CN101528916B - 规模可调的aav生产方法 - Google Patents
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Abstract
本发明描述一种无需细胞裂解而产生AAV的方法。该方法包括从上清液中获得AAV。如果AAV的衣壳具有肝素结合位点，则该方法包括修饰AAV衣壳和/或培养条件，来消除AAV肝素结合位点和细胞之间的结合，从而使AAV进入上清液，即培养基。这样，本发明的方法提供高得率的AAV上清液，与使用细胞裂解步骤产生的AAV相比，它能使与细胞膜和胞内物质分离的产物纯度更高。
Description
针对联邦资助研究或开发的声明
本申请描述的工作至少部分由国立健康研究院资助，NHLBI资助号是P01-HL-059407。美国政府拥有本发明的某些权利。
发明背景
本发明描述一种新的获得和生产AAV的方法。
腺病毒相关病毒(AAV)是细小病毒家族的一员，它无包膜，是二十面体病毒，具有4.7千碱基对(kb)至6kb的单链线性DNA基因组。AAV被归于依赖病毒属，因为它被发现是纯化的腺病毒群中的污染物。AAV的生活周期包括潜伏期(感染后AAV基因组整合到宿主基因组)和感染期的(腺病毒或者单纯疱疹病毒感染后，整合的AAV基因组接着被拯救、复制以及包装成能感染的病毒)。AAV具有非病原性，宽寄主范围(包括非分裂细胞)的传染性，以及能够整合的性质，是吸引人的运输载体。
描述了多种不同的AAV序列和从组织中分离它们的方法。已描述来自猿或人组织来源的AAV 1-6、AA V7、AAV9和AA V9及其他AAV序列。参见例如，国际专利公开WO 02/33269，WO 02/386122(AAV8)，和国际专利公开WO 2005/033321。于是，出现了这样一种趋势，即不再严格按照血清学交叉反应性(血清型)来定义AAV。最近的一些文献按照种系发生的关联性定义AAV之间的关系，并提出了称为“亚型(clades)”的分组。参见例如，Gao等人，J Virol 78(12)：6381-6388(2004年6月)；国际专利公开WO2005/033321。
AAV2是细胞伴随病毒(cell-associated)，因此认为它主要位于其生产细胞中，如今生产AAV的方法主要基于这样的观点。所以，大多数现有AAV生产策略是从生产细胞系的细胞沉淀中分离载体颗粒。这些策略都采用某些方法来将载体从细胞沉淀中释放出来，例如采用超声处理、酶法、化学或物理裂解。不幸地是，这会让所有的胞内蛋白质和碎片释放到病毒收获物中。因此还需要后续的纯化过程。因为生产和纯化的效率都比较低，所以就需要在开始时采用大量的生产细胞。
因此，需要高效的生产和纯化AAV的方法。
发明概述
本发明提供生产AAV的方法，不需要终止病毒生产细胞的培养。该方法包括收获释放到上清液中的AAV，无需收集细胞沉淀或裂解细胞。在实施方案之一中，所述方法包括修饰或改造AAV衣壳、细胞和/或培养条件，以此大量减少或消除AAV肝素结合位点与生产细胞之间的结合，从而使AAV进入上清液即培养基。这样，本发明的方法提供包含高得率AAV的上清液，与采用细胞收集和/或细胞裂解步骤的AAV生产方法相比，它能使与细胞膜和胞内物质分离的产物纯度更高。
本发明的技术可用于高效且不同规模的AAV生产，从而显著提高经济和时间成本效率。本发明技术能进行小规模的AAV生产，还可以在商业上用于研究用的万有试剂盒(all-inclusive kit)。由于可以多次从上清液中收获AAV，采用连续的系统或生物反应器就能够使得病毒颗粒的产量达到临床应用或制药应用所需，由此摆脱细胞基料对产能的限制。任选地与采用不含或低含量血清或蛋白质的生长培养基结合，则纯化可大大简化。与现有生产方法和/或存在大量胞内物质的情况相比，本本发明技术能够与更高效的纯化和浓缩方法联合使用。
从本发明的详细说明中还可以看到本发明的其他优势。
附图简要说明
图1显示血清存在(S)或不存在(SF)的条件下，硫酸乙酰肝素蛋白多糖(HSPG)作用于AAV2和AAV2HSPG的效果，以AAV2/8的生产为阳性对照。
图2显示阳性对照AAV2/8的上清液中的(I)、松散细胞伴随的(looselycell associated)(II)和紧密细胞伴随的(tightly cell associated)(III)耐DNA酶(DNase-resistant)AAV颗粒(drp)分配的定量。
图2B-2E显示血清存在(血清或S)或不存在(无血清或SF)的条件下，AAV2和AAV2HSPG-的上述分配。
图3显示单个15cm培养皿转染所得AAV分离株得率。各非肝素结合分离株的总得率都高于2×1012GC，最高达1×1013GC。由图可见，AAV2和分离株hu.51结合肝素，因而其产量受到了限制。
图4是柱状图，显示多种AAV免疫的T细胞活化结果。Balb/c小鼠用1×1011GC的AAV2/6、AAV2/6.1、AAV2/6.2、AAV2/6.1.2、AAV2/1或AAV2载体免疫。13天后，每组取3只小鼠的脾细胞并混合。进行ELISPOT试验，用Balb/c AAV表位IPQYGYLTL[SEQ ID NO：1]体外刺激等量脾细胞。
图5是柱状图，显示在293细胞培养系统中，所示各AAV病毒载体三重瞬时转染后，各载体释放到上清液的分配比例。
发明的详细说明
本发明提供一种无需细胞裂解的生产AAV的方法。该方法包括从病毒生产培养物的上清液中收获AAV。
对于不表现出与硫酸乙酰肝素蛋白多糖或肝素的亲合力的AAVs(大多数AAV具有这种性质)，大部分耐DNA酶的感染性颗粒在培养物上清液中或者仅松弛地与细胞相伴。未有诱导产生的病毒性裂解、渗透压裂解(osmolytic)或其他类型的细胞裂解。
本发明提供规模可调的生产AAV的技术，AAV可用于多种基因转移和/或疫苗应用。当本发明方法与低蛋白污染或无蛋白污染的培养基联合使用进行收获时，还能显著降低对纯化步骤的压力。这种生产方法可与合适的纯化和浓缩方法联合使用，包括，例如用于纯化和浓缩的色谱法、过滤或沉淀。
由于目前的AAV生产策略使用细胞沉淀作为分离颗粒的基料，这些方法可称为一种可反复重来的过程，但无法实现连续的收集。
与目前的方法不同，在实施方式之一中，本发明方法以上清液作为多种AAV的主要来源。这允许对同一批生产细胞进行重复或连续的收获，从而大量生产AAV以满足临床或临床前研究或治疗性应用所需。在目前收集细胞沉淀随后进行纯化的方法中，需要大量颗粒才能提供可用的病毒滴度。所以，存在一个阈值，低于该阀值，回收有用数量的颗粒在技术上将是不可行的。在实施方式之一中，某种AAV载体能由生产该病毒的细胞分泌出至少约10％耐DNA酶的载体颗粒或基因组(drp vg)。这样的drp vg代表了包装在AAV衣壳中的基因组序列(例如，小基因、表达盒和/或AAV核酸序列)。在另一个实施方案中，某种AAV载体分泌至少约20％drp vg。在另一个实施方案中，某种AAV载体分泌至少约40％drp vg。基于本发明更高效的生产策略，能够调节生产规模来满足对少量和大量病毒颗粒的需求。由此，病毒生产可以根据预期需求量来定制，而无需细胞裂解或终止细胞培养。
例如，已发现，在一种基于293细胞的三重转染生产系统中，AAV8载体的病毒颗粒中超过40％(平均值)分泌进入上清液。还发现：基于AAV7和rh8R的其他一些载体的分泌比率也在这一范围内。还发现，在这一系统中，有些载体的病毒颗粒有超过30％(平均值)分泌进入上清液中，例如，AAV1[衣壳蛋白见SEQ ID NO：2]、AAV6[AAV6衣壳蛋白质见SEQ ID NO：3]、AAV6.1[SEQ ID NO：3，该衣壳蛋白质含K531E改变]、AAV6.1.2[SEQID NO：3，K531E，F 129L]、rh.32.33[衣壳蛋白质见SEQ ID NO：4]、rh.10[衣壳蛋白质见SEQ ID NO：5]和rh64R1[rh64衣壳蛋白质见SEQ ID NO：6，R697W]和rh8R[rh8衣壳蛋白质见SEQ ID NO：7，D531E]。在另一个实施例中发现，在上述系统中，三重转染生产中，有些AAV载体的病毒颗粒中超过20％(平均值)分泌进入上清液。还发现，另有一些AAV载体，例如基于AAV9[衣壳蛋白质见SEQ ID NO：8]的载体，病毒颗粒中超过10％分泌进入上述系统的上清液中。其他例子还包括病毒颗粒中超过10％分泌进入上清液的AAVs，它们也可用于本发明的方法。在实施方案之一中，据此产生的载体来自于天然从产生该载体的细胞中分泌出来的AAVs。
在另一个实施方案中，AAVs接受了令其能被分泌的修饰。在实施方案之一中，发明人发现：具有肝素结合域且转导(感染)能力被肝素封闭(blocked)的AAV无法测得其分泌。这种AAV的例子是AAV2[衣壳蛋白质见SEQ ID NO：9](它在生产过程中主要呈细胞伴随形式)和AAV3[衣壳蛋白质见SEQID NO：10]。因此，在一个实施方案中，本发明方法包括修饰或改造AAV衣壳、细胞、和/或培养条件来显著减少或消除AAV肝素结合位点与该生产者细胞之间的结合，从而使AAV进入上清液即培养基。
本发明的方法提供包含高得率AAV的上清液，与细胞裂解后回收AAV的方法相比，能更高纯度地使产物与细胞膜、蛋白质和胞内物质分离。在一个实施方案中，本发明从没有进行溶菌的上清液开始，因此简化了后续纯化。在正常培养物的上清液中发现含量极其有限的细胞碎片，蛋白质污染物显著减少。在一个实施方案中，使用无血清培养基以避免由生长培养基带入的血清或其他蛋白质的污染。
一方面，本发明提供在病毒生产培养物中生产AAV的方法。多种AAV的序列已被公开。参见例如，AAV 1(US专利号6,759,237)、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、rh32.33、rh.10、hu.11，其他人源或非人源AAV，参见例如，国际专利公开WO 02/33269、WO02/386122(AAV8)和GenBank，以及校正了单碱基误差(singleton errors)的序列，例如，AAV6.2、AAV6.1、AAV6.1.2、rh64R1和rh8R[参见例如，WO2006/110689，2006年10月19日公开)。或者，可如本发明所述对其它AAV序列，包括本领域技术人员使用常规技术[参见，例如，国际专利公开号WO 2005/033321和GenBank]或其它方式鉴定的序列，进行修饰。
某些AAV序列天然缺失上述肝素结合位点。对于肝素结合位点缺失的AAV，例如，AAV8[衣壳蛋白质见SEQ ID NO：11]，不需要修饰或改造AAV序列、细胞或培养基。利用各种AAV结合试验和结合AAV的肝素及其部分，可以方便地测定AAV衣壳结合肝素的能力。而且，本领域技术人员能够方便地测定肝素封闭AAV感染/转导的能力。例如，确定肝素封闭AAV感染/转导能力的适当实验可见于C.Halbert et al，J Virol，75(14)：6615-6624(July 2001)and C.E.Walsh and H.Chao，Haemophilia，8(Suppl.2)，p.60-67(2002)。
其他AAV序列，例如AAV6，具有肝素结合位点，但是AAV6的感染能力因肝素的存在被部分抑制，而不是封闭(blocked)。AAV6 vp1衣壳序列， 据记载，具有调节肝素结合的单个氨基酸残基，即第531位的天然赖氨酸[SEQ ID NO：3]。[AAV6序列记载于国际专利申请PCT/US06/13375，根据该国际申请中的方案编号(参见表格等)。在这种情况下，不需要修饰该AAV序列，因为发现它只与细胞松散伴随。
在一个实施方案中，对于具有肝素结合位点且感染/转染细胞的能力被肝素封闭的AAV，本发明对该AAV进行修饰，以减少或消除肝素结合，从而增加分泌到上清液中的病毒颗粒的数量。在一个实施方案中，肝素结合域是Arg-Xaa-Xaa-Arg(RxxR)[SEQ ID NO：12]基序，与记载中AAV2中的一样(即，AAV2vp 1衣壳蛋白质的585到588位氨基酸，SEQ ID NO：9，Kern等人，J Virol 77：11072-81；Opie等人，J Virol 77：6995-7006(基于WO02/33269中的编号)]。Xaa表示任意氨基酸。发明人已鉴定到含有RxxR基序的其它AAV衣壳，包括几种亚型B的AAV。这种具有RxxR基序的AAV衣壳的例子包括hu.51[SEQ ID NO：13]、hu.34[SEQ ID NO：14]、hu.35[SEQID NO：15]、hu.45[SEQ ID NO：16]，和hu.47[SEQ ID NO：17]。本领域技术人员能够从已知AAV序列中方便地鉴别到其他具有RxxR结构域的AAV。另外，使用本领域技术人员已知的技术可以容易地鉴定到其他肝素结合位点。在另一个实施例中，AAV3结合肝素，但它不含RxxR结构域。
发明人已发现：对肝素结合序列的一个或多个氨基酸残基进行改变从而引入非保守性氨基酸变化，不仅能够消除(ablate)肝素结合，还能够显著降低T细胞活化。这正是本申请人的另一申请-“具有降低的衣壳免疫原性的修饰AAV载体及其用途”-的主题，它要求美国临时申请号60/795,965(申请日为2006年4月28日)的优先权，此处将其引为参考。
在一个实施方案中，本发明提供在病毒培养物中生产AAV的方法，其中，所述AAV接受了去肝素结合域修饰。
在一个实施方案中，采用定点突变技术修饰编码AAV衣壳肝素结合位点的核酸序列，其中，对负责调节肝素结合的一个或多个氨基酸残基密码子进行改变，从而在编码氨基酸中制造非保守性改变。非保守氨基酸改变的例子包括：例如，用不同化学结构(性质)的氨基酸去取代某氨基酸，由此影响到蛋白质功能。下表说明最常见的氨基酸及其性质。
| 氨基酸 | 缩写 | 疏水性 | 极性 | 电荷 | 芳香族 或脂肪 族 | 密码子 |
| 丙氨酸 | Ala，A | X | - | - | - | GCU、GCC、GCA、 GCG |
| 半胱氨酸 | Cys，C | X | - | - | - | UGU、UGC |
| 天冬氨酸 | Asp，D | - | X | 负电 | - | GAU、GAC |
| 谷氨酸 | Glu，E | - | X | 负电 | - | GAA、GAG |
| 苯丙氨酸 | Phe，F | X | - | - | 芳香族 | UUU、UUC |
| 甘氨酸 | Gly，G | X | - | - | - | GGU、GGC、GGA、 GGG |
| 组氨酸 | His，H | - | X | 正电 | 芳香族 | CAU、CAC |
| 异亮氨酸 | Ile，I | X | - | - | 脂肪族 | AUU、AUC、AUA |
| 赖氨酸 | Lys，K | - | X | 正电 | - | AAA、AAG |
| 亮氨酸 | Leu，L | X | - | - | 脂肪族 | UUA、UUG、CUU、 CUA、CUC、CUG |
| 甲硫氨酸 | Met，M | X | - | - | - | AUG |
| 天冬酰胺 | Asn，N | - | X | - | - | AAU、AAC |
| 脯氨酸 | Pro，P | X | - | - | - | CCU、CCC、CCA、 CCG |
| 谷氨酰胺 | Gln，Q | - | X | - | - | CAA、CAG |
| 精氨酸 | Arg，R | - | X | 正电 | - | CGU、CGC、CGA、 CGG、AGA、AGG |
| 丝氨酸 | Ser，S | - | X | - | - | UCU、UCC、UCA、 UCG、AGU、AGC |
| 苏氨酸 | Thr，T | X | X | - | - | ACU、ACC、ACA、 ACG |
| 缬氨酸 | Val，V | X | - | - | 脂肪族 | GUU、GUC、GUA、 GUG |
[0037]
| 色氨酸 | Trp，W | X | - | - | 芳香族 | UGG |
| 酪氨酸 | Tyr，Y | X | X | - | 芳香族 | UAU、UAC |
例如，用定点突变技术修饰编码肝素结合位点的核酸序列。例如，在RxxR基序[SEQ ID NO：3]中，改变基序的首个和/或末尾精氨酸的密码子，将其中之一(或两个)改变为非保守氨基酸。已发现，改变基序中这两个精氨酸中的任何一个都能阻止肝素的结合。如本文所述，如果肝素结合基序是RxxR，经修饰硫酸肝素糖蛋白结合位点的第一个氨基酸可以从Arg变为Ser或GIu。在另一个实施方案中，经修饰的硫酸肝素糖蛋白结合位点的末尾氨基酸从Arg变为Thr。在另一个实施方案中，AAV6vpl衣壳蛋白质[SEQ IDNO：3]的第531位赖氨酸被变为非保守氨基酸。本领域技术人员也可选择除本文所述之外的其它非保守氨基酸改变。
类似地，可用本领域已知技术鉴定其他肝素结合域，并用定点突变或其它合适技术改变精氨酸的编码序列来修饰这些肝素结合域。参见例如，Sambrook等人，Molecular Cloning：A Laboratory Manual，Cold Spring Harbor出版社(Cold Spring Cold Spring Harbor，NY)。
另外，可以用其他改变肝素结合域序列的方法来防止肝素结合。在另一个实施方案，通过不同于改变肝素结合位点序列的方法来消除肝素与包含肝素结合位点的AAV的结合。例如，可以为AAV衣壳提供一个分子，该分子能够在生产者细胞中屏蔽肝素结合位点。
在另一个实施方案中，可以通过改造生产细胞来消除或显著减少肝素的产生，例如，用RNA对肝素生物合成中的重要基因进行突变或用RNA靶向所述重要基因，这些可以是瞬时性的也可以是长效的。在另一个实施方案，可以采用肝素生物合成天然缺陷型生产细胞系。
病毒的细胞培养物使用的细胞(稳定地或者瞬时地)至少含有产生AAV颗粒所需的最基本元件，生产包含AAV耐DNA酶基因组的病毒颗粒包括将表达盒包装入AAV衣壳。所需最基本元件包括，待包装入AAV衣壳的表达盒、AAV cap和AAV rep或其功能片段，以及辅助功能因子(helperfunctions)。
已描述了多种适合用于产生AAV的细胞和细胞系。细胞自身可从各种 生物有机体中选择，包括原核生物(例如细菌)细胞，和真核细胞，包括昆虫细胞，酵母细胞和哺乳动物细胞。特别合适的宿主细胞选自各种哺乳动物细胞，包括但不限于以下细胞：A549、WEHI、3T3、10T1/2、BHK、MDCK、COS 1、COS 7、BSC 1、BSC 40、BMT 10、VERO、WI38、HeLa、HEK 293细胞(它们表达功能腺病毒E1)、Saos、C2C12、L细胞、HT1080、HepG2和初级成纤维细胞、肝细胞和成肌细胞，它们来源于人、猴子、小鼠、大鼠、兔子和仓鼠等哺乳动物。本发明对提供细胞的哺乳动物的种类没有限制，对于哺乳动物细胞的类型也没有限制，即不限于成纤维细胞、肝细胞、肿瘤细胞等。
AAV序列可从多种来源获得。例如：适当的AAV序列可如WO2005/033321所述获得或来自已知来源，例如，美国典型菌种保藏中心，或者载体核心研发机构(academic vector core facilities)。或者，根据已发表序列利用已知技术合成合适的序列。此处提供了合适的AAV序列例子。
通常，表达盒的至少包括：5′反向末端重复(ITR)，编码所需治疗剂、免疫原或抗原的核酸序列以及3′AAV ITR，所述编码核酸序列与调控其表达的调控序列操作性连接。在一个实施方案中，使用血清型2AAV的5′ITR和/或3′ITR。然而，也可选用其他适当来源的5′ITR和3′ITR。要被包装到衣壳蛋白中从而形成AAV病毒体(颗粒)的是所述表达盒。
除了表达盒之外，细胞还包含与表达盒中的AAV ITR序列来自相同或与之交叉互补的来源的rep序列和驱动AAV衣壳在细胞中表达的序列(cap序列)。所述AAV cap和rep序列可以分别选自不同的AAV亲本序列，用本领域已知的适当方式导入宿主细胞中。虽然可以使用全长rep基因，但已发现，其小片段，即rep78/68和rep52/40，足以允许AAV的复制和包装。
细胞还需要辅助功能因子来进行本发明AAV的包装。任选地，所述辅助功能因子可由疱疹病毒提供。在另一个实施方案，必要的辅助功能因子分别由人或非人灵长类腺病毒源提供，例如可从多种来源获得的那些，所述来源包括美国典型菌种保藏中心(ATCC)，Manassas，VA(US)。已经知道多种合适的腺病毒的序列。参见例如，黑猩猩腺病毒C1和C68[US专利6,083,716]；Pan 5、Pan6和Pan7[WO 02/33645]，杂交腺病毒[例如WO 05/001103中所述]和GenBank。
在一个实施方案中，宿主细胞至少包含表达E1a基因产物、E1b基因产物、E2a基因产物和/或E4ORF6基因产物所需的基本必要腺病毒DNA序列。宿主细胞可包含其他腺病毒基因，比如VAI RNA，但这些基因不是必需的。在一个实施方案中，使用的细胞不含除E1、E2a和/或E4 ORF6之外的腺病毒基因；不包含任何可能在rAAV产生过程中造成杂病毒同源重组的其他病毒基因；并且，它能够被DNA感染或转染并表达转入的基因。
可用于本发明的一种细胞是用编码rep序列和编码cap的序列稳定转化，并用腺病毒E1、E2a和E4ORF6 DNA和携带上述表达盒的构建体转染的宿主细胞。也可类似地使用稳定表达rep和/或cap的细胞系，例如B-50(国际专利申请公开WO 99/15685)或美国专利5,658,785中描述的细胞系。另一种合适的宿主细胞包含表达E4ORF6所需的基本必要腺病毒DNA。还可用本发明新的经修饰cap序列构造其他细胞系。
本发明宿主细胞的制备包括诸如选定DNA序列的组装等技术。这种组装可利用常规方法完成。相关技术包括公知的cDNA和基因组克隆，可参考Sambrook等人，Molecular Cloning：A Laboratory Manual，Cold SpringHarbor出版社，Cold Spring Harbor NY.，包括聚合酶链式反应、合成法，以及能够提供所需核苷酸序列的其他各种合适的方法。
AAV生产必需的元件(例如，腺病毒E1a、E1b、E2a和/或E4ORF6基因产物、rep或其片段、cap、表达盒，以及其他各种所需辅助功能因子)可以以各种传递所携序列的遗传元件的形式各自或联合进入宿主细胞。
本文中，遗传元件(载体)包括：例如，裸DNA、质粒、噬菌体、转座子、粘粒、游离基因、非病毒运载体(例如，脂类载体)中的蛋白质、病毒等等，它们传递所携带的序列。选定的载体可以用各种适当的方法进行递送，包括转染、电穿孔、脂质体运送、膜融合技术、高速DNA包被小球(highvelocity DNA coated pellets)、病毒感染和原生质体融合。本发明各实施方案使用的方法都是核酸操作领域技术人员熟知的，包括遗传工程、重组工程和合成技术。参见例如，Sambrook等人，Molecular Cloning：A LaboratoryManual，Cold Spring Harbor出版社，Cold Spring Harbor，NY.。参见例如， K.Fisher等人，J.Virol.，70：520-532(1993)和美国专利号5,478,745。
在一个实施方案中，一个或多个腺病毒基因稳定整合到宿主细胞的基因组中或者作为游离基因稳定表达。各腺病毒基因的启动子可以各自选自组成型启动子、诱导型启动子或者天然的腺病毒启动子。例如，可利用有机体或细胞的某种特定生理状态(即，利用分化状态或者正在复制的细胞或者休眠细胞)或者通过添加外源因子来调节这些启动子。
在一个实施方案中，稳定的宿主细胞含有处于诱导型启动子调控下的所述必要元件。然而，所述必要元件也可由组成型启动子来调控。
诱导型启动子使得基因的表达可被如下控制：提供外源化合物，环境因素(诸如温度)，或者特定的生理状态(例如，急性期，细胞的特定分化状态或仅限于正在复制的细胞)。诱导型启动子和系统有多种商业来源，包括但不限于，Invitrogen、Clontech和Ariad。还公开了许多其他系统，本领域技术人员可方便地做出选择。通过外源提供促动剂来调节的启动子的例子包括：锌诱导羊金属硫蛋白(MT)启动子，地塞米松(Dex)诱导小鼠乳腺瘤病毒(MMTV)启动子，T7聚合酶启动子系统[WO 98/10088]；蜕皮激素昆虫启动子[No等人，Proc.Natl.Acad.Sci.USA，93：3346-3351(1996)]，四环素阻遏系统[Gossen等人，Proc.Natl.Acad.Sci.USA，89：5547-5551(1992)]，四环素诱导系统[Gossen等人，Science，268：1766-1769(1995)，也参见Harvey等人，Curr.Opin.Chem.Biol，2：512-518(1998)]，RU486诱导系统[Wang等人，Nat.Biotech，15：239-243(1997)和Wang等，Gene Ther.，4：432-441(1997)]和雷帕霉素诱导系统[Magari等，J.CHn.Invest.，100：2865-2872(1997)]。本发明可用的其他诱导型启动子是受特定生理状态调控的启动子，所述状态例如温度、急性期、细胞特定分化状态调控或仅限于正在复制的细胞。
在另一个实施方案，使用天然的启动子。当需要基因产物的表达拟似天然表达时，可以使用天然启动子。必须暂时或者根据发育(developmentally)或者组织特异性地或者应答特异的转录刺激来调控转基因表达时，可以使用天然启动子。在另一实施方案中，还可以采用其他天然表达控制元件，比如增强子元件、多腺苷酸化位点或Kozak共有序列来模拟天然表达。
转基因的另一个实施方案包括与组织特异性启动子操作性相连的转基因。例如，如果要求在骨骼肌中表达，应该使用在肌肉中有活性的启动子。此类启动子包括来自编码骨β肌动蛋白、肌球蛋白轻链2A、抗肌萎缩蛋白、肌肉肌酸激酶基因的启动子，以及比天然启动子活性更高的合成肌肉启动子(见Li等，Nat.Biotech.，17：241-245(1999))。组织特异性启动子的例子已知的有肝特异性的(白蛋白，Miyatake等，J.Virol.，71：5124-32(1997)；乙型肝炎病毒核心启动子，Sandig等，Gene Ther.，3：1002-9(1996)；甲胎蛋白(AFP)，Arbuthnot等，Hum.Gene Ther.，7：1503-14(1996))，骨钙素(Stein等，Mol.Biol.Rep.，24：185-96(1997))；骨唾液蛋白的(Chen等，J.Bone Miner.Res.，11：654-64(1996))，淋巴细胞(CD2，Hansal等，J.Immunol，161：1063-8(1998)；免疫球蛋白重链；T细胞受体α链)，神经元的例如神经元特异的烯醇酶(NSE)启动子(Andersen等，Cell.MoI Nenrobiol，13：503-15(1993))，神经丝轻链基因的(Piccioli等，Proc.Natl.Acad.Sci.USA，88：5611-5(1991))，和神经元特异性vgf基因(Piccioli等，Neuron，15：373-84(1995))的，及其他。
适当的可活化启动子和组成型启动子的例子是本领域技术人员所熟知的。在另一个替换方式中，选定的稳定宿主细胞可以包含组成型启动子调控下的一个或多个选定元件，和一个或多个诱导型启动子调控下的其它一个或多个其他选定元件。例如，稳定的宿主细胞可由293细胞(它包含E1辅助因子，由组成型启动子调控)来制备，但是该细胞包含受诱导型启动子调控的rep和/或cap蛋白。本领域技术人员能够构建出其他的稳定宿主细胞。
或者，可以用适当的遗传元件将需要在宿主细胞中培养从而将表达盒包装进AAV所需的元件中的一个或多个以转入方式(in trans)提供给宿主细胞。
一旦选定了合适的细胞培养系统，细胞在允许AAV包装的条件下于适当的培养基中培养，收集培养物上清液，从中分离AAV。在一个实施方案中，本发明提供了一种生产规模可调的系统，它允许在维持细胞的培养长达整个连续生产过程，即，不需要为了收集而进行细胞破坏和/或细胞裂解。 在一个实施方案中，这样的系统维持着一个活性细胞培养。在另一个实施方案，细胞培养物中包含活细胞和非活细胞的混合群。在培养过程中，培养基的加入可以是在培养过程中和/或在收集上清液时进行，从而形成连续的生产过程。培养基、新鲜细胞、和/或诸如调节剂等必要营养物和其他元素的加入可以重复至少两次，如两次到100次，或者多于100次，具体视细胞培养周期而定。
虽然本发明的方法能够进行病毒的连续生产，当完成一次生产周期后，较好的是，在进行生产细胞破坏之前从中提取所有残留的AAV。可使用常规方法进行提取。典型的此类方法包括除去上清液，通过冻/融或超声处理技术裂解细胞，然后用去垢剂处理(例如benzonase)。纯化通常通过三次CsCl梯度离心，渗析和浓缩来进行。
在一个实施方案中，本发明提供将细胞培养在适当培养基中形成的细胞培养。任选地，在生产前，或在生产中的适当时机提供激活或诱导所需基因产物表达所需的各种组分或病毒粒子生产所需的各种组分。这些组分可以随培养基一同加入或另行加入。例如，可将携带有一个或多个所需组分的一个或多个适当的遗传元件(例如质粒)转染到合意的细胞系中。
在一个实施方案中，培养基为无血清培养基，比如Dulbecco′s改良的Eagle培养基(DMEM)，它包含无机盐(如CaCl2(无水)、Fe(NO3)9H2O、KCl、MgSO4(无水)、NaCl和NaH2PO4H2O)，氨基酸(如L-精氨酸HCl、L-胱氨酸2HCl、谷氨酰胺、甘氨酸、组氨酸HCl H2O、异亮氨酸、赖氨酸HCl、甲硫氨酸、苯基丙氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸2Na 2H2O和缬氨酸)，维生素(如D-Ca泛酸盐、氯化胆碱、叶酸、i-环己六醇、烟酰胺、核黄素和硫胺HCl)以及其他组分(如D-葡萄糖、酚红和丙酮酸钠)。可选用其他合适的无血清培养基。
较好的是，本发明在无血清条件下(即在无血清培养基中)产生的细胞还具有这样的优点，即，它可以在无血清或者无血清相关成分的条件下进行培养。这样，简化了分离并降低了成本，且提高了安全性，系统具有良好的可靠性(合成培养基对再现性来说是最好的)。本发明的细胞，特别是那些基于初级细胞的细胞，能够进行正常(就人类而言)的翻译后修饰、翻译中修 饰(peri-translational modification)和组装。这意味着它们非常适于制备用于治疗药物和疫苗组合物的病毒蛋白质和病毒。
在另一个实施方案，可以选用含血清的培养基。此外或与此不同，可在培养过程前或中间将培养基与必需的营养物、活化(诱导)剂或血清(DMEM+10％胎牛血清)混合。在另一个实施方案，可以使用无蛋白的培养基。
提供(例如用蠕动泵或其他合适的方法)新鲜培养基和各种必要的诱导剂或活化剂的流速与从培养容器除去用过的培养基的流速相同。保持培养体积恒定，稀释比可以通过改变泵速来改变。启动后，培养物维持在所选细胞培养物的合适的温度范围(例如，大约室温到37℃)，同时搅拌。根据所选的细胞类型，培养可以是有氧的或厌氧的。
估计，对于稳定表达细胞系来说，转染后或培养开始后约24小时需要添加培养基。然而，可以定期地对培养物进行采样，以确定宿主细胞的浓度和上清液中AAV的浓度，从而更加准确地预测上清液收集的时机和培养基添加的时机。
这样，在一个实施方案中，本发明提供AAV病毒的连续生产系统。在一个实施方案中，采用分批培养。例如，各批培养可以采用悬浮细胞和/或贴壁细胞，加料分批培养，充排式培养(fill and draw)。多种分批培养系统是本领域技术人员所知的，它们使用例如生物反应器、发酵罐、微载体系统、静态烧瓶(static flasks)、细胞工厂、摇瓶(roller bottles)、一次性培养袋(disposable bags)(例如，Wave TM系统)、不锈钢容器和玻璃容器。也可以使用其他系统进行AAV病毒生产，例如灌注系统，如中空纤维生物反应器，微载体系统、贴壁细胞培养系统(cell cube system(Corning))，旋转滤膜，填充床生物反应器(例如纤维小管(fibre cell))，细胞微囊技术。
在一个连续系统中，本领域技术人员都知道如何在不同阶段取样，测定传染力、基因组滴定或用其他适当方法来测定病毒浓度。一旦达得适当的浓度，就可将上清液引入到视需要而定的净化系统中。同时，向细胞培养物中添加适量的置换培养基和各种其他的必要元素。
上清液中的AAV可用本领域已知的各种合适的技术进行收获。例如， 单柱技术(monolith column)(例如离子交换、亲合层析或IMAC模式)，色谱法(例如捕获色谱法、固定化色谱法和扩展床色谱法)，过滤和沉淀，都能用于纯化和浓缩。这些方法可单用，也可联用。在一个实施方案中，捕获色谱法(包括柱式系统或膜式系统)与过滤和沉淀联用。本领域技术人员能够轻易地选择合适的沉淀法，例如利用聚乙二醇(PEG)8000和NH3SO4，可由。其后，沉淀物可用benzonase处理，使用适当技术纯化。
在一个实施方案中，有利地，用本发明方法生产的细胞培养上清液中的AAV明显高于留在细胞中的AAV。在某些实施方案中，上清液含有至少60％得率的AAV。
目前，发明人已发现有超过30种重组AAV，从上清液中收获这些AAV，比之从细胞小球中收获，提高了AAV的生产效率。
于是，本发明还提供按照本发明的方法或用途获得的用于治疗组合物或疫苗组合物的病毒，这些病毒或病毒蛋白质不含有任何非人哺乳动物的蛋白质性物质，本发明还提供包含这样的病毒和/或病毒蛋白质的药物制剂。这样的病毒例子包括申请人另一同日申请，“具有降低的衣壳免疫原性的修饰的AAV载体及其用途”中描述的例子，该申请要求美国临时专利申请60/795,965的(申请日2006年4月28日)的优先权。
于是，在一个实施方案中，本发明提供用于如本发明所述生产AAV的试剂盒。这样的试剂盒可以包含一个或多个下列元件。可以提供能够指导AAV病毒颗粒包装的生产细胞。这样的生产细胞可以经改造而含有AAV生产的全部必要元件。或者，这样的生产细胞可以经改变而不能够表达能与肝素结合位点结合的肝素。其他合适的元件可以包括：转染试剂，用于载体构建的质粒元件，收集、纯化、浓缩或者收获组装后AAV颗粒所必需的元件，用于通过正选或负选纯化病毒颗粒的试剂，用于浓缩病毒制剂的试剂和/或用于酶法消化病毒制剂中污染物的试剂。
下列实施例是对按照本发明在细胞培养物上清液中生产AAV颗粒的方法说明。
实施例1：
实验：
用CaPO4和生产AAV所需因子的多种质粒一同转染293细胞，所述质粒为AAV2rep、腺病毒辅助构建体以及两侧为ITR的转基因盒。所述AAV2rep质粒还包含所研究特定病毒的cap序列。所有实验中，唯有cap序列可变。这些实验对数种转基因盒进行了重复。在含血清的DMEM中，转染后二十四小时，培养基被替换为含有或不含有血清的新鲜培养基。转染后三(3)天，从293贴壁细胞的培养基中取样品(I)。接着，将细胞脱壁(scrape)，转移到一个容器中。在离心除去细胞沉淀以后，从脱壁后上清液中取样品(II)。接下来，通过三次连续的冻融循环(-80℃到37℃)裂解细胞。除去细胞碎片，从培养基中取样品(III)。采用TaqmanTMPCR，通过耐DNA酶基因组滴定来定量样品中的AAV。上述转染得到的总生产得率等于样品III的病毒颗粒浓度。其中包括三部分：培养上清液部分，细胞沉淀部分和经细胞脱壁和此后的离心而释放的部分。各部分的绝对值如下获得。
上清液的颗粒数＝样品I的颗粒数
脱壁和离心出来的那部分颗粒数(与细胞松散伴随)＝样品II的颗粒数减去样品I的颗粒数
细胞沉淀中的那部分颗粒数＝样品III的颗粒数减去样品II的颗粒数。
结果：
RxxR[SEQ ID NO：12]基序(区域)的存在不仅将AAV颗粒主要局限在细胞沉淀中，还可能因饱和效应限制了由细胞底物到病毒颗粒的生产。这种限制在非肝素结合性AAV2同源病毒或AAV8中未观察到(图1和2)。血清的存在(S)或不存在(SF)(图2)看来对非肝素结合性AAV的生产没有显著影响。另一方面，肝素结合性AAV2的饱和效应在血清存在下似乎有所减轻。
在另一个实施例中，在实验室规模下采用现有方法，预期40个15cm的培养皿将总共产出约4×1013的AAV2/7颗粒(平均值)。本发明，包括上清液，能达到单个培养皿收获4.7×1012颗粒。
此外，结合无血清培养基的使用，本发明技术显著减轻后续纯化工作的负担。更具体地说，将本发明上清液收集法生产的AAV2/1和AAV2/8与 使用现有方法产生并经CsCl梯度纯化的AAV2/1和AAV2/8进行比较。对于两种病毒，在一系列浓度范围上，本发明上清液收集方法得到的AAV2/1和AAV2/8颗粒明显具有更高的传染性。
对于许多AAV分离株来说，用腺病毒辅助质粒AF6、包装所需表达AAV rep-cap的反式质粒(trans plasmid)和提供载体基因组的AAV2.CMV.eGFP顺式质粒(cis plasmid)，可由单个15cm培养皿转染生产耐DNA酶颗粒，该生产具有可再现性。这些非肝素结合性分离株小规模纯化的滴度可累计达每个培养皿1012至1013个含基因组颗粒(图3)。这些数量足够实验室体外或体内实验所需。
实施例2：
对血清型1、2、3、5、6、7、8和9，以及新载体rh32.33、rh.10、hu.11、AAV6.2、AAV6.1、AAV6.1.2、rh64R1和rh8R载体进入上清液的情况进行了研究。AAV2，AAV2/3和AAV2/5被发现分泌最少(少于总耐DNA酶载体基因组或颗粒(drp Vg)的10％)。AAV2/9在病毒载体生产期间释放到上清液中的程度一般(超过总drp vg的10％，低于20％)。三重转染后，其他所有被检载体以293细胞进行生产过程中都分泌超过20％的病毒颗粒到上清液中。
比较从上清液中获得的载体、纯化(CsCl纯化，仅AAV2用肝素纯化)获得的载体以及从细胞沉淀裂解物中获得的载体的传染性。在用1、2、6、8或9以及AAV2HSPG-分离株进行的293细胞AAV载体转导试验中发现，从上清液中获得的AAV的传染性等同或高于后两部分的载体。
载体向上清液中的释放似乎与它的肝素亲合力相关。通过对天然AAV2(SEQ ID NO：9)的RGNR[SEQ ID NO：18]肝素结合基序进行基因突变(变成SGNT，SEQ ID NO：19)以除去这一亲合力，使得释放到上清液中的那部分载体增加超过40％。在AAV8衣壳(非肝素结合性，SEQ ID NO：11)的同源位置上导入致肝素结合的两个精氨酸产生AAV8RQNR载体，在收获病毒载体时，所得载体几乎完全集结于细胞沉淀。与其亲本载体AAV8形成对比，亲本AAV8的可回收基因组颗粒中(平均)超过40％释放到上清液中。
实施例3：
进行免疫研究，评定具有不同衣壳的各种AAV载体对T细胞活化的效果。该研究比较了天然AAV6衣壳和三种经修饰的AAV，前者已知在第531位赖氨酸具有一个肝素结合域，三种经修饰的AAV的衣壳含有定点修饰。使用的这些AAV被称为AAV2/6.2(含有不在K531位置上的修饰)，AAV2/6.1(具有的AAV6衣壳[SEQ ID NO：3]在531位被修饰而包含一个谷氨酸(即非保守性氨基酸改变)；和AAV2/6.1.2，所含AAV6衣壳同时含有AAV6.2和AAV6.1的衣壳修饰。这些载体的序列和产生记载于国际专利申请PCT/US06/13375。以AAV1作为阴性对照，AAV2作为阳性对照。
Balb/c小鼠(雄性)用1×1011GC的AAV2/6、AAV2/6.1、AAV2/6.2、AAV2/6.1.2、AAV2/1或AAV2载体进行肌肉注射免疫。十三(13)天后，取每组三只小鼠的脾细胞，混合。进行ELISPOT试验，取等量的脾细胞用Balb/c AAV抗原表位IPQYGYLTL[SEQ ID NO：1]进行体外刺激。参见图4。
结果显示，含未修饰AAV6衣壳的病毒载体诱导的T细胞水平与AAV2衣壳诱导的相当。相比之下，除去了肝素结合域的经修饰AAV6载体(AAV2/6.1和AAV2/6.1.2)所诱导的T细胞应答与负对照(AAV1)没有显著区别。
这证明：将介导肝素与AAV衣壳结合的氨基酸残基变为非保守氨基酸残基不仅消除了肝素结合，而且明显降低T细胞活化。
实施例4
评定几种血清型的AAVs结合阴离子交换膜(Mustang Q，Pall Scientific)的能力，缓冲液pH从6.0到9.0，用0到500mM的盐梯度跟踪洗脱。高pH缓冲液对每一被检血清型的结合和洗脱来说都是最适合的(表1)。三种血清型(AAV8，AAV7和Rh8Rc)在100到150mM梯度范围内洗脱，另两种(AAV9和Rh64R1)一加梯度洗脱液就开始洗脱。上样物质的回收率为50％(AAV7)到100％(Rh64R1)。
表1新的AAV血清型在Mustang Q膜上的结合和洗脱特征
| 结合和洗脱的最 佳pH | 洗脱起点(mM) | 最佳洗脱产率 | |
| AAV7 | 8 | 145 | 50％ |
| AAV8 | 9 | 110 | 71％ |
| AAV9 | 8.5 | 10 | 63％ |
| Rh8Rc | 9 | 110 | 67％ |
| Rh64R1 | 9 | 10 | 100％ |
数据显示，阴离子交换膜技术适用于多种AAV血清型的纯化。由于膜大孔结构提供的高流速和高结合容量，该技术尤其适合从细胞培养上清液中纯化AAV。数据表明，为了获得适当的盐浓度从而使AAV结合到阴离子交换膜上，有必要进行上清液稀释或改换缓冲液。
本说明书引用的全部出版物此处以参考文献引入。尽管以特别优选的方式勾画出本发明，但要承认可以出现不背离本发明精神的修饰。
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Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Thr Ser Asn Gly Glu
305 310 315 320
Thr Thr Val Ala Asn Asn Leu Thr Ser Thr Val Gln Ile Phe Ala Asp
325 330 335
Ser Ser Tyr Glu Leu Pro Tyr Val Met Asp Ala Gly Gln Glu Gly Ser
340 345 350
Leu Pro Pro Phe Pro Asn Asp Val Phe Met Val Pro Gln Tyr Gly Tyr
355 360 365
Cys Gly Ile Val Thr Gly Glu Asn Gln Asn Gln Thr Asp Arg Asn Ala
370 375 380
Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn
385 390 395 400
Asn Phe Glu Met Ala Tyr Asn Phe Glu Lys Val Pro Phe His Ser Met
405 410 415
Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Leu Asp
420 425 430
Gln Tyr Leu Trp His Leu Gln Ser Thr Thr Ser Gly Glu Thr Leu Asn
435 440 445
Gln Gly Asn Ala Ala Thr Thr Phe Gly Lys Ile Arg Ser Gly Asp Phe
450 455 460
Ala Phe Tyr Arg Lys Asn Trp Leu Pro Gly Pro Cys Val Lys Gln Gln
465 470 475 480
Arg Phe Ser Lys Thr Ala Ser Gln Asn Tyr Lys Ile Pro Ala Ser Gly
485 490 495
Gly Asn Ala Leu Leu Lys Tyr Asp Thr His Tyr Thr Leu Asn Asn Arg
500 505 510
Trp Ser Asn Ile Ala Pro Gly Pro Pro Met Ala Thr Ala Gly Pro Ser
515 520 525
Asp Gly Asp Phe Ser Asn Ala Gln Leu Ile Phe Pro Gly Pro Ser Val
530 535 540
Thr Gly Asn Thr Thr Thr Ser Ala Asn Asn Leu Leu Phe Thr Ser Glu
545 550 555 560
Gly Glu Ile Ala Ala Thr Asn Pro Arg Asp Thr Asp Met Phe Gly Gln
565 570 575
Ile Ala Asp Asn Asn Gln Asn Ala Thr Thr Ala Pro Ile Thr Gly Asn
580 585 590
Val Thr Ala Met Gly Val Leu Pro Gly Met Val Trp Gln Asn Arg Asp
595 600 605
Ile Tyr Tyr Gln Gly Pro Ile Trp Ala Lys Ile Pro His Ala Asp Gly
610 615 620
His Phe His Pro Ser Pro Leu Ile Gly Gly Phe Gly Leu Lys His Pro
625 630 635 640
Pro Pro Gln Ile Phe Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ala
645 650 655
Thr Thr Phe Thr Ala Ala Arg Val Asp Ser Phe Ile Thr Gln Tyr Ser
660 665 670
Thr Gly Gln Val Ala Val Gln Ile Glu Trp Glu Ile Glu Lys Glu Arg
675 680 685
Ser Lys Arg Arg Asn Pro Glu Val Gln Phe Thr Ser Asn Tyr Gly Asn
690 695 700
Gln Ser Ser Met Leu Trp Ala Pro Asp Thr Thr Gly Lys Tyr Thr Glu
705 710 715 720
Pro Arg Val Ile Gly Ser Arg Tyr Leu Thr Asn His Leu
725 730
<210>5
<211>738
<212>PRT
<213>vp1，克隆rh.10
<400>5
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser
1 5 10 15
Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro
20 25 30
Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile
145 150 155 160
Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln
165 170 175
Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro
180 185 190
Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly
195 200 205
Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser
210 215 220
Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val
225 230 235 240
Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
245 250 255
Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp
260 265 270
Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn
275 280 285
Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn
290 295 300
Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn
305 310 315 320
Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala
325 330 335
Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln
340 345 350
Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe
355 360 365
Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn
370 375 380
Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr
385 390 395 400
Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr
405 410 415
Gln Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser
420 425 430
Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu
435 440 445
Ser Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu
450 455 460
Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala Lys Asn Trp
465 470 475 480
Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser
485 490 495
Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His
500 505 510
Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr
515 520 525
His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met
530 535 540
Phe Gly Lys Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val
545 550 555 560
Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr
565 570 575
Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn Ala Ala
580 585 590
Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val
595 600 605
Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile
610 615 620
Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe
625 630 635 640
Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val
645 650 655
Pro Ala Asp Pro Pro Thr Thr Phe Ser Gln Ala Lys Leu Ala Ser Phe
660 665 670
Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu
675 680 685
Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr
690 695 700
Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Asp
705 710 715 720
Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg
725 730 735
Asn Leu
<210>6
<211>738
<212>PRT
<213>vp1，克隆rh.64
<400>6
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser
1 5 10 15
Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro
20 25 30
Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile
145 150 155 160
Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln
165 170 175
Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro
180 185 190
Pro Ala Ala Pro Ser Ser Val Gly Ser Gly Thr Met Ala Ala Gly Gly
195 200 205
Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser
210 215 220
Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val
225 230 235 240
Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
245 250 255
Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp
260 265 270
Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn
275 280 285
Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn
290 295 300
Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn
305 310 315 320
Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala
325 330 335
Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln
340 345 350
Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe
355 360 365
Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn
370 375 380
Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr
385 390 395 400
Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Ser Phe Ser Tyr
405 410 415
Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser
420 425 430
Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu
435 440 445
Ser Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu
450 455 460
Phe Ser Gln Ala Gly Pro Ser Asn Met Ser Ala Gln Ala Arg Asn Trp
465 470 475 480
Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser
485 490 495
Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His
500 505 510
Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr
515 520 525
Asn Lys Asp Asp Glu Asp Arg Phe Phe Pro Ser Ser Gly Ile Leu Met
530 535 540
Phe Gly Lys Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Asn Val
545 550 555 560
Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr
565 570 575
Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala
580 585 590
Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val
595 600 605
Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile
610 615 620
Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe
625 630 635 640
Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val
645 650 655
Pro Ala Asp Pro Pro Thr Ala Phe Asn Gln Ala Lys Leu Asn Ser Phe
660 665 670
Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Val Trp Glu
675 680 685
Leu Gln Lys Glu Asn Ser Lys Arg Arg Asn Pro Glu Ile Gln Tyr Thr
690 695 700
Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Glu
705 710 715 720
Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg
725 730 735
Asn Leu
<210>7
<211>736
<212>PRT
<213>vp1，克隆rh.8
<400>7
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser
1 5 10 15
Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro
20 25 30
Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly
145 150 155 160
Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro
180 185 190
Ala Ala Pro Ser Gly Leu Gly Pro Asn Thr Met Ala Ser Gly Gly Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp Asn
260 265 270
Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg
275 280 285
Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn
290 295 300
Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile
305 310 315 320
Gln Val Lys Glu Val Thr Thr Asn Glu Gly Thr Lys Thr Ile Ala Asn
325 330 335
Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu
340 345 350
Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro
355 360 365
Ala Asp Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn
370 375 380
Gly Ser Gln Ala Leu Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe
385 390 395 400
Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr
405 410 415
Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu
420 425 430
Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Val
435 440 445
Arg Thr Gln Thr Thr Gly Thr Gly Gly Thr Gln Thr Leu Ala Phe Ser
450 455 460
Gln Ala Gly Pro Ser Ser Met Ala Asn Gln Ala Arg Asn Trp Val Pro
465 470 475 480
Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Asn Gln Asn
485 490 495
Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Ala Lys Phe Lys Leu Asn
500 505 510
Gly Arg Asp Ser Leu Met Asn Pro Gly Val Ala Met Ala Ser His Lys
515 520 525
Asp Asp Asp Asp Arg Phe Phe Pro Ser Ser Gly Val Leu Ile Phe Gly
530 535 540
Lys Gln Gly Ala Gly Asn Asp Gly Val Asp Tyr Ser Gln Val Leu Ile
545 550 555 560
Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Glu
565 570 575
Tyr Gly Ala Val Ala Ile Asn Asn Gln Ala Ala Asn Thr Gln Ala Gln
580 585 590
Thr Gly Leu Val His Asn Gln Gly Val Ile Pro Gly Met Val Trp Gln
595 600 605
Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His
610 615 620
Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu
625 630 635 640
Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala
645 650 655
Asp Pro Pro Leu Thr Phe Asn Gln Ala Lys Leu Asn Ser Phe Ile Thr
660 665 670
Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln
675 680 685
Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn
690 695 700
Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Glu Gly Val
705 710 715 720
Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
725 730 735
<210>8
<211>736
<212>PRT
<213>人腺病毒9型
<400>8
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser
1 5 10 15
Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro
20 25 30
Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly
145 150 155 160
Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro
180 185 190
Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly
195 200 205
Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn
260 265 270
Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg
275 280 285
Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn
290 295 300
Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile
305 310 315 320
Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn
325 330 335
Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu
340 345 350
Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro
355 360 365
Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp
370 375 380
Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe
385 390 395 400
Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu
405 410 415
Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu
420 425 430
Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser
435 440 445
Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser
450 455 460
Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro
465 470 475 480
Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn
485 490 495
Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn
500 505 510
Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys
515 520 525
Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly
530 535 540
Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile
545 550 555 560
Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser
565 570 575
Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln
580 585 590
Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln
595 600 605
Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His
610 615 620
Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met
625 630 635 640
Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala
645 650 655
Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr
660 665 670
Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln
675 680 685
Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn
690 695 700
Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val
705 710 715 720
Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
725 730 735
<210>9
<211>735
<212>PRT
<213>人腺病毒2型
<400>9
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser
1 5 10 15
Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro
20 25 30
Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly
145 150 155 160
Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro
180 185 190
Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr
260 265 270
Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285
Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp
290 295 300
Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val
305 310 315 320
Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu
325 330 335
Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr
340 345 350
Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp
355 360 365
Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser
370 375 380
Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser
385 390 395 400
Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu
405 410 415
Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg
420 425 430
Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr
435 440 445
Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln
450 455 460
Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly
465 470 475 480
Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn
485 490 495
Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly
500 505 510
Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp
515 520 525
Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys
530 535 540
Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr
545 550 555 560
Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr
565 570 575
Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr
580 585 590
Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp
595 600 605
Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr
610 615 620
Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys
625 630 635 640
His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn
645 650 655
Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln
660 665 670
Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys
675 680 685
Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr
690 695 700
Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr
705 710 715 720
Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
725 730 735
<210>10
<211>736
<212>PRT
<213>人腺病毒3型
<400>10
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser
1 5 10 15
Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro
20 25 30
Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Gly
130 135 140
Ala Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly
145 150 155 160
Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro
180 185 190
Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr
260 265 270
Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285
Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp
290 295 300
Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val
305 310 315 320
Arg Gly Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu
325 330 335
Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr
340 345 350
Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp
355 360 365
Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser
370 375 380
Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser
385 390 395 400
Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu
405 410 415
Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg
420 425 430
Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr
435 440 445
Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser
450 455 460
Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro
465 470 475 480
Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn
485 490 495
Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn
500 505 510
Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys
515 520 525
Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly
530 535 540
Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile
545 550 555 560
Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln
565 570 575
Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr
580 585 590
Thr Gly Thr Val Asn His Gln Gly Ala Leu Pro Gly Met Val Trp Gln
595 600 605
Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His
610 615 620
Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu
625 630 635 640
Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala
645 650 655
Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr
660 665 670
Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln
675 680 685
Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn
690 695 700
Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val
705 710 715 720
Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
725 730 735
<210>11
<211>738
<212>PRT
<213>人腺病毒8型
<400>11
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser
1 5 10 15
Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro
20 25 30
Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile
145 150 155 160
Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln
165 170 175
Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro
180 185 190
Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly
195 200 205
Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser
210 215 220
Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val
225 230 235 240
Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His
245 250 255
Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp
260 265 270
Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn
275 280 285
Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn
290 295 300
Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn
305 310 315 320
Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala
325 330 335
Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln
340 345 350
Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe
355 360 365
Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn
370 375 380
Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr
385 390 395 400
Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr
405 410 415
Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser
420 425 430
Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu
435 440 445
Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr Gln Thr Leu Gly
450 455 460
Phe Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp
465 470 475 480
Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly
485 490 495
Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His
500 505 510
Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr
515 520 525
His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile
530 535 540
Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val
545 550 555 560
Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr
565 570 575
Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala
580 585 590
Pro Gln Ile Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val
595 600 605
Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile
610 615 620
Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe
625 630 635 640
Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val
645 650 655
Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe
660 665 670
Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu
675 680 685
Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr
690 695 700
Ser Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu
705 710 715 720
Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg
725 730 735
Asn Leu
<210>12
<211>4
<212>PRT
<213>人腺病毒
<220>
<221>MISC_FEATURE
<222>(2)..(2)
<223>可为任意氨基酸
<220>
<221>MISC_FEATURE
<222>(3)..(3)
<223>可为任意氨基酸
<400>12
Arg Xaa Xaa Arg
1
<210>13
<211>735
<212>PRT
<213>vp1，克隆hu.51
<400>13
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser
1 5 10 15
Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro
20 25 30
Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Gly Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly
145 150 155 160
Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro
180 185 190
Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr
260 265 270
Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285
Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp
290 295 300
Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val
305 310 315 320
Lys Glu Val Thr Gln Asn Asp Gly Thr Thr ThrIle Ala Asn Asn Leu
325 330 335
Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr
340 345 350
Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp
355 360 365
Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser
370 375 380
Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser
385 390 395 400
Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu
405 410 415
Asp Val Pro Phe His Ser Gly Tyr Ala His Ser Gln Ser Leu Asp Arg
420 425 430
Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Thr Thr
435 440 445
Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln
450 455 460
Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly
465 470 475 480
Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn
485 490 495
Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly
500 505 510
Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp
515 520 525
Asn Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys
530 535 540
Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr
545 550 555 560
Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr
565 570 575
Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr
580 585 590
Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp
595 600 605
Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr
610 615 620
Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys
625 630 635 640
His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn
645 650 655
Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln
660 665 670
Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys
675 680 685
Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr
690 695 700
Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr
705 710 715 720
Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
725 730 735
<210>14
<211>735
<212>PRT
<213>vp1，克隆hu.34
<400>14
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser
1 5 10 15
Glu Gly Ile Arg Gln Arg Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro
20 25 30
Glu Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly
145 150 155 160
Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro
180 185 190
Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr
260 265 270
Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285
Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp
290 295 300
Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val
305 310 315 320
Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu
325 330 335
Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr
340 345 350
Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp
355 360 365
Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Glu Ser
370 375 380
Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser
385 390 395 400
Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu
405 410 415
Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Gly Arg
420 425 430
Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr
435 440 445
Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln
450 455 460
Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly
465 470 475 480
Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn
485 490 495
Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly
500 505 510
Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp
515 520 525
Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys
530 535 540
Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr
545 550 555 560
Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr
565 570 575
Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr
580 585 590
Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp
595 600 605
Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr
610 615 620
Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys
625 630 635 640
His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn
645 650 655
Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln
660 665 670
Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys
675 680 685
Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr
690 695 700
Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr
705 710 715 720
Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
725 730 735
<210>15
<211>735
<212>PRT
<213>vp1，克隆hu.35
<400>15
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser
1 5 10 15
Glu Gly Ile Arg Gln Arg Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro
20 25 30
Glu Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly
145 150 155 160
Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro
180 185 190
Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr
260 265 270
Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285
Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp
290 295 300
Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val
305 310 315 320
Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu
325 330 335
Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr
340 345 350
Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp
355 360 365
Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser
370 375 380
Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser
385 390 395 400
Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu
405 410 415
Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Gly Arg
420 425 430
Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr
435 440 445
Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln
450 455 460
Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly
465 470 475 480
Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn
485 490 495
Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly
500 505 510
Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp
515 520 525
Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys
530 535 540
Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr
545 550 555 560
Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr
565 570 575
Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr
580 585 590
Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp
595 600 605
Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr
610 615 620
Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys
625 630 635 640
His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn
645 650 655
Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln
660 665 670
Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys
675 680 685
Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr
690 695 700
Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr
705 710 715 720
Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
725 730 735
<210>16
<211>735
<212>PRT
<213>vp1，克隆hu.45
<400>16
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser
1 5 10 15
Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro
20 25 30
Lys Pro Ala Glu Arg His Arg Asp Asp Ser Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly
145 150 155 160
Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro
180 185 190
Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr
260 265 270
Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285
Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp
290 295 300
Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val
305 310 315 320
Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu
325 330 335
Thr Ser Thr Val Gln Val Phe Thr Asp Ser Gly Tyr Gln Leu Pro Tyr
340 345 350
Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp
355 360 365
Val Phe Met Val Pro Gln Tyr Gly Tyr Pro Thr Leu Asn Asn Gly Ser
370 375 380
Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser
385 390 395 400
Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu
405 410 415
Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg
420 425 430
Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Thr Thr
435 440 445
Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln
450 455 460
Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly
465 470 475 480
Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn
485 490 495
Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly
500 505 510
Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Val Ala Ser His Lys Asp
515 520 525
Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys
530 535 540
Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr
545 550 555 560
Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr
565 570 575
Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr
580 585 590
Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp
595 600 605
Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr
610 615 620
Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys
625 630 635 640
His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn
645 650 655
Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln
660 665 670
Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys
675 680 685
Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr
690 695 700
Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr
705 710 715 720
Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu
725 730 735
<210>17
<211>735
<212>PRT
<213>vp1，克隆hu.47
<400>17
Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser
1 5 10 15
Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro
20 25 30
Lys Pro Ala Glu Arg His Arg Asp Asp Ser Arg Gly Leu Val Leu Pro
35 40 45
Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro
50 55 60
Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp
65 70 75 80
Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala
85 90 95
Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly
100 105 110
Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro
115 120 125
Leu Gly Leu Val Gly Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg
130 135 140
Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly
145 150 155 160
Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr
165 170 175
Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro
180 185 190
Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly
195 200 205
Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser
210 215 220
Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile
225 230 235 240
Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu
245 250 255
Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Ser His Tyr
260 265 270
Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His
275 280 285
Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp
290 295 300
Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val
305 310 315 320
Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu
325 330 335
Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr
340 345 350
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Claims (25)
1.生产重组AAV的方法，所述方法包括：
(a)维持包含AAV生产细胞和生长培养基的细胞培养物，所述细胞包含编码肝素结合位点缺失的AAV衣壳的AAV序列，该编码序列受引导所述AAV衣壳在所述生产细胞内表达的序列调控的调控，所述生产细胞还包含有待包装到所述AAV衣壳内的表达盒、一项或多项将表达盒包装入AAV衣壳所需的腺病毒辅助功能因子和足以允许复制的AAV rep蛋白；
(b)从培养物中收集上清液，无需收集细胞沉淀或裂解细胞；和
(c)从上清液中分离重组AAV，无需收集细胞沉淀或裂解细胞。
2.如权利要求1所述的方法，进一步包括在上清液收集过程中或者之后加入培养基以实现连续生产过程的步骤。
3.如权利要求1或2所述的方法，其中，所述方法的步骤至少重复两次。
4.如权利要求3所述的方法，其中，所述方法的步骤至少重复2到100次。
5.如权利要求1所述的方法，其中，所述AAV是AAV8。
6.如权利要求1所述的方法，其中，所述AAV接受了去天然肝素结合位点的修饰。
7.如权利要求6所述的方法，其中，所述肝素结合位点的特征为氨基酸序列RxxR，X为任何氨基酸。
8.如权利要求7所述的方法，其中，所述AAV选自由AAV2、hu.51、hu.34、hu.35、hu.45和hu.47组成的组。
9.如权利要求7所述的方法，其中，所述肝素结合位点在RxxR序列的第一个氨基酸被修饰。
10.如权利要求9所述的方法，其中，经修饰肝素结合位点的第一个氨基酸由Arg变成Ser或Glu。
11.如权利要求7所述的方法，其中，所述肝素结合位点在RxxR序列的最后一个氨基酸被修饰。
12.如权利要求11所述的方法，其中，经修饰肝素结合位点的最后一个氨基酸由Arg变成Thr。
13.如权利要求1所述的方法，其中，所述培养步骤在无血清培养基中进行。
14.如权利要求1所述的方法，其中，在HEK293细胞中培养AAV。
15.如权利要求1的所述方法，其中，所述分离步骤进一步包括通过选自色谱法、过滤和沉淀的方法浓缩AAV。
16.如权利要求15所述的方法，其中，所述色谱法是柱式色谱。
17.如权利要求15所述的方法，其中，所述色谱法是膜式色谱。
18.生产重组AAV的方法，所述方法包括以下步骤：
(a)在经改造的哺乳动物生产细胞中培养AAV，所述哺乳动物生产细胞经改造而消除或显著减少肝素的产生，所述细胞包含编码AAV衣壳的AAV序列，该编码序列受引导所述AAV衣壳在所述生产细胞内表达的序列调控，所述生产细胞还包含有待包装到所述AAV衣壳内的表达盒、一项或多项将表达盒包装入AAV衣壳所需的腺病毒辅助功能和足以允许复制的AAV rep蛋白；
(b)从培养物中收集上清液，无需收集细胞沉淀或裂解细胞；和
(c)从上清液中分离重组AAV，无需收集细胞沉淀或裂解细胞。
19.按照权利要求1至18中任一项所述方法从所述细胞培养物中收集得到的细胞培养上清液。
20.如权利要求19所述的细胞培养上清液，其中，所述上清液包含得率至少60％的AAV。
21.如权利要求1所述的方法，其中，所述细胞被以编码腺病毒辅助功能因子的序列稳定转化。
22.如权利要求1所述的方法，其中，所述腺病毒辅助功能因子在可活化或可诱导的启动子的调控下表达。
23.如权利要求1所述的方法，其中，所述细胞被以编码AAV rep蛋白和/或AAV cap蛋白的序列稳定转化。
24.如权利要求23所述的方法，其中，所述AAV rep蛋白和/或AAVcap蛋白在可活化或可诱导的启动子的调控下表达。
25.如权利要求1所述的方法，其中，所述细胞被以待包装AAV基因组稳定转化。
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| PCT/US2007/010055 WO2007127264A2 (en) | 2006-04-28 | 2007-04-27 | Scalable production method for aav |
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| WO2005005610A2 (en) * | 2003-06-30 | 2005-01-20 | The Regents Of The University Of California | Mutant adeno-associated virus virions and methods of use thereof |
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| US20230116968A1 (en) | 2023-04-20 |
| WO2007127264A3 (en) | 2008-04-17 |
| JP5268890B2 (ja) | 2013-08-21 |
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