CN112899241A - 蜱传脑炎病毒报告病毒TBEV miRFP670nano的构建方法及应用 - Google Patents
蜱传脑炎病毒报告病毒TBEV miRFP670nano的构建方法及应用 Download PDFInfo
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Abstract
本发明公开了蜱传脑炎病毒报告病毒TBEV miRFP670nano的构建方法及应用。本发明构建出了含有miRFP670nano报告基因的报告病毒,使可以通过检测miRFP670nano荧光蛋白信号来监测TBEV、研究病毒毒力,主要目的是为日后的TBEV的病毒示踪和活体成像做准备。
Description
技术领域
本发明涉及蜱传脑炎病毒(TBEV)miRFP670nano报告病毒的构建,特别涉及稳定表达含有miRFP670nano荧光蛋白的报告病毒的构建及其应用。
背景技术
在中国,第一例人类蜱传脑炎(TBE)病例于1943年被发现。TBE在中国的分布与其蜱媒的分布密切相关。我国TBE病例均为远东亚型,由全沟硬蜱传播。内蒙古大兴安岭、黑海龙江小兴安岭、吉林省长白山林区是东北地区典型的TBE疫区。西北地区的新疆天山北坡林区和阿尔泰南坡林区也有间歇性的TBE报告。此外,TBE在云南(中国西南部)和西藏(中国西部)也有报道。
蜱传脑炎病毒(TBEV)是黄病毒蜱传类群中最重要的一种。TBEV有一个约11千碱基长的正链RNA基因组,基因组包括5'UTR和3'UTR以及一个开放阅读框(Open readingframe,ORF),基因组总共编码3414个氨基酸,并翻译成单个多蛋白,该多蛋白经共转录和转录后加工成三种结构蛋白(Structural protein,SP)和七种非结构蛋白(Non-structuralprotein,nSP)。其中,结构蛋白有C、PrM和E蛋白,非结构蛋白有NS1、NS2A、 NS2B、NS3、NS4A、NS4B和NS5。相比于其他黄病毒尤其是蚊媒病毒,蜱传病毒研究相对较少,很多功能是基于其他黄病毒的研究推理出的。
miRFP670nano是最近从蓝藻色素中衍生出的近红外蛋白,可以用作内部蛋白质标签,有效结合内源性胆绿素并在各种哺乳动物细胞产生荧光,在体外和哺乳动物细胞中表现出高细胞亮度,对变性和降解还具有高度的稳定性。miRFP670nano的分子量只有17kda,是哺乳动物细胞中最小的单体近红外荧光蛋白,其亮度是目前最先进的双结构域近红外荧光蛋白的两倍。
发明内容
本发明的目的是构建出含有miRFP670nano荧光蛋白的蜱传脑炎病毒的报告病毒,使其可以在细胞中稳定表达,并且可以通过检测miRFP670nano荧光蛋白信号来研究病毒毒力,可作为抗病毒药物筛选的有力工具,为日后的TBEV的病毒示踪和活体成像做准备。
本发明采用的技术方案是:蜱传脑炎病毒报告病毒TBEV miRFP670nano的构建方法,包括如下步骤:
1)SnaBI-C38片段的克隆:以TBEV FL为模板,引物1和引物2为特异性引物,PCR 扩增蜱传脑炎病毒SnaBI-C38片段;
引物1:5’-TACGTATTAGTCATCGCTATTAC-3’;
引物2:5’-CTCTCCGCTTCCCACGAGTCCATTTGGC-3’;
PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环,最后68℃延伸7min。
2)T2A和P2A序列与miRFP670nano基因的连接:将T2A序列和P2A序列设计在引物上,并分两轮PCR将T2A和P2A序列连接到miRFP670nano基因上。
第一轮PCR:合成的基因miRFP670nano为模板,T2A miRFP670nano F1和P2AmiRFP670nano R1为引物进行PCR,扩增好目的片段后,回收目的条带。
引物T2A miRFP670nano F1:
5’-CATGCGGTGACGTCGAGGAGAATCCTGGACCTATGGCAAACCTGGACAAG-3’;
引物P2A miRFP670nano R1:
5’-CTGCTTCAGCAGGCTGAAGTTAGTAGCTCCGCTTCCGCTCTGCTGGATGGCGATG-3’;
第一轮PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环;最后68℃延伸7min。
第二轮PCR:第一轮PCR回收的目的条带为模板,T2A miRFP670nano F2和P2AmiRFP670nano R2为引物进行PCR,扩增好目的片段后,回收目的条带。
引物T2A miRFP670nano F2:
5’-GGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTC-3’;
引物P2A miRFP670nano R2:
5’-AGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGGCTGAAGTTAG-3’;
第二轮PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环;最后68℃延伸7min。
将第二轮扩增好后,回收的目的条带连接到pGEM T载体上,命名为miRFP670nano2*2A pGEM T。
3)T2A-miRFP670nano-P2A片段的克隆:以miRFP670nano 2*2A pGEM T为模板,引物3和引物4为特异性引物,扩增出T2A-miRFP670nano-P2A片段,此时该片段与前后两部分有重叠序列;
引物3:5’-CAAATGGACTCGTGGGAAGCGGAGAGGGCAGAG-3’;
引物4:5’-CTTCCCGGCCATAGGTCCAGGGTTCTCCTC-3’;
PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环,最后68℃延伸7min。
4)C1-AflⅡ片段的克隆:以TBEV FL为模板,引物5和引物6为特异性引物,扩增出C1-AflⅡ片段;
引物5:5’-GAACCCTGGACCTATGGCCGGGAAGGCCATTC-3’;
引物6:5’-CTTAAGCAACACTCCAGTCTGG-3’;
PCR反应条件为:95℃预变性5min;98℃10s,56℃30s,68℃2min为一个循环,进行35个循环;最后68℃延伸7min。
5)构建SnaB I-C38-T2A-miRFP670nano-P2A-C1-AflⅡ片段:以引物1和引物6为特异性引物,步骤1)获得的SnaBI-C38片段、步骤3)获得的T2A-miRFP670nano-P2A片段和步骤4)获得的C1-AflⅡ片段为模板,通过重叠延伸PCR,将此三个片段连接在一起,得到SnaB I-C38-T2A-miRFP670nano-P2A-C1-AflⅡ片段;
PCR反应条件为:95℃预变性5min;98℃10s,56℃30s,68℃3min为一个循环,进行35个循环;最后68℃延伸7min。
6)含有miRFP670nano荧光蛋白的TBEV报告病毒的构建:将步骤5)获得的SnaB I-C38-T2A-miRFP670nano-P2A-C1-AflⅡ片段和TBEV的感染性克隆用SnaBI和AflⅡ双酶切,将酶切片段跑胶回收,再用T4连接酶进行连接。构建含有miRFP670nano荧光蛋白的 TBEV报告病毒TBEV miRFP670nano 2*2A。
本发明的有益效果是:
1、本发明通过将构建的含有miRFP670nano荧光蛋白的TBEV miRFP670nano质粒转染至BHK 21细胞,进行荧光蛋白活性检测,验证了本发明TBEV miRFP670nano的可用性。
2、本发明获得的TBEV miRFP670nano质粒转染后可以用于检测蜱传脑炎病毒的复制情况。
3、本发明构建了含有miRFP670nano荧光蛋白的TBEV报告病毒,为进一步研究TBEV的抗病毒药物的筛选奠定了基础。
附图说明
图1是本发明TBEV miRFP670nano的构建图。
图2是检测TBEV miRFP670nano质粒的荧光蛋白情况;
具体实施方式
实施例1
(一)稳定表达含有miRFP670nano荧光蛋白的蜱传脑炎病毒报告病毒(TBEVmiRFP670nano)的构建方法
构建方法如图1所示。
蜱传脑炎病毒TBEV感染性克隆,采用本实验室保存的TBEV FL。
1、SnaBI-C38片段的克隆
以TBEV FL为模板,引物1和引物2为特异性引物,PCR扩增蜱传脑炎病毒SnaBI-C38片段。
引物1:5’-TACGTATTAGTCATCGCTATTAC-3’;
引物2:5’-CTCTCCGCTTCCCACGAGTCCATTTGGC-3’;
PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环,最后68℃延伸7min。
2、T2A和P2A序列与miRFP670nano基因的连接
通过两轮PCR扩增,将T2A和P2A序列与miRFP670nano基因进行连接,具体为:
将T2A序列和P2A序列设计在引物上,并分两轮PCR将T2A和P2A序列连接到miRFP670nano基因上。
第一轮PCR:合成的基因miRFP670nano为模板,T2A miRFP670nano F1和P2AmiRFP670nano R1为引物进行PCR,扩增好目的片段后,回收目的条带。
引物T2A miRFP670nano F1:
5’-CATGCGGTGACGTCGAGGAGAATCCTGGACCTATGGCAAACCTGGACAAG-3’;
引物P2A miRFP670nano R1:
5’-CTGCTTCAGCAGGCTGAAGTTAGTAGCTCCGCTTCCGCTCTGCTGGATGGCGATG-3’;
第一轮PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环;最后68℃延伸7min。
第二轮PCR:第一轮PCR回收的目的条带为模板,T2A miRFP670nano F2和P2AmiRFP670nano R2为引物进行PCR,扩增好目的片段后,回收目的条带。
引物T2A miRFP670nano F2:
5’-GGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTC-3’;
引物P2A miRFP670nano R2:
5’-AGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGGCTGAAGTTAG-3’;
第二轮PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环;最后68℃延伸7min。
将第二轮PCR扩增回收的目的条带连接到pGEM T载体上,命名为miRFP670nano 2*2A pGEM T。
3、构建T2A-miRFP670nano-P2A片段
以miRFP670nano 2*2A pGEM T为模板,引物3和引物4为特异性引物,扩增出T2A-miRFP670nano-P2A片段,此时该片段与前后两部分有重叠序列。
引物3:5’-CAAATGGACTCGTGGGAAGCGGAGAGGGCAGAG-3’;
引物4:5’-CTTCCCGGCCATAGGTCCAGGGTTCTCCTC-3’;
PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环,最后68℃延伸7min。
4、C1-AflⅡ片段的克隆
以TBEV FL为模板,引物5和引物6为特异性引物,扩增出片段C1-AflⅡ。
引物5:5’-GAACCCTGGACCTATGGCCGGGAAGGCCATTC-3’;
引物6:5’-CTTAAGCAACACTCCAGTCTGG-3’;
PCR反应条件为:95℃预变性5min;98℃10s,56℃30s,68℃2min为一个循环,进行35个循环;最后68℃延伸7min。
5、构建SnaB I-C38-T2A-miRFP670nano-P2A-C1-AflⅡ片段
以引物1和引物6为特异性引物,步骤1)获得的SnaBI-C38片段、步骤3)获得的T2A-miRFP670nano-P2A片段和步骤4)获得的C1-AflⅡ片段为模板,通过重叠延伸 PCR,将此三个片段连接在一起,得到SnaB I-C38-T2A-miRFP670nano-P2A-C1-AflⅡ片段;
PCR反应条件为:95℃预变性5min;98℃10s,56℃30s,68℃3min为一个循环,进行35个循环;最后68℃延伸7min。
6、含有miRFP670nano荧光蛋白的TBEV报告病毒的构建
将步骤5)获得的SnaB I-C38-T2A-miRFP670nano-P2A-C1-AflⅡ片段和TBEV的感染性克隆用SnaBI和AflⅡ双酶切3h,将酶切片段跑胶回收,再用T4连接酶进行连接。构建含有miRFP670nano荧光蛋白的TBEV报告病毒TBEV miRFP670nano 2*2A。
(二)验证
以TBEV感染性克隆为模板,成功构建出TBEV miRFP670nano报告病毒。在C蛋白的第38个氨基酸后面插入miRFP670nano报告基因,TBEV miRFP670nano报告病毒在miRFP670nano报告基因前后两面均插入了2A序列。2A序列是为了方便miRFP670nano基因和其后的C蛋白的切割。构建好的报告病毒测序,测得的DNA序列如SEQ ID NO.1所示,整个基因组均无突变。
(三)含有miRFP670nano荧光蛋白的蜱传脑炎病毒TBEV miRFP670nano的应用 1、转染细胞获得病毒
将BHK 21细胞接种至细胞培养小皿,放入37℃培养箱培养过夜。待细胞密度达到80%,利用Lip3000进行TBEV miRFP670nano感染性克隆的转染操作。转染前将细胞培养基换成含有2%FBS的DMEM(含双抗)。取2个1.5ml EP管,分别加入125μl Opti-MEM 培养基,标记管A和管B。管A加入7.5μl Lip3000,轻轻混匀;管B依次加入TBEV miRFP670nano质粒2.5μg和lip3000 5μl,轻轻混匀。将管B中混合物逐滴加入管A,边加边混匀,室温孵育5min。将混合液逐滴滴入BHK-21细胞。转染后每天观察细胞的病变效应,第3-4天收取上清病毒悬液。
2、感染细胞扩增病毒
接种BHK21细胞至细胞培养小皿,放入37℃培养箱培养过夜。待细胞密度达到50%,进行感染实验。感染前配制好病毒稀释液。感染时首先吸出培养基并用D-Hanks洗两次细胞。每个小皿加500ul病毒稀释液,并用封口膜包好,孵育1.5h,期间每隔30min震荡一次细胞。1.5h后补加1mlD+2%FBS培养基。放置37℃,5%CO2培养箱,扩增病毒。
3、miRFP670nano荧光蛋白活性检测
(1)细胞准备:提前一天将细胞铺玻璃底小皿,放入37℃培养箱培养过夜。待细胞密度达到50~80%,进行感染实验;
(2)感染4天后收样,收样时加入0.5mL 4%多聚甲醛固定液,室温固定30min;
(3)用PBS(或PBST)洗3次,5~10min/次;
(4)透化,400ul用3%Triton-100孵育细胞10min;
(5)染核,以Hoechst 33258染核,10min
(6)用PBS(或PBST)洗3次,5~10min/次;
(7)加一滴抗荧光猝灭剂,到共聚焦显微镜上观察荧光。
结果如图2所示,与对照组相比,转染了报告病毒质粒的细胞有红色点状聚集样式,未转染报告病毒的细胞无荧光。说明在报告病毒中miRFP670nano报告基因已经成功表达。
<110> 辽宁大学
<120>蜱传脑炎病毒报告病毒TBEV miRFP670nano的构建方法及应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 14822
<212> DNA
<213> TBEV miRFP670nano
<400> 1
AGATTTTCTT GCACGTGTGT GCGTTTGCTC CGGATAGCAA CAGCAGCGAC AGGTTTGAGA 60
GAGATAATTT TTCGCTTGAC CAGTCGTGAA CGTGTTGAGA AAAAGACAGC TTAGGAGAAC 120
AAGAGCTGGG GATGGCCGGG AAGGCCATTC TGAAAGGAAA GGGGGGCGGT CCCCCTCGAC 180
GAGTGTCGAA AGAGACCGCG AGGAAGACGC GTCAATCTAG GGTCCAAATG CCAAATGGAC 240
TCGTGGGAAG CGGAGAGGGC AGAGGAAGTC TGCTAACATG CGGTGACGTC GAGGAGAATC 300
CTGGACCTAT GGCAAACCTG GACAAGATGC TGAATACCAC AGTAACAGAG GTGCGGCAGT 360
TCCTGCAGGT GGACAGAGTG TGCGTGTTCC AGTTTGAGGA GGATTATAGC GGAGTGGTGG 420
TGGTGGAGGC CGTGGACGAT AGGTGGATCT CCATCCTGAA GACCCAGGTG CGGGATAGAT 480
ACTTCATGGA GACAAGGGGC GAGGAGTATT CTCACGGCCG CTACCAGGCC ATCGCCGACA 540
TCTACACCGC AAACCTGACA GAGTGCTACA GGGATCTGCT GACACAGTTT CAGGTGAGAG 600
CAATCCTGGC CGTGCCCATC CTGCAGGGCA AGAAGCTGTG GGGCCTGTTG GTGGCACACC 660
AGCTGGCGGC CCCTAGACAG TGGCAGACCT GGGAGATCGA CTTTCTGAAG CAGCAGGCCG 720
TGGTGGTGGG CATCGCCATC CAGCAGAGCG GAAGCGGAGC TACTAACTTC AGCCTGCTGA 780
AGCAGGCTGG AGACGTGGAG GAGAACCCTG GACCTATGGC CGGGAAGGCC ATTCTGAAAG 840
GAAAGGGGGG CGGTCCCCCT CGACGAGTGT CGAAAGAGAC CGCGAGGAAG ACGCGTCAAT 900
CTAGGGTCCA AATGCCAAAT GGACTCGTGT TGATGCGCAT GTTGGGGATT TTATGGCATG 960
CCGTGGCCGG CACCGCTAGG AGTCCCGTGT TGAAGTCTTT CTGGAATTCA GTCCCACTGA 1020
AACAGGCCAT GGCAGCACTC CGGAAAATTA AAAAGGCAGT GAGCACCCTG ATGGTAGGTC 1080
TGCAAAGACG TGGCAAAAGA AGGTCAGCAG CAGACTGGAC AAGTTGGTTG CTGGTTCTGG 1140
TTTTGGTGGG GGTGACACTT GCAGCCACAG TGCGGAAAGA AAGGGATGGC ACTACCGTGA 1200
TCAGAGCTGA AGGAAAAGAT GCGGCAACCC AGGTGCGTGT GGAAAATGGC ACCTGTGTGA 1260
TCCTGGCCAC GGACATGGGA TCATGGTGTG ATGACTCACT AACCTATGAG TGTGTGACCA 1320
TAGACCAGGG GGAGGAACCA GTTGACGTGG ATTGCTTCTG CAGGAATGTT GATGGAGTTT 1380
ACCTGGAGTA TGGACGGTGT GGAAAACAAG AAGGATCAAG AACAAGGCGC TCAGTGCTGA 1440
TCCCGTCCCA TGCCCAGGGA GACCTCACAG GAAGGGGACA CAAATGGTTA GAAGGGGATT 1500
CACTGCGGAC GCATCTCACT AGGGTTGAAG GATGGGTCTG GAAGAACAAA GTGCTCACCC 1560
TGGCGGTGAT CGCCATTGTG TGGCTGACCG TGGAAAGCGT GGTGACCAGG GTCGCCGTAG 1620
TGGTGGTGCT CTTGTGCCTG GCTCCGGTTT ATGCCTCACG ATGCACACAT TTGGAAAACA 1680
GAGATTTTGT TACTGGCACT CAGGGAACCA CTCGTGTGAC TCTGGTGCTG GAACTGGGAG 1740
GATGCGTCAC CATAACAGCC GAGGGGAAGC CATCGATGGA CGTGTGGCTT GACTCCATTT 1800
ATCAGGAGAA TCCTGCCAAA ACACGTGAGT ACTGCCTGCA CGCAAAGCTG TCGGACACCA 1860
AGGTCGCGGC CAGATGCCCC ACAATGGGAC CTGCCACTTT GGCTGAAGAG CACCAGAGTG 1920
GCACAGTGTG CAAGAGAGAC CAGAGTGATC GAGGCTGGGG TAATCATTGT GGATTATTTG 1980
GAAAAGGCAG CATTGTGACC TGCGTCAAGG CGTCCTGTGG GGCAAAAAAG AAGGCCACAG 2040
GACACGTGTA TGATGCCAAC AAAATTGTGT ACACGGTTAA AGTAGAGCCG CATACGGGGG 2100
ATTACGTCGC CGCTAATGAG ACCCATAGTG GAAGAAAAAC AGCATCCTTC ACGGTTTCCT 2160
CGGAAAAAAC CATCTTGACC ATGGGAGACT ACGGAGATGT GTCCTTGTTG TGTCGAGTAG 2220
CTAGCGGTGT TGACCTTGCT CAGACTGTCA TTCTGGAACT TGACAAGACT TCAGAACACC 2280
TACCGACGGC CTGGCAGGTT CACCGGGACT GGTTCAATGA TCTGGCCCTG CCGTGGAAAC 2340
ATGAAGGGGC ACAGAACTGG AACAATGCTG AACGGCTAGT TGAGTTTGGA GCCCCACATG 2400
CTGTGAAAAT GGATGTGTAT AACCTTGGAG ACCAGACTGG AGTGTTGCTT AAGTCACTTG 2460
CTGGTGTTCC AGTGGCGCAC ATTGATGGAA CCAAGTACCA CCTGAAAAGT GGCCACGTGA 2520
CATGCGAGGT AGGACTAGAA AAACTCAAGA TGAAAGGTCT CACATACACA ATGTGCGACA 2580
AGACGAAATT CACGTGGAAG AGAATCCCAA CTGACAGTGG ACATGATACA GTGGTCATGG 2640
AAGTTGCATT CTCTGGAACC AAACCCTGTA GGATCCCGGT GAGGGCCGTG GCACACGGCT 2700
CTCCAGATGT GAATGTGGCT ATGTTGATAA CACCCAACCC CACAATCGAA ACCAATGGTG 2760
GTGGTTTCAT AGAAATGCAG TTACCGCCAG GAGACAACAT CATCTATGTC GGGGAACTGA 2820
GTCACCAATG GTTCCAAAAA GGGAGTAGCA TTGGAAGGGT CTTTCAAAAA ACCAGGAAAG 2880
GTATAGAACG ACTGACAGTG ATCGGAGAAC ACGCCTGGGA TTTTGGCTCA ACTGGTGGAT 2940
TCCTGACCTC GGTTGGCAAG GCGCTACACA CAGTTCTTGG TGGTGCCTTC AACAGCCTGT 3000
TTGGAGGAGT AGGGTTCTTG CCTAAGATCC TAGTGGGAGT GGTCCTGGCC TGGTTGGGCC 3060
TGAACATGAG GAATCCGACC ATGTCCATGA GCTTCCTCCT GGCCGGAGGA CTGGTTCTGG 3120
CCATGACACT CGGAGTCGGA GCTGATGTTG GCTGTGCTGT GGACACCGAA CGGATGGAGC 3180
TCCGTTGTGG TGAGGGTCTG GTTGTATGGA GGGAGGTTTC CGAGTGGTAT GACAATTATG 3240
CATACTACCC TGAGACACCA GGAGCTCTTG CTTCGGCCAT AAAAGAGACC TTCGAGGAGG 3300
GAACTTGTGG TATAGTGCCC CAAAACAGAC TTGAAATGGC CATGTGGAGA AGCTCGGCGA 3360
CAGAACTGAA TCTGGCCTTG GCGGAGGGAG ACGCAAATCT CACAGTGGTG GTGGACAAAC 3420
TTGATCCCAC AGACTATCGA GGTGGCATTC CTGGGCTGCT AAAGAAGGGG AAAGACATAA 3480
AGGTTTCTTG GAAGAGCTGG GGCCACTCAA TGATCTGGAG TGTCCCCGAG GCCCCCCGTC 3540
GGTTTATGGT GGGAACAGAG GGAAGCAGTG AGTGTCCACT AGAGAGAAGG AAAACAGGTG 3600
TCTTCACGGT GGCAGAGTTT GGGGTTGGCC TGAGAACAAA AGTATTCTTG GACTTCAGAC 3660
AGGAATCAAC ACATGAGTGT GACACAGGAG TGATGGGAGC CGCTGTCAAG AATGGCATGG 3720
CAGTCCACAC AGACCAGAGC CTCTGGATGA AATCCGTGAG AAATGACACA GGGACCTACA 3780
TAGTAAGTAT CAAGGTTACA AGACAGGTTT AAGGAGACCA ATAGAAACTG GGCTTGTCGA 3840
GACAGAGAAG ACTCTTGCGT TTCTGATAGG CACCTATTGG TCTTACTGAC ATCCACTTTG 3900
CCTTTCTCTC CACAGGTGGA ACTTCTGGTT ACTGACCTGA GAAATTGCTC ATGGCCGGCT 3960
AGCCACACCA TTGACAATGC TGAGGTGGTG GACTCAGAAC TCTTCCTTCC AGCCAGTCTG 4020
GCAGGGCCTA GATCCTGGTA TAACAGGATA CCCGGGTACT CAGAACAAGT GAAAGGACCA 4080
TGGAAGTACT CGCCCATCCG AGTGACAAGA GAAGAGTGCC CTGGCACGAG GGTCACCATA 4140
AATGCCGACT GTGACAAAAG GGGGGCTTCT GTGAGGAGTA CCACAGAGAG TGGCAAGGTG 4200
ATTCCAGAGT GGTGCTGCCG AACGTGCACA TTACCTCCAG TGACGTTCCG TACGGGGACA 4260
GACTGTTGGT ATGCCATGGA AATACGACCA GTTCATGACC AGGGAGGGCT TGTTCGCTCA 4320
ATGGTGGTGG CAGACAATGG AGAGCTGCTT AGTGAGGGGG GCATTCCCGG GATAGTGGCT 4380
TTGTTTGTGG TCCTTGAGTA CGTCATCCGG AGGAGGCCAG CCACTGGAAC AACGGCCATG 4440
TGGGGAGGCA TTGTTGTCCT TGCATTGCTC GTCACTGGTC TGGTGAGAAT CGAAAGCCTG 4500
GTGCGTTATG TCGTGGCAGT TGGGATCACA TTTCATCTTG AGCTAGGGCC AGAGATTGTG 4560
GCTCTGACAC TGTTACAGGC TGTGTTTGAG TTGAGGGTTG GCCTGCTCAG CGCTTTTGCA 4620
CTACGCAGCA ACCTCACTGT CAGAGAGATG GTAACCATCT ACTTCCTTCT GCTGGTTTTG 4680
GAGTTGGGAT TGCCAGGTGA GGGTCTTGGG GCCCTATGGA AATGGGGAGA TGCATTGGCC 4740
ATGGGGGCAT TGATTTTCAG AGCCTGCACG GCAGAGGAAA AGACTGGTGT TGGACTCCTG 4800
CTCATGGCTC TCATGACACA GCAAGACCTG GCGATTGCAC ACTATGGACT CATGCTCTTC 4860
CTGGGCACGG CCTCATGTTG TTCAATCTGG AAACTGATTC GAGGACACAG AGAACAGAAG 4920
GGATTGACCT GGGTTGTCCC CCTGGCCGGG CTACTCGGAG GAGAGGGCTC TGGAGTCAGA 4980
CTGCTGGCTT TTTGGGAACT GGCCATCCAT GGAAGGAGAC GGTCATTCAG TGAACCACTG 5040
ACTGTTGTGG GAGTCATGCT AACCCTGGCC AGCGGCATGA TGCGGCACAC CTCTCAGGAG 5100
GCCCTTTGCG CGCTCGCCGT GGCCTCGTTC CTTCTGCTCA TGCTGGTCCT AGGGACAAGG 5160
AAGATGCAGC TAGTGGCTGA ATGGAGTGGC TGTGTGGAGT GGCACCCAGA ACTGATGAAT 5220
GAAGGTGGAG AGGTGAGCCT GCGGGTCCGG CAGGACTCAA TGGGAAACTT CCACCTGACA 5280
GAGCTTGAGA AAGAGGAGAG AGTGATGGCT TTCTGGCTGC TGGCAGGACT GGCGGCTTCG 5340
GCCTTCCACT GGTCTGGCAT TCTTGGTGTG ATGGGATTGT GGACGCTGTC GGAAATGCTG 5400
AGGACGGCTC GAAGATCAGA TTTGGTCTTC TCTGGACAGG GGGGACGTGA GCGTGGTGAC 5460
AGGCCCTTTG AGGTCAAGGA TGGCGTCTAT AGAATCTTCA GCCCAGGACT GCTCTGGGGG 5520
CAGCGCCAGG TGGGAGTTGG CTATGGCTCC AAAGGTGTCC TACACACGAT GTGGCATGTG 5580
ACGAGAGGGG CGGCGCTGTC CATTGATGAC GCCGTCGCAG GGCCCTATTG GGCTGACGTC 5640
AAAGAGGACG TTGTATGCTA TGGTGGAGCC TGGAGTCTTG AGGAGAAGTG GAAAGGTGAG 5700
ACAGTGCAGG TTCATGCCTT CCCACCGGGG AGAGCCCATG AGGTGCATCA ATGTCAGCCC 5760
GGGGAACTGC TCCTGGACAC AGGTAGGAGG ATAGGGGCAG TGCCAATTGA TCTGGCAAAA 5820
GGGACATCTG GCAGCCCCAT CCTCAACTCC CAAGGAGTGG TTGTGGGACT GTATGGGAAT 5880
GGACTGAAGA CCAATGAAAC CTACGTCAGC AGCATTGCTC AAGGTGAGGC TGAAAAAAGT 5940
CGACCCAATC TCCTGCCGGC CGTCATTGGC ACAGGCTGGA CAGCAAAAGG GCAGATCACA 6000
GTGCTGGACA TGCACCCAGG CTCTGGGAAG ACCCACAGAG TCCTCCCGGA GCTCATTCGC 6060
CAATGCATTG ACAGACGCCT AAGGACATTG GTGTTGGCCC CAACCCGTGT GGTGCTCAAG 6120
GAAATGGAGC GTGCCTTGAA TGGGAAGAGA GTCATGTTCC ATTCTCCGGC AGTTGGAGAT 6180
CAGCAGGTGG GAGGGGCCAT CGTCGACGTG ATGTGCCATG CAACCTATGT CAATAGACGC 6240
CTGCTCCCGC AGGGGAGACA GAATTGGGAA GTGGCAATCA TGGATGAAGC CCATTGGACG 6300
GATCCACACA GCATAGCCGC TCGGGGTCAC CTGTACACCT TGGCTAAGGA AAACAAATGT 6360
GCCCTGGTTC TTATGACAGC AACGCCACCC GGGAAGAGCG AACCCTTCCC AGAGTCCAAC 6420
GGGGCAATCA CCAGTGAAGA GAAGCAGATC CCTGATGGGG AGTGGCGTGA TGGGTTCGAC 6480
TGGATCACCG AGTATGAGGG GCGTACCGCA TGGTTCGTTC CCTCGATTGC AAAAGGTGGT 6540
ACTATAGCCC GCACCCTGAG ACAAAAAGGA AAAAGCGTGA TCTGTCTGAA CAGCAAGACA 6600
TTTGAAAAGG ACTACTCCAG AGTGAGAGAT GAGAAACCCG ACTTCGTGGT CACAACCGAC 6660
ATATCTGAAA TGGGGGCCAA CCTCGATGTG AGCCGTGTCA TAGACGGGCG AACAAACATC 6720
AAACCGGAAG AGGTTGATGG GAGAGTTGAG CTCACAGGGA CCAGACGTGT GACCACGGCC 6780
TCTGCGGCCC AACGCCGTGG GAGAGTCGGA AGACAGGAGG AAAGAACAGA TGAATACATA 6840
TACTCTGGAC AGTGTGATGA TGATGATAGT GGACTTGTGC AGTGGAAGGA AGCGCAGATA 6900
CTTCTTGACA ACATAACAAC ACTGCGGGGG CCTGTGGCCA CCTTTTATGG ACCAGAGCAG 6960
GACAAGATGC CAGAGGTGGC AGGTCATTTC CGCCTCACAG AAGAGAAAAG AAAGCACTTT 7020
CGACATCTTC TCACCCATTG TGACTTCACG CCATGGTTGG CATGGCACGT CGCAGCAAAC 7080
GTGTCTAGTG TGACAAGCCG GAACTGGACT TGGGAAGGCC CTGAGGAGAA CACTGTGGAT 7140
GAGGCCAATG GAGATCTGGT CACCTTCAGG AGCCCGAATG GGGCTGAAAG AACACTGAGG 7200
CCAGTATGGA GGGATGCGCG CATGTTCAGA GAAGGACGTG ACATCAGAGA GTTCATCGCG 7260
TATGCCTCAG GGAGACGCAG CTTTGGAGAT GTGTTGAGCG GAATGTCCGG TGTTCCTGAG 7320
CTTCTGCGCC ATAGATGTGT TAGCGCCATG GATGTCTTCT ACACACTGAT GCATGAGGAG 7380
CCTGGCAGCA GGGCAATGAA GATGGCCGAG AGAGATGCTC CAGAGGCTTT TTTGACGGTG 7440
GTAGAGATGA TGGTGCTCGG CCTGGCCACT CTTGGGGTCG TCTGGTGCTT TGTTGTTCGC 7500
ACCTCAATCA GTCGCATGAT GCTTGGCACG CTGGTACTGC TGGCCTCACT GGCGCTCCTG 7560
TGGGCCGGTG GTGTAAGCTA CGGGATTATG GCAGGAGTGG CCCTCATTTT CTACACGTTG 7620
TTGACGGTGC TGCAGCCTGA AGCGGGGAAA CAGAGGAGCA GTGATGACAA CAAGTTGGCC 7680
TACTTCCTGT TGACGCTCTG CAGTCTAGCT GGACTGGTAG CCGCCAATGA AATGGGATTT 7740
CTGGAGAAGA CTAAGGCGGA CCTGTCCACG GTGTTGTGGA GTGAACATGA AGAGTTGCGG 7800
TCGTGGGAAG AGTGGACCAA CATCGACATC CAGCCTGCAC GTTCCTGGGG AACTTACGTG 7860
CTGGTGGTCT CTTTGTTCAC ACCATACATA ATTCACCAAC TTCAGACCAA GATCCAACAA 7920
CTCGTCAACA GCGCTGTTGC AACTGGGGCT CAGGCCATGC GAGACCTCGG AGGAGGGGCT 7980
CCATTCTTTG GGGTAGCAGG GCATGTAATG GCTTTGGGAG TGGTATCGCT AGTTGGTGCA 8040
ACGCCAACAT CCTTGGTGGT TGGTGTTGGT CTGGCGGCGT TCCACCTGGC CATTGTGGTG 8100
TCCGGACTAG AGGCTGAGTT GACACAAAGA GCCCACAAAG TCTTCTTCTC GGCAATGGTG 8160
CGCAATCCCA TGGTGGATGG AGACGTCATC AATCCATTTG GAGAGGGAGA GGCAAAACCT 8220
GCTCTGTATG AGAGGAAAAT GAGCCTGGTC TTGGCGATAG TGCTTTGCTT GATGTCGGTG 8280
GTCATGAACA GAACGGTGCC TTCTATCACT GAGGCTTCTG CTGTGGGACT GGCGGCAGCG 8340
GGACAACTGC TCAGACCAGA GGCGGATACC CTGTGGACGA TGCCAGTGGC CTGTGGCCTG 8400
AGCGGCGTGG TCAGGGGTAG CCTCTGGGGA TTCTTGCCCC TCGGGCATAG ACTCTGGCTA 8460
AGGGCCTCTG GGAGTAGGCG TGGTGGCTCT GAGGGGGACA CTCTCGGTGA CTTGTGGAAA 8520
CGGAAACTCA ATGGCTGTAC CAAAGAAGAG TTCTTCGCCT ATAGACGCAC TGGCATCCTG 8580
GAGACGGAAA GGGACAAGGC ACGGGAACTC CTCAGGAGAG GGGAGACCAA CATGGGGCTG 8640
GCTGTGTCAC GGGGCACGGC TAAACTTGCC TGGCTTGAGG AACGAGGTTA CGCAACTCTC 8700
AAGGGTGAGG TCGTGGACCT TGGATGTGGA AGAGGCGGCT GGTCCTACTA TGCGGCCTCT 8760
AGACCGGCTG TCATGAGTGT CAAAGCCTAC ACAATTGGTG GAAAGGGACA CGAGACCCCA 8820
AAGATGGTGA CAAGCTTGGG TTGGAACCTG ATCAAGTTCA GAGCGGGAAT GGATGTGTTC 8880
AGCATGCAGC CACACCGAGC TGATACCATT ATGTGTGACA TCGGAGAAAG CAACCCAGAT 8940
GCCGTGGTGG AGGGTGAGAG GACACGGAAA GTGATACTAC TCATGGAACA GTGGAAAAAC 9000
CGCAATCCCA CGGCTACCTG TGTGTTCAAG GTGTTGGCCC CATACCGCCC AGAGGTCATA 9060
GAAGCACTAC ACAGATTCCA ACTGCAGTGG GGCGGAGGAC TGGTGAGGAC CCCTTTCTCA 9120
AGGAATTCAA CCCATGAAAT GTATTACTCG ACTGCTGTCA CTGGAAACAT TGTGAATTCA 9180
GTTAACATCC AATCAAGAAA ACTCTTGGCC CGGTTCGGGG ACCAGAGGGG ACCCACCAGG 9240
GTGCCTGAGC TGGACCTCGG AGTTGGGACT CGATGCGTTG TCTTGGCTGA GGACAAGGTG 9300
AAGGAAAAAG ATGTGCAGGA GAGGATCAGT GCGCTGCGAG AGCAGTATGG TGAGACCTGG 9360
CATATGGACA GAGAGCATCC GTACAGGACC TGGCAGTACT GGGGCAGCTA CCGCACCGCG 9420
CCAACCGGGT CAGCGGCGTC ACTGATCAAT GGAGTCGTGA AGCTTCTCAG CTGGCCATGG 9480
AACGCGCGGG AGGATGTCGT GCGAATGGCC ATGACTGACA CCACAGCCTT TGGACAGCAG 9540
CGAGTGTTCA AAGAGAAGGT TGACACCAAG GCTCAGGAAC CTCAGCCTGG CACAAAGGTC 9600
ATCATGAGAG CAGTGAATGA CTGGATTCTG GAACGATTGG CACGGAAAAG CAAACCACGA 9660
ATGTGCAGCA GAGAGGAGTT CATAGCGAAA GTGAAATCCA ATGCGGCTCT GGGGGCTTGG 9720
TCTGATGAGC AGAACAGGTG GTCAAGTGCA AAAGAGGCTG TAGAGGATCC CGCATTCTGG 9780
CAGCTCGTGG ATGAAGAGAG AGAGAGACAC CTTGCTGGGA GATGCGCCCA CTGTGTGTAC 9840
AACATGATGG GCAAAAGAGA AAAGAAGCTT GGAGAGTTTG GAGTGGCCAA AGGAAGCCGG 9900
GCCATATGGT ACATGTGGCT GGGGAGCCGC TTTCTGGAGT TCGAAGCTCT TGGCTTTTTG 9960
AATGAGGACC ACTGGGCCTC TAGGGGGTCC AGTGGATCTG GAGTGGAGGG AATAAGCTTG 10020
AATTACCTGG GCTGGTATCT CAAAGGGTTG TCAACACTGG AAGGAGGCCT TTTCTACGCG 10080
GATGACACAG CCGGCTGGGA CACCAAGGTC ACCAACGCAG ACCTAGAGGA TGAAGAACAG 10140
CTCCTACGCT ACATGGAGGG TGAACACAAG CAACTGGCGG CTACAATAAT GCAGAAGGCA 10200
TACCACGCCA AGGTGGTAAA AGTAGCCCGG CCCTCCCGAG ATGGGGGCTG TGTCATGGAT 10260
GTCATCACAA GAAGAGACCA AAGAGGCTCA GGGCAGGTTG TGACTTATGC CCTCAACACT 10320
CTAACCAACA TAAAGGTTCA GCTGATCCGT ATGATGGAGG GGGAGGGAGT CATTGAGGCC 10380
TCGGACGCAC ATAATCCAAG ATTACTCCGA GTGGAACGAT GGCTGAGGGA TCATGGAGAA 10440
GAACGTCTCG GAAGAATGCT CGTGAGCGGT GATGACTGTG TGGTGAGACC GGTGGATGAC 10500
AGGTTCAGTA GGGCACTCTA TTTTCTGAAT GACATGGCCA AAACCAGGAA AGACATTGGA 10560
GAGTGGGAGC ATTCGGTTGG CTTCTCGAAC TGGGAGGAGG TTCCCTTTTG CTCACACCAC 10620
TTCCACGAGT TGGTGATGAA GGATGGGCGT GCTCTCATAG TGCCATGCCG AGACCAAGAT 10680
GAACTGGTTG GAAGAGCCCG CGTCTCACCA GGGTGCGGCT GGAGTGTCCG TGAAACCGCC 10740
TGCCTTTCAA AAGCTTATGG GCAGATGTGG CTGCTGAGTT ACTTCCACCG GCGCGACTTG 10800
CGGACGCTTG GACTTGCCAT CTGTTCGGCG GTGCCCATTG ACTGGGTCCC CACTGGCCGC 10860
ACGACCTGGA GCATCCATGC TAGCGGAGCC TGGATGACCA CAGAGGACAT GTTGGATGTC 10920
TGGAACAGGG TGTGGATCCT GGACAACCCC TTCATGCACA GTAAAGAAAA GATTGTGGAA 10980
TGGAGGGATG TCCCGTATCT CCCCAAATCC CATGATATGC TGTGTTCCTC TCTTGTTGGG 11040
AGAAAAGAGA GGGCAGAGTG GGCCAAGAAC ATCTGGGGAG CAGTAGAGAA AGTCAGGAAG 11100
ATGATCGGAC AAGAGAAGTT TAAGGACTAC CTTTCCTGCA TGGACCGGCA CGACTTGCAC 11160
TGGGAGCTCA AACTGGAGAG CTCAATAATT TAAAACCAGA CTGTGACTGA GCACAACCTG 11220
GAGTGCTCGT TAAACATTGT CCAGAACCAA AAACCACAGC AAACAATTTA AAAAACACCC 11280
CCAGAGTGCC CCACGGCAAC ACGTCAGTGA GAGTGGCGAC GGGAAAATGG TCGATCCCGA 11340
CGTAGGGCAC TCTGTAAAAC TTTGTGAGAC CCCCGGCACC ATGATAAGGC CGAACATGGT 11400
GCAAGAACGG GAGGCCCCCG GAAGCATGCT TCCGGGAGGA GGGAAGAGAG AAATTGGCAA 11460
CTCTCTTCGG GATTTTTCCT CCTCCTATAC CAAATTCCCC CTCAACAGAG GGGGGGCGGT 11520
TCTTGTTCTC CCTGAGCCAC CATCACCCAG ACACAGATAG TCTGACAAGG AGGTGACGTG 11580
TGACTCGGAA AAACACCCGC TGGGTCGGCA TGGCATCTCC ACCTCCTCGC GGTCCGACCT 11640
GGGCTACTTC GGTAGGCTAA GGGAGAAGAA CTTGTTTATT GCAGCTTATA ATGGTTACAA 11700
ATAAAGCAAT AGCATCACAA ATTTCACAAA TAAAGCATTT TTTTCACTGC ATTCTAGTTG 11760
TGGTTTGTCC AAACTCATCA ATGTATCTTA TCATGTCTCT CGAGCAAGAC GTTTCCCGTT 11820
GAATATGGCT CATAACACCC CTTGTATTAC TGTTTATGTA AGCAGACAGT TTTATTGTTC 11880
ATGATGATAT ATTTTTATCT TGTGCAATGT AACATCAGAG ATTTTGAGAC ACAACGTGGC 11940
TTTGTTGAAT AAATCGAACT TTTGCTGAGT TGAAGGATCA GATCACGCAT CTTCCCGACA 12000
ACGCAGACCG TTCCGTGGCA AAGCAAAAGT TCAAAATCAC CAACTGGTCC ACCTACAACA 12060
AAGCTCTCAT CAACCGTGGC TCCCTCACTT TCTGGCTGGA TGATGGGGCG ATTCAGGCCT 12120
GGTATGAGTC AGCAACACCT TCTTCACGAG GCAGACCTCA GCGCTAGCGG AGTGTATACT 12180
GGCTTACTAT GTTGGCACTG ATGAGGGTGT CAGTGAAGTG CTTCATGTGG CAGGAGAAAA 12240
AAGGCTGCAC CGGTGCGTCA GCAGAATATG TGATACAGGA TATATTCCGC TTCCTCGCTC 12300
ACTGACTCGC TACGCTCGGT CGTTCGACTG CGGCGAGCGG AAATGGCTTA CGAACGGGGC 12360
GGAGATTTCC TGGAAGATGC CAGGAAGATA CTTAACAGGG AAGTGAGAGG GCCGCGGCAA 12420
AGCCGTTTTT CCATAGGCTC CGCCCCCCTG ACAAGCATCA CGAAATCTGA CGCTCAAATC 12480
AGTGGTGGCG AAACCCGACA GGACTATAAA GATACCAGGC GTTTCCCCCT GGCGGCTCCC 12540
TCGTGCGCTC TCCTGTTCCT GCCTTTCGGT TTACCGGTGT CATTCCGCTG TTATGGCCGC 12600
GTTTGTCTCA TTCCACGCCT GACACTCAGT TCCGGGTAGG CAGTTCGCTC CAAGCTGGAC 12660
TGTATGCACG AACCCCCCGT TCAGTCCGAC CGCTGCGCCT TATCCGGTAA CTATCGTCTT 12720
GAGTCCAACC CGGAAAGACA TGCAAAAGCA CCACTGGCAG CAGCCACTGG TAATTGATTT 12780
AGAGGAGTTA GTCTTGAAGT CATGCGCCGG TTAAGGCTAA ACTGAAAGGA CAAGTTTTGG 12840
TGACTGCGCT CCTCCAAGCC AGTTACCTCG GTTCAAAGAG TTGGTAGCTC AGAGAACCTT 12900
CGAAAAACCG CCCTGCAAGG CGGTTTTTTC GTTTTCAGAG CAAGAGATTA CGCGCAGACC 12960
AAAACGATCT CAAGAAGATC ATCTTATTAA GGGGTCTGAC GCTCAGTGGA ACGAAAACTC 13020
ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA 13080
TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA 13140
CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT 13200
TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG 13260
TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA 13320
GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC 13380
TATTAATTGT TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT 13440
TGTTGCCATT GCTGCAGGCA TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG 13500
CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT 13560
TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT 13620
GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT 13680
GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC 13740
TTGCCCGGCG TCAACACGGG ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT 13800
CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG 13860
TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT 13920
TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG 13980
GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA 14040
TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC 14100
GCGCACATTT CCCCGAAAAG TGCCACCTGA CGTCTAAGAA ACCATTATTA TCATGACATT 14160
AACCTATAAA AATAGGCGTA TGCACGAGGC CCTTTCGTCT TCAAGAATTT TATAAGGTAC 14220
CGAATTCTTG ATTATTGACT AGTTATTAAT AGTAATCAAT TACGGGGTCA TTAGTTCATA 14280
GCCCATATAT GGAGTTCCGC GTTACATAAC TTACGGTAAA TGGCCCGCCT GGCTGACCGC 14340
CCAACGACCC CCGCCCATTG ACGTCAATAA TGACGTATGT TCCCATAGTA ACGCCAATAG 14400
GGACTTTCCA TTGACGTCAA TGGGTGGAGT ATTTACGGTA AACTGCCCAC TTGGCAGTAC 14460
ATCAAGTGTA TCATATGCCA AGTACGCCCC CTATTGACGT CAATGACGGT AAATGGCCCG 14520
CCTGGCATTA TGCCCAGTAC ATGACCTTAT GGGACTTTCC TACTTGGCAG TACATCTACG 14580
TATTAGTCAT CGCTATTACC ATGGTGATGC GGTTTTGGCA GTACATCAAT GGGCGTGGAT 14640
AGCGGTTTGA CTCACGGGGA TTTCCAAGTC TCCACCCCAT TGACGTCAAT GGGAGTTTGT 14700
TTTGGCACCA AAATCAACGG GACTTTCCAA AATGTCGTAA CAACTCCGCC CCATTGACGC 14760
AAATGGGCGG TAGGCGTGTA CGGTGGGAGG TCTATATAAG CAGAGCTGGT TTAGTGAACC 14820
GT 14822
Claims (9)
1.蜱传脑炎病毒报告病毒TBEV miRFP670nano的构建方法,其特征在于,包括如下步骤:
1)SnaBI-C38片段的克隆:以TBEV FL为模板,引物1和引物2为特异性引物,PCR扩增蜱传脑炎病毒SnaBI-C38片段;所述引物1和引物2的序列为:
引物1:
5’-TACGTATTAGTCATCGCTATTAC-3’;
引物2:
5’-CTCTCCGCTTCCCACGAGTCCATTTGGC-3’;
2)T2A和P2A序列与miRFP670nano基因的连接:将T2A序列和P2A序列设计在引物上,并分两轮PCR将T2A和P2A序列连接到miRFP670nano基因上;第一轮PCR:合成的基因miRFP670nano为模板,T2A miRFP670nano F1和P2A miRFP670nano R1为引物进行PCR,扩增好目的片段后,回收目的条带;进行第二轮PCR:第一轮PCR回收的目的条带为模板,T2AmiRFP670nano F2和P2A miRFP670nano R2为引物进行PCR,扩增好目的片段后,回收目的条带,将第二轮回收的目的条带连接到pGEM T载体上,命名为miRFP670nano 2*2A pGEM T;所述T2A miRFP670nano F1、P2A miRFP670nano R1、T2A miRFP670nano F2和P2AmiRFP670nano R2的序列为:
引物T2A miRFP670nano F1:
5’-CATGCGGTGACGTCGAGGAGAATCCTGGACCTATGGCAAACCTGGACAAG-3’;
引物P2A miRFP670nano R1:
5’-CTGCTTCAGCAGGCTGAAGTTAGTAGCTCCGCTTCCGCTCTGCTGGATGGCGATG-3’;
引物T2A miRFP670nano F2:
5’-GGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTC-3’;
引物P2A miRFP670nano R2:
5’-AGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGGCTGAAGTTAG-3’;
3)T2A-miRFP670nano-P2A片段克隆:以miRFP670nano 2*2A pGEM T为模板,引物3和引物4为特异性引物,扩增出T2A-miRFP670nano-P2A片段;所述引物3和引物4的序列为:
引物3:
5’-CAAATGGACTCGTGGGAAGCGGAGAGGGCAGAG-3’;
引物4:
5’-CTTCCCGGCCATAGGTCCAGGGTTCTCCTC-3’;
4)C1-AflⅡ片段的克隆:以TBEV FL为模板,引物5和引物6为特异性引物,扩增出C1-AflⅡ片段;所述引物5和引物6的序列为:
引物5:
5’-GAACCCTGGACCTATGGCCGGGAAGGCCATTC-3’;
引物6:
5’-CTTAAGCAACACTCCAGTCTGG-3’;
5)构建SnaB I-C38-T2A-miRFP670nano-P2A-C1-AflⅡ片段:以引物1和引物6为特异性引物,步骤1)获得的SnaBI-C38片段、步骤3)获得的T2A-miRFP670nano-P2A片段和步骤4)获得的C1-AflⅡ片段为模板,通过重叠延伸PCR,将三个片段连接在一起,得到SnaB I-C38-T2A-miRFP670nano-P2A-C1-AflⅡ片段;
6)将扩增好的SnaB I-C38-T2A-miRFP670nano-P2A-C1-AflⅡ片段和TBEV的感染性克隆用SnaBI和AflⅡ双酶切,将酶切片段跑胶回收,再用T4连接酶进行连接,获得蜱传脑炎病毒报告病毒TBEV miRFP670nano。
2.根据权利要求1所述的构建方法,其特征在于,所述蜱传脑炎病毒报告病毒TBEVmiRFP670nano的DNA序列如SEQ ID NO.1所示。
3.根据权利要求1所述的构建方法,其特征在于,步骤1)SnaBI-C38片段的克隆中,PCR反应体系为:TBEV FL 1ul,引物1和引物2各1ul,KOD buffer 5ul,dNTP 5ul,MgSO43ul,KOD1ul,ddH2O 33ul,总体系50ul;PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环,最后68℃延伸7min。
4.根据权利要求1所述的构建方法,其特征在于,步骤2)miRFP670nano 2*2A pGEM T的克隆中,
第一轮PCR反应体系为:miRFP670nano 1ul,引物T2A miRFP670nano F1和P2AmiRFP670nano R1各1ul,KOD buffer 5ul,dNTP 5ul,MgSO4 3ul,KOD 1ul,ddH2O 33ul,总体系50ul;PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环,最后68℃延伸7min;
第二轮PCR反应体系为:第一轮PCR回收的目的条带1ul,引物T2A miRFP670nano F2和P2A miRFP670nano R2各1ul,KOD buffer 5ul,dNTP 5ul,MgSO4 3ul,KOD 1ul,ddH2O33ul,总体系50ul;PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环,最后68℃延伸7min。
5.根据权利要求1所述的构建方法,其特征在于,步骤3)构建T2A-miRFP670nano-P2A片段中,PCR反应体系为:miRFP670nano 2*2A pGEM T片段1ul,引物3和引物4各1ul,KODbuffer 5ul,dNTP 5ul,MgSO4 3ul,KOD 1ul,ddH2O 33ul,总体系50ul;PCR反应条件为:95℃预变性5min;98℃10s,58℃30s,68℃1min为一个循环,进行35个循环;最后68℃延伸7min。
6.根据权利要求1所述的构建方法,其特征在于,步骤4)C1-AflⅡ片段的克隆中,PCR反应体系为:TBEV FL 1ul,引物5和引物6各1ul,KOD buffer 5ul,dNTP 5ul,MgSO43ul,KOD1ul,ddH2O 33ul,总体系50ul;PCR反应条件为:95℃预变性5min;98℃10s,56℃30s,68℃2min作为一个循环,进行35个循环;最后68℃延伸7min。
7.根据权利要求1所述的构建方法,其特征在于,步骤5)构建SnaB I-C38-T2A-miRFP670nano-P2A-C1-AflⅡ片段中,PCR反应体系为:SnaBI-C38片段2ul,T2A-miRFP670nano-P2A片段2ul,C1-AflⅡ片段2ul,引物1和引物6各1ul,KOD buffer 5ul,dNTP5ul,MgSO4 3ul,KOD 1ul,ddH2O 28ul,总体系50ul;PCR反应条件为:95℃预变性5min;98℃10s,56℃30s,68℃3min为一个循环,进行35个循环;最后68℃延伸7min。
8.按照权利要求1-7任意一项所述的方法构建的蜱传脑炎病毒报告病毒TBEVmiRFP670nano在抗病毒药物筛选中的应用。
9.按照权利要求1-7任意一项所述的方法构建的蜱传脑炎病毒报告病毒TBEVmiRFP670nano在检测蜱传脑炎病毒的复制情况中的应用。
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