CN112813087A - 一种SalI限制性内切酶的制备方法 - Google Patents
一种SalI限制性内切酶的制备方法 Download PDFInfo
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- CN112813087A CN112813087A CN202110020913.XA CN202110020913A CN112813087A CN 112813087 A CN112813087 A CN 112813087A CN 202110020913 A CN202110020913 A CN 202110020913A CN 112813087 A CN112813087 A CN 112813087A
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Abstract
本发明公开了一种SalI限制性内切酶的制备方法，包括以下步骤：一、合成SalIM和SalI的编码基因，分别如SEQ ID NO.1所示和SEQ ID NO.2所示，分别构建至相应载体中，获得带有SalIM基因的重组载体和融合了纯化标签的SalI蛋白表达质粒；二、将带有SalIM基因的重组载体转入大肠杆菌中，获得具备基底SalIM表达的重组菌，保护基因组DNA不被SalI内切酶切割，使大肠杆菌能够继续正常生长增殖，从而提高SalI的表达量。三、制作成具备基底SalIM表达的重组菌感受态细胞；四、将融合了纯化标签的SalI蛋白表达质粒转入具备基底SalIM表达的重组菌感受态细胞中，获得阳性单克隆，培养进行SalI限制性内切酶的表达；五、SalI限制性内切酶的纯化。
Description
技术领域
本发明涉及分子生物学和细胞工程领域，特别涉及一种SalI限制性内切酶的制备方法。
背景技术
SalI是白色链霉菌G(Streptomyces albus G)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GTCGAC并分别在双链5’端之后第一个G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。
传统蛋白表达方法将SalI表达的构建质粒直接转入大肠杆菌中进行表达。但是，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因组复制，其基底表达会导致克隆菌种无法正常增殖。因此，难以拿到大量的SalI蛋白。目前尚无国内外文献介绍SalI的重组表达与纯化方法。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.,Takasaki,Y.,Sato,C.,Ando,S.,and Tanaka,I.(2009)Structures of restriction endonuclease HindIII incomplex with its cognate DNA and divalent cations.Actacrystallographica.Section D,Biological crystallography 65,1326-1333)。
因此，本领域的技术人员致力于开发一种能提高SalI表达量的重组表达与纯化方法。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是如何提高SalI蛋白在原核表达菌株中的表达量。
为实现上述目的，本发明的一个方面提供了一种SalI限制性内切酶的制备方法，在原核表达菌株中同时表达SalI和SalIM。可选地，SalI和SalIM改造去除了序列中的SalI酶切位点。
进一步地，SalIM的DNA及氨基酸序列如SEQ ID No.1，SEQ ID No.3所示，或者其氨基酸序列与SEQ ID No.3具有至少95％及以上的相似性；所述SalI的DNA及氨基酸序列如SEQ ID No.2，SEQ ID No.4所示，或者其氨基酸序列与SEQ ID No.4具有至少95％及以上的相似性。
进一步地，原核表达菌株包括大肠杆菌。可选地，原核表达菌株为大肠杆菌BL21(DE3)。
进一步地，该方法包括：先在所述原核表达菌株中转化入含有SalIM的重组质粒，使得原核表达菌株进行SalIM表达并进行基因组保护修饰；然后在含有SalIM的重组质粒的原核表达菌株中进一步转化入含有SalI的重组质粒，获得含有SalIM的重组质粒和含有SalI的重组质粒的原核表达菌株。
进一步地，将含有SalIM的重组质粒的原核表达菌株制备成感受态细胞，并在所述感受态细胞中转化入含有SalI的重组质粒。
进一步地，含有SalIM的重组质粒不含纯化标签，含有SalI的重组质粒带有纯化标签。进一步地，纯化标签选自MBP、GST或6*HIS。
进一步地，含有SalIM的重组质粒使用pET16b载体，含有SalI的重组质粒使用pET28a载体。
进一步地，该方法还包括：将含有SalIM的重组质粒和含有SalI的重组质粒的原核表达菌株进行诱导表达后，通过亲和介质、离子交换层析和/或凝胶色谱层析进行SalI的纯化。
进一步地，所述诱导表达为，加入终浓度0.5mM的IPTG，在20～23℃下诱导表达19-22小时。可选地，在22℃下诱导。
通过本发明的制备方法，能得到10mg/L的SalI内切酶总质量，远高于直接将含有SalI的重组质粒直接转化大肠杆菌BL21(DE3)并进行表达和纯化后的总质量。并且，获得的SalI纯度高，比活大于4*106U/mg。
以下将结合附图对本发明的构思、具体步骤及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是SalI的酶切位点示意图；
图2是本发明的一个实施例的简要操作流程图；
图3是本发明实施1中构建好pET16b-SalIM重组质粒的大肠杆菌感受态细胞与普通的BL21(DE3)(唯地生物)，转化SalI表达质粒涂布平板后的菌种生长效果；
图4是本发明实施2中经过纯化后的SalI蛋白的SDS-PAGE图；
图5是本发明实施例3中的酶切实验后的琼脂糖凝胶电泳图，其中Marker为分子梯度，SalI+BamHI(T)为第一组反应，SalII为第二组反应，SalI(T)+BamHI(T)为第三组反应。T代表限制性内切酶购自ThermoFisher Scientific。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现，本发明的保护范围并非仅限于文中提到的实施例。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。所采用的试剂，若无特殊说明，均为市售或公开渠道可以获得的试剂。
在本发明中，可选用本领域已知的各种载体，如市售的载体，包括质粒等。
本发明的具体实施方式中，通过利用表达SalIM(SalI甲基化酶)的特殊BL21(DE3)菌种，进行SalI的表达。大肠杆菌中基底表达的SalIM特异识别大肠杆菌基因组上的双链序列GTCGAC，使两条链上A-5的特异性甲基化，保护基因组DNA不被SalI内切酶切割，使大肠杆菌能够继续正常生长增殖。
具体地，先将SalIM表达基因构建至原核载体，并将其转入大肠杆菌BL21(DE3)，后以此菌株制备感受态细胞。然后将SalI内切酶构建至不同抗性的原核表达载体，转入表达SalIM的感受态细胞中，进行表达纯化并最终得到高纯度的SalI蛋白。
SalIM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SalIM的残留。
本发明的一个具体实施方式的构建及表达、纯化、鉴定的流程图如图2所示。
实施例1构建含有SalIM重组质粒和SalI重组质粒的重组大肠杆菌菌株
1)构建SalIM重组质粒和SalI重组质粒
本实施例中，SalIM的DNA序列如SEQ ID No.1所示，其氨基酸序列如SEQ ID No.3所示；SalI的DNA序列如SEQ ID No.2所示，其氨基酸序列如SEQ ID No.4所示。两个序列在设计时排除了SalI酶切位点，并经过了密码子与优化。通过基因合成获得SalIM和SalI的DNA序列。
通过重组酶介导的的构建反应，将SalIM的序列通过NcoI和BamHI酶切位点构建至原核表达载体pET16b上，获得pET16b-SalIM重组质粒。将SalI的序列通过NdeI和HindIII酶切位点构建至原核表达载体pET28a上，获得pET28a-SalI重组质粒。其中，在SEQ ID No.1所示的序列上加上两个酶切位点的引物对SMF和SMR的序列如SEQ ID No.5和SEQ ID No.6所示；在SEQ ID No.2所示的序列上加上两个酶切位点的引物对SREF和SRER的序列如SEQ IDNo.7和SEQ ID No.8所示。重组酶购自诺唯赞。经过测序确认构建后的原核表达载体氨基酸翻译读码框正确。
2)制备含有pET16b-SalIM重组质粒的大肠杆菌感受态细胞
在不具备SalIM天然表达特性的大肠杆菌中转入构建的pET16b-SalIM重组质粒，使大肠杆菌具备基底的SalIM表达，SalIM特异识别大肠杆菌基因组上的双链序列GTCGAC，使两条链上A-5的特异性甲基化，保护基因组DNA不被重组表达的SalI内切酶切割，使大肠杆菌能够继续正常生长增殖。
大肠杆菌感受态细胞-80℃取出后于冰上放置10min，后加入pET16b-SalIM重组质粒，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。获得阳性克隆即含有pET16b-SalIM重组质粒的大肠杆菌BL21(DE3)菌株。
将含有pET16b-SalIM重组质粒的大肠杆菌BL21(DE3)菌株制备成感受态细胞，具体操作如下：
接菌：在含有pET16b-SalIM重组质粒的大肠杆菌BL21(DE3)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(离心管需预冷)，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，然后置于液氮浴中快速冷冻。将制作好的感受态细胞于-80℃冰箱保存备用。
3)制备含有pET16b-SalIM重组质粒和pET28a-SalI重组质粒的重组大肠杆菌菌株的大肠杆菌菌株
取制备好的含有pET16b-SalIM重组质粒的大肠杆菌BL21(DE3)感受态细胞，转入pET28a-SalI重组质粒，并挑取平板上生长的单克隆进行验证，阳性克隆即为含有pET16b-SalIM重组质粒和pET28a-SalI重组质粒的重组大肠杆菌BL21(DE3)菌株。
将上述构建好的含有pET16b-SalIM重组质粒和pET28a-SalI重组质粒的重组大肠杆菌BL21(DE3)菌株，以及直接在普通的BL21(DE3)(唯地生物)中转化pET28a-SalI重组质粒后菌株，涂布平板后的菌种生长效果如图3所示。可见，SalI在普通BL21(DE3)中的基底表达会导致克隆菌种无法正常增殖(图3左)，而转入SalIM后，菌种的增殖正常(图3右)。
实施例2SalI的表达及纯化
以1L表达为例：取实施例1中制备的含有pET16b-SalIM重组质粒和pET28a-SalI重组质粒的重组大肠杆菌BL21(DE3)的单克隆，接种至含有100ug/ml氨苄(Ampicillin)和25ug/ml卡那(Kanamycin)双抗性的10ml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，220rpm 37℃，摇床培养至摇至OD 0.6-0.8，再加入终浓度0.5mM的IPTG进行诱导，22℃，表达21小时。
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，之后进行凝胶色谱层析。离子交换层析，蛋白在约在300mM 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68ml。最终SDS-PAGE确定纯度，浓缩通过A280吸光值结合消光系数确定最终蛋白质浓度。
SDS-PAGE结果如图4所示，使用2.5μg上样，经SDS-PAGE检测纯度达到90％以上。以1L表达体系为例，最终得到的SalI内切酶总质量约10mg/L。而按照常规的方法，将SalI重组质粒直接转化大肠杆菌BL21(DE3)并进行表达和纯化后，SalI内切酶重质量不足1mg/L。
SalIM虽然也过量表达，但由于构建的重组SalIM没有亲和纯化标签，因此，通过亲和纯化及后面的过柱纯化步骤，基本完全去除了SalIM和其他杂蛋白的污染，在最终纯化得到的产物中检测不到SalIM活性。
实施例3SalI限制性内切酶的酶活的测定
取实施例2中表达和纯化后的SalI限制性内切酶进行酶活测定。
通过酶切实验确定表达纯化的SalI限制性内切酶的活性是否正常。进行三组反应，第一组使用实施例2中的SalI和购买的BamHI进行双酶切，第二组使用实施例2中的SalI进行单酶切，第三组使用购买的SalI和BamHI进行双酶切。反应条件为：20μl反应体系，10USalI，10U BamHI，反应缓冲体系：100mM NaCl,50mM Tris-HCl,10mM MgCl2,100μg/ml BSA(pH 7.9@25℃)，5μg质粒(构建有基因的商用pFastbac载体)，37℃反应10min。其中，BamHI购自ThermoFisher Scientific。酶切后的琼脂糖凝胶电泳图如图5所示，实施例2制备的SalI进行单酶切或双酶切，均能正常切割质粒，表明SalI限制性内切酶功能正常。
一个单位被定义为在37℃下1小时内消化1μg线性化质粒DNA(含有1个相应内切酶位点)，总反应体积为20μl所需的酶量。所用酶活鉴定质粒的DNA如SEQ ID No.9所示。
取不同稀释比的SalI，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。测定的SalI的比活大于4*106U/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作出诸多修改和变化。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> 一种SalI限制性内切酶的制备方法
<130> CN100-20001PICN
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 1761
<212> DNA
<213> Artificial Sequence（人工序列）
<400> 1
atgcattctg aagcacgtga agcagaagct cgtcgtctgc tgctgcagga aactctggat 60
gcagagcgta cgttcctgga acgtaaccag tggggtcagt tcgctacccc gccgtctctg 120
gctggtgaaa tcatgcgtta cactatcgac ctgcatgagg aaacccgcat caacttcctg 180
gaaccgtctt gcggctctgg ctcctttttt tccgcactgc tgcgcaacct gggtgacaaa 240
aaaatcgagc acgcggttgg cgtggaactg gatccgcgtt tcagcaaagc agcttccgac 300
ctgtggactg gtcagggtct gcgcgtgatc gaaggtgact tcacctcccc ttctctggtg 360
tctggtccgg ttgcctctct gctggttgct aacccgccgt acgttcgtca ccaccacctg 420
ggtatcgatc agaaacgtga cctggtcgca cgctgcgcag atcagctggg tatcaaaccg 480
agcggcctgt ctggtctgta tctgtatttc gtgctgctgt ctcatcgcct gctgcgtgcg 540
gatgctgttt ctacctggct gattccatcc gaatttatgg atgtcaacta cggcaccgct 600
ctgaaagaat atctggcgac ccgtgttcag ctggtgcgta tccaccagta cgatgccgct 660
gaagttcagt tcgacgatgc actggtgacc tcttccgtgg ttgtctttcg taactctcct 720
ccgcgtccgg gtcatacggc tgaattctct ttcggcggta ctctgtctga gccgaaagtt 780
acccaccaga ttccgtctgc ggctctgacc ccggaagcca aatggtctcg ctatgttacc 840
ggcgttatgc cggcagacat caatctgaaa cagactggtc cgaaactgag cgacttcttc 900
aaaatccgtc gtggtctggc aaccggttct aacgcgttct tcattattcc acgctctgaa 960
gctgagcgtc tgggcatcaa acgtaacttc ctgcgcccaa tcctgccatc tccgcgcaaa 1020
ctgaaaggtg acgcaatcac tgcagatgcg tccggctggc ctgacatccc agaacaactg 1080
gcgctgctgg attgtcctct gccgattgaa gacctgctgc tggaaaaccc agcactggcg 1140
gcttatctga gcacggctga tgagaaaatc cgcggcggct atctggtaag caaacgttct 1200
ccgtggtaca aacaagagca gcgtgaacca gctccgatcc tgctgaccta catgggtcgt 1260
ggtaaggatg accaacaccc tctgcgtttc atccgcaacg attctgacgc ggtcgctact 1320
aacatgtatc tgatgctgta cccgaccgct ctgctgcaac gctacctggc aggcgatccg 1380
gaacgtatta aacaggtgca taaggccctg ctggccatca ctgctgctga tctgcgtggc 1440
ggtggtcgtg tttatggcgg tggcctgcac aaaatggaac cgaaagaact ggcggcactg 1500
ccagctgacg gcattgctac tctggacccg gtgctgcgtg aagatatcag catggtctct 1560
gtgcctcctc gtaaacgtac cggtcgtcct cagatgcctg gtccgtccgc tagcgaagtt 1620
cgtgcttggg cccgtgcgaa cggcgtttgt gttccagatc gtggccgtct gcgtccggaa 1680
gtttgggacg catggcgtca ggctcatgcg ggtgaagcct ctccgctgaa catcgacgct 1740
ggtgatcagg ttgcgctgtg g 1761
<210> 2
<211> 945
<212> DNA
<213> Artificial Sequence（人工序列）
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attaacgcgg ataaaccgca ccgttggaac gatgatgttc aggctagcgt tcgtctgtac 60
aaccagtggt tcctggatgc agcgccgaaa gcgtaccgag atacccgtca gctgaccatc 120
gatgaagttg aacaggcttt ccagcgtacc gcgaacatga ccagcatcac cccggaagtt 180
ctgaaagcgc acccgaaaac cctggctacc ctgcgtatgt ctactgcacc gccgatcgcg 240
cgtgatcgtc tggtgggtct gtcccacggt agcaaaagcc tgctggatac catggaaaaa 300
ggtaaactgc cgccgcgtat gaaaggcgat gttctggata cccacctggc taaaatgtgt 360
gcggttctga ccgatctgct ggatctggac ctgttccact ggtatccgac cggtgaaccg 420
gcggaaccgc gtcagcgtga actggcggca accgtggttg cggatcgact gtgcggcgcg 480
atcgcggacc cgatcgttcg caacgcgcag gaacgtcgtc agctggcgct gatcgaagaa 540
tggctgctgg cgcgtggtta caccaagaaa actcacagcg catccctgcc gctgaacacc 600
atgcagccgg gtaccttctc tttccgtcag aacgttgttg tgggttccga tctgccggtt 660
aacatcccgg ttgacgcggt gatccagccg cacacgccgc actctcataa actgccgatc 720
ctcatcgaag cgaaaagcgc gggcgatttc accaacacta acaaacgtcg taaagaagaa 780
gcgaccaaaa tccaccagct gcagctgaaa tacggtaacg aaatcagctt aaccctgttc 840
ctgtgtggct actttaatac cggttacctg ggttacagcg cggcggaagg tctggattgg 900
gtttgggaac accgtattga tgatctggaa gcagcgggtg cgtaa 945
<210> 3
<211> 587
<212> PRT
<213> Artificial Sequence（人工序列）
<400> 3
Met His Ser Glu Ala Arg Glu Ala Glu Ala Arg Arg Leu Leu Leu Gln
1 5 10 15
Glu Thr Leu Asp Ala Glu Arg Thr Phe Leu Glu Arg Asn Gln Trp Gly
20 25 30
Gln Phe Ala Thr Pro Pro Ser Leu Ala Gly Glu Ile Met Arg Tyr Thr
35 40 45
Ile Asp Leu His Glu Glu Thr Arg Ile Asn Phe Leu Glu Pro Ser Cys
50 55 60
Gly Ser Gly Ser Phe Phe Ser Ala Leu Leu Arg Asn Leu Gly Asp Lys
65 70 75 80
Lys Ile Glu His Ala Val Gly Val Glu Leu Asp Pro Arg Phe Ser Lys
85 90 95
Ala Ala Ser Asp Leu Trp Thr Gly Gln Gly Leu Arg Val Ile Glu Gly
100 105 110
Asp Phe Thr Ser Pro Ser Leu Val Ser Gly Pro Val Ala Ser Leu Leu
115 120 125
Val Ala Asn Pro Pro Tyr Val Arg His His His Leu Gly Ile Asp Gln
130 135 140
Lys Arg Asp Leu Val Ala Arg Cys Ala Asp Gln Leu Gly Ile Lys Pro
145 150 155 160
Ser Gly Leu Ser Gly Leu Tyr Leu Tyr Phe Val Leu Leu Ser His Arg
165 170 175
Leu Leu Arg Ala Asp Ala Val Ser Thr Trp Leu Ile Pro Ser Glu Phe
180 185 190
Met Asp Val Asn Tyr Gly Thr Ala Leu Lys Glu Tyr Leu Ala Thr Arg
195 200 205
Val Gln Leu Val Arg Ile His Gln Tyr Asp Ala Ala Glu Val Gln Phe
210 215 220
Asp Asp Ala Leu Val Thr Ser Ser Val Val Val Phe Arg Asn Ser Pro
225 230 235 240
Pro Arg Pro Gly His Thr Ala Glu Phe Ser Phe Gly Gly Thr Leu Ser
245 250 255
Glu Pro Lys Val Thr His Gln Ile Pro Ser Ala Ala Leu Thr Pro Glu
260 265 270
Ala Lys Trp Ser Arg Tyr Val Thr Gly Val Met Pro Ala Asp Ile Asn
275 280 285
Leu Lys Gln Thr Gly Pro Lys Leu Ser Asp Phe Phe Lys Ile Arg Arg
290 295 300
Gly Leu Ala Thr Gly Ser Asn Ala Phe Phe Ile Ile Pro Arg Ser Glu
305 310 315 320
Ala Glu Arg Leu Gly Ile Lys Arg Asn Phe Leu Arg Pro Ile Leu Pro
325 330 335
Ser Pro Arg Lys Leu Lys Gly Asp Ala Ile Thr Ala Asp Ala Ser Gly
340 345 350
Trp Pro Asp Ile Pro Glu Gln Leu Ala Leu Leu Asp Cys Pro Leu Pro
355 360 365
Ile Glu Asp Leu Leu Leu Glu Asn Pro Ala Leu Ala Ala Tyr Leu Ser
370 375 380
Thr Ala Asp Glu Lys Ile Arg Gly Gly Tyr Leu Val Ser Lys Arg Ser
385 390 395 400
Pro Trp Tyr Lys Gln Glu Gln Arg Glu Pro Ala Pro Ile Leu Leu Thr
405 410 415
Tyr Met Gly Arg Gly Lys Asp Asp Gln His Pro Leu Arg Phe Ile Arg
420 425 430
Asn Asp Ser Asp Ala Val Ala Thr Asn Met Tyr Leu Met Leu Tyr Pro
435 440 445
Thr Ala Leu Leu Gln Arg Tyr Leu Ala Gly Asp Pro Glu Arg Ile Lys
450 455 460
Gln Val His Lys Ala Leu Leu Ala Ile Thr Ala Ala Asp Leu Arg Gly
465 470 475 480
Gly Gly Arg Val Tyr Gly Gly Gly Leu His Lys Met Glu Pro Lys Glu
485 490 495
Leu Ala Ala Leu Pro Ala Asp Gly Ile Ala Thr Leu Asp Pro Val Leu
500 505 510
Arg Glu Asp Ile Ser Met Val Ser Val Pro Pro Arg Lys Arg Thr Gly
515 520 525
Arg Pro Gln Met Pro Gly Pro Ser Ala Ser Glu Val Arg Ala Trp Ala
530 535 540
Arg Ala Asn Gly Val Cys Val Pro Asp Arg Gly Arg Leu Arg Pro Glu
545 550 555 560
Val Trp Asp Ala Trp Arg Gln Ala His Ala Gly Glu Ala Ser Pro Leu
565 570 575
Asn Ile Asp Ala Gly Asp Gln Val Ala Leu Trp
580 585
<210> 4
<211> 315
<212> PRT
<213> Artificial Sequence（人工序列）
<400> 4
Met Ile Asn Ala Asp Lys Pro His Arg Trp Asn Asp Asp Val Gln Ala
1 5 10 15
Ser Val Arg Leu Tyr Asn Gln Trp Phe Leu Asp Ala Ala Pro Lys Ala
20 25 30
Tyr Arg Asp Thr Arg Gln Leu Thr Ile Asp Glu Val Glu Gln Ala Phe
35 40 45
Gln Arg Thr Ala Asn Met Thr Ser Ile Thr Pro Glu Val Leu Lys Ala
50 55 60
His Pro Lys Thr Leu Ala Thr Leu Arg Met Ser Thr Ala Pro Pro Ile
65 70 75 80
Ala Arg Asp Arg Leu Val Gly Leu Ser His Gly Ser Lys Ser Leu Leu
85 90 95
Asp Thr Met Glu Lys Gly Lys Leu Pro Pro Arg Met Lys Gly Asp Val
100 105 110
Leu Asp Thr His Leu Ala Lys Met Cys Ala Val Leu Thr Asp Leu Leu
115 120 125
Asp Leu Asp Leu Phe His Trp Tyr Pro Thr Gly Glu Pro Ala Glu Pro
130 135 140
Arg Gln Arg Glu Leu Ala Ala Thr Val Val Ala Asp Arg Leu Cys Gly
145 150 155 160
Ala Ile Ala Asp Pro Ile Val Arg Asn Ala Gln Glu Arg Arg Gln Leu
165 170 175
Ala Leu Ile Glu Glu Trp Leu Leu Ala Arg Gly Tyr Thr Lys Lys Thr
180 185 190
His Ser Ala Ser Leu Pro Leu Asn Thr Met Gln Pro Gly Thr Phe Ser
195 200 205
Phe Arg Gln Asn Val Val Val Gly Ser Asp Leu Pro Val Asn Ile Pro
210 215 220
Val Asp Ala Val Ile Gln Pro His Thr Pro His Ser His Lys Leu Pro
225 230 235 240
Ile Leu Ile Glu Ala Lys Ser Ala Gly Asp Phe Thr Asn Thr Asn Lys
245 250 255
Arg Arg Lys Glu Glu Ala Thr Lys Ile His Gln Leu Gln Leu Lys Tyr
260 265 270
Gly Asn Glu Ile Ser Leu Thr Leu Phe Leu Cys Gly Tyr Phe Asn Thr
275 280 285
Gly Tyr Leu Gly Tyr Ser Ala Ala Glu Gly Leu Asp Trp Val Trp Glu
290 295 300
His Arg Ile Asp Asp Leu Glu Ala Ala Gly Ala
305 310 315
<210> 5
<211> 39
<212> DNA
<213> Artificial Sequence（人工序列）
<400> 5
ctttaagaag gagatatacc atgcattctg aagcacgtg 39
<210> 6
<211> 40
<212> DNA
<213> Artificial Sequence（人工序列）
<400> 6
ctttgttagc agccggatcc ttaccacagc gcaacctgat 40
<210> 7
<211> 39
<212> DNA
<213> Artificial Sequence（人工序列）
<220>
<223> 7
<400> 7
tggtgccgcg cggcagccat attaacgcgg ataaaccgc 39
<210> 8
<211> 35
<212> DNA
<213> Artificial Sequence（人工序列）
<400> 8
tgctcgagtg cggccgcatt acgcacccgc tgctt 35
<210> 9
<211> 6032
<212> DNA
<213> Artificial Sequence（人工序列）
<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 cgtaagcatc gttgctgctc 2520
cataacatca aacatcgacc cacggcgtaa cgcgcttgct gcttggatgc ccgaggcata 2580
gactgtacaa aaaaacagtc ataacaagcc atgaaaaccg ccactgcgcc gttaccaccg 2640
ctgcgttcgg tcaaggttct ggaccagttg cgtgagcgca tacgctactt gcattacagt 2700
ttacgaaccg aacaggctta tgtcaactgg gttcgtgcct tcatccgttt ccacggtgtg 2760
cgtcacccgg caaccttggg cagcagcgaa gtcgaggcat ttctgtcctg gctggcgaac 2820
gagcgcaagg tttcggtctc cacgcatcgt caggcattgg cggccttgct gttcttctac 2880
ggcaaggtgc tgtgcacgga tctgccctgg cttcaggaga tcggaagacc tcggccgtcg 2940
cggcgcttgc cggtggtgct gaccccggat gaagtggttc gcatcctcgg ttttctggaa 3000
ggcgagcatc gtttgttcgc ccaggactct agctatagtt ctagtggttg gctacgtata 3060
ctccggaata ttaatagatc atggagataa ttaaaatgat aaccatctcg caaataaata 3120
agtattttac tgttttcgta acagttttgt aataaaaaaa cctataaata ttccggatta 3180
ttcataccgt cccaccatcg ggcgcggatc catgtggata ctggctctct ccttgttcca 3240
gagcttcgcg aatgttttca gtgaagagcc ccactccagc ctctactttg tcaatgcatc 3300
gctgcaagag gtagtgtttg caagcacatc ggggacgctg gtgccctgcc cggctgcagg 3360
catccctcct gtgactctca gatggtacct agcaacgggc gaggagatct acgatgtccc 3420
cgggaaccgc cacgtccatc ccaatggcac tctccaaatt ttcccctttc ctccttcaag 3480
cttcagcacc ttaatccatg ataatactta ctattgcaca gctgaaaacc cttcagggaa 3540
aattagaagt caggatgtcc acatcaaggc tgttttacgg gagccctata cagtccgtgt 3600
ggaggaccag aaaaccatga gaggcaatgt cgcggtgttc aagtgcatta tcccctcctc 3660
ggtggaggcg tacgtcactg tcgtctcatg ggagaaagac acggtttcac ttgtctcagg 3720
atctagattt ctcatcacat ccacgggagc cttgtatatt aaagatgtac agaacgaaga 3780
tgggctgtac aactaccgct gcatcacgcg gcacagatac acaggggaga cgagacaaag 3840
caacagcgcg agactgttcg tgtcagaccc agcaaactca gccccatcca tcctggacgg 3900
gtttgaccac cgcaaagcca tggcagggca gcgcgtggag ctgccttgca aagcacttgg 3960
gcaccccgag ccagactacc gctggctgaa ggacaacatg cccctggaac tttctggaag 4020
gttccaaaag acagtgactg ggttgctcat tgagaacagc cgcccctcag actcaggcag 4080
ctatgtgtgt gaagtatcca accgatatgg cactgccaag gtgataggcc gcctgtacgt 4140
gaaacagcca ctgaaagcca ccatcagtcc cagaaaggtt aaaagcagcg tgggcagcca 4200
ggtctcctta tcctgcagtg tgacaggaaa tgaagaccag gaactctcct ggtaccgaaa 4260
tggcgaaatc ctcaaccctg gaaaaaacgt gaggatcaca ggactcaacc acgcaaacct 4320
tataatggat cacatggtca agagtgatgg gggtgcctac cagtgctttg tgcgcaagga 4380
caagctatct gctcaagact atgtccaggt ggtccttgaa gacggaactc ccaaaatcat 4440
ttctgccttt agcgagaaag tggtgagccc ggcagagcca gtgtccctcg tgtgcaatgt 4500
gaagggtaca cccttgccca cggtcacctg gaccctggac gatgacccca tcctcaaggg 4560
cagcggtcac cgcatcagcc aaatgatcac gtccgagggg aacgtggtca gctacctgaa 4620
tatctccagc tcccaggtcc gggatggggg tgtctaccgc tgcactgcca acaactcggc 4680
tggagtcgtc ctgtaccagg ctcgaataaa cgtaagaggg cctgcaagca tcagaccaat 4740
gaaaaacatc actgcgatag cggggcgtga cacgtacatc cactgccgcg taattggcta 4800
tccgtattac tccatcaagt ggtacaagaa cgctaacctg cttcctttca accaccgcca 4860
ggtggcgttt gagaacaatg ggactctgaa actctctgat gtgcagaaag aagttgacga 4920
gggagagtac acgtgtaatg tgctggtaca gccacagctc tccaccagcc agagtgtcca 4980
cgtgacagtg aaagttccac ctttcatcca accttttgag ttcccaagat tctccatcgg 5040
gcagcgggtt ttcatcccat gtgtggtggt ctcaggggac ttacccatca ccatcacctg 5100
gcagaaggat ggccggccaa ttccagcaag cctcggagta accattgaca acattgactt 5160
caccagctcc ctgaggatct ccaacctctc cctaatgcac aatgggaatt acacctgcat 5220
tgcgagaaac gaggcagcag ccgtggaaca ccagagtcag ctgattgtga gagttccccc 5280
taagttcgtg gtacagcccc gggaccagga cgggatctat ggcaaagcag tggtcgacga 5340
gctcactagt cgcggccgct ttcgaatcta gacatcacca tcaccatcac tagagcttgt 5400
cgagaagtac tagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt 5460
aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt 5520
taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac 5580
aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc 5640
ttatcatgtc tggatctgat cactgcttga gcctaggaga tccgaaccag ataagtgaaa 5700
tctagttcca aactattttg tcatttttaa ttttcgtatt agcttacgac gctacaccca 5760
gttcccatct attttgtcac tcttccctaa ataatcctta aaaactccat ttccacccct 5820
cccagttccc aactattttg tccgcccaca gcggggcatt tttcttcctg ttatgttttt 5880
aatcaaacat cctgccaact ccatgtgaca aaccgtcatc ttcggctact ttttctctgt 5940
cacagaatga aaatttttct gtcatctctt cgttattaat gtttgtaatt gactgaatat 6000
caacgcttat ttgcagcctg aatggcgaat gg 6032
Claims (9)
1.一种SalI限制性内切酶的制备方法，其特征在于，在原核表达菌株中同时表达SalI和SalIM。
2.如权利要求1所述的制备方法，其特征在于，所述SalIM的DNA及氨基酸序列如SEQ IDNo.1，SEQ ID No.3所示，或者其氨基酸序列与SEQ ID No.3具有至少95％及以上的相似度；所述SalI的DNA及氨基酸序列如SEQ ID No.2，SEQ ID No.4所示，或者其氨基酸序列与SEQID No.4具有至少95％及以上的相似度。
3.如权利要求1所述的制备方法，其特征在于，所述原核表达菌株包括大肠杆菌。
4.如权利要求1所述的制备方法，其特征在于，先在所述原核表达菌株中转化入含有SalIM的重组质粒，使得原核表达菌株进行SalIM基底表达并进行基因组保护修饰；然后在含有SalIM的重组质粒的原核表达菌株中进一步转化入含有SalI的重组质粒，获得含有SalIM的重组质粒和含有SalI的重组质粒的原核表达菌株。
5.如权利要求4所述的制备方法，其特征在于，将含有SalIM的重组质粒的原核表达菌株制备成感受态细胞，并在所述感受态细胞中转化入含有SalI的重组质粒。
6.如权利要求4所述的制备方法，其特征在于，所述含有SalIM的重组质粒不含纯化标签，所述含有SalI的重组质粒带有纯化标签。
7.如权利要求4所述的制备方法，其特征在于，所述含有SalIM的重组质粒使用pET16b载体，所述含有SalI的重组质粒使用pET28a载体。
8.如权利要求6所述的制备方法，其特征在于，将含有SalIM的重组质粒和含有SalI的重组质粒的原核表达菌株进行诱导表达后，通过亲和介质、离子交换层析和/或凝胶色谱层析进行SalI的纯化。
9.如权利要求8所述的制备方法，其特征在于，所述诱导表达为，加入终浓度0.5mM的IPTG，在20～23℃下诱导表达19-22小时。
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