CN1520259A - 利用球状载体将第八因子/第九因子进行肝脏基因转移治疗血友病的方法 - Google Patents
利用球状载体将第八因子/第九因子进行肝脏基因转移治疗血友病的方法 Download PDFInfo
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Abstract
本发明是利用球状载体将第八因子/第九因子进行肝脏基因转移治疗血友病的方法,用于血友病治疗的非病毒球状载体,由包含修饰的乙肝病毒env L蛋白的双脂层构成,这样的结构既减弱或消除了S肽引起的免疫反应,又保证了肝靶向性的信号仍暴露在球体的表面。本发明还包括用于治疗A型和B型血友病的第八因子或第九因子的表达结构,该表达结构可以是单链或双链DNA。本发明还包括注射非病毒球状载体治疗血友病的方法,包含恰当表达顺序核酸结构的球状载体通过静脉或动脉注射入体内,通过观察病人体内蛋白质或RNA的表达或疾病表征的改善了解植入基因产品的表达情况。
Description
技术领域
本发明是指利用球状载体将第八因子/第九因子进行肝脏基因转移治疗血友病的方法。
背景技术
血友病是最常见的遗传疾病之一,因第八因子缺失引起的A型血友病发病率为每5,000个新生男婴中有1名;而因第九因子缺失导致的B型血友病发病率为每30,000个新生男婴中有一名发病,而且在所有研究过的人种中,发病率都很一致。长期以来,富集的凝血因子被用来治疗血友病,这种疗法的目的是控制出血而且需要终生重复的静脉注射。由于对血浆制品风险意识的提高,开发新的有效治疗方法的重要性也在不断提升。
人们已经在开发基因治疗的方法来治疗血友病。因其特性,血友病是很适于用基因治疗方法的一种疾病:相关的蛋白已被很好地研究,相关的基因已被克隆因而易于获得,大型和小型的相关动物模型也已建立,更重要的是,基因产品不需要器官特异性的表达,而且,由于蛋白的功能是通过蛋白激活来调控的,所以蛋白表达的水平不需要严格的调控。另外,很低的蛋白表达水平就可以在功能上改善疾病。血友病基因治疗最主要的障碍是基因表达的产品需要对病人终身维持。因而,有效的疗法可能需要将基因治疗载体重复导入。
利用逆转录病毒和腺病毒相关病毒(AAV)作为载体的血友病基因治疗的临床试验正在进行,以腺病毒和慢性病毒(LENTIVIRUS)为载体的基因治疗实验也有人偿试过。但是以病毒为载体的基因治疗的通病是它的装载核酸的容量有限,并可导致免疫反应。使用DNA或RNA,在有或没有合成脂质体的条件下,都只有低效的基因转移,非病毒方法只能产生短期、非靶向性的基因表达。
Yamada等人(2001a)最近描述了一种新的对肝有特异性的、表达修饰乙肝病毒表面蛋白的球状载体。产生这种包含乙肝膜蛋白球体的方法为同行所熟知(Kuroda等人1992年和Yamada等人,2001b,在此引用全文)。简述如下:通过将鸡溶菌酶基因的信号序列在框地(inframe)接入乙肝表面env L蛋白(SEQ ID I)基因之前,包含pre-S1加pre-S2加S肽的修饰的乙肝表面env L蛋白就可在酵母中有效地表达出来,信号序列在翻译的过程中将蛋白导入内质网,饱含蛋白的球体从内质网中分裂出来,聚集在酵母的细胞质中,这些球体由内质网的脂双层和修饰的env L蛋白为主要蛋白成份组成。通过上述方法形成的颗粒非常稳定,并可通过重复使用氧化铯和蔗糖梯度离心的方法纯化,而这些纯化方法为同业所熟知。
质粒DNA可以通过电极通导(electroporation)法加入含env L蛋白的颗粒,这些包含DNA的颗粒被证实可以促进DNA特异地进入肝细胞(通过在细胞培养水平和直接注射进移植人肝癌细胞的裸鼠的实验)。Yamada等人(2001a)建议这样的球状载体可用于将核酸或其它分子特异性地导入肝脏。
发明内容
本发明的主要目的是提供一种用于血友病治疗的非病毒球状载体,该球状载体,由包含修饰的乙肝病毒env L蛋白的双脂层构成,这样的结构既减弱或消除了S肽引起的免疫反应,又保证了肝靶向性的信号仍暴露在球体的表面。本发明的另一目的是提供一种用于治疗A型和B型血友病的第八因子或第九因子的表达结构,该表达结构可以是单链或双链DNA。含有但不限于下列启动子:非组织特异,如细胞肥大病毒(cytomegalovirus),Rous肉瘤,和组织特异的启动子,如α-胚胎蛋白、球蛋白、白蛋白和α1-微球蛋白,该表达结构还可以包含其它的基因表达调控因子,包括但不限于强化因子(enhancers),基因内区(introns),多聚A序列和RNA靶向顺序,促进质粒复制的因子,如SV40复制起始因子也可包括在内,来自AAV的末端重复片断(ITR)序列也可包括在表达结构里,用来促进表达结构的稳定性或用于通过AAV Rep蛋白促进与宿主DNA的嵌合。另外,类似ITR顺序的真核DNA转座子/转座子酶(transposon/transposases)系统也可用来促进与宿主DNA的嵌合。
本发明的再一目的是提供一种注射非病毒球载状体治疗血友病的方法,包含恰当表达顺序核酸结构的球状载体通过静脉或动脉注射入体内,通过观察病人体内蛋白质或RNA的表达或疾病表征的改善了解植入基因产品的表达情况。
对本发明的技术方案详细叙述如下:
本发明一个非病毒的球状载体,它由下列部份组成:
一个在球状体表面暴露着乙肝病毒外壳(envelope)env蛋白的球状膜和;
一个核酸表达结构,包含完整的第八因子或第九因子的表达序列和在肝细胞中有功能的启动子序列。
其中的env蛋白含有突变以降低其抗原性。
其中的表达结构为DNA。
其中的表达结构为双链质粒DNA。
其中的表达结构为RNA。
其中的启动子为非组织特异的启动子。
其中的非组织特异启动子可来自以下来源:细胞肥大病毒(cytomegalovirus)启动子,Rous肉瘤启动子,辅酶Q(Ubiquitin)启动子,鸡β-肌动蛋白(β-actin)启动子和延长因子(elongation factor)(EF)-1α启动子。
其中的启动子为肝脏特异的启动子。
其中的肝脏特异启动子来自以下来源:α-胚胎蛋白启动子,球蛋白启动子,α1-微球蛋白和白蛋白。
其中的表达结构包括来自腺病毒相关病毒的反向(Inverted)末端序列(AAV-ITR)。
其中的表达结构包括真核细胞的转座子(transposon)和转座子酶(transposase)序列。
其中的表达结构包括第八因子的表达序列。
其中的第八因子包含无意(silent)突变以增加其表达能力。
其中的表达结构包括第九因子的表达序列。
本发明一个非病毒的球状载体,它由以下部份组成:
一个在球状体表面暴露着乙肝病毒外壳(envelope)env蛋白的球状膜和;
一个包含了完整第八因子或第九因子的蛋白质。
其中的env蛋白包含了突变以降低其抗原性。
本发明一个治疗血友病的方法,它由以下部份组成:
将由表面暴露着乙肝病毒env蛋白球状膜的非病毒球状载体输入具有血友病的个体的血循环中以及;
一个由完整的第八因子或第九因子表达序列和在肝细胞中有功能的启动子序列组成的核酸表达结构,以及,
监测该病人疾病改善的情况。
其中输入血循环的方法通过静脉注射。
其中输入血循环的方法通过注射入肝动脉或腹腔动脉(hepatic orportal artery)。
本发明一种利用球状载体将第八因子/第九因子进行肝脏基因转移治疗血友病的方法,其优点和达到的功效是:該球状载体结构既减弱或消除了S肽引起的免疫反应,又保证了肝靶向性的信号仍暴露在球体的表面。該注射非病毒球载状体治疗血友病的方法,改善了解植入基因產品的表连情况。疗效好,无副作用。
具体实施方式
血友病是最常见的遗传疾病之一,可由任何一个凝血因子的突变或缺失引起。最常见的为第八因子或第九因子的缺损。目前的治疗方法需要终身补充凝血因子,涉及重复的静脉输入,同时也使病人承受血制品带来的危险。
基於下列原因,血友病很适于基因治疗。第一,相关基因已克隆并易获得;第二,相关蛋白特性了解清楚,它们的激活是通过蛋白的裂解来调控而不是在转录或转译水平来调控,因而蛋白表达水平不需要严密的控制;第三,低水平的蛋白表达已被证明足以引起表征的恢复;第四,虽然肝脏是产生大多数第八、第九因子的生理器官,在身体哪个部位产生这些因子相对并不重要;第五,不少动物模型可用于分析各种治疗方法的效果。即便如此,到目前为止,还没有一个有效的基因转移载体或治疗血友病的方法被开发出来。
本发明为治疗血友病的球状载体,由天然脂球体和在球体内表达第八或第九因子的表达结构组成。该脂球体最好用酵母或昆虫细胞,如Sf9细胞产生,并将修饰的乙肝病毒dnv L蛋白整合于膜层中。球体的产生根据Kuroda(1992)疫苗的产生方法加上Yamada(2001b)的改进,简述如下:乙肝病毒env L蛋白由三个区域组成:有108或119个残基的pre-S1区域涉及与肝细胞的直接作用;有55个残基的pre-S2区域涉及白蛋白协调的聚合反应以及主要的有226个残基的S蛋白区域。试图在各种真核细胞里产生L蛋白的尝试都没有成功。可能是由于N端出现的pre-S1短蛋白链。为克服这一问题,我们将L蛋白N端的表达序列用鸡溶菌酶信号序列取代,以将N端导入内质网。这一杂合的序列被嵌入酵母(S.cerevisiae)表达载体,然后通过常用的转化方法导入酵母。杂合的L蛋白被大量地生产出来(可达整个酵母蛋白的42%),并被准确地插入膜层。球状体从内质网上分裂出来,形成23纳米的圆球或线状颗粒,并将蛋白包含在颗粒的膜层中。酵母细胞用玻璃球破碎以释放出这些球体,然后这些球体通过数轮不连续的氯化铯和蔗糖梯度离心纯化。从昆虫细胞生产和纯化球状体也通过类似的过程。通过匀浆(homogenization)和差速(differential)离心,一个粗的膜组分可以象从酵母中一样从昆虫细胞中得到。这个粗的膜组分可以象处理酵母细胞一样,通过氯化铯和蔗糖梯度离心分离出纯化的球状体。上述方法可廉价、大量地生产球状体以适应基因转移的需要。这些球状体在低温保存情况下长期稳定,但在重复融解、冷冻情况下会变得不稳定。
球状载体可用来将任何核酸结构,单、双链DNA或RNA或其他基因产品导入肝脏。本发明的一个优选具体化例(preferred embodiment)中,核酸为一双链DNA质粒。该结构至少含有人第八因子(SEQ1 D2)或第九因子(SEQ1 D3)来治疗A型或B型血友病,并含有促进血友病基因转录的启动子。这一结构还可含有其他的调控因子或强化因子(enhancer)来调节基因表达,基因内区(intron)或多聚A序列以促进RNA加工(processing)和基因表达,RNA靶向序列,AAV-ITR或真核转座子(transposon)序列以促进表达盘(cassettes)的稳定和与宿主基因组的整合,以及病毒复制起始序列以促进质粒在宿主中的扩增,这些序列为同业人士所熟知。球状体核酸结构中可包含的序列不象基于病毒基因组的其它基因转移方法那样受限制,因为球状体可容纳相当大量的核酸。
相当数量的启动子(promoter)序列可在肝脏中起作用,这些包括非组织专一的启动子(promoter)如CMV、RSV、辅酶Q(Ubiquitin)、鸡β-肌动蛋白(β-actin)和延长因子(elongation factor)(EF)-1α;也包括组织特异性启动子(promoter)如α-胚胎蛋白,球蛋白α1-微球蛋白及白蛋白。
AAV-ITR序列可被置于所有除了复制起始点以外所有表达和调控序列的两端,这些AAV-ITR序列被证实可以增加基因治疗方法里转入结构的稳定性。另外,这些AAV-ITR序列在AAV-Rep蛋白的帮助下可促进与人类基因组定点的整合,而AAV-Rep蛋白的提供可通过直接将蛋白整合入含核酸结构的球状体或将其表达盘(cassettes)包入同一球状体。
与AAV-ITR序列相似,真核转座子(transposon)序列可结合于所有表达和调控序列之外,促进整合的转座子酶(transposase)可从同一表达盘(cassette)中表达,也可用位于同一球状体中的独立表达盘(cassette)中加以表达。
表达第八因子时需特别注意以下这点。研究表明人类第八因子包含一序列(核甘酸1741到1771,SEQ1 D2)能降低异源(heterologous)蛋白的表达(Fallaux等人,1996),该序列AT含量高,被证实与一细胞核因子结合,并抑制细胞中报告结构(reporterconstruct)的表达。切除该序列或将该序列随机突变产生的是没有活性的第八因子。但是,在基因产品中引入不导致氨基酸顺序变化的silent突变的方法是为同行所熟知可以促进第八因子的表达的。
在一个优选具体化例(preferred embodiment),本发明的核酸结构是通过电极通导(electroporation)的过程进入球状体的。核酸结构与球状体充分混合,加上水达到最终所需体积,然后转入一电极通导糟(electroporation curette)电压和电阻因curette(糟)的大小(空隙长度)和糟(curette)中物质的体积而有很大差异。行业中人士熟知如何调节这些参数以达到最大程度地转移核酸进入球状体并将球状体的损伤降到最低。
另外,核酸可以通过同业中熟知的用包含核酸脂质体与球状体融合的方法将核酸导入球状体(Dzau等人,1996)发明中的核酸结构通过混合(vortexing)包埋入脂质体,脂质体的组成可是已知的磷脂和膜组分(如磷酸胆碱,胆固醇)或市场上可买到的特有的膜组分混合物(如Gibco BRL生成的Lipofectamine)。包埋了核酸的脂质体与来源于酵母或昆虫细胞的球状体在37℃下共同培育10-30分钟后将自行融合。
此外,第八因子或第九因子蛋白本身可以直接包入本发明中的球状体。第八(SEQ ID4)或第九(SEQ ID5)因子蛋白可以通过多种为同业人士所熟知的方法获得。含有这些蛋白的高浓度溶液可与纯化的球状体混合,通过渗透压冲击(Osmotic shock)或超声(sonication)的方法促进蛋白进入球体。蛋白也可通过混合(Vortexing)或超声(sonication)先包入人工膜,这些包含蛋白的人工膜然后可以与源于乙肝病毒的球状体融合。
本发明包含核酸或蛋白的非病毒球状载体可由静脉或动脉注射进入人体。为增加输送效率,球状载体可以直接注射进肝动脉或腹腔动脉(hepatic or portal artery)。病人被定期观测第八或第九因子的出现或体征的改善。注射非病毒球状体的量因启动子的强弱、整合序列、每个球状体包含质粒的量以及许多为同业所熟知的其它因素而改变。由于检测病人健康状况的方法为人熟知,有效的剂量很容易被确定。
序列表
<110>肯尼斯·R·.陈
星岛雅彦
<120>利用球状载体将第八因子/第九因子进行肝脏基因转移治
疗血友病的方法
<130>6627-PA1170
<150>60/286,314
<151>2001-04-25
<160>5
<170>PatentIn version 3.1
<210>1
<211>9029
<212>DNA
<213>Homo sapiens
<400>1
gcttagtgct gagcacatcc agtgggtaaa gttccttaaa atgctctgca aagaaattgg 60
gacttttcat taaatcagaa attttacttt tttcccctcc tgggagctaa agatatttta 120
gagaagaatt aaccttttgc ttctccagtt gaacatttgt agcaataagt catgcaaata 180
gagctctcca cctgcttctt tctgtgcctt ttgcgattct gctttagtgc caccagaaga 240
tactacctgg gtgcagtgga actgtcatgg gactatatgc aaagtgatct cggtgagctg 300
cctgtggacg caagatttcc tcctagagtg ccaaaatctt ttccattcaa cacctcagtc 360
gtgtacaaaa agactctgtt tgtagaattc acggatcacc ttttcaacat cgctaagcca 420
aggccaccct ggatgggtct gctaggtcct accatccagg ctgaggttta tgatacagtg 480
gtcattacac ttaagaacat ggcttcccat cctgtcagtc ttcatgctgt tggtgtatcc 540
tactggaaag cttctgaggg agctgaatat gatgatcaga ccagtcaaag ggagaaagaa 600
gatgataaag tcttccctgg tggaagccat acatatgtct ggcaggtcct gaaagagaat 660
ggtccaatgg cctctgaccc actgtgcctt acctactcat atctttctca tgtggacctg 720
gtaaaagact tgaattcagg cctcattgga gccctactag tatgtagaga agggagtctg 780
gccaaggaaa agacacagac cttgcacaaa tttatactac tttttgctgt atttgatgaa 840
gggaaaagtt ggcactcaga aacaaagaac tccttgatgc aggataggga tgctgcatct 900
gctcgggcct ggcctaaaat gcacacagtc aatggttatg taaacaggtc tctgccaggt 960
ctgattggat gccacaggaa atcagtctat tggcatgtga ttggaatggg caccactcct 1020
gaagtgcact caatattcct cgaaggtcac acatttcttg tgaggaacca tcgccaggcg 1080
tccttggaaa tctcgccaat aactttcctt actgctcaaa cactcttgat ggaccttgga 1140
cagtttctac tgttttgtca tatctcttcc caccaacatg atggcatgga agcttatgtc 1200
aaagtagaca gctgtccaga ggaaccccaa ctacgaatga aaaataatga agaagcggaa 1260
gactatgatg atgatcttac tgattctgaa atggatgtgg tcaggtttga tgatgacaac 1320
tctccttcct ttatccaaat tcgctcagtt gccaagaagc atcctaaaac ttgggtacat 1380
tacattgctg ctgaagagga ggactgggac tatgctccct tagtcctcgc ccccgatgac 1440
agaagttata aaagtcaata tttgaacaat ggccctcagc ggattggtag gaagtacaaa 1500
aaagtccgat ttatggcata cacagatgaa acctttaaga ctcgtgaagc tattcagcat 1560
gaatcaggaa tcttgggacc tttactttat ggggaagttg gagacacact gttgattata 1620
tttaagaatc aagcaagcag accatataac atctaccctc acggaatcac tgatgtccgt 1680
cctttgtatt caaggagatt accaaaaggt gtaaaacatt tgaaggattt tccaattctg 1740
ccaggagaaa tattcaaata taaatggaca gtgactgtag aagatgggcc aactaaatca 1800
gatcctcggt gcctgacccg ctattactct agtttcgtta atatggagag agatctagct 1860
tcaggactca ttggccctct cctcatctgc tacaaagaat ctgtagatca aagaggaaac 1920
cagataatgt cagacaagag gaatgtcatc ctgttttctg tatttgatga gaaccgaagc 1980
tggtacctca cagagaatat acaacgcttt ctccccaatc cagctggagt gcagcttgag 2040
gatccagagt tccaagcctc caacatcatg cacagcatca atggctatgt ttttgatagt 2100
ttgcagttgt cagtttgttt gcatgaggtg gcatactggt acattctaag cattggagca 2160
cagactgact tcctttctgt cttcttctct ggatatacct tcaaacacaa aatggtctat 2220
gaagacacac tcaccctatt cccattctca ggagaaactg tcttcatgtc gatggaaaac 2280
ccaggtctat ggattctggg gtgccacaac tcagactttc ggaacagagg catgaccgcc 2340
ttactgaagg tttctagttg tgacaagaac actggtgatt attacgagga cagttatgaa 2400
gatatttcag catacttgct gagtaaaaac aatgccattg aaccaagaag cttctcccag 2460
aattcaagac accctagcac taggcaaaag caatttaatg ccaccacaat tccagaaaat 2520
gacatagaga agactgaccc ttggtttgca cacagaacac ctatgcctaa aatacaaaat 2580
gtctcctcta gtgatttgtt gatgctcttg cgacagagtc ctactccaca tgggctatcc 2640
ttatctgatc tccaagaagc caaatatgag actttttctg atgatccatc acctggagca 2700
atagacagta ataacagcct gtctgaaatg acacacttca ggccacagct ccatcacagt 2760
ggggacatgg tatttacccc tgagtcaggc ctccaattaa gattaaatga gaaactgggg 2820
acaactgcag caacagagtt gaagaaactt gatttcaaag tttctagtac atcaaataat 2880
ctgatttcaa caattccatc agacaatttg gcagcaggta ctgataatac aagttcctta 2940
ggacccccaa gtatgccagt tcattatgat agtcaattag ataccactct atttggcaaa 3000
aagtcatctc cccttactga gtctggtgga cctctgagct tgagtgaaga aaataatgat 3060
tcaaagttgt tagaatcagg tttaatgaat agccaagaaa gttcatgggg aaaaaatgta 3120
tcgtcaacag agagtggtag gttatttaaa gggaaaagag ctcatggacc tgctttgttg 3180
actaaagata atgccttatt caaagttagc atctctttgt taaagacaaa caaaacttcc 3240
aataattcag caactaatag aaagactcac attgatggcc catcattatt aattgagaat 3300
agtccatcag tctggcaaaa tatattagaa agtgacactg agtttaaaaa agtgacacct 3360
ttgattcatg acagaatgct tatggacaaa aatgctacag ctttgaggct aaatcatatg 3420
tcaaataaaa ctacttcatc aaaaaacatg gaaatggtcc aacagaaaaa agagggcccc 3480
attccaccag atgcacaaaa tccagatatg tcgttcttta agatgctatt cttgccagaa 3540
tcagcaaggt ggatacaaag gactcatgga aagaactctc tgaactctgg gcaaggcccc 3600
agtccaaagc aattagtatc cttaggacca gaaaaatctg tggaaggtca gaatttcttg 3660
tctgagaaaa acaaagtggt agtaggaaag ggtgaattta caaaggacgt aggactcaaa 3720
gagatggttt ttccaagcag cagaaaccta tttcttacta acttggataa tttacatgaa 3780
aataatacac acaatcaaga aaaaaaaatt caggaagaaa tagaaaagaa ggaaacatta 3840
atccaagaga atgtagtttt gcctcagata catacagtga ctggcactaa gaatttcatg 3900
aagaaccttt tcttactgag cactaggcaa aatgtagaag gttcatatga cggggcatat 3960
gctccagtac ttcaagattt taggtcatta aatgattcaa caaatagaac aaagaaacac 4020
acagctcatt tctcaaaaaa aggggaggaa gaaaacttgg aaggcttggg aaatcaaacc 4080
aagcaaattg tagagaaata tgcatgcacc acaaggatat ctcctaatac aagccagcag 4140
aattttgtca cgcaacgtag taagagagct ttgaaacaat tcagactccc actagaagaa 4200
acagaacttg aaaaaaggat aattgtggat gacacctcaa cccagtggtc caaaaacatg 4260
aaacatttga ccccgagcac cctcacacag atagactaca atgagaagga gaaaggggcc 4320
attactcagt ctcccttatc agattgcctt acgaggagtc atagcatccc tcaagcaaat 4380
agatctccat tacccattgc aaaggtatca tcatttccat ctattagacc tatatatctg 4440
accagggtcc tattccaaga caactcttct catcttccag cagcatctta tagaaagaaa 4500
gattctgggg tccaagaaag cagtcatttc ttacaaggag ccaaaaaaaa taacctttct 4560
ttagccattc taaccttgga gatgactggt gatcaaagag aggttggctc cctggggaca 4620
agtgccacaa attcagtcac atacaagaaa gttgagaaca ctgttctccc gaaaccagac 4680
ttgcccaaaa catctggcaa agttgaattg cttccaaaag ttcacattta tcagaaggac 4740
ctattcccta cggaaactag caatgggtct cctggccatc tggatctcgt ggaagggagc 4800
cttcttcagg gaacagaggg agcgattaag tggaatgaag caaacagacc tggaaaagtt 4860
ccctttctga gagtagcaac agaaagctct gcaaagactc cctccaagct attggatcct 4920
cttgcttggg ataaccacta tggtactcag ataccaaaag aagagtggaa atcccaagag 4980
aagtcaccag aaaaaacagc ttttaagaaa aaggatacca ttttgtccct gaacgcttgt 5040
gaaagcaatc atgcaatagc agcaataaat gagggacaaa ataagcccga aatagaagtc 5100
acctgggcaa agcaaggtag gactgaaagg ctgtgctctc aaaacccacc agtcttgaaa 5160
cgccatcaac gggaaataac tcgtactact cttcagtcag atcaagagga aattgactat 5220
gatgatacca tatcagttga aatgaagaag gaagattttg acatttatga tgaggatgaa 5280
aatcagagcc cccgcagctt tcaaaagaaa acacgacact attttattgc tgcagtggag 5340
aggctctggg attatgggat gagtagctcc ccacatgttc taagaaacag ggctcagagt 5400
ggcagtgtcc ctcagttcaa gaaagttgtt ttccaggaat ttactgatgg ctcctttact 5460
cagcccttat accgtggaga actaaatgaa catttgggac tcctggggcc atatataaga 5520
gcagaagttg aagataatat catggtaact ttcagaaatc aggcctctcg tccctattcc 5580
ttctattcta gccttatttc ttatgaggaa gatcagaggc aaggagcaga acctagaaaa 5640
aactttgtca agcctaatga aaccaaaact tacttttgga aagtgcaaca tcatatggca 5700
cccactaaag atgagtttga ctgcaaagcc tgggcttatt tctctgatgt tgacctggaa 5760
aaagatgtgc actcaggcct gattggaccc cttctggtct gccacactaa cacactgaac 5820
cctgctcatg ggagacaagt gacagtacag gaatttgctc tgtttttcac catctttgat 5880
gagaccaaaa gctggtactt cactgaaaat atggaaagaa actgcagggc tccctgcaat 5940
atccagatgg aagatcccac ttttaaagag aattatcgct tccatgcaat caatggctac 6000
ataatggata cactacctgg cttagtaatg gctcaggatc aaaggattcg atggtatctg 6060
ctcagcatgg gcagcaatga aaacatccat tctattcatt tcagtggaca tgtgttcact 6120
gtacgaaaaa aagaggagta taaaatggca ctgtacaatc tctatccagg tgtttttgag 6180
acagtggaaa tgttaccatc caaagctgga atttggcggg tggaatgcct tattggcgag 6240
catctacatg ctgggatgag cacacttttt ctggtgtaca gcaataagtg tcagactccc 6300
ctgggaatgg cttctggaca cattagagat tttcagatta cagcttcagg acaatatgga 6360
cagtgggccc caaagctggc cagacttcat tattccggat caatcaatgc ctggagcacc 6420
aaggagccct tttcttggat caaggtggat ctgttggcac caatgattat tcacggcatc 6480
aagacccagg gtgcccgtca gaagttctcc agcctctaca tctctcagtt tatcatcatg 6540
tatagtcttg atgggaagaa gtggcagact tatcgaggaa attccactgg aaccttaatg 6600
gtcttctttg gcaatgtgga ttcatctggg ataaaacaca atatttttaa ccctccaatt 6660
attgctcgat acatccgttt gcacccaact cattatagca ttcgcagcac tcttcgcatg 6720
gagttgatgg gctgtgattt aaatagttgc agcatgccat tgggaatgga gagtaaagca 6780
atatcagatg cacagattac tgcttcatcc tactttacca atatgtttgc cacctggtct 6840
ccttcaaaag ctcgacttca cctccaaggg aggagtaatg cctggagacc tcaggtgaat 6900
aatccaaaag agtggctgca agtggacttc cagaagacaa tgaaagtcac aggagtaact 6960
actcagggag taaaatctct gcttaccagc atgtatgtga aggagttcct catctccagc 7020
agtcaagatg gccatcagtg gactctcttt tttcagaatg gcaaagtaaa ggtttttcag 7080
ggaaatcaag actccttcac acctgtggtg aactctctag acccaccgtt actgactcgc 7140
taccttcgaa ttcaccccca gagttgggtg caccagattg ccctgaggat ggaggttctg 7200
ggctgcgagg cacaggacct ctactgaggg tggccactgc agcacctgcc actgccgtca 7260
cctctccctc ctcagctcca gggcagtgtc cctccctggc ttgccttcta cctttgtgct 7320
aaatcctagc agacactgcc ttgaagcctc ctgaattaac tatcatcagt cctgcatttc 7380
tttggtgggg ggccaggagg gtgcatccaa tttaacttaa ctcttaccta ttttctgcag 7440
ctgctcccag attactcctt ccttccaata taactaggca aaaagaagtg aggagaaacc 7500
tgcatgaaag cattcttccc tgaaaagtta ggcctctcag agtcaccact tcctctgttg 7560
tagaaaaact atgtgatgaa actttgaaaa agatatttat gatgttaaca tttcaggtta 7620
agcctcatac gtttaaaata aaactctcag ttgtttatta tcctgatcaa gcatggaaca 7680
aagcatgttt caggatcaga tcaatacaat cttggagtca aaaggcaaat catttggaca 7740
atctgcaaaa tggagagaat acaataacta ctacagtaaa gtctgtttct gcttccttac 7800
acatagatat aattatgtta tttagtcatt atgaggggca cattcttatc tccaaaacta 7860
gcattcttaa actgagaatt atagatgggg ttcaagaatc cctaagtccc ctgaaattat 7920
ataaggcatt ctgtataaat gcaaatgtgc atttttctga cgagtgtcca tagatataaa 7980
gccatttggt cttaattctg accaataaaa aaataagtca ggaggatgca attgttgaaa 8040
gctttgaaat aaaataacaa tgtcttcttg aaatttgtga tggccaagaa agaaaatgat 8100
gatgacatta ggcttctaaa ggacatacat ttaatatttc tgtggaaata tgaggaaaat 8160
ccatggttat ctgagatagg agatacaaac tttgtaattc taataatgca ctcagtttac 8220
tctctccctc tactaatttc ctgctgaaaa taacacaaca aaaatgtaac aggggaaatt 8280
atataccgtg actgaaaact agagtcctac ttacatagtt gaaatatcaa ggaggtcaga 8340
agaaaattgg actggtgaaa acagaaaaaa cactccagtc tgccatatca ccacacaata 8400
ggatccccct tcttgccctc cacccccata agattgtgaa gggtttactg ctccttccat 8460
ctgcctgacc ccttcactat gactacacag aatctcctga tagtaaaggg ggctggaggc 8520
aaggataagt tatagagcag ttggaggaag catccaaaga ttgcaaccca gggcaaatgg 8580
aaaacaggag atcctaatat gaaagaaaaa tggatcccaa tctgagaaaa ggcaaaagaa 8640
tggctacttt tttctatgct ggagtatttt ctaataatcc tgcttgaccc ttatctgacc 8700
tctttggaaa ctataacata gctgtcacag tatagtcaca atccacaaat gatgcaggtg 8760
caaatggttt atagccctgt gaagttctta aagtttagag gctaacttac agaaatgaat 8820
aagttgtttt gttttatagc ccggtagagg agttaacccc aaaggtgata tggttttatt 8880
tcctgttatg tttaacttga taatcttatt ttggcattct tttcccattg actatataca 8940
tctctatttc tcaaatgttc atggaactag ctcttttatt ttcctgctgg tttcttcagt 9000
aatgagttaa ataaaacatt gacacatac 9029
<210>2
<211>2804
<212>DNA
<213>Homo sapiens
<400>2
accactttca caatctgcta gcaaaggtta tgcagcgcgt gaacatgatc atggcagaat 60
caccaggcct catcaccatc tgccttttag gatatctact cagtgctgaa tgtacagttt 120
ttcttgatca tgaaaacgcc aacaaaattc tgaatcggcc aaagaggtat aattcaggta 180
aattggaaga gtttgttcaa gggaaccttg agagagaatg tatggaagaa aagtgtagtt 240
ttgaagaagc acgagaagtt tttgaaaaca ctgaaagaac aactgaattt tggaagcagt 300
atgttgatgg agatcagtgt gagtccaatc catgtttaaa tggcggcagt tgcaaggatg 360
acattaattc ctatgaatgt tggtgtccct ttggatttga aggaaagaac tgtgaattag 420
atgtaacatg taacattaag aatggcagat gcgagcagtt ttgtaaaaat agtgctgata 480
acaaggtggt ttgctcctgt actgagggat atcgacttgc agaaaaccag aagtcctgtg 540
aaccagcagt gccatttcca tgtggaagag tttctgtttc acaaacttct aagctcaccc 600
gtgctgagac tgtttttcct gatgtggact atgtaaattc tactgaagct gaaaccattt 660
tggataacat cactcaaagc acccaatcat ttaatgactt cactcgggtt gttggtggag 720
aagatgccaa accaggtcaa ttcccttggc aggttgtttt gaatggtaaa gttgatgcat 780
tctgtggagg ctctatcgtt aatgaaaaat ggattgtaac tgctgcccac tgtgttgaaa 840
ctggtgttaa aattacagtt gtcgcaggtg aacataatat tgaggagaca gaacatacag 900
agcaaaagcg aaatgtgatt cgaattattc ctcaccacaa ctacaatgca gctattaata 960
agtacaacca tgacattgcc cttctggaac tggacgaacc cttagtgcta aacagctacg 1020
ttacacctat ttgcattgct gacaaggaat acacgaacat cttcctcaaa tttggatctg 1080
gctatgtaag tggctgggga agagtcttcc acaaagggag atcagcttta gttcttcagt 1140
accttagagt tccacttgtt gaccgagcca catgtcttcg atctacaaag ttcaccatct 1200
ataacaacat gttctgtgct ggcttccatg aaggaggtag agattcatgt caaggagata 1260
gtgggggacc ccatgttact gaagtggaag ggaccagttt cttaactgga attattagct 1320
ggggtgaaga gtgtgcaatg aaaggcaaat atggaatata taccaaggta tcccggtatg 1380
tcaactggat taaggaaaaa acaaagctca cttaatgaaa gatggatttc caaggttaat 1440
tcattggaat tgaaaattaa cagggcctct cactaactaa tcactttccc atcttttgtt 1500
agatttgaat atatacattc tatgatcatt gctttttctc tttacagggg agaatttcat 1560
attttacctg agcaaattga ttagaaaatg gaaccactag aggaatataa tgtgttagga 1620
aattacagtc atttctaagg gcccagccct tgacaaaatt gtgaagttaa attctccact 1680
ctgtccatca gatactatgg ttctccacta tggcaactaa ctcactcaat tttccctcct 1740
tagcagcatt ccatcttccc gatcttcttt gcttctccaa ccaaaacatc aatgtttatt 1800
agttctgtat acagtacagg atctttggtc tactctatca caaggccagt accacactca 1860
tgaagaaaga acacaggagt agctgagagg ctaaaactca tcaaaaacac tactcctttt 1920
cctctaccct attcctcaat cttttacctt ttccaaatcc caatccccaa atcagttttt 1980
ctctttctta ctccctctct cccttttacc ctccatggtc gttaaaggag agatggggag 2040
catcattctg ttatacttct gtacacagtt atacatgtct atcaaaccca gacttgcttc 2100
catagtggag acttgctttt cagaacatag ggatgaagta aggtgcctga aaagtttggg 2160
ggaaaagttt ctttcagaga gttaagttat tttatatata taatatatat ataaaatata 2220
taatatacaa tataaatata tagtgtgtgt gtgtatgcgt gtgtgtagac acacacgcat 2280
acacacatat aatggaagca ataagccatt ctaagagctt gtatggttat ggaggtctga 2340
ctaggcatga tttcacgaag gcaagattgg catatcattg taactaaaaa agctgacatt 2400
gacccagaca tattgtactc tttctaaaaa taataataat aatgctaaca gaaagaagag 2460
aaccgttcgt ttgcaatcta cagctagtag agactttgag gaagaattca acagtgtgtc 2520
ttcagcagtg ttcagagcca agcaagaagt tgaagttgcc tagaccagag gacataagta 2580
tcatgtctcc rttaactagc ataccccgaa gtggagaagg gtgcagcagg ctcaaaggca 2640
taagtcattc caatcagcca actaagttgt ccttttctgg tttcgtgttc accatggaac 2700
attttgatta tagttaatcc ttctatcttg aatcttctag agagttgctg accaactgac 2760
gtatgtttcc ctttgtgaat taataaactg gtgttctggt tcat 2804
<210>3
<211>1286
<212>DNA
<213>Hepatitis B virus
<400>3
gtcgagtata aaaacaatga gatctttgtt gatcttggtt ttgtgtttct tgccattggc 60
tgctttgggt aaggttcgac aaggcatggg aggttggtct tccaaacctc gataaggcat 120
ggggacgaat ctttctgttc ccaatcctct gggattcttt cccgatcacc agttggaccc 180
tgcgttcgga gccaactcaa acaatccaga ttgggacttc aaccccaaca aggatcaatg 240
gccagaggca aatcaggtag gagcgggagc attcgggcca gggttcaccc caccacacgg 300
cggtcttttg gggtggagcc ctcaggctca gggcatattg acaacagtgc cagcagcacc 360
tcctcctgcc tccaccaatc ggcagtcagg aagacagcct actcccatct ctccacctct 420
aagagacagt catcctcagg ccatgcagtg gaattccaca acattccacc aagctctgct 480
agatcccaga gtgaggggcc tatattttcc tgctggtggc tccagttccg gaacagtaaa 540
ccctgttccg actactgcct cacccatatc tggggaccct gcaccgaaca tggagaacac 600
aacatcagga ttcctaggac ccctgctcgt gttacaggcg gggtttttct tgttgacaag 660
aatcctcaca ataccacaga gtctagactc gtggtggact tctctcaatt ttctaggggg 720
agcacccacg tgtcctggcc aaaattcgca gtccccaacc tccaatcact caccaacctc 780
ttgtcctcca atttgtcctg gctatcgctg gatgtgtctg cggcgtttta tcatattcct 840
cttcatcctg ctgctatgcc tcatcttctt gttggttctt ctggactacc aaggtatgtt 900
gcccgtttgt cctctacttc caggaacatc aaccaccagc acggggccat gcaagacctg 960
cacgattcct gctcaaggaa cctctatgtt tccctcttgt tgctgtacaa aaccttcgga 1020
cggaaactgc acttgtattc ccatcccatc atcctgggct ttcgcaagat tcctatggga 1080
gtgggcctca gtccgtttct cctggctcag tttactagtg ccatttgttc agtggttcgt 1140
agggctttcc cccactgttt ggctttcagt tatatggatg atgtggtatt gggggccaag 1200
tctgtacaac atcttgagtc cctttttacc tctattacca attttctttt gtctttgggt 1260
atacatttaa attgaattga attgaa 1286
<210>4
<211>2351
<212>PRT
<213>Homo sapiens
<400>4
Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe
1 5 10 15
Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser
20 25 30
Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg
35 40 45
Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val
50 55 60
Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile
65 70 75 80
Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln
85 90 95
Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser
100 105 110
His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser
115 120 125
Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp
130 135 140
Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu
145 150 155 160
Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser
165 170 175
Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile
180 185 190
Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr
195 200 205
Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly
210 215 220
Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp
225 230 235 240
Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr
245 250 255
Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val
260 265 270
Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile
275 280 285
Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser
290 295 300
Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met
305 310 315 320
Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His
325 330 335
Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro
340 345 350
Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp
355 360 365
Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser
370 375 380
Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr
385 390 395 400
Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro
405 410 415
Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn
420 425 430
Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met
435 440 445
Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu
450 455 460
Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu
465 470 475 480
Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro
485 490 495
His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys
500 505 510
Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe
515 520 525
Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp
530 535 540
Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg
545 550 555 560
Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu
565 570 575
Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val
580 585 590
Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu
595 600 605
Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp
610 615 620
Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val
625 630 635 640
Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp
645 650 655
Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe
660 665 670
Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr
675 680 685
Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro
690 695 700
Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly
705 710 715 720
Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp
725 730 735
Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys
740 745 750
Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro
755 760 765
Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp
770 775 780
Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys
785 790 795 800
Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser
805 810 815
Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr
820 825 830
Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn
835 840 845
Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly
850 855 860
Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu
865 870 875 880
Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lya Lys Leu Asp Phe Lys
885 890 895
Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn
900 905 910
Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met
915 920 925
Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys
930 935 940
Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu
945 950 955 960
Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu
965 970 975
Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe
980 985 990
Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala
995 1000 1005
Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser
1010 1015 1020
Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser
1025 1030 1035
Leu Leu Ile Glu Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu
1040 1045 1050
Ser Asp Thr Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg
1055 1060 1065
Met Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met
1070 1075 1080
Ser Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln
1085 1090 1095
Lys Lys Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met
1100 1105 1110
Ser Phe Phe Lys Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile
1115 1120 1125
Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro
1130 1135 1140
Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu
1145 1150 1155
Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys
1160 1165 1170
Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro
1175 1180 1185
Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu
1190 1195 1200
Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu
1205 1210 1215
Lys Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile
1220 1225 1230
His Thr Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu
1235 1240 1245
Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr
1250 1255 1260
Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn
1265 1270 1275
Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu Glu
1280 1285 1290
Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu
1295 1300 1305
Lys Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln
1310 1315 1320
Asn Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg
1325 1330 1335
Leu Pro Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp
1340 1345 1350
Asp Thr Ser Thr Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro
1355 1360 1365
Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala
1370 1375 1380
Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser
1385 1390 1395
Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser
1400 1405 1410
Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe
1415 1420 1425
Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys
1430 1435 1440
Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys
1445 1450 1455
Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly
1460 1465 1470
Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser
1475 1480 1485
Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp
1490 1495 1500
Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys Val His
1505 1510 1515
Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser
1520 1525 1530
Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr
1535 1540 1545
Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val
1550 1555 1560
Pro Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser
1565 1570 1575
Lys Leu Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln
1580 1585 1590
Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys
1595 1600 1605
Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys
1610 1615 1620
Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys
1625 1630 1635
Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg
1640 1645 1650
Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu
1655 1660 1665
Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr
1670 1675 1680
Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile
1685 1690 1695
Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys
1700 1705 1710
Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr
1715 1720 1725
Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser
1730 1735 1740
Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr
1745 1750 1755
Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu
1760 1765 1770
His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp
1775 1780 1785
Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser
1790 1795 1800
Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly
1805 1810 1815
Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr
1820 1825 1830
Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu
1835 l840 1845
Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu
1850 1855 1860
Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His
1865 1870 1875
Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln
1880 1885 1890
Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp
1895 1900 1905
Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn
1910 1915 1920
Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His
1925 1930 1935
Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met
1940 1945 1950
Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser
1955 1960 1965
Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr
1970 1975 1980
Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr
1985 1990 1995
Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly
2000 2005 2010
Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly
2015 2020 2025
Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro
2030 2035 2040
Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala
2045 2050 2055
Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His
2060 2065 2070
Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser
2075 2080 2085
Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile
2090 2095 2100
Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser
2105 2110 2115
Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr
2120 2125 2130
Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn
2135 2140 2145
Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile
2150 2155 2160
Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg
2165 2170 2175
Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys
2180 2185 2190
Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln
2195 2200 2205
Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser
2210 2215 2220
Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp
2225 2230 2235
Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe
2240 2245 2250
Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys
2255 2260 2265
Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser
2270 2275 2280
Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys
2285 2290 2295
Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val
2300 2305 2310
Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His
2315 2320 2325
Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu
2330 2335 2340
Gly Cys Glu Ala Gln Asp Leu Tyr
2345 2350
<210>5
<211>461
<212>PRT
<213>Homo sapiens
<400>5
Met Gln Arg Val Asn Met Ile Met Ala Glu Ser Pro Gly Leu Ile Thr
1 5 10 15
Ile Cys Leu Leu Gly Tyr Leu Leu Ser Ala Glu Cys Thr Val Phe Leu
20 25 30
Asp His Glu Asn Ala Asn Lys Ile Leu Asn Arg Pro Lys Arg Tyr Asn
35 40 45
Ser Gly Lys Leu Glu Glu Phe Val Gln Gly Asn Leu Glu Arg Glu Cys
50 55 60
Met Glu Glu Lys Cys Ser Phe Glu Glu Ala Arg Glu Val Phe Glu Asn
65 70 75 80
Thr Glu Arg Thr Thr Glu Phe Trp Lys Gln Tyr Val Asp Gly Asp Gln
85 90 95
Cys Glu Ser Asn Pro Cys Leu Asn Gly Gly Ser Cys Lys Asp Asp Ile
100 105 110
Asn Ser Tyr Glu Cys Trp Cys Pro Phe Gly Phe Glu Gly Lys Asn Cys
115 120 125
Glu Leu Asp Val Thr Cys Asn Ile Lys Asn Gly Arg Cys Glu Gln Phe
130 135 140
Cys Lys Asn Ser Ala Asp Asn Lys Val Val Cys Ser Cys Thr Glu Gly
145 150 155 160
Tyr Arg Leu Ala Glu Asn Gln Lys Ser Cys Glu Pro Ala Val Pro Phe
165 170 175
Pro Cys Gly Arg Val Ser Val Ser Gln Thr Ser Lys Leu Thr Arg Ala
180 185 190
Glu Thr Val Phe Pro Asp Val Asp Tyr Val Asn Ser Thr Glu Ala Glu
195 200 205
Thr Ile Leu Asp Asn Ile Thr Gln Ser Thr Gln Ser Phe Asn Asp Phe
210 215 220
Thr Arg Val Val Gly Gly Glu Asp Ala Lys Pro Gly Gln Phe Pro Trp
225 230 235 240
Gln Val Val Leu Asn Gly Lys Val Asp Ala Phe Cys Gly Gly Ser Ile
245 250 255
Val Asn Glu Lys Trp Ile Val Thr Ala Ala His Cys Val Glu Thr Gly
260 265 270
Val Lys Ile Thr Val Val Ala Gly Glu His Asn Ile Glu Glu Thr Glu
275 280 285
His Thr Glu Gln Lys Arg Asn Val Ile Arg Ile Ile Pro His His Asn
290 295 300
Tyr Asn Ala Ala Ile Asn Lys Tyr Asn His Asp Ile Ala Leu Leu Glu
305 310 315 320
Leu Asp Glu Pro Leu Val Leu Asn Ser Tyr Val Thr Pro Ile Cys Ile
325 330 335
Ala Asp Lys Glu Tyr Thr Asn Ile Phe Leu Lys Phe Gly Ser Gly Tyr
340 345 350
Val Ser Gly Trp Gly Arg Val Phe His Lys Gly Arg Ser Ala Leu Val
355 360 365
Leu Gln Tyr Leu Arg Val Pro Leu Val Asp Arg Ala Thr Cys Leu Arg
370 375 380
Ser Thr Lys Phe Thr Ile Tyr Asn Asn Met Phe Cys Ala Gly Phe His
385 390 395 400
Glu Gly Gly Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro His Val
405 410 415
Thr Glu Val Glu Gly Thr Ser Phe Leu Thr Gly Ile Ile Ser Trp Gly
420 425 430
Glu Glu Cys Ala Met Lys Gly Lys Tyr Gly Ile Tyr Thr Lys Val Ser
435 440 445
Arg Tyr Val Asn Trp Ile Lys Glu Lys Thr Lys Leu Thr
450 455 460
Claims (19)
1.一个非病毒的球状载体,其特征在于:它由下列部份组成:
一个在球状体表面暴露着乙肝病毒外壳(envelope)env蛋白的球状膜和;
一个核酸表达结构,包含完整的第八因子或第九因子的表达序列和在肝细胞中有功能的启动子序列。
2.依据权利要求1所述之该球状载体,其中的env蛋白含有突变以降低其抗原性。
3.依据权利要求1所述之该球状载体,其中的表达结构为DNA。
4.依据权利要求1所述之该球状载体,其中的表达结构为双链质粒DNA。
5.依据权利要求1所述之该球状载体,其中的表达结构为RNA。
6.依据权利要求1所述之该球状载体,其中的启动子为非组织特异的启动子。
7.依据权利要求6所述之该球状载体,其中的非组织特异启动子可来自以下来源:细胞肥大病毒(cytomegalovirus)启动子,Rous肉瘤启动子,辅酶Q(Ubiquitin)启动子,鸡β-肌动蛋白(β-actin)启动子和延长因子(elongation factor)(EF)-1α启动子。
8.依据权利要求1所述之该球状载体,其中的启动子为肝脏特异的启动子。
9.依据权利要求8所述之该球状载体,其中的肝脏特异启动子来自以下来源:α-胚胎蛋白启动子,球蛋白启动子,α1-微球蛋白和白蛋白。
10.依据权利要求1所述之该球状载体,其中的表达结构包括来自腺病毒相关病毒的反向(Inverted)末端序列(AAV-ITR)。
11.依据权利要求1所述之该球状载体,其中的表达结构包括真核细胞的转座子(transposon)和转座子酶(transposase)序列。
12.依据权利要求1所述之该球状载体,其中的表达结构包括第八因子的表达序列。
13.依据权利要求12所述之该球状载体,其中的第八因子包含无意(silent)突变以增加其表达能力。
14.依据权利要求1所述之该球状载体,其中的表达结构包括第九因子的表达序列。
15.一个非病毒的球状载体,其特征在于:它由以下部份组成:
一个在球状体表面暴露着乙肝病毒外壳(envelope)env蛋白的球状膜和;
一个包含了完整第八因子或第九因子的蛋白质。
16.依据权利要求15所述之该球状载体,其中的env蛋白包含了突变以降低其抗原性。
17.一个治疗血友病的方法,其特征在于:它由以下部份组成:
将由表面暴露着乙肝病毒env蛋白球状膜的非病毒球状载体输入具有血友病的个体的血循环中以及;
一个由完整的第八因子或第九因子表达序列和在肝细胞中有功能的启动子序列组成的核酸表达结构,以及;
监测该病人疾病改善的情况。
18.依据权利要求17所述之该方法,其中输入血循环的方法通过静脉注射。
19.依据权利要求17所述之该方法,其中输入血循环的方法通过注射入肝动脉或腹腔动脉(hepatic or portal artery)。
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US28631401P | 2001-04-25 | 2001-04-25 | |
US60/286,314 | 2001-04-25 |
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CN1520259A true CN1520259A (zh) | 2004-08-11 |
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CNA028107349A Pending CN1520259A (zh) | 2001-04-25 | 2002-04-25 | 利用球状载体将第八因子/第九因子进行肝脏基因转移治疗血友病的方法 |
Country Status (4)
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US (1) | US20030044982A1 (zh) |
CN (1) | CN1520259A (zh) |
AU (1) | AU2002254731A1 (zh) |
WO (1) | WO2002086091A2 (zh) |
Cited By (1)
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CN102573890A (zh) * | 2009-07-31 | 2012-07-11 | 拜耳医药保健有限公司 | 经过修饰的因子ix多肽及其用途 |
Families Citing this family (2)
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JP2004277355A (ja) * | 2003-03-17 | 2004-10-07 | Beacle Inc | 血友病治療用薬剤及びそれを用いた血友病治療方法 |
JP5685529B2 (ja) | 2008-06-13 | 2015-03-18 | プロイェクト、デ、ビオメディシナ、シーマ、ソシエダッド、リミターダProyecto De Biomedicina Cima, S.L. | 生物学的活性を有する化合物の投与のための複合体 |
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PL168787B1 (pl) * | 1989-08-03 | 1996-04-30 | Smithkline Beecham Biolog | Sposób wytwarzania czastki kompozytowej PL |
EP0746625B1 (en) * | 1992-11-09 | 2004-03-31 | THE UNITED STATES GOVERNMENT as represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES | Targetable vector particles |
EP0830381A4 (en) * | 1995-06-09 | 2000-11-22 | William N Drohan | CHITINE HYDROGELS, PRODUCTION METHOD AND USE |
US6221349B1 (en) * | 1998-10-20 | 2001-04-24 | Avigen, Inc. | Adeno-associated vectors for expression of factor VIII by target cells |
US6135942A (en) * | 1998-11-30 | 2000-10-24 | Leptin; Maria | Nucleic acids proteins of a D. melanogaster insulin-like gene and uses thereof |
-
2002
- 2002-04-25 US US10/132,829 patent/US20030044982A1/en not_active Abandoned
- 2002-04-25 AU AU2002254731A patent/AU2002254731A1/en not_active Abandoned
- 2002-04-25 WO PCT/US2002/013164 patent/WO2002086091A2/en not_active Application Discontinuation
- 2002-04-25 CN CNA028107349A patent/CN1520259A/zh active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102573890A (zh) * | 2009-07-31 | 2012-07-11 | 拜耳医药保健有限公司 | 经过修饰的因子ix多肽及其用途 |
Also Published As
Publication number | Publication date |
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WO2002086091A2 (en) | 2002-10-31 |
AU2002254731A1 (en) | 2002-11-05 |
WO2002086091A3 (en) | 2003-02-27 |
US20030044982A1 (en) | 2003-03-06 |
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