctc tatggtcttc gtgtcctggc 4320
tgcccccgct gaagctgaac ggcatcattc ggatccaaag ccaccatcag tcccagaaag 4380
gttaaaagca gcgtgggcag ccaggtctcc ttatcctgca gtgtgacagg aaatgaagac 4440
caggaactct cctggtaccg aaatggcgaa atcctcaacc ctggaaaaaa cgtgaggatc 4500
acaggactca accacgcaaa ccttataatg gatcacatgg tcaagagtga tgggggtgcc 4560
taccagtgct ttgtgcgcaa ggacaagcta tctgctcaag actatgtcca ggtggtcctt 4620
gaagacggaa ctcccaaaat catttctgcc tttagcgaga aagtggtgag cccggcagag 4680
ccagtgtccc tcgtgtgcaa tgtgaagggt acacccttgc ccacggtcac ctggaccctg 4740
gacgatgacc ccatcctcaa gggcagcggt caccgcatca gccaaatgat cacgtccgag 4800
gggaacgtgg tcagctacct gaatatctcc agctcccagg tccgggatgg gggtgtctac 4860
cgctgcactg ccaacaactc ggctggagtc gtcctgtacc aggctcgaat aaacgtaaga 4920
gggcctgcaa gcatcagacc aatgaaaaac atcactgcga tagcggggcg tgacacgtac 4980
atccactgcc gcgtaattgg ctatccgtat tactccatca agtggtacaa gaacgctaac 5040
ctgcttcctt tcaaccaccg ccaggtggcg tttgagaaca atgggactct gaaactctct 5100
gatgtgcaga aagaagttga cgagggagag tacacgtgta atgtgctggt acagccacag 5160
ctctccacca gccagagtgt ccacgtgaca gtgaaagttc cacctttcat ccaacctttt 5220
gagttcccaa gattctccat cgggcagcgg gttttcatcc catgtgtggt ggtctcaggg 5280
gacttaccca tcaccatcac ctggcagaag gatggccggc caattccagc aagcctcgga 5340
gtaaccattg acaacattga cttcaccagc tccctgagga tctccaacct ctccctaatg 5400
cacaatggga attacacctg cattgcgaga aacgaggcag cagccgtgga acaccagagt 5460
cagctgattg tgagagttcc ccctaagttc gtggtacagc cccgggacca ggacgggatc 5520
tatggcaaag cagtgattct caattgctct gcagagggtt atcctgtgcc tacaattgtg 5580
tggaaattct caaaaggtgc tggggtcccc cagttccagc caattgcctt gaatggccga 5640
atccaggtcc tgagtaatgg ctcactcttg atcaagcatg ttgtagaaga agacagtggc 5700
tactacctct gcaaggtcag caacgatgtg ggcgcagacg tcagcaagtc catgtacctc 5760
acagtgaaaa ttcctgccat gataacctct taccccaaca ccaccctggc cactcagggt 5820
caaaggaagg aaatgagctg cacagccaat ggggagaagc ccatcattgt ccgctgggag 5880
aaagaggaca ggatcattaa ccctgaaatg gcccgctacc tggtatccac caaggaggtg 5940
ggagaggaag tgatatctac gctgcagatt ttgccaacag tgagagaaga ttccggtttc 6000
ttctcctgcc atgctatcaa ttcatacggg gaggaccgtg gaataattca actcacagtg 6060
caagaacccc cagatcctcc cgagattgag atcaaagatg tcaaagctcg caccatcacg 6120
ctcaggtgga ctatggggtt tgatggcaac agccccatca caggctatga cattgaatgc 6180
aaaaataaat cagactcctg ggattctgct caaagaacca aagatgtttc ccctcagctg 6240
aactcggcca ccatcattga tatccaccct tcctccacct acagcatccg catgtacgcc 6300
aagaaccgga ttggcaagag tgagcccagc aacgagatca ccatcacggc ggatgaggca 6360
gctcctgatg gtccacctca ggaagttcac ttggaaccca cctcatctca gagtatcagg 6420
gttacctgga aggctcccaa gaaacactta caaaacggga tcattcgcgg ctaccaaata 6480
ggctaccggg agtacagcac ggggggtaac ttccagttca acatcatcag tatcgacacc 6540
accggggaca gcgaagtgta caccctggac aacctgaata agttcacgca gtatggcctg 6600
gtagtacagg cttgcaaccg ggccggcaca ggaccttctt ctcaggagat catccatggc 6660
ggtccgaagc gcgcggaatt caaaggccta cgtcgacgag ctcactagtc gcggccgctt 6720
tcgaatctag acatcaccat caccatcact agagcttgtc gagaagtact agaggatcat 6780
aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc 6840
cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta 6900
taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 6960
gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggatctgatc 7020
actgcttgag cctaggagat ccgaaccaga taagtgaaat ctagttccaa actattttgt 7080
catttttaat tttcgtatta gcttacgacg ctacacccag ttcccatcta ttttgtcact 7140
cttccctaaa taatccttaa aaactccatt tccacccctc ccagttccca actattttgt 7200
ccgcccacag cggggcattt ttcttcctgt tatgttttta atcaaacatc ctgccaactc 7260
catgtgacaa accgtcatct tcggctactt tttctctgtc acagaatgaa aatttttctg 7320
tcatctcttc gttattaatg tttgtaattg actgaatatc aacgcttatt tgcagcctga 7380
atggcgaatg g 7391
Claims (10)
1.一种重组NcoI限制性内切酶的制备方法，其特征在于，包括以下步骤：
步骤一、合成NcoIM编码基因，构建至原核表达载体，获得NcoIM表达质粒；合成NcoI编码基因，构建至带有纯化标签的原核表达载体，获得融合了纯化标签的NcoI蛋白表达质粒；所述NcoIM编码基因的DNA序列和所述NcoI编码基因的DNA序列分别根据所述NcoIM编码基因的氨基酸序列如SEQ ID NO:3所示和所述NcoI编码基因的氨基酸序列如SEQ ID NO:4所示优化且排除NcoI酶切位点获得；
步骤二、将所述NcoIM表达质粒转入不具备NcoIM天然表达特性的大肠杆菌感受态细胞中，获得具备基底NcoIM表达的重组菌；
步骤三、将所述步骤二获得的具备基底NcoIM表达的重组菌制作成具备基底NcoIM表达的重组菌感受态细胞；
步骤四、将所述步骤一得到的融合了纯化标签的NcoI蛋白表达质粒转入所述步骤三获得的具备基底NcoIM表达的重组菌感受态细胞中，获得阳性单克隆，培养进行NcoI限制性内切酶的表达；
步骤五、NcoI限制性内切酶的纯化。
2.如权利要求1所述的重组NcoI限制性内切酶的制备方法，其特征在于，所述NcoIM编码基因的DNA及氨基酸序列如SEQ ID NO:1，SEQ ID NO:3所示，所述NcoI编码基因的DNA及氨基酸序列如SEQ ID NO:2，SEQ ID NO:4所示；或者所述NcoIM编码基因的对应氨基酸序列与SEQ ID NO:3有95％及以上的相似性，所述NcoI编码基因的对应氨基酸序列与SEQ IDNO:4有95％的相似性。
3.如权利要求1所述的重组NcoI限制性内切酶的制备方法，其特征在于，所述步骤一中的NcoIM的编码基因构建至原核表达载体pET16B；NcoI的编码基因构建至带有纯化标签的原核表达载体pET28A。
4.如权利要求3所述的重组NcoI限制性内切酶的制备方法，其特征在于，所述步骤一具体为：使用高保真酶扩增NcoIM编码基因，同时以NcoI、BamHI限制性内切酶，双酶切pET16B载体后，通过同源重组方式插入至pET16B，确保读码框正确；使用高保真酶扩增NcoI编码基因，同时以NdeI、NotI限制性内切酶，双酶切pET29A载体后，通过同源重组方式插入至pET29A，确保读码框正确。
5.如权利要求1所述的重组NcoI限制性内切酶的制备方法，其特征在于，所述步骤一中的纯化标签为MBP、GST、6*HIS中的一种或者多种。
6.如权利要求1所述的重组NcoI限制性内切酶的制备方法，其特征在于，所述步骤二中的不具备NcoIM天然表达特性的大肠杆菌感受态细胞为BL21(DE3)感受态细胞。
7.如权利要求1所述的重组NcoI限制性内切酶的制备方法，其特征在于，所述步骤五中采用亲和纯化层析、离子交换层析，或者亲和纯化层析、凝胶色谱层析，或者亲和纯化层析、离子交换层析、凝胶色谱层析进行NcoI限制性内切酶的纯化。
8.如权利要求1所述的重组NcoI限制性内切酶的制备方法，其特征在于，还包括步骤六、NcoI限制性内切酶的酶活的测定。
9.如权利要求8所述的重组NcoI限制性内切酶的制备方法，其特征在于，所述步骤六具体为：取不同稀释比的NcoI加入足量单位BamHI，在37℃下一小时，消化1μg鉴定用质粒，最终通过琼脂糖电泳确认酶的比活；所述鉴定所用质粒具有一个NcoI及BamHI识别位点。
10.如权利要求9所述的重组NcoI限制性内切酶的制备方法，其特征在于，所述鉴定所用质粒的DNA序列如SEQ ID NO:9所示。
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