CN111979225B - 低温VSW3 RNA聚合酶体外转录合成全长无中断cas9 mRNA的应用 - Google Patents
低温VSW3 RNA聚合酶体外转录合成全长无中断cas9 mRNA的应用 Download PDFInfo
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- CN111979225B CN111979225B CN201910440379.0A CN201910440379A CN111979225B CN 111979225 B CN111979225 B CN 111979225B CN 201910440379 A CN201910440379 A CN 201910440379A CN 111979225 B CN111979225 B CN 111979225B
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- rna polymerase
- rna
- cas9
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Abstract
本发明公开低温VSW3 RNA聚合酶体外转录合成全长无中断cas9 mRNA的应用,所述VSW3 RNA聚合酶体外转录合成全长无中断cas9 mRNA的过程包括以下步骤:(1)构建含VSW3 RNA聚合酶特异性启动子的cas9质粒;(2)PCR法制备转录模板;(3)VSW3 RNA聚合酶转录合成加帽cas9 mRNA;(4)模板DNA消除和mRNA加尾;运用VSW3 RNA聚合酶在低温下转录合成cas9 mRNA不仅能够提高RNA产物的稳定性,更重要的是该酶转录合成cas9 mRNA无中断RNA产物,提高了全长RNA的纯度,省掉分离除去中断RNA产物纯化步骤,减少纯化过程造成的损失,在保证RNA纯度的前提下提高cas9 mRNA产量,是满足cas9 mRNA大量合成需求,且能保证质量和降低生产成本的最佳选择。
Description
技术领域
本发明属于长链RNA合成研究生产领域,具体涉及基因编辑工具cas9 mRNA的合成,应用低温VSW3 RNA聚合酶在体外可以高效的转录合成全长无中断cas9 mRNA,克服了目前通用的T7 RNA聚合酶转录合成cas9 mRNA时不可避免地发生中断的问题,显著提高产物纯度和产量,从而有效降低生产成本。
背景技术
最近几年,随着小RNA、mRNA、长链非编码RNA、RNA适配体、核酶等RNA研究技术的飞速发展,RNA在生物学上越来越受到重视(Burnett J C.,2012)。越来越多的生物学研究和应用需要用到大量的RNA,质量和纯度要求也越来越高,其中各类mRNA的需求成倍增加,其中最具代表的是广泛运用于基因编辑的cas9 mRNA。CRISPR-Cas9是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御,可用来对抗入侵的病毒及外源DNA。而CRISPR-Cas9基因编辑技术,则是对靶向基因进行特定DNA修饰的技术,这项技术也是目前用于基因编辑中前沿的方法(Hsu PD,2014)。
2013年,来自麻省理工学院,哈佛医学院,Broad研究院等处的研究人员和青年华人学者张峰完成了人类细胞全基因组范围内CRISPR-Cas9敲除筛选,这对于CRISPR-Cas9技术的发展具有重要意义(Ran FA,2013)。作者通过用慢病毒载体构建CRISPR-Cas9敲除(GeCKO)文库,在基因组范围内,能靶向18080个基因(64,751个独特靶向序列)进行敲除。此项技术立即用于药物的靶点筛选,并再新药筛选中,取得喜人的突破(Shalem O,2013)。以CRISPR-Cas9基础的基因编辑技术在一系列基因治疗的应用领域都展现出极大的应用前景,例如血液病、肿瘤和其他遗传疾病。目前,该技术成果已应用于人类细胞、斑马鱼、小鼠以及细菌的基因组精确修饰(Yuxuan Wu,2017;Ling Li,2019);与此同时,越来越多的RNAs开始作为药物分子进入临床实验阶段,有的已经被FDA批准上市,作为药物运用于临床无疑对RNA产品的质量也提出了更高的要求(Dowdy S F.,2017)。
目前,包括cas9基因在细胞内的表达基本都是通过含有该基因序列的表达载体转染到细胞中进行复制并转录成mRNA,最终翻译表达为目的蛋白。但是与目的基因真核表达质粒相比,直接向细胞内转入mRNA有明显优势。首先,转染mRNA比质粒DNA安全,没有质粒DNA可能整合到基因组中的风险;其次,质粒在细胞内长期存在,持续表达目的蛋白会造成脱靶效应风险提高。所以,如果我们只是想表达某种蛋白,在达到研究或治疗目的后,这种蛋白能很快被细胞代谢降解,直接转入mRNA无疑是最好的选择。因为mRNA翻译成目的蛋白的半衰期在2-4天左右,这为很多蛋白和酶包括cas9在细胞内实施基因编辑功能留下了足够效用时间(R.Alexander,2018)。
目前合成小于100nt的RNA主要以化学合成为主(JunichiYano,2012),但长度大于100nt的RNA已经达到化学合成的极限,成本急剧增加,科研人员只能依靠RNA聚合酶体外转录合成的方法来合成大于100nt的RNA(McKenna S A.,2007)。当今用于体外合成RNA的标准酶工具是来自大肠杆菌噬菌体T7 RNA聚合酶,这个酶在上世纪70年代被鉴定(Chamberlin,M.),在随后的数十年中,人们对T7 RNA聚合酶进行了大量的优化和改造,以满足大家提出的对RNA合成的各种需求,但是T7本身的一些特性是很难根本解决的,比如说对起始转录碱基强烈偏好性(“GG”),未知的转录终止现象等(Maslak,M.,1994;Sousa R,2003)。
本人通过对噬菌体单亚基RNA聚合酶研究的深入了解,发现来源于噬菌体的单亚基RNA聚合酶呈现丰富的种属多样性,转录性质各具特色,近年来我们一直在发掘新的单亚基RNA聚合酶,特别是低温RNA聚合酶,最终我们发现了低温噬菌体VSW-3(Zhang C,2017)。其表达的单亚基VSW3 RNA聚合酶,能够在较低温度下大量合成RNA,且RNA合成能力可以与T7 RNA聚合酶媲美。而T7RP在合成cas9 mRNA时会不可避免的合成RNA中断产物,需要进一步通过如高效液相色谱(HPLC)等纯化手段来去除中断RNA产物。
发明内容
本发明的目的是针对现有技术存在的问题,提供低温VSW3 RNA聚合酶体外转录合成全长无中断cas9 mRNA的应用。
为实现上述目的,本发明采用的技术方案是:
低温VSW3 RNA聚合酶体外转录合成全长无中断cas9 mRNA的应用,所述VSW3 RNA聚合酶体外转录合成全长无中断cas9 mRNA的过程包括以下步骤:
(1)构建含VSW3 RNA聚合酶特异性启动子的cas9质粒;
(2)PCR法制备转录模板;
(3)VSW3 RNA聚合酶转录合成加帽cas9 mRNA;
(4)模板DNA消除和mRNA加尾;
其中,VSW3聚合酶的基因序列如序列表SEQ ID NO.1所示,氨基酸序列如序列表SEQ ID NO.2所示,所述VSW3 RNA聚合酶特异性启动子的碱基序列如序列表SEQ ID NO.3所示,所述转录模板的碱基序列如序列表SEQ ID NO.4所示。
进一步的,所述步骤(1)的具体步骤为:通过PCR和无缝克隆技术用VSW3 RNA聚合酶特异性识别的启动子替换T7RP启动子;在cas9基因羧基端的SV40核定位信号肽后插入终止密码子。以确保冗余蛋白标签元件不被翻译。
进一步的,所述步骤(2)的具体步骤为:以步骤(1)构建的cas9质粒为PCR模板,设计一对引物,扩增出包含完整VSW3RP启动子,cas9基因和SV40 NLS碱基序列的转录模板DNA,所述引物如序列表SEQ ID NO.5-6所示。
进一步的,所述步骤(3)的具体步骤为:以步骤(2)所得的转录模板DNA纯化后作为VSW3 RNA聚合酶合成全长无中断的加帽cas9 mRNA的转录模板,转录反应体系成分包含:5×转录buffer、四种核糖核苷三磷酸ATP、GTP、CTP、UTP,抗-反向帽类似物-ARCA,RNase抑制剂,焦磷酸酶,转录模板DNA。
进一步的,转录模板DNA使用试剂盒进行纯化。
进一步的,所述步骤(4)的具体步骤为:用DNaseI消化除去转录反应中的模板DNA,RNA产物经过纯化,最后用poly A加尾酶对RNA进行加尾纯化。
与现有技术相比,本发明的有益效果是:
低温单亚基RNA聚合酶VSW3RP在体外转录合成全长cas9 mRNA且无中断RNA产物,解决了运用T7RP转录合成cas9 mRNA时不可避免的发生中断的问题。这不仅有利于提高cas9 mRNA纯度,而且简化了纯化步骤,降低因纯化造成的损失和降解风险,同时也为其它长链RNA合成提供了一种新的有效的低温候选酶工具。
附图说明
图1为本发明构建的cas9 mRNA转录模板质粒结构示意图(A)和最终合成的cas9mRNA结构示意图(B);
图2为VSW3 RNA聚合酶与T7RP、syn5RP、KP34RP分别转录合成cas9 mRNA的结果比较;图2-A为VSW3RP与T7RP合成cas9 mRNA的对比图,图2-B为VSW3 RNA、syn5RP、KP34RP合成cas9 mRNA的对比图;
图3为用VSW3 RNA聚合酶合成的全长无中断加帽cas9 mRNA产物结果;
图4为应用VSW3RP合成的cas9 mRNA注射到斑马鱼受精卵中,敲除色素基因的结果。
具体实施方式
下面将结合本发明中的附图,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:
低温VSW3 RNA聚合酶在合成全长无中断cas9 mRNA中的生产应用,运用VSW3RP体外转录合成全长cas9 mRNA无中断RNA产物,完整的cas9 mRNA合成过程包括以下步骤:
(1)构建含有VSW3RP启动子的cas9质粒;
本发明中cas9质粒购买自addgene(http://www.addgene.org/72247/),编号:72247。基于无缝克隆原理设计两对引物,在将T7RP启动子序列替换为VSW3RP启动子(5’-TAATTGGGCCACCTATA-3’)的同时,在SV40核定位信号肽的3’端插入终止密码子,最终构建的转录模板序列见序列表SEQ ID NO.1和图1,我们最终合成的cas9 mRNA包含了有助于翻译起始的mRNA帽子结构和KOZAK序列,完整的Cas9蛋白基因和羧基端的SV40 NLS核定位信号肽序列以及紧邻的终止密码子;两对引物序列如下:
cas9gene-F:TGGGCCACCTATAGTAGAGCCGCCACCATGGATAAAAAGTATTCTATTG(SEQ IDNO.7)
cas9gene-R:TGGTCTTTCTAGTCGCTCGAGACTTTCCTCTTCTTCT(SEQ ID NO.8)
cas9plasmid-F:GCGACTAGAAAGACCATGACGGTGATTATAAAGATC(SEQ ID NO.9)cas9plasmid-R:
ACTATAGGTGGCCCAATTAGCGGCCGCGGATCTCTAGCGGATCTGACG(SEQ ID NO.9)
无缝克隆操作方法可参考碧云天公司的无缝克隆试剂盒说明书(D7010S)。
(2)PCR扩增转录模板;
以步骤(1)构建的cas9质粒为PCR模板,设计一对引物,能够扩增出包含完整VSW3RP启动子,cas9基因和SV40NLS碱基序列的转录模板DNA。本发明中我们使用的引物为:
Trans_Template-cas9-F:GTTTAGTGAACCGTCAGATCCGCTAGAGATC(SEQ ID NO.5)
Trans_Template-cas9-R:CTCAATGGTGATGGTGATGATGACCGGTCAT(SEQ ID NO.6)
PCR反应体系如下表:
PCR扩增程序:98℃预变性2min、98℃变性15s、58℃退火15s、72℃延伸75s、35个循环、72℃终延伸5min,4℃保存。
(3)PCR产物纯化
a.向50ul PCR产物中加入450ul去离子水混匀后加入250ul酚氯仿,剧烈震荡15秒后静止2min后常温13000rpm离心10min,小心吸取400ul上清到新的1.5ml的无酶离心管,加入45ul的3M醋酸钠混匀,最后加入1ml的无水乙醇混匀后-20摄氏度放置1h
b.13000rpm、4℃离心30min;倒弃上清,加1ml预冷的80%乙醇,13000rpm、4℃离心20min;
c.用真空泵吸尽残留液体,每管加30ul DEPC水溶解沉淀,测定浓度后置于-20℃备用。
(4)cas9 mRNA转录合成并加帽加尾
以步骤(3)制备的PCR产物作为VSW3 RNA聚合酶合成加帽cas9 mRNA的转录模板。合成加帽cas9 mRNA的反应体系包括:5×转录buffer、四种核糖核苷三磷酸ATP、GTP、CTP、UTP,抗-反向帽类似物-ARCA(NEB#S1404),RNase抑制剂,焦磷酸酶,转录模板(PCR产物)。体外转录合成100ul Cas9-mRNA的反应体系如下:
转录反应在20℃下进行,转录时间为14小时,结果见图2-A和图2-B,如图2-A所示,T7RP不管是在37℃还是在20℃下合成cas9 mRNA都不可避免的发生了两处转录中断,而VSW3RP不仅无中断产物,而且最终的cas9 mRNA产量和纯度都是最高的;如图2-B所示,虽然syn5RP能够合成无中断的全长cas9 mRNA,但是产量明显低于VSW3RP,KP34RP在合成全长cas9 mRNA时,合成了一条接近全长的中断RNA产物,一方面KP34RP在37℃转录存在降解弥散,无清晰分辨紧邻的两个条带;另外一方面在进行琼脂糖电泳时,上样量过多也会导致相邻两个RNA条带未能分开,且总的RNA产量也是明显低于VSW3RP的。
(5)DNaseI除模板DNA
按照10ul转录体系加入1ul DNaseI的比例,100ul就加入10ul DNaseI,混匀后37℃水浴30分钟,每隔15min轻轻上下颠倒混匀一次。
(6)RNA纯化
本发明使用NEB生产的RNA纯化试剂盒(#T2040S)进行RNA纯化,具体步骤见NEBRNA纯化试剂盒说明书
(7)mRNA加尾
为了提高mRNA的稳定性和蛋白翻译效率,在合成好的加帽cas9 mRNA的3’端通过E.coli Poly(A)加尾酶(NEB,#M0276S)进行加尾处理,结果见附图3。
加尾反应体系如下:
成分 | 质量/体积 |
RNA | 1-10μg |
10X加尾缓冲液 | 2μl |
ATP(10mM) | 2μl |
Poly(A)聚合酶 | 2ul |
补充DEPC水 | 20ul |
混匀后37度水浴30分钟,每隔15min轻轻上下颠倒混匀一次。最终再次使用NEB生产的RNA纯化试剂盒纯化后,溶解于1mM的柠檬酸钠缓冲液中(PH=6.4),RNA浓度控制在1ug/ul左右,分装后置于-80℃保存,避免反复冻融。
(8)cas9 mRNA细胞实验检测
将我们用VSW3RP合成的cas9 mRNA和购买的靶向敲除色素基因的sgRNA共注射到斑马鱼受精卵中,成功获得了敲除色素基因斑马鱼,结果见附图4。
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。
序列表
<110> 武汉核圣生物技术有限公司
<120> 低温VSW3 RNA聚合酶体外转录合成全长无中断cas9 mRNA的应用
<130> 1
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2394
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgaaccaga tcgagctaga acaggaaatg attgacggtg gccgggcgaa gatgttcggc 60
tcattcaatc gcaacgaaga gcaaggagcg gcgcacaaca acccatacgc cgcagcggtg 120
taccggcgat tcgtgcaacc tctggccgat caaatcgacg cctactgcgg tgaggtcaag 180
cgcggcgtga tggcggcagg caaagccctg ctgcgcccgc atgacccgat ggtgttggcg 240
ttcatgaccg ttcgcatggt catggacacc acgctgcaat cgaaggacaa cgcaccaacc 300
gctgtggccc gagccttggg ccagagcatc tacggggaga ctctgctcgc caagtttgag 360
caggtcgaac ccgacctata cttcacgctg gtcaatgact ttgagcggcg tatgaccaag 420
tcggagcggc accggctgac ggttttcaag atgcaggccg agaagaacgg cgtaccgctg 480
cctgtgtggt cgccagagga caagttggcc atcggcacta tcttgctcta ccttgcccgc 540
gatgtcgggc tggtggagat cacagaggtg cgcaagggca agaagactgt gcgcgagtac 600
aacatgacgc cggatgtggc gggcatgctt gacaacatca aggactttgt ggcaggggcc 660
agcccgatgg tgctgccttg tgtggtgcct ccggtgccat ggactgatgc caacaacgga 720
ggataccaca caccgggcat gcgccgcata agcccctgct gcatccgtgg gcgaccgcga 780
gtcgaagacc tgaccgatgt accggacatc ccgttgcgtg cgctcaacat cctccagagc 840
cgcccatggc gcatcaatcg catggtgttg gacgcggtgg atctggtggg ccagcggttc 900
gacgtgggtg aggtgctggc acaggccgag ctgccgaagc cgaagtcgct tctgtggctg 960
gacgatgtgc cgaaggaaga aatgaacccc gcgcaactgg ccgagttcgg tgcgtggaag 1020
atcgagatgc gcgagtggta caccgagaac aagagcaggg gcgtgcagtg gggccggtac 1080
tatgaggcgc tgcgagtagc ccgcaagttc aaggacttgc cgttctggtt cgtgtaccaa 1140
tacgactacc gaggccgagc atatgcgaac acgaggggcg ttagcccgca aggttcagat 1200
ctccagaagg cgctgcttat ggcagacgtt ggcgtcccaa tcgccgacga acgagccaag 1260
ttctggttct acacagccgg agcaaaccgg ttcgggtacg acaaagccac actggcagag 1320
aggtacgaat ggactgtaga acgctcggaa atgatctgtg ctattgctgc cgatcccgta 1380
gccaacaggc aatggacgga ggcggacaac ccgttccagt ttctcgcatg gtgcttcgag 1440
ttcgcccagt acacggcaat gcccgagagc ttcttatctc gcctcgctct tggacaggat 1500
gggagctgca acgggctaca gcacttctca gcgatgttgc gcgacgaagt gggtggactc 1560
gcgaccaact tagtgccctc tacaacgcag caggacatct atcgactggt agctgtggag 1620
acaacgcggt tgttacaagc tatgcctcac gagaactgcg agttcacgct gaagtggaag 1680
ctgcacagcc tgtcccgcga cttagtcaaa cgaagcgtta tgactttgcc gtatggatcg 1740
acgaggttca gttgtgctga cttcatctac accgagtaca tggcgaagca caaggcgccg 1800
gagttcgcca agggcgacta ccagaaggcc gctcgctggc tgagcgtacc ggtgtgggac 1860
gcaatcggca acgtagtggt caaggcaaga gaggcgatgg catggcttca gaacgcctct 1920
gacgagctga tagacgccgg gatcgacgag atctactggc ggtcgccaag cggattcatg 1980
gttcggcaac ggtacggcaa ggaagaattc gttcttgtca agactcgatt ggctggcgga 2040
gtcagaattc ggccaaccat caagctggag ctagaggaac catgcaagcg ccggcaccgg 2100
aacgggatag ctcccaactt cgttcacagc cacgacgccg cgcacatgca cctcctgatc 2160
tgcgccgccg aggatcatgg gctgggccat ctggcattca tccatgacga ctacggtacg 2220
actgcggatg gtactgaaac gctccacaag ctcatcaggg cgacgttcgt tgccatgtac 2280
gagcaagggt gcccattgac cgcattccgc gacacatacg gcatcacaga agatctcccg 2340
gaacgcggtg atctcgacct gaatctggtt cacgattcca cgtatttctt cgcc 2394
<210> 2
<211> 798
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Asn Gln Ile Glu Leu Glu Gln Glu Met Ile Asp Gly Gly Arg Ala
1 5 10 15
Lys Met Phe Gly Ser Phe Asn Arg Asn Glu Glu Gln Gly Ala Ala His
20 25 30
Asn Asn Pro Tyr Ala Ala Ala Val Tyr Arg Arg Phe Val Gln Pro Leu
35 40 45
Ala Asp Gln Ile Asp Ala Tyr Cys Gly Glu Val Lys Arg Gly Val Met
50 55 60
Ala Ala Gly Lys Ala Leu Leu Arg Pro His Asp Pro Met Val Leu Ala
65 70 75 80
Phe Met Thr Val Arg Met Val Met Asp Thr Thr Leu Gln Ser Lys Asp
85 90 95
Asn Ala Pro Thr Ala Val Ala Arg Ala Leu Gly Gln Ser Ile Tyr Gly
100 105 110
Glu Thr Leu Leu Ala Lys Phe Glu Gln Val Glu Pro Asp Leu Tyr Phe
115 120 125
Thr Leu Val Asn Asp Phe Glu Arg Arg Met Thr Lys Ser Glu Arg His
130 135 140
Arg Leu Thr Val Phe Lys Met Gln Ala Glu Lys Asn Gly Val Pro Leu
145 150 155 160
Pro Val Trp Ser Pro Glu Asp Lys Leu Ala Ile Gly Thr Ile Leu Leu
165 170 175
Tyr Leu Ala Arg Asp Val Gly Leu Val Glu Ile Thr Glu Val Arg Lys
180 185 190
Gly Lys Lys Thr Val Arg Glu Tyr Asn Met Thr Pro Asp Val Ala Gly
195 200 205
Met Leu Asp Asn Ile Lys Asp Phe Val Ala Gly Ala Ser Pro Met Val
210 215 220
Leu Pro Cys Val Val Pro Pro Val Pro Trp Thr Asp Ala Asn Asn Gly
225 230 235 240
Gly Tyr His Thr Pro Gly Met Arg Arg Ile Ser Pro Cys Cys Ile Arg
245 250 255
Gly Arg Pro Arg Val Glu Asp Leu Thr Asp Val Pro Asp Ile Pro Leu
260 265 270
Arg Ala Leu Asn Ile Leu Gln Ser Arg Pro Trp Arg Ile Asn Arg Met
275 280 285
Val Leu Asp Ala Val Asp Leu Val Gly Gln Arg Phe Asp Val Gly Glu
290 295 300
Val Leu Ala Gln Ala Glu Leu Pro Lys Pro Lys Ser Leu Leu Trp Leu
305 310 315 320
Asp Asp Val Pro Lys Glu Glu Met Asn Pro Ala Gln Leu Ala Glu Phe
325 330 335
Gly Ala Trp Lys Ile Glu Met Arg Glu Trp Tyr Thr Glu Asn Lys Ser
340 345 350
Arg Gly Val Gln Trp Gly Arg Tyr Tyr Glu Ala Leu Arg Val Ala Arg
355 360 365
Lys Phe Lys Asp Leu Pro Phe Trp Phe Val Tyr Gln Tyr Asp Tyr Arg
370 375 380
Gly Arg Ala Tyr Ala Asn Thr Arg Gly Val Ser Pro Gln Gly Ser Asp
385 390 395 400
Leu Gln Lys Ala Leu Leu Met Ala Asp Val Gly Val Pro Ile Ala Asp
405 410 415
Glu Arg Ala Lys Phe Trp Phe Tyr Thr Ala Gly Ala Asn Arg Phe Gly
420 425 430
Tyr Asp Lys Ala Thr Leu Ala Glu Arg Tyr Glu Trp Thr Val Glu Arg
435 440 445
Ser Glu Met Ile Cys Ala Ile Ala Ala Asp Pro Val Ala Asn Arg Gln
450 455 460
Trp Thr Glu Ala Asp Asn Pro Phe Gln Phe Leu Ala Trp Cys Phe Glu
465 470 475 480
Phe Ala Gln Tyr Thr Ala Met Pro Glu Ser Phe Leu Ser Arg Leu Ala
485 490 495
Leu Gly Gln Asp Gly Ser Cys Asn Gly Leu Gln His Phe Ser Ala Met
500 505 510
Leu Arg Asp Glu Val Gly Gly Leu Ala Thr Asn Leu Val Pro Ser Thr
515 520 525
Thr Gln Gln Asp Ile Tyr Arg Leu Val Ala Val Glu Thr Thr Arg Leu
530 535 540
Leu Gln Ala Met Pro His Glu Asn Cys Glu Phe Thr Leu Lys Trp Lys
545 550 555 560
Leu His Ser Leu Ser Arg Asp Leu Val Lys Arg Ser Val Met Thr Leu
565 570 575
Pro Tyr Gly Ser Thr Arg Phe Ser Cys Ala Asp Phe Ile Tyr Thr Glu
580 585 590
Tyr Met Ala Lys His Lys Ala Pro Glu Phe Ala Lys Gly Asp Tyr Gln
595 600 605
Lys Ala Ala Arg Trp Leu Ser Val Pro Val Trp Asp Ala Ile Gly Asn
610 615 620
Val Val Val Lys Ala Arg Glu Ala Met Ala Trp Leu Gln Asn Ala Ser
625 630 635 640
Asp Glu Leu Ile Asp Ala Gly Ile Asp Glu Ile Tyr Trp Arg Ser Pro
645 650 655
Ser Gly Phe Met Val Arg Gln Arg Tyr Gly Lys Glu Glu Phe Val Leu
660 665 670
Val Lys Thr Arg Leu Ala Gly Gly Val Arg Ile Arg Pro Thr Ile Lys
675 680 685
Leu Glu Leu Glu Glu Pro Cys Lys Arg Arg His Arg Asn Gly Ile Ala
690 695 700
Pro Asn Phe Val His Ser His Asp Ala Ala His Met His Leu Leu Ile
705 710 715 720
Cys Ala Ala Glu Asp His Gly Leu Gly His Leu Ala Phe Ile His Asp
725 730 735
Asp Tyr Gly Thr Thr Ala Asp Gly Thr Glu Thr Leu His Lys Leu Ile
740 745 750
Arg Ala Thr Phe Val Ala Met Tyr Glu Gln Gly Cys Pro Leu Thr Ala
755 760 765
Phe Arg Asp Thr Tyr Gly Ile Thr Glu Asp Leu Pro Glu Arg Gly Asp
770 775 780
Leu Asp Leu Asn Leu Val His Asp Ser Thr Tyr Phe Phe Ala
785 790 795
<210> 3
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
taattgggcc acctata 17
<210> 4
<211> 4158
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gggagagccg ccaccatgga taaaaagtat tctattggtt tagacatcgg cactaattcc 60
gttggatggg ctgtcataac cgatgaatac aaagtacctt caaagaaatt taaggtgttg 120
gggaacacag accgtcattc gattaaaaag aatcttatcg gtgccctcct attcgatagt 180
ggcgaaacgg cagaggcgac tcgcctgaaa cgaaccgctc ggagaaggta tacacgtcgc 240
aagaaccgaa tatgttactt acaagaaatt tttagcaatg agatggccaa agttgacgat 300
tctttctttc accgtttgga agagtccttc cttgtcgaag aggacaagaa acatgaacgg 360
caccccatct ttggaaacat agtagatgag gtggcatatc atgaaaagta cccaacgatt 420
tatcacctca gaaaaaagct agttgactca actgataaag cggacctgag gttaatctac 480
ttggctcttg cccatatgat aaagttccgt gggcactttc tcattgaggg tgatctaaat 540
ccggacaact cggatgtcga caaactgttc atccagttag tacaaaccta taatcagttg 600
tttgaagaga accctataaa tgcaagtggc gtggatgcga aggctattct tagcgcccgc 660
ctctctaaat cccgacggct agaaaacctg atcgcacaat tacccggaga gaagaaaaat 720
gggttgttcg gtaaccttat agcgctctca ctaggcctga caccaaattt taagtcgaac 780
ttcgacttag ctgaagatgc caaattgcag cttagtaagg acacgtacga tgacgatctc 840
gacaatctac tggcacaaat tggagatcag tatgcggact tatttttggc tgccaaaaac 900
cttagcgatg caatcctcct atctgacata ctgagagtta atactgagat taccaaggcg 960
ccgttatccg cttcaatgat caaaaggtac gatgaacatc accaagactt gacacttctc 1020
aaggccctag tccgtcagca actgcctgag aaatataagg aaatattctt tgatcagtcg 1080
aaaaacgggt acgcaggtta tattgacggc ggagcgagtc aagaggaatt ctacaagttt 1140
atcaaaccca tattagagaa gatggatggg acggaagagt tgcttgtaaa actcaatcgc 1200
gaagatctac tgcgaaagca gcggactttc gacaacggta gcattccaca tcaaatccac 1260
ttaggcgaat tgcatgctat acttagaagg caggaggatt tttatccgtt cctcaaagac 1320
aatcgtgaaa agattgagaa aatcctaacc tttcgcatac cttactatgt gggacccctg 1380
gcccgaggga actctcggtt cgcatggatg acaagaaagt ccgaagaaac gattactccc 1440
tggaattttg aggaagttgt cgataaaggt gcgtcagctc aatcgttcat cgagaggatg 1500
accgcctttg acaagaattt accgaacgaa aaagtattgc ctaagcacag tttactttac 1560
gagtatttca cagtgtacaa tgaactcacg aaagttaagt atgtcactga gggcatgcgt 1620
aaacccgcct ttctaagcgg agaacagaag aaagcaatag tagatctgtt attcaagacc 1680
aaccgcaaag tgacagttaa gcaattgaaa gaggactact ttaagaaaat tgaatgcttc 1740
gattctgtcg agatctccgg ggtagaagat cgatttaatg cgtcacttgg tacgtatcat 1800
gacctcctaa agataattaa agataaggac ttcctggata acgaagagaa tgaagatatc 1860
ttagaagata tagtgttgac tcttaccctc tttgaagatc gggaaatgat tgaggaaaga 1920
ctaaaaacat acgctcacct gttcgacgat aaggttatga aacagttaaa gaggcgtcgc 1980
tatacgggct ggggagcctt gtcgcggaaa cttatcaacg ggataagaga caagcaaagt 2040
ggtaaaacta ttctcgattt tctaaagagc gacggcttcg ccaataggaa ctttatggcc 2100
ctgatccatg atgactcttt aaccttcaaa gaggatatac aaaaggcaca ggtttccgga 2160
caaggggact cattgcacga acatattgcg aatcttgctg gttcgccagc catcaaaaag 2220
ggcatactcc agacagtcaa agtagtggat gagctagtta aggtcatggg acgtcacaaa 2280
ccggaaaaca ttgtaatcga gatggcacgc gaaaatcaaa cgactcagaa ggggcaaaaa 2340
aacagtcgag agcggatgaa gagaatagaa gagggtatta aagaactggg cagccagatc 2400
ttaaaggagc atcctgtgga aaatacccaa ttgcagaacg agaaacttta cctctattac 2460
ctacaaaatg gaagggacat gtatgttgat caggaactgg acataaaccg tttatctgat 2520
tacgacgtcg atcacattgt accccaatcc tttttgaagg acgattcaat cgacaataaa 2580
gtgcttacac gctcggataa gaaccgaggg aaaagtgaca atgttccaag cgaggaagtc 2640
gtaaagaaaa tgaagaacta ttggcggcag ctcctaaatg cgaaactgat aacgcaaaga 2700
aagttcgata acttaactaa agctgagagg ggtggcttgt ctgaacttga caaggccgga 2760
tttattaaac gtcagctcgt ggaaacccgc gccatcacaa agcatgttgc gcagatacta 2820
gattcccgaa tgaatacgaa atacgacgag aacgataagc tgattcggga agtcaaagta 2880
atcactttaa agtcaaaatt ggtgtcggac ttcagaaagg attttcaatt ctataaagtt 2940
agggagataa ataactacca ccatgcgcac gacgcttatc ttaatgccgt cgtagggacc 3000
gcactcatta agaaataccc gaagctagaa agtgagtttg tgtatggtga ttacaaagtt 3060
tatgacgtcc gtaagatgat cgcgaaaagc gaacaggaga taggcaaggc tacagccaaa 3120
tacttctttt attctaacat tatgaatttc tttaagacgg aaatcactct ggcaaacgga 3180
gagatacgca aacgaccttt aattgaaacc aatggggaga caggtgaaat cgtatgggat 3240
aagggccggg acttcgcgac ggtgagaaaa gttttgtcca tgccccaagt caacatagta 3300
aagaaaactg aggtgcagac cggagggttt tcaaaggaat cgattcttcc aaaaaggaat 3360
agtgataagc tcatcgctcg taaaaaggac tgggacccga aaaagtacgg tggcttcgat 3420
agccctacag ttgcctattc tgtcctagta gtggcaaaag ttgagaaggg aaaatccaag 3480
aaactgaagt cagtcaaaga attattgggg ataacgatta tggagcgctc gtcttttgaa 3540
aagaacccca tcgacttcct tgaggcgaaa ggttacaagg aagtaaaaaa ggatctcata 3600
attaaactac caaagtatag tctgtttgag ttagaaaatg gccgaaaacg gatgttggct 3660
agcgccggag agcttcaaaa ggggaacgaa ctcgcactac cgtctaaata cgtgaatttc 3720
ctgtatttag cgtcccatta cgagaagttg aaaggttcac ctgaagataa cgaacagaag 3780
caactttttg ttgagcagca caaacattat ctcgacgaaa tcatagagca aatttcggaa 3840
ttcagtaaga gagtcatcct agctgatgcc aatctggaca aagtattaag cgcatacaac 3900
aagcacaggg ataaacccat acgtgagcag gcggaaaata ttatccattt gtttactctt 3960
accaacctcg gcgctccagc cgcattcaag tattttgaca caacgataga tcgcaaacga 4020
tacacttcta ccaaggaggt gctagacgcg acactgattc accaatccat cacgggatta 4080
tatgaaactc ggatagattt gtcacagctt gggggtgacg gatcccccaa gaagaagagg 4140
aaagtctcga gcgactag 4158
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
agctggttta gtgaaccgtc agatc 25
<210> 6
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
actcaatggt gatggtgatg atgacc 26
<210> 7
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgggccacct atagtagagc cgccaccatg gataaaaagt attctattg 49
<210> 8
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tggtctttct agtcgctcga gactttcctc ttcttct 37
<210> 9
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gcgactagaa agaccatgac ggtgattata aagatc 36
<210> 10
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
actataggtg gcccaattag cggccgcgga tctctagcgg atctgacg 48
Claims (5)
1.低温VSW3 RNA聚合酶体外转录合成全长无中断cas9 mRNA的应用,其特征在于,所述VSW3 RNA聚合酶体外转录合成全长无中断cas9 mRNA的过程包括以下步骤:
(1)构建含VSW3 RNA聚合酶特异性启动子的cas9质粒;
(2)PCR法制备转录模板;
(3)在20℃条件下,VSW3 RNA聚合酶转录合成加帽cas9 mRNA;
(4)模板DNA消除和mRNA加尾;
其中,VSW3聚合酶的基因序列如序列表SEQ ID NO.1所示,氨基酸序列如序列表SEQ IDNO.2所示,所述VSW3 RNA聚合酶特异性启动子的碱基序列如序列表SEQ ID NO.3所示,所述转录模板的碱基序列如序列表SEQ ID NO.4所示;
以步骤(1)构建的cas9质粒为PCR模板,设计一对引物,扩增出包含完整VSW3RP启动子,cas9基因和SV40 NLS碱基序列的转录模板DNA,所述引物如序列表SEQ ID NO.5-6所示。
2.如权利要求1所述的应用,其特征在于,所述步骤(1)的具体步骤为:通过PCR和无缝克隆技术用VSW3 RNA聚合酶特异性识别的启动子替换T7RP启动子;在cas9基因羧基端的SV40核定位信号肽后插入终止密码子。
3.如权利要求1所述的应用,其特征在于,所述步骤(3)的具体步骤为:以步骤(2)所得的转录模板DNA纯化后作为VSW3 RNA聚合酶合成全长无中断的加帽cas9 mRNA的转录模板,转录反应体系成分包含:5×转录buffer、四种核糖核苷三磷酸ATP、GTP、CTP、UTP,抗-反向帽类似物-ARCA,RNase抑制剂,焦磷酸酶,转录模板DNA。
4.如权利要求3所述的应用,其特征在于,转录模板DNA使用试剂盒进行纯化。
5.如权利要求1所述的应用,其特征在于,所述步骤(4)的具体步骤为:用DNaseI消化除去转录反应中的模板DNA,RNA产物经过纯化,最后用poly A加尾酶对RNA进行加尾纯化。
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荧光假单胞菌低温噬菌体VSW-3生物学特性及比较基因组分析;张春晶;《中国优秀硕士学位论文全文数据库(电子期刊)基础科学辑》(第1期);第29页 * |
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