CN114410687A - 一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 - Google Patents
一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 Download PDFInfo
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Abstract
本发明公开了一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体；包含：a、微基因座控制区，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不含HS1区的微型调控元件；b、beta珠蛋白的基因序列，该基因序列如SEQ ID NO.3所示；c、beta珠蛋白的基因序列上游侧翼的启动子序列；d、beta珠蛋白的基因序列下游的侧翼序列；e、来自泡沫病毒(Foamy virus)的绝缘子序列。与现有技术相比，本发明对载体结构进行了优化，发现对Beta珠蛋白基因(HBB)的2号内含子优化后，既可以在一定程度提高基因的表达，又可以大幅度提高病毒包装产量，使其临床使用和产业化成为可能。
Description
技术领域
本发明属于基因工程技术领域，涉及一种慢病毒载体，尤其涉及一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体。
背景技术
迄今为止，人类共发现有7000余种罕见病，但仅5％的罕见病有药可用。目前，几乎所有的罕见病药物都需要终身使用。基因治疗则为罕见病提供了一次性治愈的可能。地中海贫血(地贫)是一种全球分布最广、累积患病人群最多的单基因遗传性血红蛋白病，是罕见病中最常见的人类遗传性疾病。
基因治疗具备良好的安全性是其能够成为常规疗法，使更多患者受益的前提。慢病毒载体以不可控的方式插入到基因组(约75％整合至转录活跃的基因编码区域)，伴随有较高的激活癌基因和失活抑癌基因的风险。事实上，慢病毒载体曾在基因治疗地中海贫血的过程中激活HMGA2基因，导致转导的造血干细胞克隆大量扩增。该克隆的异常扩增是由于慢病毒基因治疗载体中使用的绝缘子元件，也即鸡beta珠蛋白脱氧核糖核酸酶I超敏感部位4核心部位(core chicken hypersensitivity site 4，cHS4)造成的(Cavazzana-Calvo,M.etal.Transfusion independence and HMGA2 activation after gene therapy ofhumanbeta-thalassaemia.Nature 467,318-22(2010).)。
绝缘子元件是具有阻止基因沉默或者防止所在区域基因激活功能的基因元件。cHS4绝缘子含有隐秘的RNA剪接信号，造成克隆扩增，影响基因治疗的安全性，并且使得慢病毒的滴度大幅下降(Cavazzana-Calvo,M.et al.Transfusion independence andHMGA2activation after gene therapy of human beta-thalassaemia.Nature 467,318-22(2010).Negre,O.et al.Preclinical evaluation of efficacy and safety ofanimproved lentiviral vector for the treatment of beta-thalassemia andsicklecell disease.Curr Gene Ther 15,64-81(2015).)。本发明所包含的绝缘子元件来自泡沫病毒(Foamy virus)，仅36bp的绝缘子序列，不含有隐秘的RNA剪接信号，并具有维持基因表达和防止所在区域的癌基因激活的功能。
Beta珠蛋白基因(HBB)在发育过程中的特异性表达由一段位于其上游的基因座控制区(LCR)调控(Kim,A.&Dean,A.Chromatin loop formation in the beta-globinlocusand its role in globin gene transcription.Mol Cells 34,1-5(2012).)。该段序列长达16kb，远远超出了慢病毒载体的运载能力。为了构建用于基因治疗的beta珠蛋白基因表达盒，必须从LCR区域中挑选出具有调控beta珠蛋白基因表达功能的、大小合适的核心元件。LCR由HS2、HS3、HS4等脱氧核糖核酸酶I(DNase I)超敏感位点(hypersensitivesites)组成。这些超敏感位点长度依然太大，必须对其进一步缩减才能包装进慢病毒载体。由于这些超敏感位点功能不同，必须相互配合才能发挥精准的调控功能，因此，必须找到有利beta珠蛋白基因高效和特异表达的组合。例如，由HS4、HS3、HS2脱氧核糖核酸酶I超敏感位点的核心元件组成的微基因座(miniLCR)导致病毒效价显著降低(Weber,L.etal.AnOptimized Lentiviral Vector Efficiently Corrects the Human SickleCellDisease Phenotype.Mol Ther Methods Clin Dev10,268-280(2018)。本发明的筛选的微基因座不会导致病毒效价的显著降低。
目前，基于慢病毒载体治疗地中海贫血技术仍未真正实现临床使用及商业化的原因有二：一，现有技术生产出的治疗用病毒载体滴度过低，导致生产规模和生产成本过大，难以生产出足量的病毒载体供应临床使用和商业化；二，现有技术生产出的治疗用病毒载体进行治疗后，其beta珠蛋白的表达量十分有限，难以起到有效的治疗效果。本发明对载体结构进行了优化，发现对Beta珠蛋白基因(HBB)的2号内含子优化后，既可以在一定程度提高基因的表达，又可以大幅度提高病毒包装产量，使其临床使用和产业化成为可能。
发明内容
现有技术中将慢病毒载体用于地中海贫血与镰刀型贫血基因治疗存在安全隐患，也并不十分高效；本发明的目的在于针对现有技术载体滴度过低，无法达到临床应用水平；基因表达量不足，无法起到治疗效果；以及载体缺乏绝缘子序列，容易激活临近基因表达，存在较大安全性风险等缺陷，提供一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体。
本发明的目的是通过以下技术方案实现的：
本发明涉及一种包含beta珠蛋白表达盒的慢病毒载体，所述表达盒包含：
a、微基因座控制区，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不含HS1区的微型调控元件；
b、beta珠蛋白的基因序列；beta珠蛋白基因的2号内含子经优化，所述基因beta珠蛋白的基因序列如SEQ ID NO.2所示；
c、beta珠蛋白的基因序列上游侧翼的启动子序列；
d、beta珠蛋白的基因序列下游的侧翼序列；
e、来自泡沫病毒(Foamy virus)的绝缘子序列。
作为本发明的一个实施方案，所述微型基因座控制区是由HS2、HS3、HS4脱氧核糖核酸酶I超敏感位点序列经过筛选后，组合而成；或是由HS3、HS2脱氧核糖核酸酶I超敏感位点序列经过筛选后，组合而成。
作为本发明的一个实施方案，所述微基因座控制区是从beta珠蛋白的基因座控制区LCR的HS4、HS3、HS2区域分别截取的脱氧核糖核苷酸序列组成的微型基因座调控元件。
作为本发明的一个实施方案，所述微型基因座调控元件的序列如SEQ ID NO.1所示。
作为本发明的一个实施方案，所述启动子序列如SEQ ID NO.4所示。
作为本发明的一个实施方案，所述侧翼序列如SEQ ID NO.5所示。
作为本发明的一个实施方案，所述绝缘子序列如SEQ ID NO.6所示。
本发明涉及一种包含beta珠蛋白表达盒的慢病毒载体在制备地中海贫血和镰刀型贫血的基因治疗药物中的应用。
与现有技术相比，本发明具有如下有益效果：
1)获得一种安全、高效的地中海贫血和镰刀型贫血的基因治疗方法；
2)提高了beta珠蛋白的基因的表达；
3)提高了病毒包装效率，增加了病毒滴度；
4)提高了病毒载体的安全性，降低了临床使用的安全风险；
5)本发明所使用的来自来自泡沫病毒的绝缘子序列，可以维持基因表达的同时防止所在区域基因激活。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为载体设计示意图；其中，a为LGO4载体设计示意图，b为LGO2载体设计示意图；
图2为各载体血红蛋白T87Q相对表达示意图；
图3为各载体滴度对比示意图；
图4为不同载体感染干细胞后，形成BFU-E(爆式红细胞集落形成单位)的数量示意图。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例
本实施例涉及一种人源beta珠蛋白的表达盒，进而获得含该表达盒的用于基因治疗的慢病毒载体LGO4。LGO4载体设计如图1(a)所示，该表达盒包含以下几种元件：(1)微基因座控制区(miniLCR)，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不含HS1区的微型调控元件，具体为从beta珠蛋白16kb的基因座控制区LCR的HS4、HS3、HS2区域分别截取长度约380bp、1900bp、720bp的脱氧核糖核苷酸序列组成总长3kb(序列如SEQ ID NO.1所示)的微型基因座调控元件；(2)beta珠蛋白的基因序列。其中外显子2含T87Q突变，2号内含子序列经优化；该beta珠蛋白的基因序列如SEQ ID NO.2所示；LGO2载体的设计图如图1(b)，其选用的beta珠蛋白的基因序列如SEQ ID NO.3所示；(3)265-bp的beta珠蛋白的基因序列上游侧翼的启动子序列(序列如SEQ ID NO.4所示)；(4)beta珠蛋白的基因序列下游侧翼300-bp序列(序列如SEQ ID NO.5所示)；(5)将该表达盒置于慢病毒载体；在慢病毒载体的LTR U3区域添加一段来自泡沫病毒(Foamy virus)的仅36bp的绝缘子序列(序列如SEQID NO.6所示：aagggagacatctagtgatataagtgtgaactacac)。该绝缘子序列具有维持基因长期表达和阻止整合位点附近基因激活的功能，且不会降低慢病毒载体的滴度。含3kb微基因座控制区的慢病毒载体的序列如SEQ ID NO.7所示。将本发明载体质粒(13μg)与表达膜蛋白的质粒(如3.75μg pMD2G(序列如SEQ ID NO.8所示))、表达GagPol长链蛋白的质粒(通常为13μg pMDlg/pRRE(序列如SEQ ID NO.9所示))、表达REV蛋白(通常为3μg pRSV-REV(序列如SEQ ID NO.10所示))的质粒共转染病毒4X106生产细胞(如293T)，生产出表达beta珠蛋白的慢病毒LGO4。LGO2载体设计如图1(a)所示，其他步骤基本同上(参照CN201910824134)，获得表达beta珠蛋白的慢病毒LGO2。使用表达绿色荧光蛋白的载体质粒(pLV/PGK-EGFP，引用文献DOI:10.1093/nar/gkt1163)替换本发明载体质粒，其它步骤同上，获得对照病毒载体。慢病毒通过超速离心或者柱层析的方法的富集或者纯化。
在体外使用相同量的各个病毒载体感染人造血干细胞，感染完成后诱导干细胞向红系分化，在第14天提取各组细胞RNA，使用荧光定量PCR进行RT-Q-PCR分析整合的血红蛋白T87QmRNA的表达水平，反应了血红蛋白T87Q的相对表达量(图2)，LG04号载体表达量最高。
使用293T细胞包装LG02和LG04载体，纯化后，感染293T，检测病毒感染滴度(图3)，结果显示，优化后的内含子可以提高病毒包装滴度。
使用相同量的各个病毒载体感染人造血干细胞后，将干细胞接种在含有特点细胞因子的固体培养基内，培养14天后，显微镜下统计爆式红细胞集落形成单位，集落数量代表干细胞分化能力，如图4，可以看出，LG04号载体感染对造血干细胞分化能力基本无影响。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。
序列表
<110> 上海本导基因技术有限公司
<120> 一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体
<130> DD17030
<141> 2021-12-15
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3013
<212> DNA
<213> beta珠蛋白的微型基因座调控元件( beta-globin)
<400> 1
gatctgaccc cgtatgtgag catgtgtcct ctaacagcac aggccttttg ccacctagct 60
gtccaggggt gccttaaaat ggcaaacaag gtttgttttc ttttcctgtt ttcatgcctt 120
cctcttccat atccttgttt catattaata catgtgtata gatcctaaaa atctatacac 180
atgtattaat aaagcctgat tctgccgctt ctaggtatag aggccacctg caagataaat 240
atttgattca caataactaa tcattctatg gcaattgata acaacaaata tatatatata 300
tatatatata cgtatatgtg tatatatata tatatattca ggaaataata tattctagaa 360
tatgtcacat tctgtctcag gcatccattt tctttatgat gccgtttgag gtggagtttt 420
agtcaggtgg tcagcttctc cttttttttg ccatctgccc tgtaagcatc ctgctgggga 480
cccagatagg agtcatcact ctaggctgag aacatctggg cacacaccct aagcctcagc 540
atgactcatc atgactcagc attgctgtgc ttgagccaga aggtttgctt agaaggttac 600
acagaaccag aaggcggggg tggggcactg accccgacag gggcctggcc agaactgctc 660
atgcttggac tatgggaggt cactaatgga gacacacaga aatgtaacag gaactaagga 720
aaaactgttc attaaaaaaa gtctaaccag ctgcattcga ctttgactgc agcagctggt 780
tagaaggttc tactggagga gggtcccagc ccattgctaa attaacatca ggctctgaga 840
ctggcagtat atctctaaca gtggttgatg ctatcttctg gaacttgcct gctacattga 900
gaccactgac ccatacatag gaagcccata gctctgtcct gaactgttag gccactggtc 960
cagagagtgt gcatctcctt tgatcctcat aataacccta tgagatagac acaattatta 1020
ctcttacttt atagatgatg atcctgaaaa cataggagtc aaggcacttg cccctagctg 1080
ggggtatagg ggagcagtcc catgtagtag tagaatgaaa aatgctgcta tgctgtgcct 1140
cccccacctt tcccatgtct gccctctact catggtctat ctctcctggc tcctgggagt 1200
catggactcc acccagcacc accaacctga cctaaccacc tatctgagcc tgccagccta 1260
taacccatct gggccctgat agctggtggc cagccctgac cccaccccac cctccctgga 1320
acctctgata gacacatctg gcacaccagc tcgcaaagtc accgtgaggg tcttgtgttt 1380
gctgagtcaa aattccttga aatccaagtc cttagagact cctgctccca aatttacagt 1440
catagacttc ttcatggctg tctcctttat ccacagaatg attcctttgc ttcattgccc 1500
catccatctg atcctcctca tcagtgcagc acagggccca tgagcagtag ctgcagagtc 1560
tcacataggt ctggcactgc ctctgacatg tccgacctta ggcaaatgct tgactcttct 1620
gagctcagtc ttgtcatggc aaaataaaga taataatagt gtttttttat ggagttagcg 1680
tgaggatgga aaacaatagc aaaattgatt agactataaa aggtctcaac aaatagtagt 1740
agattttatc atccattaat ccttccctct cctctcttac tcatcccatc acgtatgcct 1800
cttaattttc ccttacctat aataagagtt attcctctta ttatattctt cttatagtga 1860
ttctggatat taaagtggga atgaggggca ggccactaac gaagaagatg tttctcaaag 1920
aagccattct ccccacatag atcatctcag cagggttcag gaagataaag gaggatcaag 1980
gtcgaaggta ggaactaagg aagaacactg ggcaagtgga tccaggttgt ccaacgctga 2040
aagtaggaat ctaagcacta gtctctggat gctaggaggg cctctgcatg ggtaactctt 2100
tcaactagcc aggggctgga ctgtggagaa accatttcca gatagaagtg aggagattcc 2160
agcaggaaac acttaagaga ggatccctgg aagttcgggg caggaggctc cctgtcacat 2220
gaaggaaacc tgctcagcgt aggctctagg ttcttcccta ctcttatcca atggggcttc 2280
tgattttaag ccagtcttca caaagcccca gatttctaca tgctggagcc ctgaatgggc 2340
aggaagcatt gcggtttcca tttcattcag agctctttca taccctgctt ccccatagtt 2400
tgtctcaaca tttctgttga taatctgatt catgcaacca agaattatga gagagccttc 2460
agtcatgcct aggcctgcat ttattgttgt gcatatgatg gggtagcaga caagaaaacg 2520
ttaacagcat agactttctc agagctatat gagtttggga attaatcaag accagccttt 2580
tgctcagggt agaaatcccc tatagggatt tattcaatga tgggttaaaa gcttctacat 2640
agtttccatg tcacagccag ggctgggcag tctcctgtta tttcttttaa aataaatata 2700
tcatttaaat gcataaataa gcaaaccctg ctcgggaatg ggagggagag tctctggagt 2760
ccaccccttc tcggccctgg ctctgcagat agtgctatca aagccctgac agagccctgc 2820
ccattgctgg gccttggagt gagtcagcct agtagagagg cagggcaagc catctcatag 2880
ctgctgagtg ggagagagaa aagggctcat tgtctataaa ctcaggtcat ggctattctt 2940
attctcacac taagaaaaag aatgagatgt ctacatatac cctgcgtccc ctcttgtgta 3000
ctggggtccc caa 3013
<210> 2
<211> 476
<212> DNA
<213> beta珠蛋白的基因序列( beta-globin，2号内含子优化)
<400> 2
gcgagtctat gggacccttg atgttttctt tccccttctt ttctatggtt aagttcatgt 60
cataggaagg ggataagtaa cagggtacac atattgacca aatcagggta attttgcatt 120
tgtaatttta aaaaatgctt tcttctttta atatactttt ttgtttatct tatttctaat 180
actttcccta atctctttct ttcagggcaa taatgataca atgtatcatg cctctttgca 240
ccattctaaa gaataacagt gataatttct gggttaaggc aatagcaata tttctgcata 300
taaatatttc tgcatataaa ttgtaactga tgtaagaggt ttcatattgc taatagcagc 360
tacaatccag ctaccattct gcttttattt tatggttggg ataaggctgg attattctga 420
gtccaagcta ggcccttttg ctaatcatgt tcatacctct tatcttcctc ccacag 476
<210> 3
<211> 1050
<212> DNA
<213>beta珠蛋白的基因序列( beta-globin)
<400> 3
atggtgcacc tgactcctga ggagaagtct gccgttactg ccctgtgggg caaggtgaac 60
gtggatgaag ttggtggtga ggccctgggc aggttggtat caaggttaca agacaggttt 120
aaggagacca atagaaactg ggcatgtgga gacagagaag actcttgggt ttctgatagg 180
cactgactct ctctgcctat tggtctattt tcccaccctt aggctgctgg tggtctaccc 240
ttggacccag aggttctttg agtcctttgg ggatctgtcc actcctgatg ctgttatggg 300
caaccctaag gtgaaggctc atggcaagaa agtgctcggt gcctttagtg atggcctggc 360
tcacctggac aacctcaagg gcacctttgc ccagctgagt gagctgcact gtgacaagct 420
gcacgtggat cctgagaact tcagggcgag tctatgggac ccttgatgtt ttctttcccc 480
ttcttttcta tggttaagtt catgtcatag gaaggggata agtaacaggg tacacatatt 540
gaccaaatca gggtaatttt gcatttgtaa ttttaaaaaa tgctttcttc ttttaatata 600
cttttttgtt tatcttattt ctaatacttt ccctaatctc tttctttcag ggcaataatg 660
atacaatgta tcatgcctct ttgcaccatt ctaaagaata acagtgataa tttctgggtt 720
aaggcaatag caatatttct gcatataaat atttctgcat ataaattgta actgatgtaa 780
gaggtttcat attgctaata gcagctacaa tccagctacc attctgcttt tattttatgg 840
ttgggataag gctggattat tctgagtcca agctaggccc ttttgctaat catgttcata 900
cctcttatct tcctcccaca gctcctgggc aacgtgctgg tctgtgtgct ggcccatcac 960
tttggcaaag aattcacccc accagtgcag gctgcctatc agaaagtggt ggctggtgtg 1020
gctaatgccc tggcccacaa gtatcactaa 1050
<210> 4
<211> 265
<212> DNA
<213> beta珠蛋白的启动子序列( beta-globin)
<400> 4
ggtgtctgtt tgaggttgct agtgaacaca gttgtgtcag aagcaaatgt aagcaataga 60
tggctctgcc ctgactttta tgcccagccc tggctcctgc cctccctgct cctgggagta 120
gattggccaa ccctagggtg tggctccaca gggtgaggtc taagtgatga cagccgtacc 180
tgtccttggc tcttctggca ctggcttagg agttggactt caaaccctca gccctccctc 240
taagatatat ctcttggccc catac 265
<210> 5
<211> 300
<212> DNA
<213> beta珠蛋白的侧翼序列( beta-globin)
<400> 5
gccaatgtgc attagctgtt tgcagcctca ccttctttca tggagtttaa gatatagtgt 60
attttcccaa ggtttgaact agctcttcat ttctttatgt tttaaatgca ctgacctccc 120
acattccctt tttagtaaaa tattcagaaa taatttaaat acatcattgc aatgaaaata 180
aatgtttttt attaggcaga atccagatgc tcaaggccct tcataatatc ccccagttta 240
gtagttggac ttagggaaca aaggaacctt taatagaaat tggacagcaa gaaagcgagc 300
<210> 6
<211> 36
<212> DNA
<213>来自泡沫病毒的绝缘子序列(Foamy virus)
<400> 6
aagggagaca tctagtgata taagtgtgaa ctacac 36
<210> 7
<211> 11290
<212> DNA
<213>含微基因座控制区的慢病毒载体(Artificial Sequence)
<400> 7
aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 60
cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 120
cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 180
tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 240
tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 300
ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta 360
atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa 420
tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat 480
gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta 540
cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttggcc 600
attgcatacg ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt 660
accgccatgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt 720
agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg 780
ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac 840
gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt 900
ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa 960
atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta 1020
catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg 1080
gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg 1140
gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc 1200
attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt 1260
agtgaaccgg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta 1320
gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc 1380
cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa 1440
atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca gaggagctct 1500
ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg cggcgactgg 1560
tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg tgcgagagcg 1620
tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta aggccagggg 1680
gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta gaacgattcg 1740
cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg ggacagctac 1800
aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca gtagcaaccc 1860
tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta gacaagatag 1920
aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat cttcagacct 1980
ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa 2040
attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa 2100
agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg 2160
ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag 2220
cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc 2280
tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa 2340
cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg 2400
aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag 2460
tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa 2520
aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg 2580
aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga 2640
ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag 2700
ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc 2760
gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac 2820
ggatctcgac ggtatcggtt aacttttaaa agaaaagggg ggattggggg gtacagtgca 2880
ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa 2940
attacaaaaa ttcaaaattt tatcgatcac gagactagcc tcgacgatgg tcgccaatgt 3000
gcattagctg tttgcagcct caccttcttt catggagttt aagatatagt gtattttccc 3060
aaggtttgaa ctagctcttc atttctttat gttttaaatg cactgacctc ccacattccc 3120
tttttagtaa aatattcaga aataatttaa atacatcatt gcaatgaaaa taaatgtttt 3180
ttattaggca gaatccagat gctcaaggcc cttcataata tcccccagtt tagtagttgg 3240
acttagggaa caaaggaacc tttaatagaa attggacagc aagaaagcga gcttagtgat 3300
acttgtgggc cagggcatta gccacaccag ccaccacttt ctgataggca gcctgcactg 3360
gtggggtgaa ttctttgcca aagtgatggg ccagcacaca gaccagcacg ttgcccagga 3420
gctgtgggag gaagataaga ggtatgaaca tgattagcaa aagggcctag cttggactca 3480
gaataatcca gccttatccc aaccataaaa taaaagcaga atggtagctg gattgtagct 3540
gctattagca atatgaaacc tcttacatca gttacaattt atatgcagaa atatttatat 3600
gcagaaatat tgctattgcc ttaacccaga aattatcact gttattcttt agaatggtgc 3660
aaagaggcat gatacattgt atcattattg ccctgaaaga aagagattag ggaaagtatt 3720
agaaataaga taaacaaaaa agtatattaa aagaagaaag cattttttaa aattacaaat 3780
gcaaaattac cctgatttgg tcaatatgtg taccctgtta cttatcccct tcctatgaca 3840
tgaacttaac catagaaaag aaggggaaag aaaacatcaa gggtcccata gactcgccct 3900
gaagttctca ggatccacgt gcagcttgtc acagtgcagc tcactcagct gggcaaaggt 3960
gcccttgagg ttgtccaggt gagccaggcc atcactaaag gcaccgagca ctttcttgcc 4020
atgagccttc accttagggt tgcccataac agcatcagga gtggacagat ccccaaagga 4080
ctcaaagaac ctctgggtcc aagggtagac caccagcagc ctaagggtgg gaaaatagac 4140
caataggcag agagagtcag tgcctatcag aaacccaaga gtcttctctg tctccacatg 4200
cccagtttct attggtctcc ttaaacctgt cttgtaacct tgataccaac ctgcccaggg 4260
cctcaccacc aacttcatcc acgttcacct tgccccacag ggcagtaacg gcagacttct 4320
cctcaggagt caggtgcacc atggtgtctg tttgaggttg ctagtgaaca cagttgtgtc 4380
agaagcaaat gtaagcaata gatggctctg ccctgacttt tatgcccagc cctggctcct 4440
gccctccctg ctcctgggag tagattggcc aaccctaggg tgtggctcca cagggtgagg 4500
tctaagtgat gacagccgta cctgtccttg gctcttctgg cactggctta ggagttggac 4560
ttcaaaccct cagccctccc tctaagatat atctcttggc cccatacctc gaggatctga 4620
ccccgtatgt gagcatgtgt cctctaacag cacaggcctt ttgccaccta gctgtccagg 4680
ggtgccttaa aatggcaaac aaggtttgtt ttcttttcct gttttcatgc cttcctcttc 4740
catatccttg tttcatatta atacatgtgt atagatccta aaaatctata cacatgtatt 4800
aataaagcct gattctgccg cttctaggta tagaggccac ctgcaagata aatatttgat 4860
tcacaataac taatcattct atggcaattg ataacaacaa atatatatat atatatatat 4920
atacgtatat gtgtatatat atatatatat tcaggaaata atatattcta gaatatgtca 4980
cattctgtct caggcatcca ttttctttat gatgccgttt gaggtggagt tttagtcagg 5040
tggtcagctt ctcctttttt ttgccatctg ccctgtaagc atcctgctgg ggacccagat 5100
aggagtcatc actctaggct gagaacatct gggcacacac cctaagcctc agcatgactc 5160
atcatgactc agcattgctg tgcttgagcc agaaggtttg cttagaaggt tacacagaac 5220
cagaaggcgg gggtggggca ctgaccccga caggggcctg gccagaactg ctcatgcttg 5280
gactatggga ggtcactaat ggagacacac agaaatgtaa caggaactaa ggaaaaactg 5340
ttcattaaaa aaagtctaac cagctgcatt cgactttgac tgcagcagct ggttagaagg 5400
ttctactgga ggagggtccc agcccattgc taaattaaca tcaggctctg agactggcag 5460
tatatctcta acagtggttg atgctatctt ctggaacttg cctgctacat tgagaccact 5520
gacccataca taggaagccc atagctctgt cctgaactgt taggccactg gtccagagag 5580
tgtgcatctc ctttgatcct cataataacc ctatgagata gacacaatta ttactcttac 5640
tttatagatg atgatcctga aaacatagga gtcaaggcac ttgcccctag ctgggggtat 5700
aggggagcag tcccatgtag tagtagaatg aaaaatgctg ctatgctgtg cctcccccac 5760
ctttcccatg tctgccctct actcatggtc tatctctcct ggctcctggg agtcatggac 5820
tccacccagc accaccaacc tgacctaacc acctatctga gcctgccagc ctataaccca 5880
tctgggccct gatagctggt ggccagccct gaccccaccc caccctccct ggaacctctg 5940
atagacacat ctggcacacc agctcgcaaa gtcaccgtga gggtcttgtg tttgctgagt 6000
caaaattcct tgaaatccaa gtccttagag actcctgctc ccaaatttac agtcatagac 6060
ttcttcatgg ctgtctcctt tatccacaga atgattcctt tgcttcattg ccccatccat 6120
ctgatcctcc tcatcagtgc agcacagggc ccatgagcag tagctgcaga gtctcacata 6180
ggtctggcac tgcctctgac atgtccgacc ttaggcaaat gcttgactct tctgagctca 6240
gtcttgtcat ggcaaaataa agataataat agtgtttttt tatggagtta gcgtgaggat 6300
ggaaaacaat agcaaaattg attagactat aaaaggtctc aacaaatagt agtagatttt 6360
atcatccatt aatccttccc tctcctctct tactcatccc atcacgtatg cctcttaatt 6420
ttcccttacc tataataaga gttattcctc ttattatatt cttcttatag tgattctgga 6480
tattaaagtg ggaatgaggg gcaggccact aacgaagaag atgtttctca aagaagccat 6540
tctccccaca tagatcatct cagcagggtt caggaagata aaggaggatc aaggtcgaag 6600
gtaggaacta aggaagaaca ctgggcaagt ggatccaggt tgtccaacgc tgaaagtagg 6660
aatctaagca ctagtctctg gatgctagga gggcctctgc atgggtaact ctttcaacta 6720
gccaggggct ggactgtgga gaaaccattt ccagatagaa gtgaggagat tccagcagga 6780
aacacttaag agaggatccc tggaagttcg gggcaggagg ctccctgtca catgaaggaa 6840
acctgctcag cgtaggctct aggttcttcc ctactcttat ccaatggggc ttctgatttt 6900
aagccagtct tcacaaagcc ccagatttct acatgctgga gccctgaatg ggcaggaagc 6960
attgcggttt ccatttcatt cagagctctt tcataccctg cttccccata gtttgtctca 7020
acatttctgt tgataatctg attcatgcaa ccaagaatta tgagagagcc ttcagtcatg 7080
cctaggcctg catttattgt tgtgcatatg atggggtagc agacaagaaa acgttaacag 7140
catagacttt ctcagagcta tatgagtttg ggaattaatc aagaccagcc ttttgctcag 7200
ggtagaaatc ccctataggg atttattcaa tgatgggtta aaagcttcta catagtttcc 7260
atgtcacagc cagggctggg cagtctcctg ttatttcttt taaaataaat atatcattta 7320
aatgcataaa taagcaaacc ctgctcggga atgggaggga gagtctctgg agtccacccc 7380
ttctcggccc tggctctgca gatagtgcta tcaaagccct gacagagccc tgcccattgc 7440
tgggccttgg agtgagtcag cctagtagag aggcagggca agccatctca tagctgctga 7500
gtgggagaga gaaaagggct cattgtctat aaactcaggt catggctatt cttattctca 7560
cactaagaaa aagaatgaga tgtctacata taccctgcgt cccctcttgt gtactggggt 7620
ccccaactcg agggaattcc gataatcaac ctctggatta caaaatttgt gaaagattga 7680
ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 7740
tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt 7800
tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg 7860
tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg 7920
ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc 7980
gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc 8040
tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct 8100
tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg 8160
ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg 8220
ccgcctcccc gcatcgggaa ttcgagctcg gtacctttaa gaccaatgac ttacaaggca 8280
gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc 8340
caacgaagac aaaagggaga catctagtga tataagtgtg aactacacga tctgcttttt 8400
gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta 8460
gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc 8520
cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa 8580
atctctagca gcatctagct agaattaatt ccgtgtattc tatagtgtca cctaaatcgt 8640
atgtgtatga tacataaggt tatgtattaa ttgtagccgc gttctaacga caatatgtac 8700
aagcctaatt gtgtagcatc tggcttactg aagcagaccc tatcatctct ctcgtaaact 8760
gccgtcagag tcggtttggt tggacgaacc ttctgagttt ctggtaacgc cgtcccgcac 8820
ccggaaatgg tcagcgaacc aatcagcagg gtcatcgcta gcctagggac gtacccaatt 8880
cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact 8940
gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct 9000
ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg 9060
gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca 9120
gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct 9180
ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 9240
tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac 9300
gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct 9360
ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt 9420
ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac 9480
aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg 9540
gggaaatgtg gcacgtagaa agccagtccg cagaaacggt gctgaccccg gatgaatgtc 9600
agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca ggtagcttgc 9660
agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag cgaaccggaa 9720
ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa ctggatggct 9780
ttcttgccgc caaggatctg atggcgcagg ggatcaagct ctgatcaaga gacaggatga 9840
ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg 9900
gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg 9960
ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc 10020
ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct 10080
tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa 10140
gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg 10200
gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa 10260
gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat 10320
gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg 10380
agcatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc 10440
atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac 10500
cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg 10560
gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc 10620
tatcgccttc ttgacgagtt cttctgaatt attaactcga gctgtcagac caagtttact 10680
catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 10740
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 10800
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 10860
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 10920
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 10980
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 11040
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 11100
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 11160
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 11220
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 11280
gcagggtcgg 11290
<210> 8
<211> 5824
<212> DNA
<213> pMD2G质粒(Artificial Sequence)
<400> 8
ggatcccctg agggggcccc catgggctag aggatccggc ctcggcctct gcataaataa 60
aaaaaattag tcagccatga gcttggccca ttgcatacgt tgtatccata tcataatatg 120
tacatttata ttggctcatg tccaacatta ccgccatgtt gacattgatt attgactagt 180
tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 240
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 300
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 360
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 420
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 480
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 540
gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 600
ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 660
tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 720
tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 780
ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccc ctcgaagctt 840
acatgtggta ccgagctcgg atcctgagaa cttcagggtg agtctatggg acccttgatg 900
ttttctttcc ccttcttttc tatggttaag ttcatgtcat aggaagggga gaagtaacag 960
ggtacacata ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct 1020
tcttttaata tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc 1080
agggcaataa tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat 1140
aatttctggg ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg 1200
taactgatgt aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct 1260
tttattttat ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta 1320
atcatgttca tacctcttat cttcctccca cagctcctgg gcaacgtgct ggtctgtgtg 1380
ctggcccatc actttggcaa agcacgtgag atctgaattc aacagagatc gatctgtttc 1440
cttgacacta tgaagtgcct tttgtactta gcctttttat tcattggggt gaattgcaag 1500
ttcaccatag tttttccaca caaccaaaaa ggaaactgga aaaatgttcc ttctaattac 1560
cattattgcc cgtcaagctc agatttaaat tggcataatg acttaatagg cacagccata 1620
caagtcaaaa tgcccaagag tcacaaggct attcaagcag acggttggat gtgtcatgct 1680
tccaaatggg tcactacttg tgatttccgc tggtatggac cgaagtatat aacacagtcc 1740
atccgatcct tcactccatc tgtagaacaa tgcaaggaaa gcattgaaca aacgaaacaa 1800
ggaacttggc tgaatccagg cttccctcct caaagttgtg gatatgcaac tgtgacggat 1860
gccgaagcag tgattgtcca ggtgactcct caccatgtgc tggttgatga atacacagga 1920
gaatgggttg attcacagtt catcaacgga aaatgcagca attacatatg ccccactgtc 1980
cataactcta caacctggca ttctgactat aaggtcaaag ggctatgtga ttctaacctc 2040
atttccatgg acatcacctt cttctcagag gacggagagc tatcatccct gggaaaggag 2100
ggcacagggt tcagaagtaa ctactttgct tatgaaactg gaggcaaggc ctgcaaaatg 2160
caatactgca agcattgggg agtcagactc ccatcaggtg tctggttcga gatggctgat 2220
aaggatctct ttgctgcagc cagattccct gaatgcccag aagggtcaag tatctctgct 2280
ccatctcaga cctcagtgga tgtaagtcta attcaggacg ttgagaggat cttggattat 2340
tccctctgcc aagaaacctg gagcaaaatc agagcgggtc ttccaatctc tccagtggat 2400
ctcagctatc ttgctcctaa aaacccagga accggtcctg ctttcaccat aatcaatggt 2460
accctaaaat actttgagac cagatacatc agagtcgata ttgctgctcc aatcctctca 2520
agaatggtcg gaatgatcag tggaactacc acagaaaggg aactgtggga tgactgggca 2580
ccatatgaag acgtggaaat tggacccaat ggagttctga ggaccagttc aggatataag 2640
tttcctttat acatgattgg acatggtatg ttggactccg atcttcatct tagctcaaag 2700
gctcaggtgt tcgaacatcc tcacattcaa gacgctgctt cgcaacttcc tgatgatgag 2760
agtttatttt ttggtgatac tgggctatcc aaaaatccaa tcgagcttgt agaaggttgg 2820
ttcagtagtt ggaaaagctc tattgcctct tttttcttta tcatagggtt aatcattgga 2880
ctattcttgg ttctccgagt tggtatccat ctttgcatta aattaaagca caccaagaaa 2940
agacagattt atacagacat agagatgaac cgacttggaa agtaactcaa atcctgcaca 3000
acagattctt catgtttgga ccaaatcaac ttgtgatacc atgctcaaag aggcctcaat 3060
tatatttgag tttttaattt ttatggaatt caccccacca gtgcaggctg cctatcagaa 3120
agtggtggct ggtgtggcta atgccctggc ccacaagttt cactaagctc gcttccttgc 3180
tgtccaattt ctattaaagg ttccttggtt ccctaagtcc aactactaaa ctgggggata 3240
ttatgaaggg ccttgagcat ctggattctg cctaataaaa aacatttatt ttcattgcaa 3300
tgatgtattt aaattatttc tgaatatttt actaaaaagg gaatgtggga ggtcagtgca 3360
tttaaaacat aaagaaatga agagctagtt caaaccttgg gaaaatacac tatatcttaa 3420
actccatgaa agaaggtgag gctgcaaaca gctaatgcac attggcaaca gccctgatgc 3480
ctatgcctta ttcatccctc agaaaaggat tcaagtagag gcttgatttg gaggttaaag 3540
tttggctatg ctgtatttta cattacttat tgttttagct gtcctcatga atgtcttttc 3600
actacccatt tgcttatcct gcatctctca gccttgactc cactcagttc tcttgcttag 3660
agataccacc tttcccctga agtgttcctt ccatgtttta cggcgagatg gtttctcctc 3720
gcctggccac tcagccttag ttgtctctgt tgtcttatag aggtctactt gaagaaggaa 3780
aaacaggggg catggtttga ctgtcctgtg agcccttctt ccctgcctcc cccactcaca 3840
gtgacccgga atccctcgac atggcagtct agcactagtg cggccgcaga tctgcttcct 3900
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 3960
aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 4020
aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 4080
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 4140
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 4200
cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 4260
ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 4320
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 4380
agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 4440
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 4500
acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 4560
gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 4620
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 4680
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 4740
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 4800
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 4860
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 4920
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 4980
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 5040
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 5100
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 5160
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 5220
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 5280
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 5340
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 5400
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 5460
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 5520
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 5580
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 5640
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 5700
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 5760
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 5820
acgt 5824
<210> 9
<211> 8895
<212> DNA
<213> pMDlg/pRRE 质粒(Artificial Sequence)
<400> 9
ggatcccctg agggggcccc catgggctag aggatccggc ctcggcctct gcataaataa 60
aaaaaattag tcagccatga gcttggccca ttgcatacgt tgtatccata tcataatatg 120
tacatttata ttggctcatg tccaacatta ccgccatgtt gacattgatt attgactagt 180
tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt 240
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 300
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 360
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 420
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 480
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 540
gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt 600
ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac 660
tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg 720
tgggaggtct atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat 780
ccacgctgtt ttgacctcca tagaagacac cgggaccgat ccagcctccc ctcgaagctt 840
acatgtggta ccgagctcgg atcctgagaa cttcagggtg agtctatggg acccttgatg 900
ttttctttcc ccttcttttc tatggttaag ttcatgtcat aggaagggga gaagtaacag 960
ggtacacata ttgaccaaat cagggtaatt ttgcatttgt aattttaaaa aatgctttct 1020
tcttttaata tacttttttg tttatcttat ttctaatact ttccctaatc tctttctttc 1080
agggcaataa tgatacaatg tatcatgcct ctttgcacca ttctaaagaa taacagtgat 1140
aatttctggg ttaaggcaat agcaatattt ctgcatataa atatttctgc atataaattg 1200
taactgatgt aagaggtttc atattgctaa tagcagctac aatccagcta ccattctgct 1260
tttattttat ggttgggata aggctggatt attctgagtc caagctaggc ccttttgcta 1320
atcatgttca tacctcttat cttcctccca cagctcctgg gcaacgtgct ggtctgtgtg 1380
ctggcccatc actttggcaa agcacgtgag atctgaattc gagatctgcc gccgccatgg 1440
gtgcgagagc gtcagtatta agcgggggag aattagatcg atgggaaaaa attcggttaa 1500
ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag 1560
aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg 1620
gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag 1680
tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag 1740
acaagataga ggaagagcaa aacaaaagta agaaaaaagc acagcaagca gcagctgaca 1800
caggacacag caatcaggtc agccaaaatt accctatagt gcagaacatc caggggcaaa 1860
tggtacatca ggccatatca cctagaactt taaatgcatg ggtaaaagta gtagaagaga 1920
aggctttcag cccagaagtg atacccatgt tttcagcatt atcagaagga gccaccccac 1980
aagatttaaa caccatgcta aacacagtgg ggggacatca agcagccatg caaatgttaa 2040
aagagaccat caatgaggaa gctgcagaat gggatagagt gcatccagtg catgcagggc 2100
ctattgcacc aggccagatg agagaaccaa ggggaagtga catagcagga actactagta 2160
ctagtaccct tcaggaacaa ataggatgga tgacacataa tccacctatc ccagtaggag 2220
aaatctataa aagatggata atcctgggat taaataaaat agtaagaatg tatagcccta 2280
ccagcattct ggacataaga caaggaccaa aggaaccctt tagagactat gtagaccgat 2340
tctataaaac tctaagagcc gagcaagctt cacaagaggt aaaaaattgg atgacagaaa 2400
ccttgttggt ccaaaatgcg aacccagatt gtaagactat tttaaaagca ttgggaccag 2460
gagcgacact agaagaaatg atgacagcat gtcagggagt ggggggaccc ggccataaag 2520
caagagtttt ggctgaagca atgagccaag taacaaatcc agctaccata atgatacaga 2580
aaggcaattt taggaaccaa agaaagactg ttaagtgttt caattgtggc aaagaagggc 2640
acatagccaa aaattgcagg gcccctagga aaaagggctg ttggaaatgt ggaaaggaag 2700
gacaccaaat gaaagattgt actgagagac aggctaattt tttagggaag atctggcctt 2760
cccacaaggg aaggccaggg aattttcttc agagcagacc agagccaaca gccccaccag 2820
aagagagctt caggtttggg gaagagacaa caactccctc tcagaagcag gagccgatag 2880
acaaggaact gtatccttta gcttccctca gatcactctt tggcagcgac ccctcgtcac 2940
aataaagata ggggggcaat taaaggaagc tctattagat acaggagcag atgatacagt 3000
attagaagaa atgaatttgc caggaagatg gaaaccaaaa atgatagggg gaattggagg 3060
ttttatcaaa gtaggacagt atgatcagat actcatagaa atctgcggac ataaagctat 3120
aggtacagta ttagtaggac ctacacctgt caacataatt ggaagaaatc tgttgactca 3180
gattggctgc actttaaatt ttcccattag tcctattgag actgtaccag taaaattaaa 3240
gccaggaatg gatggcccaa aagttaaaca atggccattg acagaagaaa aaataaaagc 3300
attagtagaa atttgtacag aaatggaaaa ggaaggaaaa atttcaaaaa ttgggcctga 3360
aaatccatac aatactccag tatttgccat aaagaaaaaa gacagtacta aatggagaaa 3420
attagtagat ttcagagaac ttaataagag aactcaagat ttctgggaag ttcaattagg 3480
aataccacat cctgcagggt taaaacagaa aaaatcagta acagtactgg atgtgggcga 3540
tgcatatttt tcagttccct tagataaaga cttcaggaag tatactgcat ttaccatacc 3600
tagtataaac aatgagacac cagggattag atatcagtac aatgtgcttc cacagggatg 3660
gaaaggatca ccagcaatat tccagtgtag catgacaaaa atcttagagc cttttagaaa 3720
acaaaatcca gacatagtca tctatcaata catggatgat ttgtatgtag gatctgactt 3780
agaaataggg cagcatagaa caaaaataga ggaactgaga caacatctgt tgaggtgggg 3840
atttaccaca ccagacaaaa aacatcagaa agaacctcca ttcctttgga tgggttatga 3900
actccatcct gataaatgga cagtacagcc tatagtgctg ccagaaaagg acagctggac 3960
tgtcaatgac atacagaaat tagtgggaaa attgaattgg gcaagtcaga tttatgcagg 4020
gattaaagta aggcaattat gtaaacttct taggggaacc aaagcactaa cagaagtagt 4080
accactaaca gaagaagcag agctagaact ggcagaaaac agggagattc taaaagaacc 4140
ggtacatgga gtgtattatg acccatcaaa agacttaata gcagaaatac agaagcaggg 4200
gcaaggccaa tggacatatc aaatttatca agagccattt aaaaatctga aaacaggaaa 4260
atatgcaaga atgaagggtg cccacactaa tgatgtgaaa caattaacag aggcagtaca 4320
aaaaatagcc acagaaagca tagtaatatg gggaaagact cctaaattta aattacccat 4380
acaaaaggaa acatgggaag catggtggac agagtattgg caagccacct ggattcctga 4440
gtgggagttt gtcaataccc ctcccttagt gaagttatgg taccagttag agaaagaacc 4500
cataatagga gcagaaactt tctatgtaga tggggcagcc aatagggaaa ctaaattagg 4560
aaaagcagga tatgtaactg acagaggaag acaaaaagtt gtccccctaa cggacacaac 4620
aaatcagaag actgagttac aagcaattca tctagctttg caggattcgg gattagaagt 4680
aaacatagtg acagactcac aatatgcatt gggaatcatt caagcacaac cagataagag 4740
tgaatcagag ttagtcagtc aaataataga gcagttaata aaaaaggaaa aagtctacct 4800
ggcatgggta ccagcacaca aaggaattgg aggaaatgaa caagtagatg ggttggtcag 4860
tgctggaatc aggaaagtac tatttttaga tggaatagat aaggcccaag aagaacatga 4920
gaaatatcac agtaattgga gagcaatggc tagtgatttt aacctaccac ctgtagtagc 4980
aaaagaaata gtagccagct gtgataaatg tcagctaaaa ggggaagcca tgcatggaca 5040
agtagactgt agcccaggaa tatggcagct agattgtaca catttagaag gaaaagttat 5100
cttggtagca gttcatgtag ccagtggata tatagaagca gaagtaattc cagcagagac 5160
agggcaagaa acagcatact tcctcttaaa attagcagga agatggccag taaaaacagt 5220
acatacagac aatggcagca atttcaccag tactacagtt aaggccgcct gttggtgggc 5280
ggggatcaag caggaatttg gcattcccta caatccccaa agtcaaggag taatagaatc 5340
tatgaataaa gaattaaaga aaattatagg acaggtaaga gatcaggctg aacatcttaa 5400
gacagcagta caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg 5460
gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt 5520
acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc 5580
agtttggaaa ggaccagcaa agctcctctg gaaaggtgaa ggggcagtag taatacaaga 5640
taatagtgac ataaaagtag tgccaagaag aaaagcaaag atcatcaggg attatggaaa 5700
acagatggca ggtgatgatt gtgtggcaag tagacaggat gaggattaac acatggaatt 5760
ccggagcggc cgcaggagct ttgttccttg ggttcttggg agcagcagga agcactatgg 5820
gcgcagcctc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc 5880
agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct 5940
ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac 6000
agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga 6060
atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt 6120
gggacagaga aattaacaat tacacaagct tccgcggaat tcaccccacc agtgcaggct 6180
gcctatcaga aagtggtggc tggtgtggct aatgccctgg cccacaagtt tcactaagct 6240
cgcttccttg ctgtccaatt tctattaaag gttccttggt tccctaagtc caactactaa 6300
actgggggat attatgaagg gccttgagca tctggattct gcctaataaa aaacatttat 6360
tttcattgca atgatgtatt taaattattt ctgaatattt tactaaaaag ggaatgtggg 6420
aggtcagtgc atttaaaaca taaagaaatg aagagctagt tcaaaccttg ggaaaataca 6480
ctatatctta aactccatga aagaaggtga ggctgcaaac agctaatgca cattggcaac 6540
agccctgatg cctatgcctt attcatccct cagaaaagga ttcaagtaga ggcttgattt 6600
ggaggttaaa gtttggctat gctgtatttt acattactta ttgttttagc tgtcctcatg 6660
aatgtctttt cactacccat ttgcttatcc tgcatctctc agccttgact ccactcagtt 6720
ctcttgctta gagataccac ctttcccctg aagtgttcct tccatgtttt acggcgagat 6780
ggtttctcct cgcctggcca ctcagcctta gttgtctctg ttgtcttata gaggtctact 6840
tgaagaagga aaaacagggg gcatggtttg actgtcctgt gagcccttct tccctgcctc 6900
ccccactcac agtgacccgg aatccctcga catggcagtc tagcactagt gcggccgcag 6960
atctgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 7020
agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 7080
catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 7140
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 7200
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 7260
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 7320
cgtggcgctt tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 7380
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 7440
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 7500
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 7560
taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac 7620
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 7680
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 7740
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 7800
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 7860
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga 7920
ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt 7980
gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg 8040
agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga 8100
gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga 8160
agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg 8220
catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc 8280
aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc 8340
gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca 8400
taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac 8460
caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg 8520
ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc 8580
ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg 8640
tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac 8700
aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat 8760
actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 8820
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 8880
agtgccacct gacgt 8895
<210> 10
<211> 4174
<212> DNA
<213> pRSV-REV质粒(Artificial Sequence)
<400> 10
agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60
acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120
tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180
ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gaattcgatg 240
tacgggccag atatacgcgt atctgagggg actaggtgtg tttaggcgaa aagcggggct 300
tcggttgtac gcggttagga gtcccctcag gattagtagt ttcgcttttg catagggagg 360
gggaaatgta gtcttatgca atacacttgt agtcttgcaa catggtaacg atgagttagc 420
aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt 480
acgatcgtgc cttattagga aggcaacaga caggtctgac atggattgga cgaaccactg 540
aattccgcat tgcagagata attgtattta agtgcctagc tcgatacaat aaacgccatt 600
tgaccattca ccacattggt gtgcacctcc aagctcgagc tcgtttagtg aaccgtcaga 660
tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca 720
gcctcccctc gaagctagtc gattaggcat ctcctatggc aggaagaagc ggagacagcg 780
acgaagacct cctcaaggca gtcagactca tcaagtttct ctatcaaagc aacccacctc 840
ccaatcccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac 900
agagacagat ccattcgatt agtgaacgga tccttagcac ttatctggga cgatctgcgg 960
agcctgtgcc tcttcagcta ccaccgcttg agagacttac tcttgattgt aacgaggatt 1020
gtggaacttc tgggacgcag ggggtgggaa gccctcaaat attggtggaa tctcctacaa 1080
tattggagtc aggagctaaa gaatagtgct gttagcttgc tcaatgccac agctatagca 1140
gtagctgagg ggacagatag ggttatagaa gtagtacaag aagcttggca ctggccgtcg 1200
ttttacatga tctgagcctg ggagatctct ggctaactag ggaacccact gcttaagcct 1260
caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 1320
aactagagat caggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 1380
tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 1440
cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 1500
ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 1560
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 1620
cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 1680
aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 1740
cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 1800
ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 1860
aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 1920
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 1980
acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 2040
cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 2100
tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 2160
ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 2220
ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 2280
atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 2340
taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 2400
cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 2460
aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 2520
aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 2580
tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 2640
ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 2700
catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 2760
aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 2820
ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 2880
gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 2940
aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 3000
gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 3060
gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 3120
gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 3180
gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 3240
gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 3300
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 3360
ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 3420
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 3480
ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 3540
ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 3600
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 3660
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 3720
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 3780
tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 3840
tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 3900
gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 3960
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 4020
ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct 4080
gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc 4140
gagcgcagcg agtcagtgag cgaggaagcg gaag 4174
Claims (7)
1.一种包含beta珠蛋白表达盒的慢病毒载体，所述表达盒包含：
a、微基因座控制区，为一段从beta珠蛋白16kb的基因座控制区中筛选出来的不含HS1区的微型调控元件；
b、beta珠蛋白的基因序列，所述基因beta珠蛋白的基因序列如SEQ ID NO.2所示；
c、beta珠蛋白的基因序列上游侧翼的启动子序列；
d、beta珠蛋白的基因序列下游的侧翼序列；
e、来自泡沫病毒Foamy virus的绝缘子序列。
2.根据权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述微型基因座控制区是由HS2、HS3、HS4脱氧核糖核酸酶I超敏感位点序列经过筛选后，组合而成。
3.根据权利要求2所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述微型基因座调控元件的序列如SEQ ID NO.1所示。
4.根据权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述启动子序列如SEQ ID NO.4所示。
5.根据权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述侧翼序列如SEQ ID NO.5所示。
6.根据权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体，其特征在于，所述绝缘子序列如SEQ ID NO.6所示。
7.一种如权利要求1所述的包含beta珠蛋白表达盒的慢病毒载体在制备地中海贫血和镰刀型贫血的基因治疗药物中的应用。
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| CN202210013838.9A CN114410687B (zh) | 2022-01-06 | 2022-01-06 | 一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 |
| PCT/CN2022/138330 WO2023130911A1 (zh) | 2022-01-06 | 2022-12-12 | 一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 |
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| CN116271106A (zh) * | 2023-05-24 | 2023-06-23 | 中吉智药(南京)生物技术有限公司 | 慢病毒载体LentilAlpha在制备治疗α-地中海贫血药物中的应用 |
| WO2023130911A1 (zh) * | 2022-01-06 | 2023-07-13 | 上海本导基因技术有限公司 | 一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 |
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| CN117384967A (zh) * | 2020-04-24 | 2024-01-12 | 康霖生物科技（杭州）有限公司 | 一种核酸构建体 |
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| CN114410687B (zh) * | 2022-01-06 | 2024-04-19 | 上海本导基因技术有限公司 | 一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 |
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| WO2023130911A1 (zh) * | 2022-01-06 | 2023-07-13 | 上海本导基因技术有限公司 | 一种适用地中海贫血症和镰刀形贫血基因治疗的慢病毒载体 |
| CN116271106A (zh) * | 2023-05-24 | 2023-06-23 | 中吉智药(南京)生物技术有限公司 | 慢病毒载体LentilAlpha在制备治疗α-地中海贫血药物中的应用 |
| CN116271106B (zh) * | 2023-05-24 | 2023-08-11 | 中吉智药(南京)生物技术有限公司 | 慢病毒载体LentilAlpha在制备治疗α-地中海贫血药物中的应用 |
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