CN114774384A - 一种Taq DNA聚合酶突变体及其筛选方法 - Google Patents
一种Taq DNA聚合酶突变体及其筛选方法 Download PDFInfo
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- CN114774384A CN114774384A CN202210450302.3A CN202210450302A CN114774384A CN 114774384 A CN114774384 A CN 114774384A CN 202210450302 A CN202210450302 A CN 202210450302A CN 114774384 A CN114774384 A CN 114774384A
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- leu
- dna polymerase
- ala
- taq dna
- glu
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Abstract
本发明公开了一种Taq DNA聚合酶突变体及其筛选方法,该Taq DNA聚合酶突变体与未经改造的野生型Taq酶相比,特异性和活性均有明显的提高,可极大程度满足研究和医学诊断应用需求。本发明还公开了一种改进后的CSR方法,简化了乳化过程和回收ePCR产物过程,使得目前操作步骤繁琐的CSR更简化;而且摒弃之前的方法中使用氯仿、苯酚和异丙醇的有毒有害试剂,让过程更环保和容易操作。
Description
技术领域
本发明涉及酶工程技术领域,尤其涉及一种Taq DNA聚合酶突变体及其筛选方法。
背景技术
Taq DNA聚合酶与大肠杆菌DNA聚合酶I(pol I)同源,属于DNA聚合酶I家族,基因全长为2496bp,共编码832aa,分子量大小为94kD,是一种耐热的聚合酶,研究者首度将Taq应用于PCR技术,使得PCR过程实现了自动连续循环。最适温度为70~75℃(催化活性最高的温度),其催化延伸效率可达150bp/s。Taq DNA聚合酶在92.5℃酶活性可持续130min,95℃持续40min,97.5℃5-6min可保持约50%的酶活性。
Taq DNA聚合酶已经成为重要的分子生物学工具酶,在各类疾病的诊断和检测中有非常广泛的应用,RT-PCR在新冠肺炎病毒检测中已被广泛采用,是CoV-2019检测的黄金标准,Taq DNA聚合酶在其中发挥了至关重要的作用,但仍存在诸多不足之处,例如检测灵敏性和特异性仍然有待提高。
改造Taq DNA聚合酶的研究,可以显著提高Taq DNA聚合酶的活性和特异性,对于提升Taq DNA聚合酶的性能,增强Taq DNA聚合酶的应用性等方面具有重要的理论与实践意义。
分隔式自我复制(Compartmentalized self-replication,CSR)是聚合酶定向进化的一种方法。自我复制发生在一个热稳定的油包水的乳化液形成的离散、分离、没有交互的空间中,每个液滴中聚合酶只复制编码自身的基因(自我复制)。分隔式自我复制(CSR)体系在改造Taq DNA聚合酶具有通量高,工作量小,靶向性强的优势。CSR已经被证明是一种行之有效改造DNA聚合酶定向进化的方法,但其流程步骤繁琐,使用到氯仿、苯酚和异丙醇等有毒有害试剂等缺陷,因此简化CSR步骤,摒弃操有毒有害试剂的缺陷,使得CSR能更简便快捷,是该方法能够简便、自然友好地改造聚合酶。
发明内容
本发明提供了一种Taq DNA聚合酶突变体及其筛选方法,该Taq DNA聚合酶突变体与未经改造的野生型Taq酶相比,特异性和活性均有明显的提高,可极大程度满足研究和医学诊断应用需求。本发明还发明了一种改进后的CSR方法,简化了乳化过程和回收ePCR产物过程,使得目前操作步骤繁琐的CSR更简化;而且摒弃之前的方法中使用氯仿、苯酚和异丙醇的有毒有害试剂,让过程更环保和容易操作。
具体技术方案如下:
本发明提供了一种Taq DNA聚合酶突变体,其氨基酸序列如SEQ ID NO.1所示。
本发明还提供了一种所述Taq DNA聚合酶突变体的编码基因。
进一步地,所述编码基因如SEQ ID NO.2所示。
本发明还提供了一种包含所述编码基因的重组载体;进一步地,所述重组载体所用的表达载体为pET-21a。
本发明还提供了一种包含所述编码基因的基因工程菌;进一步地,所述基因工程菌的宿主细胞可为BL21等。
本发明还提供了一种筛选所述Taq DNA聚合酶突变体的分隔式自我复制方法,包括以下步骤:
(1)构建饱和突变文库:以含有Taq DNA聚合酶的环状质粒作为模板,进行PCR扩增,构建Taq DNA聚合酶定点饱和突变质粒文库;
(2)突变体文库的诱导表达:将步骤(1)获得的定点饱和突变质粒转入大肠杆菌细胞中,进行诱导表达,得到菌悬液;
(3)乳化PCR:配制ePCR预混液,将ePCR预混液加入至CSR油相中,并在乳化过程中,向混合物中放入橡胶颗粒,得到单相的乳白色粘稠的乳液;
(4)乳化PCR产物回收与扩增:采用PCR产物回收试剂盒回收乳化的PCR产物,并进行扩增反应;
(5)连接转化与测序。
进一步地,步骤(1)中,所述环状质粒的PCR体系为:模板1μL,引物F10μM1μL,引物R10μM 1μL,dNTP 2.5mM 4μL,高保真酶缓冲液5μL,高保真酶1μL,补水至50μL的反应体系;
引物F的序列为GCCGCCGTTATGTACCGNNKCTGGAAGCTCGCG;引物R的序列为AACGCGAGCTTCCAGMNNCGGTACATAAC;
PCR热循环条件为:94℃预变性3min,94℃变性30s,Tm-5℃退火20s,72℃延伸4min20s,4℃保温;进行PCR扩增35个循环。
进一步地,步骤(3)中,ePCR预混液的配方为:10×PCR buffer 40μL,dNTP 2.5mM40μL,2CSR-F 8μL,2CSR-R 8μL,菌体0.5OD,ddH2O 304μL;
2CSR-F的序列为:GCGGCAAAAACCATCAACTTCGGTGTTC;2CSR-R的序列为CTCCAGCGGTACCGCAAGTGGGTAGAC。
上述方法的改进点有两点:1、乳化过程阶段,本发明向油/表面活性剂混合物中放入橡胶颗粒,在旋涡混匀仪加入菌体/ePCR悬浮液,1min可得单相的乳白色粘稠的乳液,大大提高了乳化效率;2、破乳回收ePCR产物时阶段,本发明使用操作简便PCR产物回收试剂盒(包括但不仅限于生工PCR产物回收试剂盒)进行回收;摒弃了目前文献记述的采用繁琐、污染严重、对人体有害的有机溶剂法。
与现有技术相比,本发明具有以下有益效果:
(1)本发明采用CSR方法筛选Taq DNA聚合酶突变体,获得的突变体与未经改造的野生型Taq酶相比,特异性和活性均有明显的提高,可极大程度满足研究和医学诊断应用需求。
(2)本发明改进CSR方法后,简化了乳化过程和回收ePCR产物过程,使得目前操作步骤繁琐的CSR更简化;而且摒弃之前的方法中使用氯仿、苯酚和异丙醇的有毒有害试剂,让过程更环保和容易操作。
附图说明
图1为Taq DNA聚合酶732位点饱和突变质粒的电泳图。
图2为突变体的乳化效果图。
图3为732位点ePCR扩增产物回收的电泳图。
图4为732位点CSR筛选测序结果。
图5为突变体D732A和野生型Taq DNA聚合酶纯化蛋白电泳结果;
图6为突变体D732A和野生型Taq DNA聚合酶的qPCR结果;
图7为突变体D732A和野生型Taq DNA聚合酶的PCR电泳结果,
其中野生型为泳道1和3;突变体D732A为泳道2和4;泳道1和2:2kb的扩增效果;泳道3和4:4.1kb的扩增效果。
具体实施方式
下面结合具体实施例对本发明作进一步描述,以下列举的仅是本发明的具体实施例,但本发明的保护范围不仅限于此。
实施例1采用CSR方法筛选Taq DNA聚合酶突变体
1、饱和突变文库的构建和诱导表达
突变文库是定向进化的第一步,也是至为关键的一步,点饱和突变文库构建方法如下:
以含有的pET-21a-Taq DNA聚合酶(野生型酶来源于水生嗜热菌(Thermus aquaticus),野生酶的核苷酸序列如SEQ ID NO.3所示)的环状质粒作为模板PCR扩增,环状质粒PCR体系:模板1μL,引物F(gccgccgttatgtaccgnnkctggaagctcgcg)10μM1μL,引物R(aacgcgagcttccagmnncggtacataac),10μM各1μL,dNTP(2.5mM)4μL,高保真酶缓冲液5μL,高保真酶1μL,补水至50μL的反应体系;
环状质粒PCR热循环条件:94℃预变性3min,94℃变性30s,(Tm-5)℃退火20s,72℃延伸4min 20s,4℃保温。反应条件进行PCR扩增35个循环。扩增产物经Dpn I消化后即可用于转化。
DpnI消化原始质粒:扩增PCR产物需要经过Dpn I甲基化酶去除原始的拟突变质粒模板,反应体系为DpnI 1μL,Buffer 5μL,PCR反应产物5μL,补水至50μL;反应条件为37℃孵育1h。甲基化酶消化原始质粒,进行下一步的转化实验。
取5μL反应体系,加入50μL的BL21(DE3)Chemically Competent Cell感受态细胞中(表达性宿主细胞),冰上孵育20min;42℃热激45sec;冰上放置3min;加入950μL LB培养基,在37℃摇床(220rpm)活化1h。
采用上述方法构建了DNA聚合酶732位点饱和突变质粒文库(如图1,矩形框标记),并导入到BL21(DE3)Chemically Competent Cell感受态细胞中,以备CSR筛选。
以下操作为实现Taq DNA聚合酶突变体在大肠杆菌细胞内的表达,具体操作流程如下:
种子培养:突变文库的转化菌液(2mL)接种入20mL的LB培养液(含抗生素),培养13h。
发酵培养:将200μL的过夜培养液接种入10mL新鲜的LB的培养基(含抗生素,用50mL的锥形瓶)中37℃,250rpm培养约2h,至对数生长期(OD600=0.4-0.6)。
诱导表达:用终浓度0.05mM IPTG进行诱导,30℃下继续诱导培养7小时。
取适量菌液突变体,离心稀释使OD600=0.5,取0.5mL OD600=1.0的菌液离心用400μL PCR缓冲液重悬(冰上预冷)。
按照表1制成乳化PCR预混液(ePCR预混液)。
表1 CSR的ePCR预混液
CSR引物扩增区域不宜太长或太短,而且必须要包括需要饱和突变的位点,本文设计CSR引物,扩增短序列片段长度为474bp,引物为2CSR-F和2CSR-R(如表2所示)
表2 CSR引物序列
2、乳化PCR与其产物回收与扩增
乳化PCR是CSR的关键,以下是乳化PCR的过程:
(1)400μL水相(CSR的ePCR预混液液)加入至800μL CSR油相(4.5%(v/v)Span 80,0.4%(v/v)Tween-80和0.05%(v/v)Triton X-100在轻矿物油中),在乳化过程阶段,向油/表面活性剂混合物中放入橡胶颗粒,使用100μL移液枪在旋涡混匀仪中匀速加入CSR的ePCR预混液液,1min可得单相的乳白色粘稠的乳液,如图2。
(2)将100μL乳液混合物分装到PCR管中,并去除管道底部的气泡。
(3)将PCR管放在PCR仪中,并按照表3所述运行热循环程序。
说明:在完成热循环程序后,乳液应保持均质,并最少分成两层。
表3 PCR循环程序
乳化PCR之后,就是回收乳化PCR产物,合并4个ePCR小管,离心10min后,弃掉上层油状物,在剩余的乳液中使用6-10倍的Binding solution旋涡混匀后,加入吸附柱中,12000rpm离心1min中后,Washing solution 12000rpm,30s清洗两次,空柱离心一次,加入ddH2O即可完成回收。电泳结果如下图3所示,扩增长度约在500bp左右,符合引物扩增片段长度。
Dpn I限制酶消化回收的PCR产物,添加1/10(v/v)10×Quickcut Buffer和2μL(40单位)DpnI酶,37℃孵育至少1小时,胶回收。该步骤消除了基因组和质粒DNA的残留。省略此步骤可能会导致背景放大。说明:DpnI可消化过夜。
重复PCR扩增回收的产物,使用1-10μL上述纯化DNA作为模板,利用Taq DNA聚合酶进行常规PCR并进行了胶回收。
3、连接、转化、测序与突变体的验证
将上述胶回收的产物与载体连接并转化:连接pMD18-T,转化涂平板,挑取单菌落过夜培养,测序,结果如图4所示。
在732位置处,10个测序成功的结果显示732位置出现1次D(Wild type),1次P,2次Q,3次A,3次G。频率越高的突变体其复制能力也越强,由此可知D732Q,D732G和D732A可作为进一步验证的对象。
将野生型和突变体D732A进行表达和纯化(图5所示),在蛋白定量至50ng/μL后,进行qPCR和PCR验证(扩增片段分别为2.0kb和4.1kb),结果如图6和图7所示。
qPCR表明,突变体D732 A的Ct值更小,最终荧光强度也显著提高,其表现优于野生酶。PCR电泳结果也表明,无论扩增2kb还是4.1kb,突变体D732 A的活性和特异性有明显提升。
序列表
<110> 浙江大学杭州国际科创中心
北京全式金生物技术股份有限公司
<120> 一种Taq DNA聚合酶突变体及其筛选方法
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<170> SIPOSequenceListing 1.0
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<211> 832
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<213> 水生嗜热菌(Thermus aquaticus)
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530 535 540
Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu
545 550 555 560
His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser
565 570 575
Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln
580 585 590
Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala
595 600 605
Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly
610 615 620
Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr
625 630 635 640
Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro
645 650 655
Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670
Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
675 680 685
Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg
690 695 700
Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val
705 710 715 720
Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Ala Leu Glu Ala Arg
725 730 735
Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro
740 745 750
Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu
755 760 765
Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His
770 775 780
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala
785 790 795 800
Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro
805 810 815
Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830
<210> 2
<211> 2499
<212> DNA
<213> 水生嗜热菌(Thermus aquaticus)
<400> 2
atgcgtggta tgctgccgct tttcgaaccg aaaggtcgtg ttctgctggt ggacggccac 60
cacctggctt atcgcacctt ccacgcgttg aaaggcctga ctaccagccg cggcgagccg 120
gttcaagcag tatatggttt tgctaaaagc ctgctgaaag ctctgaaaga agatggtgac 180
gccgttatcg tagtatttga cgcaaaagcc ccgtctttcc gtcacgaagc ctacggcggt 240
tacaaagcag gtcgcgctcc gactccggaa gacttcccgc gtcaacttgc cctgatcaaa 300
gagctggtag atctgctggg ccttgcccgt ctggaagttc cgggttacga agctgatgat 360
gttctggcaa gcctcgcgaa gaaagccgaa aaagaaggct acgaagtccg tatcctgacc 420
gctgacaagg atctgtatca gctgctgtcc gatcgcatcc acgcgctgca cccggaaggt 480
tatctgatca ctcctgcttg gctgtgggaa aagtacggcc tgcgtccgga tcagtgggca 540
gattaccgtg ccctgaccgg cgatgaatct gacaacctgc ctggtgttaa aggcatcggt 600
gagaaaacgg cgcgtaaatt actggaggaa tggggctctc tggaagcgct gctgaaaaac 660
ctggaccgtc tgaagccggc gattcgtgaa aaaatccttg ctcacatgga tgatctgaaa 720
ctgtcctggg atctggcaaa ggtacgcacc gatttgccac tggaagttga tttcgccaaa 780
cgtcgtgaac cggaccgtga acgtctgcgt gcattcctgg aacgcctgga attcggtagc 840
ttgctgcacg aattcggcct gctggaaagt ccaaaagccc tggaggaagc gccgtggccg 900
ccgccggaag gcgcctttgt tggtttcgta ctgagccgta aagaaccgat gtgggctgat 960
ctgctggcac tggcagccgc acgtggcggc cgtgtacacc gtgccccgga accgtacaaa 1020
gccctgcgtg acctgaaaga agcacgtggc ctgctggcta aagacctgtc agtactcgca 1080
ctgcgtgaag gtctgggtct gccgccaggt gacgacccga tgctgctggc gtacctgctg 1140
gacccgtcta acactacgcc ggagggtgta gcccgccgct acggcggcga gtggactgaa 1200
gaagctggcg aacgtgcggc gctcagcgaa cgcctgtttg cgaacctgtg gggccgtctg 1260
gaaggcgaag aacgtcttct gtggctgtat cgcgaagttg aacgtccgct gtctgcggtt 1320
ctggcgcaca tggaagctac tggcgtacgt ctggacgtgg cttatctgcg tgcgctgtct 1380
cttgaagttg ccgaagaaat tgcacgtctg gaggcggaag tatttcgtct ggctggccac 1440
ccgttcaacc tgaactcccg tgaccagctg gaacgtgtac tgttcgacga actgggtctg 1500
ccggctattg gtaaaaccga aaaaaccggc aaacgttcca cctcagctgc tgtgctggaa 1560
gcgctgcgcg aagcccaccc tatcgtcgaa aagatcctgc agtatcgtga actgaccaaa 1620
ctgaaaagta cctacatcga ccctctcccg gacctgatcc acccacgtac tggtcgcctg 1680
cacacccgtt ttaaccagac cgcaaccgcg actggtcgcc tgagcagctc tgacccgaac 1740
ctgcagaaca tcccggtccg tactccgctg ggccagcgta tccgccgtgc atttatcgcc 1800
gaagaaggtt ggctgctcgt ggcgctggac tattcgcaga tcgaacttcg cgtgcttgca 1860
cacctgtctg gtgacgagaa ccttattcgt gtttttcagg aaggtcgtga catccatacc 1920
gaaaccgcta gttggatgtt cggcgtaccg cgtgaagcag tagatccact gatgcgtcgt 1980
gcggcaaaaa ccatcaactt cggtgttctg tacggcatgt ctgcacatcg tctgagccag 2040
gaactggcga ttccgtacga agaagcccag gcgttcattg aacgttactt ccagagcttt 2100
ccgaaggtgc gtgcttggat tgaaaaaacg ctggaagaag gccgtcgtcg tggctacgtg 2160
gaaactctct ttggccgccg ccgttatgta ccggcactgg aagctcgcgt taaaagcgtt 2220
cgtgaagcag cagaacgtat ggcattcaat atgccggttc agggtacagc agcggatctg 2280
atgaaactgg ctatggtcaa gctgttcccg cgtctggaag aaatgggtgc acgtatgctg 2340
cttcaggttc acgatgaact ggtactggaa gctccgaaag aacgtgcgga agcggtggcg 2400
cgtttggcaa aggaagttat ggaaggtgtc tacccacttg cggtaccgct ggaggtggaa 2460
gttggtattg gtgaagattg gctgtctgct aaagagtaa 2499
<210> 3
<211> 2499
<212> DNA
<213> 水生嗜热菌(Thermus aquaticus)
<400> 3
atgcgtggta tgctgccgct tttcgaaccg aaaggtcgtg ttctgctggt ggacggccac 60
cacctggctt atcgcacctt ccacgcgttg aaaggcctga ctaccagccg cggcgagccg 120
gttcaagcag tatatggttt tgctaaaagc ctgctgaaag ctctgaaaga agatggtgac 180
gccgttatcg tagtatttga cgcaaaagcc ccgtctttcc gtcacgaagc ctacggcggt 240
tacaaagcag gtcgcgctcc gactccggaa gacttcccgc gtcaacttgc cctgatcaaa 300
gagctggtag atctgctggg ccttgcccgt ctggaagttc cgggttacga agctgatgat 360
gttctggcaa gcctcgcgaa gaaagccgaa aaagaaggct acgaagtccg tatcctgacc 420
gctgacaagg atctgtatca gctgctgtcc gatcgcatcc acgcgctgca cccggaaggt 480
tatctgatca ctcctgcttg gctgtgggaa aagtacggcc tgcgtccgga tcagtgggca 540
gattaccgtg ccctgaccgg cgatgaatct gacaacctgc ctggtgttaa aggcatcggt 600
gagaaaacgg cgcgtaaatt actggaggaa tggggctctc tggaagcgct gctgaaaaac 660
ctggaccgtc tgaagccggc gattcgtgaa aaaatccttg ctcacatgga tgatctgaaa 720
ctgtcctggg atctggcaaa ggtacgcacc gatttgccac tggaagttga tttcgccaaa 780
cgtcgtgaac cggaccgtga acgtctgcgt gcattcctgg aacgcctgga attcggtagc 840
ttgctgcacg aattcggcct gctggaaagt ccaaaagccc tggaggaagc gccgtggccg 900
ccgccggaag gcgcctttgt tggtttcgta ctgagccgta aagaaccgat gtgggctgat 960
ctgctggcac tggcagccgc acgtggcggc cgtgtacacc gtgccccgga accgtacaaa 1020
gccctgcgtg acctgaaaga agcacgtggc ctgctggcta aagacctgtc agtactcgca 1080
ctgcgtgaag gtctgggtct gccgccaggt gacgacccga tgctgctggc gtacctgctg 1140
gacccgtcta acactacgcc ggagggtgta gcccgccgct acggcggcga gtggactgaa 1200
gaagctggcg aacgtgcggc gctcagcgaa cgcctgtttg cgaacctgtg gggccgtctg 1260
gaaggcgaag aacgtcttct gtggctgtat cgcgaagttg aacgtccgct gtctgcggtt 1320
ctggcgcaca tggaagctac tggcgtacgt ctggacgtgg cttatctgcg tgcgctgtct 1380
cttgaagttg ccgaagaaat tgcacgtctg gaggcggaag tatttcgtct ggctggccac 1440
ccgttcaacc tgaactcccg tgaccagctg gaacgtgtac tgttcgacga actgggtctg 1500
ccggctattg gtaaaaccga aaaaaccggc aaacgttcca cctcagctgc tgtgctggaa 1560
gcgctgcgcg aagcccaccc tatcgtcgaa aagatcctgc agtatcgtga actgaccaaa 1620
ctgaaaagta cctacatcga ccctctcccg gacctgatcc acccacgtac tggtcgcctg 1680
cacacccgtt ttaaccagac cgcaaccgcg actggtcgcc tgagcagctc tgacccgaac 1740
ctgcagaaca tcccggtccg tactccgctg ggccagcgta tccgccgtgc atttatcgcc 1800
gaagaaggtt ggctgctcgt ggcgctggac tattcgcaga tcgaacttcg cgtgcttgca 1860
cacctgtctg gtgacgagaa ccttattcgt gtttttcagg aaggtcgtga catccatacc 1920
gaaaccgcta gttggatgtt cggcgtaccg cgtgaagcag tagatccact gatgcgtcgt 1980
gcggcaaaaa ccatcaactt cggtgttctg tacggcatgt ctgcacatcg tctgagccag 2040
gaactggcga ttccgtacga agaagcccag gcgttcattg aacgttactt ccagagcttt 2100
ccgaaggtgc gtgcttggat tgaaaaaacg ctggaagaag gccgtcgtcg tggctacgtg 2160
gaaactctct ttggccgccg ccgttatgta ccggacctgg aagctcgcgt taaaagcgtt 2220
cgtgaagcag cagaacgtat ggcattcaat atgccggttc agggtacagc agcggatctg 2280
atgaaactgg ctatggtcaa gctgttcccg cgtctggaag aaatgggtgc acgtatgctg 2340
cttcaggttc acgatgaact ggtactggaa gctccgaaag aacgtgcgga agcggtggcg 2400
cgtttggcaa aggaagttat ggaaggtgtc tacccacttg cggtaccgct ggaggtggaa 2460
gttggtattg gtgaagattg gctgtctgct aaagagtaa 2499
<210> 4
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gcggcaaaaa ccatcaactt cggtgttc 28
<210> 5
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctccagcggt accgcaagtg ggtagac 27
<210> 6
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gccgccgtta tgtaccgnnk ctggaagctc gcg 33
<210> 7
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aacgcgagct tccagmnncg gtacataac 29
Claims (8)
1.一种Taq DNA聚合酶突变体,其特征在于,其氨基酸序列如SEQ ID NO.1所示。
2.一种如权利要求1所述的Taq DNA聚合酶突变体的编码基因。
3.如权利要求2所述的编码基因,其特征在于,其核苷酸序列如SEQ ID NO.2所示。
4.一种包含权利要求2所述编码基因的重组载体。
5.一种包含权利要求2所述编码基因的基因工程菌。
6.一种筛选如权利要求1所述Taq DNA聚合酶突变体的分隔式自我复制方法,其特征在于,包括以下步骤:
(1)构建饱和突变文库:以含有Taq DNA聚合酶的环状质粒作为模板,进行PCR扩增,构建Taq DNA聚合酶定点饱和突变质粒文库;
(2)突变体文库的诱导表达:将步骤(1)获得的定点饱和突变质粒转入大肠杆菌细胞中,进行诱导表达,得到菌悬液;
(3)乳化PCR:配制ePCR预混液,将ePCR预混液加入至CSR油相中,并在乳化过程中,向混合物中放入橡胶颗粒,得到单相的乳白色粘稠的乳液;
(4)乳化PCR产物回收与扩增:采用PCR产物回收试剂盒回收乳化的PCR产物,并进行扩增反应;
(5)连接转化与测序。
7.如权利要求6所述的Taq DNA聚合酶突变体的分隔式自我复制方法,其特征在于,步骤(1)中,所述环状质粒的PCR体系为:模板1μL,引物F10μM 1μL,引物R10μM 1μL,dNTP2.5mM 4μL,高保真酶缓冲液5μL,高保真酶1μL,补水至50μL的反应体系;
引物F的序列为GCCGCCGTTATGTACCGNNKCTGGAAGCTCGCG;引物R的序列为AACGCGAGCTTCCAGMNNCGGTACATAAC;
PCR热循环条件为:94℃预变性3min,94℃变性30s,Tm-5℃退火20s,72℃延伸4min20s,4℃保温;进行PCR扩增35个循环。
8.如权利要求6所述的Taq DNA聚合酶突变体的分隔式自我复制方法,其特征在于,步骤(3)中,ePCR预混液的配方为:10×PCR buffer 40μL,dNTP 2.5mM 40μL,2CSR-F 8μL,2CSR-R 8μL,菌体0.5OD,ddH2O 304μL;
2CSR-F的序列为:GCGGCAAAAACCATCAACTTCGGTGTTC;2CSR-R的序列为CTCCAGCGGTACCGCAAGTGGGTAGAC。
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