CN106232618A - 预测祖先病毒序列的方法及其用途 - Google Patents
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Abstract
描述了用于预测病毒或其部分的祖先序列的方法。还描述了腺伴随病毒(AAV)壳体多肽的经预测的祖先序列。本公开还提供了基因转移的方法和对受试者接种疫苗的方法,其通过施用可操作连接于AAV壳体多肽的靶抗原进行。
Description
发明领域
一般而言,本公开涉及病毒。
发明背景
规避和避免针对基因治疗载体的中和性或毒性免疫应答是所有基因转移载体类型面临的重大挑战。迄今为止的基因转移使用基于在人类和动物中循环的病毒的载体(例如腺病毒和腺伴随病毒(AAV))而得以最有效地实现。然而,如果受试者已经天然感染了病毒,那么由于细胞和体液免疫应答,用基于该病毒的载体的后续治疗导致基因转移增加的安全风险和降低的效率。病毒壳体抗原主要负责针对病毒颗粒的天然免疫和/或适应性免疫,然而,病毒基因编码的多肽也可以具有免疫原性。
发明概述
本公开描述了预测和合成祖先病毒(ancestral viral)序列或其部分的方法,并且还描述了含有此类祖先病毒序列的病毒颗粒。将本文中描述的方法应用到腺伴随病毒(AAV);因此,本公开描述了预测的祖先AAV序列和含有此种祖先AAV序列的AAV病毒颗粒。本公开还描述了相对于含有当代序列的病毒颗粒,由含有祖先序列的病毒颗粒展现的降低的血清阳性率(seroprevalance)。
在一个方面,本公开包括腺伴随病毒(AAV)壳体多肽,例如,合成的和/或人工的AAV壳体多肽,其具有选自由以下组成的组的氨基酸序列:SEQ ID NO:1、3、5、7、9、11、13、15和17。在一些实施方案中,AAV壳体多肽或包含AAV壳体多肽的病毒颗粒比AAV2壳体多肽或包含AAV2壳体多肽的病毒颗粒展现出更低的血清阳性率,并且AAV壳体多肽或包含AAV壳体多肽的病毒颗粒与AAV8壳体多肽或包含AAV8壳体多肽的病毒颗粒展现出大致相同或更低的血清阳性率。在一些实施方案中,AAV壳体多肽或包含AAV壳体多肽的病毒颗粒比AAV2壳体多肽或包含AAV2壳体多肽的病毒颗粒在更小程度上被人血清中和,并且AAV壳体多肽或包含AAV壳体多肽的病毒颗粒比AAV8壳体多肽或包含AAV8壳体多肽的病毒颗粒在相似或更低的程度上被人血清中和。在一些实施方案中,AAV壳体多肽是纯化的。本文中提供的AAV壳体多肽可以由选自由以下组成的组的核酸序列编码:SEQ ID NO:2、4、6、8、10、12、14、16和18。
在一个方面,本公开提供核酸分子,例如,合成的和/或人工的核酸分子,其编码具有选自由以下组成的组的核酸序列的腺伴随病毒(AAV)壳体多肽:SEQ ID NO:2、4、6、8、10、12、14、16和18。还提供包括此种核酸的载体和包括此种载体的宿主细胞。
在另一个方面,本公开提供了纯化的病毒颗粒,其包括本文中所述的AAV壳体多肽。在一些实施方案中,病毒颗粒包含转基因。
在其它方面,本公开提供了腺伴随病毒(AAV)壳体多肽,例如,合成的和/或人工的AAV壳体多肽,其与选自由以下组成的组的氨基酸序列具有至少95%(例如,97,98,99,或100%)的序列同一性:SEQ ID NO:19、20、21、22、23、24、25和26。在一些实施方案中,AAV壳体多肽或包含AAV壳体多肽的病毒颗粒比AAV2壳体多肽或包含AAV2壳体多肽的病毒颗粒展现出更低的血清阳性率,并且AAV壳体多肽或包含AAV壳体多肽的病毒颗粒与AAV8壳体多肽或包含AAV8壳体多肽的病毒颗粒展现出大致相同或更低的血清阳性率。在一些实施方案中,AAV壳体多肽或包含AAV壳体多肽的病毒颗粒比AAV2壳体多肽或包含AAV2壳体多肽的病毒颗粒在更小程度上被人血清中和,并且AAV壳体多肽或包含AAV壳体多肽的病毒颗粒比AAV8壳体多肽或包含AAV8壳体多肽的病毒颗粒在相似或更小程度上被人血清中和。在一些实施方案中,AAV壳体多肽是纯化的。
在另一个方面,本文中描述的AAV壳体多肽可以通过如本文所描述的核酸序列编码。在一个实施方案中,本公开提供了编码腺伴随病毒(AAV)壳体多肽的核酸分子,其中所述核酸分子与如本文所示的核酸具有至少95%(例如,97,98,99,或100%)的序列同一性。本公开还提供包括此种核酸分子的载体,也提供了包括此种载体的宿主细胞。
在另一个方面,本公开提供了包括本文中所述的AAV壳体多肽中的至少一种的病毒颗粒。在一些实施方案中,病毒颗粒包括转基因。
在某些方面,本公开提供了将如本文中所述的病毒颗粒施用于需要基因转移或疫苗接种的受试者的方法。在一些实施方案中,病毒颗粒比AAV2病毒颗粒展现出更低的血清阳性率。在一些实施方案中,病毒颗粒与AAV8病毒颗粒展现出大致相同或更小的血清阳性率。在一些实施方案中,病毒颗粒比AAV2病毒颗粒在更小程度上被人血清中和,并且AAV病毒颗粒比AAV8病毒颗粒在相似或更小程度上被人血清中和。
在一个方面,本公开提供了将可操作连接于如本文中所述的AAV壳体多肽的靶抗原施用于需要疫苗接种的受试者的方法。在一些实施方案中,AAV壳体多肽比AAV2壳体多肽展现出更小的血清阳性率。在一些实施方案中,AAV壳体多肽比AAV8壳体多肽展现出大致相同或更低的血清阳性率。在一些实施方案中,AAV壳体多肽比AAV2壳体多肽在更小程度上被人血清中和,并且AAV壳体多肽比AAV8壳体多肽在相似或更小程度上被人血清中和。
在另一个方面,本公开提供了预测祖先病毒或其部分的序列的计算机方法(insilico method)。此类方法通常包括提供来自多个当代病毒或其部分的核苷酸或氨基酸序列;使用多重序列比对(MSA)算法比对序列;对进化建模以获得多个当代病毒或其部分的预测的祖先***发生(phylogeny);在预测的祖先***发生的***发生节点(phylogenicnode)处估计在序列的每个位置上的特定核苷酸或氨基酸残基的进化概率(evolutionaryprobability)并且基于估计的在每个位置上的概率预测祖先病毒或其部分的序列。
在一些实施方案中,使用计算机处理器进行一个或多个或全部步骤。在一些实施方案中,MSA算法使用***发生信息来预测比对中的缺口是否是缺失或***的结果。在一些实施方案中,MSA算法是概率比对套件(Probabilistic Alignment Kit)(PRANK)。在一些实施方案中,使用Aikake信息标准(Aikake Information Criterion)(AIC)选择用于对进化建模的模型。在一些实施方案中,使用JTT模型及伽马分布模型(Gamma distributionmodel)(“+G”)以及πi的频率计算(“+F”)获得预测的祖先***发生。在一些实施方案中,使用JTT+G+F模型进行对进化建模的步骤。在一些实施方案中,该方法包括基于预测的序列合成祖先病毒或其部分。在一些实施方案中,该方法包括将祖先病毒或其部分组装成祖先病毒颗粒。
在一些实施方案中,该方法还包括对祖先病毒颗粒筛选下列至少一项:(1)复制;(b)基因转移性质;(c)受体结合;或(d)血清阳性率。在一些实施方案中,祖先病毒颗粒比从多个当代病毒或其部分中的至少一种组装的病毒颗粒展现出更小的血清阳性率。在一些实施方案中,祖先病毒颗粒与多个当代病毒或其部分中的至少一种组装的病毒颗粒在更低的程度上被人血清中和。在一些实施方案中,多个当代病毒或其部分属于选自以下组成的组的家族(family):腺病毒(adenovirus)(AV),人免疫缺陷病毒(human immunodeficiencyvirus)(HIV),逆转录病毒(lentivirus),慢病毒(lentivirus),单纯疱疹病毒(herpessimplex virus)(HSV),痘苗病毒(vaccinia virus),痘病毒(pox virus),流感病毒(influenza virus),呼吸道合胞病毒(respiratory syncytial virus),副流感病毒(parainfluenza virus)和泡沫病毒(foamy virus)。
因此,本公开提供了祖先病毒或其部分,其相比于当代病毒或其部分展现出降低的对当代人群中预先存在的免疫的易感性。通常,由祖先病毒或其部分在当前人群中展现的降低的对预先存在的免疫的易感性反映为降低的对中和性抗体的易感性。
除非另外定义,本文中使用的所有技术和科学术语与方法和物质组合物所属领域的普通技术人员的通常理解具有相同的含义。虽然与本文描述的方法和材料相似或等同的方法和材料可以用于方法和物质组合物的实践或测试中,但是以下描述了合适的方法和材料。此外,材料,方法和实例仅是说明性的,并不意在进行限制。将本文中提及的所有出版物、专利申请、专利和其它参考文献以其整体并入本文作为参考。
附图简述
本专利或申请文件包含以彩色制作的至少一幅附图。通过请求并支付必要的费用由当局提供具有彩色附图的本专利或专利申请公开文本的副本。
图1是显示祖先病毒/当代病毒感染和祖先宿主/当代宿主免疫应答之间的关系的示意图。
图2A至2D是显示祖先重建(reconstruction)过程的实例的一系列示意图。显示的数据是从完整的数据集摘录并代表残基564-584(AAV2-VP1编号)。
图3A至3C一起显示了使用本文中所描述的方法产生的AAV当代序列的***发生树。
图4A和4B一起显示了祖先AAV VP1多肽的比对。
图5A和5B一起显示了功能性祖先AAV VP1多肽和当代AAV VP1多肽的比对。
图6是凝胶电泳图,其证明祖先AAV VP1序列以类似于当代AAV VP1序列的方式被转录并可变剪接。
图7是显示用祖先AAV载体转导的HEK293细胞中的萤光素酶活性的图。
图8是显示祖先AAV载体比当代AAV载体对IVIG中和更具抗性的图。
图9是显示Anc80文库和Anc80L65之间的序列比较(从对角线向上%,以下的氨基酸差异#)的图。
图10A-D是实验结果的图像,其表明了Anc80L65能够组装并产生高滴度的颗粒。图A显示了Anc80L65能够产生与AAV2相当的载体产率;图B是包括Anc80L65的病毒颗粒的TEM图像;图C显示了包括Anc80L65的病毒颗粒,基于变性条件下的SDS-PAGE凝胶,所述病毒颗粒能够产生AAV cap VP1,2和3蛋白;并且图D显示了使用AAV壳体抗体B1进行的Anc80L65的Western印迹。
图11A-C是实验结果的图像,其证明了使用GFP作为读数(图A)或萤光素酶(图B),Anc80L65相对于AAV2和/或AAV8对照能够对HEK293细胞在体外感染细胞,并且在编码核LacZ转基因的AAV的IV注射后(顶行,图C:肝),在编码GFP的AAV的直接IM注射(中间行,图C:肌肉),和在编码GFP的AAV的视网膜下注射后(底行,图C:视网膜)有效靶向肝。
图12A-D的图表明在使用IVIg(图A)或来自比利时群体的血清(图B),来自波士顿群体的血清(图C)或来自食蟹猴(Cynomolgus macaques)的血清(图D)的人类群体中,Anc80L64是最小限度血清阳性率的。
图13A和13B是使用MAFFT产生的序列同一性矩阵,其显示与代表性现存的AAV的VP1蛋白的氨基酸序列比对的祖先载体的VP1蛋白的氨基酸序列(图13A),和与代表性现存的AAV的VP3蛋白的氨基酸序列比对的祖先载体的VP3蛋白的氨基酸序列(图13B)。
图14是表明以小规模(6孔皿)一式三份产生AAV载体的图。通过qPCR评估粗制病毒以确定每一载体的绝对产生。
图15是显示取平均值并且与AAV8的滴度比较的每一载体的滴度的表。
图16是照片,其显示将每一载体的1.9E3GC/细胞加入到HEK293细胞的实验结果(除了Anc126外,在Anc126的情况下实现了2.5E2-3.1E2GC/细胞的MOI)。60小时后,用荧光显微术评估了感染性。
图17是显示实验结果的图,其中裂解来自图16的相同细胞并测定萤光素酶表达。与在图16中一样,Anc126没有用其它载体控制滴度,但是范围却为2.5E2-3.1E2GC/细胞的MOI。
图18的表显示了由每一载体转导的细胞的发光,其取平均值并与AAV8的发光相比较。
图19的图提供了测定本文中所述的祖先AAV载体的相对产生和感染性的体外实验的汇总。
发明详述
基因转移(出于实验或治疗目的)依赖于载体或载体***将遗传信息穿梭到靶细胞中。认为载体或载体***是基因转移反应的效率,特异性,宿主反应,药理学和寿命的主要决定因素。目前,实现基因转移的最高效且有效的的方式是通过使用基于已经变为复制缺陷型的病毒的载体或载体***。
然而,血清阳性率研究指示相当大比例的全球人类群体已经预先暴露(例如通过自然感染)于大量的目前在基因转移中使用的病毒,并且因此具有预先存在的免疫。已知在这些预先暴露的个体中针对病毒载体的中和抗体有时显著地限制基因转移的程度,或者甚至使病毒重定向远离靶标。参见例如Calcedo等人,(2009,J.Infect.Dis.,199:381-90)和Boutin等人,(2010,Human Gene Ther.,21:704-12)。因此,本公开是基于如下的认识:祖先病毒或其部分比当代病毒或其部分在当前人类群体中展现出降低的对先前存在的免疫的易感性(例如,降低的对中和抗体的易感性)。
图1是显示祖先和当代病毒感染与祖先和当代宿主免疫应答之间的关系的示意图。图1显示了祖先AAV可以如何耐(refractory to)当代先前存在的免疫。推测当代的现存病毒(Vc)主要在宿主免疫的进化压力下通过免疫逃逸机制从祖先物种(Vanc)进化。这些物种Vanc和Vc中的每一种均具有诱导适应性免疫,包括B细胞和T细胞免疫(分别为Ianc和Ic)的能力。假设并且本文中确认,由当代病毒诱导的免疫不一定与祖先病毒种类起交叉反应,所述祖先病毒种类就表位组成而言可以与现存病毒实质上不同。
本公开提供了预测祖先病毒或其部分的序列的方法。可以产生使用本文中描述的方法预测的一个或多个祖先病毒序列并组装成病毒颗粒。如本文中证明,从预测的祖先病毒序列组装的病毒颗粒可以比当前的当代病毒颗粒展现出更小,有时显著更小的血清阳性率。因此,本文中公开的祖先病毒序列适合于在用于基因转移的载体或载体***中使用。
预测和合成祖先病毒序列的方法
为了预测祖先病毒序列,首先从多个当代病毒或其部分编译(compile)核苷酸或氨基酸序列。尽管本文中所述的方法用腺伴随病毒(AAV)壳体序列示例,但相同的方法可应用于来自AAV的其他序列(例如,整个基因组,rep序列,ITR序列)或任何其他病毒或其部分。除AAV之外的病毒包括但不限于,腺病毒(AV),人免疫缺陷病毒(HIV),逆转录病毒,慢病毒,单纯疱疹病毒(HSV),麻疹病毒,痘苗病毒,痘病毒,流感病毒,呼吸道合胞病毒,副流感病毒,泡沫病毒,或认为先前存在的免疫成问题的任何其它病毒。
可以使用来自少到两种当代病毒或部分的序列,但是,应当理解当代病毒或其部分的更大数目的序列是期望的,以便包括现代序列多样性的尽可能多的画卷(landscape),而且还因为更大数量的序列可以增加所描述和使用的算法的预测能力。例如,可以使用来自10种或更多种当代病毒或其部分的序列,可以使用来自50种或更多种当代病毒或其部分的序列,可以使用来自100种或更多种当代病毒或其部分的序列。
此类序列可以例如从许多公共数据库获得,所述公共数据库包括但不限于GenBank,UniProt,EMBL,国际核苷酸序列数据库协作(International NucleotideSequence Database Collaboration)(INSDC)或欧洲核苷酸档案库(European NucleotideArchive)。另外/或者,此类序列可以从特异于特定生物体的数据库(例如HIV数据库)中获得。当代序列可以对应于整个基因组,或可以仅使用基因组的一部分,诸如但不限于编码病毒壳体,复制蛋白,或ITR序列的一个或多个组分的序列。
接着,使用多重序列比对(MSA)算法比对当代序列。图2(a)是显示多个序列的比对的示意图。MSA算法是本领域众所周知的,并且通常被设计成适用于不同大小的数据集和不同的输入(例如,核酸或蛋白质),以及适用于以特定方式(例如,动态编程,渐进(progressive),启发式(heuristic))比对序列并在比对中应用不同评分方案(例如,基于矩阵或基于一致性,例如,最小熵,配对总和,相似性矩阵,缺口得分)。公知的MSA算法包括,例如ClustalW(Thompson et al.,1994,Nuc.Acids Res.,22:4673-90),Kalign(Lassmannet al.,2006,Nuc.Acids Res.,34:W596-99),MAFFT(Katoh et al.,2005,Nuc.AcidsRes.,33:511-8),MUSCLE(Edgar,2004,BMC Bioinform.,5:113),和T-Coffee(Notredameet al.,2000,J.Mol.Biol.,302:205-17)。
如本文中所述的,当选择MSA算法用于本文中所述的方法中时的主要特征之一是其中所述算法处理比对中的缺口的方式。可以给序列比对中的缺口分配罚分值(penaltyvalue),其依赖于或不依赖于缺口大小。在本方法中,优选的是,与由例如***和/或缺失所致的缺口的有偏的、非***发生处理形成对比,在本文中所描述的方法中使用的MSA算法将***发生信息应用于预测在比对中的缺口是否是缺失或者***的结果。在比对和进化分析中处理缺口的合适的方法描述于Loytynoja and Goldman,2008,Science,320:1632-5中,并且将缺口以适合用于本文中所述的方法中的方式应用于比对中的商业上可获得的算法是概率比对套件(Probabilistic Alignment Kit)(PRANK;Goldman Group Software;Loytynoja and Goldman,2005,PNAS USA,102:10557-62)和PRANK算法的变型。
然后,将进化模型应用到得到的比对以获得预测的祖先***发生(参见图2(b))。在本领域中有许多可用的进化模型,其中的每个对氨基酸应用替换率的略微不同的矩阵。应用进化模型的算法包括但不限于Dayhoff模型(例如PAM120,PAM160,PAM250;Dayhoff etal.,1978,In Atlas of Protein Sequence and Structure(ed.Dayhoff),pp.345-52,National Biomedical Research Foundation,Washington D.C.),JTT模型(Jones etal.,1992,Comp.Appl.Biosci.,8:275-82),WAG模型(Whelan and Goldman,2001,Mol.Biol.Evol.,18:691-9),和Blosum模型(例如Blosum45,Blosum62,Blosum80;Henikoffand Henikoff,1992,PNAS USA,89:10915-9)。
此外,例如,通过考虑一些位置是不变的(“+I”;Reeves,1992,J.Mol.Evol.,35:17-31),一些位置经历不同速率的变化(“+G”;Yang,1993,Mol.Biol.Evol.,10:1396-1401),和/或核苷酸或氨基酸的平衡频率与比对中的那些是相同的(“+F”;Cao et al.,1994,J.Mol.Evol.,39:519-27),可以对结构和功能强加到进化模型的约束本身建模。
可以使用Aikake信息标准(AIC;Akaike,1973,In Second InternationalSymposium on Information Theory,Petrov and Csaki,eds.,pp 267-81,Budapest,Akademiai Kiado),Bayesian信息标准(BIC;Schwarz,1978,Ann.Statist.6:461-4),或其变型或组合来评估一个或多个进化模型的适合度(fitness)。此外,AIC,BIC,或其变型或组合可以用于评估在进化模型中包括一个或多个参数(例如上面讨论的约束)的相对重要性。
如下文实施例部分中解释的,基于最低AIC,ProTest3(Darriba et al.,2011,Bioinformatics,27(8):1164-5)可用于确定JTT+G+F算法是最适合于AAV进化的模型。本领域技术人员将理解,JTT+G+F算法也可以用于预测与AAV壳体多肽不同的祖先病毒序列,然而,本领域技术人员也将理解,根据数据集和适合度评分,不同进化模型可以是更合适的。
一旦已经选择了进化模型并确定了它的适合度,可以构建病毒序列或其部分的***发生树。构建***发生树在本领域是已知的,并且通常使用最大似然法(maximumlikelihood method),如由PhyML(Guindon and Gascuel,2003,Systematic Biology,52:696-704)),MOLPHY(Adachi and Hasegawa,1996,ed.Tokyo Institute of StatisticalMathematics),BioNJ(Gascuel,1997,Mol.Biol.Evol.,14:685-95)或PHYLIP(Felsenstein,1973,Systematic Biology,22:240-9)执行的那些方法。本领域技术人员将理解,在氨基酸取代的模型中计算复杂性和拟合优度(goodness of fit)之间的平衡是值得期望的。
根据需要,可以评估***发生树的显著性。许多统计方法是可用的,并且常规用于评估模型的显著性,包括但不限于自助法(bootstrap),刀切法(jackknife),交叉验证(cross-validation),置换检验(permutation test),或它们的组合或变型。显著性也可以使用例如近似似然比检验(approximate likelihood-ratio test)(aLRT;Anisimova andGascuel,2006,Systematic Biology,55:539-52)进行评价。
在***发生的任何***发生节点(例如,内部***发生节点),序列可以通过估计在序列的每个位置处特定核苷酸或氨基酸残基的进化概率来重构建(如图2(c))。***发生节点是指在预测的祖先***发生内的中间进化分支点。如本文中所使用的,“进化概率”是指与不考虑例如密码子选择中的进化偏移(evolutionary shift)的模型形成对比基于进化模型在特定位置上的特定核苷酸或氨基酸存在的概率。可使用例如任何数目的最大似然法,包括但不限于通过最大似然法进行的***发生分析(Phylogenetic Analysis byMaximum Likelihood)(PAML;Yang,1997,Comp.Applic.BioSci.,13:555-6)或使用简约性进行的***发生分析(Phylogenetic Analysis Using Parsimony)(PAUP;SinauerAssoc.,Inc.,Sunderland,MA)评估考虑了在特定位置上的特定核苷酸或氨基酸残基的进化概率的示例性模型。
基于在每个位置上的特定核苷酸或氨基酸残基的评估的进化概率,可以装配祖先病毒或其部分的预测序列以形成完整或部分的合成的核酸或多肽序列。根据需要,可以计算任何残基沿着节点在给定节点处为给定状态的可能性的,并且可以鉴定沿着序列具有特定阈值之下的计算的后验概率的任何位置(图2(d))。以这种方式,可以产生祖先支架序列,其可以包括在具有低于特定阈值的概率的那些位置上的变异。
如果使用本文中的方法预测的祖先序列是核酸序列,那么可以对该序列进行密码子优化,以便它可以被有效地翻译成氨基酸序列。针对不同生物体的密码子选择表是本领域已知的。任选地,然而,密码子选择表可以基于与祖先支架序列具有同一性(例如,至少90%的序列同一性)的一种或多种当代序列设计,并且可以将如本文中描述的祖先序列针对哺乳动物(例如,人类)的密码子选择进行密码子优化。
本文中概述的用于预测祖先病毒序列的任何或全部有步骤可以使用处理器或微处理器在计算机上(例如用计算机(in silico))进行或模拟。
祖先腺伴随病毒(AAV)支架序列
使用当代壳体序列将本文中描述的方法应用到腺伴随病毒(AAV)(在以下实施例详细描述)。AAV被广泛认为是治疗性基因转移载体和遗传疫苗载体,但在人群中展现出高血清阳性率。使用本文中描述的方法,使用当代AAV序列(见图3A-3C)装配了***发生树,并在指定的***发生节点获得预测的祖先支架序列(表1)。如本文中使用的,祖先支架序列是指使用本文所述的方法(例如,使用进化概率和进化建模)构建并且知道在自然界中不存在的序列。如本文中使用,祖先支架序列不同于共有序列,其通常使用在特定位置的核苷酸或氨基酸残基的频率构建。
表1
Anc80多肽的支架序列显示于SEQ ID NO:1,其由在SEQ ID NO:2中所示的Anc80核酸的支架序列编码。Anc80的支架序列包含11个位置,在该位置处两种残基的任一种是可能的。因此,Anc80支架序列代表2048(211)种不同的序列。
为了证明本文所述的方法用于预测病毒或其部分的祖先序列的有效性,产生了在AAV Anc80节点的2048种预测的祖先序列的文库,并如本文所述,证明形成活病毒颗粒,该活病毒颗粒比用当代壳体多肽装配的病毒颗粒展现出更小的血清阳性率,在一些情况下,显著更小的血清阳性率。
制备祖先病毒颗粒的方法
在已经获得了病毒或其部分的预测的祖先序列之后,可以产生(例如合成)实际的核酸分子和/或多肽。基于例如计算机获得的序列产生人工核酸分子或多肽的方法在本领域中是已知的,并且包括例如化学合成或重组克隆。用于产生核酸分子或多肽的其它方法在本领域中是已知的,并在下面更详细地讨论。
一旦已经产生了祖先多肽,或者一旦已经产生了祖先核酸分子并表达以产生祖先多肽,可以使用例如包装宿主细胞将祖先多肽装配成祖先病毒颗粒。使用如本文所描述的一个或多个载体可以将病毒颗粒的组分(例如,rep序列,cap序列,末端反向重复(ITR)序列)瞬时或稳定地引入到包装宿主细胞中。病毒颗粒的一种或多种组分可以基于如本文所描述的预测的祖先序列,而其余组分可以基于当代序列。在一些情况下,整个病毒颗粒可以基于预测的祖先序列。
此类祖先病毒颗粒可以使用常规方法纯化。如本文所用,“纯化的”病毒颗粒指的是从制备它们的混合物中的组分取出的病毒颗粒,所述组分如但不限于病毒组分(例如,rep序列,cap序列),包装的宿主细胞,和部分或不完全装配的病毒颗粒。
一旦装配,可以针对例如复制的能力;基因转移特性;受体结合能力;和/或在群体(例如,人类群体)中的血清阳性率筛选祖先病毒颗粒。确定病毒颗粒是否可以复制在本领域中是常规的,并且通常包括用一定量的病毒颗粒感染宿主细胞,并且确定病毒颗粒的数目是否随时间增加。确定病毒颗粒是否能够执行基因转移在本领域中也是常规的并且通常包括用含有转基因(例如可检测的转基因,如报道基因,在下面更详细地讨论)的病毒颗粒感染宿主细胞。在病毒的感染和清除之后,可评估宿主细胞中转基因的存在或不存在。确定病毒颗粒是否结合到其受体在本领域中是常规的,并且这样的方法可以在体外或体内进行。
确定病毒颗粒的血清阳性率在本领域中是常规进行的并且通常包括使用免疫测定法来确定来自个体的特定群体的样品(例如,血液样品)中的一种或多种抗体的阳性率(prevalence)。血清阳性率在本领域中理解为指群体中呈血清阳性(即,已经暴露于特定病原体或免疫原)的受试者的比例,并计算为群体中产生针对特定病原体或免疫原的抗体的受试者的数目除以在检查的群体中个体的总数。免疫测定法是本领域众所周知的,并且包括但不限于,免疫点迹(immunedot),Western印迹,酶免疫测定(EIA),酶联免疫吸附测定(ELISA),或放射性免疫测定(RIA)。如本文所指出的,祖先病毒颗粒比当代病毒颗粒(即,使用当代病毒序列或其部分组装的病毒颗粒)展现出更小的血清阳性率。仅举例而言,参见Xuet al.(2007,Am.J.Obstet.Gynecol.,196:43.e1-6);Paul et al.(1994,J.Infect.Dis.,169:801-6);Sauerbrei et al.(2011,Eurosurv.,16(44):3);和Sakhria et al.(2013,PLoS Negl.Trop.Dis.,7:e2429),其中每篇确定在给定的群体中特定抗体的血清阳性率。
如本文所述,祖先病毒颗粒比当代病毒颗粒以更小的程度被个人(例如患者)的免疫***中和。确定血清样品中的中和抗体程度的几种方法是可用的。例如,中和抗体测定测量与没有抗体的对照样品相比,实验样品含有中和感染达50%或更多的抗体浓度的滴度。还可参见Fisher et al.(1997,Nature Med.,3:306-12)和Manning et al.(1998,HumanGene Ther.,9:477-85)。
就本文示例性的祖先AAV壳体多肽而言,可以例如与AAV8壳体多肽或者包括AAV8壳体多肽的病毒颗粒,或者AAV2壳体多肽或者包括AAV2壳体多肽的病毒颗粒比较血清阳性率和/或中和的程度。本领域通常理解AAV8壳体多肽或病毒颗粒在人类群体中展现出认为较低的血清阳性率和所得的中和,而AAV2壳体多肽或病毒颗粒在人类群体中展现出被认为较高的血清阳性率和所得的中和。显然,特定的血清阳性率将取决于检查的群体以及所用的免疫学方法,但也有报道称,AAV8展现出约22%直至约38%的血清阳性率,而AAV2展现出约43.5%直至约72%的血清阳性率。参见例如,Boutin et al.,2010,“Prevalence ofserum IgG and neutralizing factors against AAV types 1,2,5,6,8 and 9 in thehealthy population:implications for gene therapy using AAV vectors,”Hum.GeneTher.,21:704-12。还参见,Calcedo et al.,2009,J.Infect.Dis.,199:381-90。
预测的腺伴随病毒(AAV)祖先核酸和多肽序列
对来自编码从Anc80节点预测的祖先壳体多肽的文库的多种不同克隆进行了测序,并且代表性AAV预测祖先壳体多肽的氨基酸序列显示于SEQ ID NO:19(Anc80L27);SEQID NO:20(Anc80L59);SEQ ID NO:21(Anc80L60);SEQ ID NO:22(Anc80L62);SEQ ID NO:23(Anc80L65);SEQ ID NO:24(Anc80L33);SEQ ID NO:25(Anc80L36);和SEQ ID NO:26(Anc80L44)。本领域技术人员将理解,编码每个氨基酸序列的核酸序列可以容易地确定。
除了具有在SEQ ID NO:19、20、21、22、23、24、25或26中所示序列的预测祖先壳体多肽之外,提供与在SEQ ID NO:19、20、21、22、23、24、25或26中所示序列的预测祖先壳体多肽具有至少95%的序列同一性(例如,至少96%,至少97%,至少98%,至少99%或100%的序列同一性)的多肽。相似地,提供了核酸,其与编码祖先壳体多肽核酸分子具有至少95%的序列同一性(例如,至少96%,至少97%,至少98%,至少99%或100%的序列同一性)(即,具有至少95%的序列同一性)的核酸。
在计算百分比序列同一性时,比对两个序列并确定两个序列之间的核苷酸或氨基酸残基的相同匹配的数目。将相同匹配的数目除以比对区的长度(即,比对的核苷酸或氨基酸残基的数目),并乘以100,以获得百分比序列同一性值。应该理解的是,比对区的长度可以是一个或两个序列的一部分直至最短序列的全长大小。还应当理解,单个序列可以与一个以上的其它序列比对,因此,相对于每一比对区可以具有不同的序列同一性百分比值。
可以使用由Altschul等人(1997,Nucleic Acids Res.,25:3389 3402)所描述的算法来进行两个或多个序列的比对以确定百分比序列同一性,所述算法并入BLAST(基本局部比对搜索工具)程序,可在万维网(World Wide Web)上以ncbi.nlm.nih.gov获得。可以进行BLAST搜索,以确定使用Altschul等人的算法比对的序列(核酸或氨基酸)和任何其它序列或其部分之间的百分比序列同一性。BLASTN是用于比对和比较核酸序列之间的同一性的程序,而BLASTP是用于比对和比较氨基酸序列之间的同一性的程序。当使用BLAST程序来计算一种序列与另一序列之间的同一性百分比时,通常使用各个程序的默认参数。
代表性比对显示于图4A和4B及图5A和5B中。图4A和4B显示了称为Anc80L65(SEQID NO:23),Anc80L27(SEQ ID NO:19),Anc80L33(SEQ ID NO:24),Anc80L36(SEQ ID NO:25),Anc80L44(SEQ ID NO:26),Anc80L59(SEQ ID NO:20),Anc80L60(SEQ ID NO:21)和Anc80L62(SEQ ID NO:22)的祖先AAV VP1壳体多肽的比对。在图4A和4B中所显示的比对证实在11个位点之每处预测的变异,以及在Anc80L60(SEQ ID NO:21)的位置609E的单个非同义突变,其可以是克隆假象(artifact)。图5A和5B显示了祖先AAV VP1壳体多肽(Anc80L65(SEQ ID NO:23),Anc80L27(SEQ ID NO:19),Anc80L33(SEQ ID NO:24),Anc80L36(SEQ IDNO:25),Anc80L60(SEQ ID NO:21),Anc80L62(SEQ ID NO:22),Anc80L44(SEQ ID NO:26)和Anc80L59(SEQ ID NO:20))和当代AAV VP1壳体多肽(AAV8(SEQ ID NO:27),AAV9(SEQ IDNO:28),AAV6(SEQ ID NO:29),AAV1(SEQ ID NO:30),AAV2(SEQ ID NO:31),AAV3(SEQ IDNO:32),AAV3B(SEQ ID NO:33),和AAV7(SEQ ID NO:34)之间的比对。在图5A和5B的比对显示祖先AAV序列具有与当代AAV序列的约85%至91%之间的序列同一性。
还提供了含有编码多肽的核酸分子的载体。载体(包括表达载体)是商品化的或可通过重组技术来产生。含有核酸分子的载体可以具有可操作连接到此种核酸分子的一种或多种表达元件,并且还可以包括序列诸如编码可选择标记(例如,抗生素抗性基因)的那些序列,和/或可用于多肽纯化中的那些序列(例如,6xHis标签)。表达元件包括指导和调节核酸编码序列的表达的核酸序列。表达元件的一个实例是启动子序列。表达元件也可以包括下列一个或多个:内含子,增强子序列,反应元件,或者调节核酸分子表达的可诱导元件。表达元件可以是细菌,酵母,昆虫,哺乳动物,或病毒来源的,并且载体可以包含来自不同来源的表达元件的组合。如本文中所使用的,可操作连接意指相对于编码序列,表达元件以下述方式放置在载体中,从而指导或调节编码序列的表达。
可以将核酸分子,例如载体(例如,表达载体,病毒载体)中的核酸分子导入宿主细胞中。术语“宿主细胞”不仅指已经接受核酸分子导入的特定细胞,而且还指这种细胞的后代或潜在后代。许多合适的宿主细胞是本领域技术人员已知的;宿主细胞可以是原核细胞(例如,大肠杆菌)或真核细胞(例如,酵母细胞,昆虫细胞,植物细胞,哺乳动物细胞)。代表性的宿主细胞可以包括但不限于A549、WEHI、3T3、10T1/2、BHK、MDCK、COS 1、COS 7、BSC 1、BSC 40、BMT 10、VERO、WI38、HeLa、293cells、Saos、C2C12、L细胞、HT1080、HepG2和来源于哺乳动物(包括人,猴,小鼠,大鼠,兔和仓鼠)的原代成纤维细胞、肝细胞和成肌细胞细胞。用于将核酸分子导入宿主细胞中的方法在本领域中是公知的,并且包括但不限于,磷酸钙沉淀,电穿孔,热休克,脂转染,显微注射和病毒介导的核酸转移(例如,转导)。
关于多肽,“纯化的”是指已经从天然伴随它的细胞组分分离或纯化的多肽(即,肽或多肽)。典型地,认为多肽当它按重量计至少70%(例如,至少75%,80%,85%,90%,95%或99%)不含与它天然相关的多肽和天然存在的分子时是“纯化的”。因为化学合成的多肽本来与天然伴随它的组分分离,所以认为合成多肽是“纯化的”,但是还可以从用于合成多肽的组分(例如,氨基酸残基)取出。关于核酸分子,“分离的”指的是与在基因组中与它通常相关的其它核酸分子分离的核酸分子。此外,分离的核酸分子可以包括工程化的核酸分子,如重组或合成的核酸分子。
可以通过已知的方法如DEAE离子交换,凝胶过滤,和/或羟磷灰石层析从天然来源(例如生物样品)获得(例如,纯化)多肽。纯化的多肽也可以例如通过在表达载体中表达核酸分子或通过化学合成来获得。可以使用任何合适的方法,例如,柱层析,聚丙烯酰胺凝胶电泳,或HPLC分析测量多肽的纯度。类似地,使用常规方法,如但不限于重组核酸技术(例如,限制性酶消化和连接)或聚合酶链式反应(PCR;参见例如PCR Primer:A LaboratoryManual,Dieffenbach&Dveksler,Eds.,Cold Spring Harbor Laboratory Press,1995)可以获得(例如,分离)核酸分子。此外,可以化学合成分离的核酸分子。
使用祖先病毒或其部分的方法
如本文所述的祖先病毒或其部分,特别是相对于当代病毒或其部分展现出降低的血清阳性率的那些祖先病毒或其部分,可以用于许多的研究和/或治疗应用中。例如,如本文所述的祖先病毒或其部分可以用于人或动物医学中以用于基因治疗(例如,用于基因转移的载体或载体***中)或用于疫苗接种(例如,用于抗原呈递)。更具体地,如本文所述的祖先病毒或其部分可用于基因添加,基因增大(augmentation),多肽治疗剂的遗传递送,遗传疫苗接种,基因沉默,基因组编辑,基因疗法,RNAi递送,cDNA递送,mRNA递送,miRNA递送,miRNA海绵擦拭(miRNA sponging),遗传免疫,光遗传学基因治疗(optogenetic genetherapy),转基因作用(transgenesis),DNA疫苗接种或DNA免疫。
可以用祖先病毒或其部分在体外(例如,在培养中生长)或在体内(例如,在受试者中)转导或感染宿主细胞。本文描述了可以用祖先病毒或其部分在体外转导或感染的宿主细胞;可以用祖先病毒或其部分在体内转导或感染的宿主细胞包,但不限于,脑,肝,肌肉,肺,眼(例如,视网膜,视网膜色素上皮),肾脏,心脏,性腺(例如,睾丸,子宫,卵巢),皮肤,鼻道,消化***,胰,胰岛细胞,神经元,淋巴细胞,耳(例如,内耳),毛囊和/或腺体(例如甲状腺)。
可以修饰如本文所述的祖先病毒或部分以包括转基因(与其它病毒序列为顺式或反式)。转基因可以是例如报道基因(例如,β-内酰胺酶,β-半乳糖苷酶(LacZ),碱性磷酸酶,胸腺激酶,绿色荧光多肽(GFP),氯霉素乙酰转移酶(CAT),或萤光素酶,或是融合多肽,其包括抗原标签域如血凝素或Myc)或治疗性基因(例如,编码激素或其受体,生长因子或其受体,分化因子或其受体,免疫***调节剂(例如,细胞因子和白细胞介素)或其受体,酶,RNA(例如,抑制性RNA或催化性RNA),或靶抗原(例如,致癌抗原,自身免疫抗原)的基因)。
特定转基因将至少部分取决于正在治疗的特定疾病或缺陷。仅举例而言,可将基因转移或基因治疗应用于以下疾病的治疗:血友病,色素性视网膜炎,囊性纤维化,利伯先天性黑朦(leber congenital amaurosis),溶酶体贮积症,先天性代谢缺陷(inbornerrors of metabolism)(例如,先天性氨基酸代谢缺陷(inborn errors of amino acidmetabolism),包括苯丙酮尿(phenylketonuria),先天性有机酸代谢缺陷(inborn errorsof organic acid metabolism),包括丙酸血症(propionic academia),先天性脂肪酸代谢缺陷(inborn errors of fatty acid metabolism),包括中链酰基辅酶A脱氢酶缺乏症(medium-chain acyl-CoA dehydrogenase deficiency,MCAD)),癌症,全色盲(achromatopsia),锥-视杆营养不良(cone-rod dystrophies),黄斑变性(例如年龄相关性黄斑变性),脂多肽脂酶缺陷,家族性高胆固醇血症,脊髓性肌萎缩,杜氏肌营养不良(Duchenne’s muscular dystrophy),阿尔茨海默氏病(Alzheimer’s disease),帕金森氏病(Parkinson’s disease),肥胖,炎性肠紊乱(inflammatory bowel disorder),糖尿病,充血性心脏衰竭,高胆固醇血症(hypercholesterolemia),听力丧失,冠状动脉心脏疾病(coronary heart disease),家族性肾淀粉样变性(familial renal amyloidosis),马方综合征(Marfan’s syndrome),致死性家族性失眠(fatal familial insomnia),Creutzfeldt-Jakob病,镰状细胞病(sickle-cell disease),亨廷顿氏病(Huntington’sdisease),额颞叶退化(fronto-temporal lobar degeneration),Usher综合征,乳糖不耐受症(lactose intolerance),脂肪沉积症(lipid storage disorder)(如C型Niemann-Pick症),Batten病,无脉络膜(choroideremia),糖原贮积病Ⅱ型(庞皮病(Pompedisease)),运动失调性毛细血管扩张症(ataxia telangiectasia)(Louis-Bar综合征),先天性甲状腺功能减退症(congenital hypothyroidism),严重联合免疫缺陷(severecombined immunodeficiency,SCID),和/或肌萎缩性侧索硬化(amyotrophic lateralsclerosis,ALS)。
转基因也可以是例如可用于免疫受试者(例如,人类,动物(例如,伴侣动物,农场动物,濒危动物)的免疫原。例如,免疫原可从获自生物体(例如,病原性生物体)或其免疫原性部分或组分(例如毒素多肽或其副产物)。举例而言,可以获得免疫原性多肽的病原性生物体包括病毒(例如,微小核糖核酸病毒,肠道病毒,正粘病毒,呼肠孤病毒(reovirus),逆转录病毒),原核生物(例如,肺炎球菌(Pneumococci),葡萄球菌(Staphylococci),李斯特菌(Listeria),假单胞菌(Pseudomonas))和真核生物(例如,阿米巴病(amebiasis),疟疾(malaria),利什曼病(leishmaniasis),线虫)。将理解,本文描述的方法和通过此种方法产生的组合物不受任何特定转基因的限制。
可将通常悬浮在生理上相容的载体中的祖先病毒或其部分施用于受试者(例如人或非人哺乳动物)。合适的载体包括盐水(其可以用多种缓冲溶液(例如,磷酸盐缓冲盐水)配制),乳糖,蔗糖,磷酸钙,明胶,葡聚糖,琼脂,果胶和水。以足够的量施用祖先病毒或其部分以转导或感染细胞,并提供基因转移和表达的足够水平从而提供治疗益处且没有不适当的副作用。常规和药学可接受的施用途径包括但不限于直接递送至器官,如例如肝或肺,经口,鼻内,气管内,通过吸入,静脉内,肌内,眼内,皮下,皮内,跨粘膜,或通过其它施用途径。根据需要,可以组合施用途径。
施用于受试者的祖先病毒或其部分的剂量将主要取决于诸如正在治疗的状况,以及受试者的年龄,体重,和健康等因素。例如,待施用于人类受试者的祖先病毒或其部分的治疗有效剂量通常是在从大约0.1ml至约10ml溶液的范围中,所述溶液含有约1×101至1×1012基因组拷贝(GCS)的浓度的祖先病毒(例如,约1×103至1×109GCS)。转基因的转导和/或表达可以在施用后在多个时间点通过DNA,RNA或蛋白质测定法进行监测。在一些情况下,可以监测转基因的表达水平,以确定剂量的频率和/或量。与那些对治疗目的描述的剂量方案相似的剂量方案也可用于免疫。
本文描述的方法还可以用于对向前进化(forward evolution)建模,以便修饰或除去病毒或其部分的一种或多种免疫原性域。
根据本发明,可以采用本领域的技术范围内的常规分子生物学,微生物学,生物化学和重组DNA技术。这些技术在文献中充分解释。本发明将在以下实施例中进一步描述,所述实施例不限制权利要求书中描述的方法和组合物的范围。
实施例
实施例1:祖先序列的计算机预测
从许多公共数据库(包括GenBank)获得AAV壳体的一组75个不同的氨基酸序列,并使用PRANK-MSA算法,版本121002,用选项“-F”比对序列。
将ProtTest3(参见例如Darriba et al.,2011,Bioinformatics,27(8):1164-5;在万维网上以darwin.uvigo.es/software/prottest3可得到)用于在不同条件下(例如在ProTest3中包含的那些条件,即“+I”,“+F”,“+G”和其组合)评估多肽进化的不同模型(例如,在ProTest3中包含的那些模型,即JTT,LG,WAG,VT,CpRev,RtRev,Dayhoff,DCMut,FLU,Blosum62,VT,HIVb,MtArt,MtMam)。基于如在ProTest3中实施的Aikake信息标准(AIC;Hirotugu,1974,IEEE Transactions on Automatic Control,19:716-23)得分选择JTT模型(Jones et al.,1992,Comp.Appl.Biosci.,8:275-82)与+G和+F(Yang,1993,Mol.Biol.Evol.,10:1396-1401;及Cao et al.,1994,J.Mol.Evol.,39:519-27)。
使用PhyML(Guindon and Gascuel,2003,Systematic Biology,52:696-704)构建AAV进化的***发生。参见图3。使用具有4个离散取代类别和估计的Gamma形参数的JTT+F取代模型产生树。通过近邻交换(Nearest Neighbor Interchange)(NNI)和子树修剪(Subtree Pruning)和再移植术(SPR)改进得到的树,并通过自助法和近似似然比检验(aLRT;Anisimova and Gascuel,2006,Systematic Biology,55:539-52)使用“SH-Like”变体评估显著性。
然后,将以上构建的***发生树用于在***发生内部的每个节点估计AAV壳体的祖先状态。使用最大似然原则通过Lazarus(Sourceforge at sf.net)中包括的最大似然***发生分析(Phylogenetic Analysis by Maximum Likelihood,PAML)软件(Yang,1997,Comp.Applic.BioSci.,13:555-6;在万维网abacus.gene.ucl.ac.uk/software/paml.html上可得到)重构建祖先壳体序列。更具体地,设置了Lazarus/PAML重构建,使用JTT+F取代模型使用4-gamma分布类别以产生氨基酸的重构建。将AAV5用作外类群(outgroup)。最后,加入了“I”选项以在PAML重构建完成后放置***缺失(indel)(即二元编码并使用Fitch算法通过最大简约性(Maximum Parsimony)放置)。
因为以最大似然的方式进行重构建,所以可以计算在给定节点的给定位置的任何残基的可能性。要做到这一点,编写了其他脚本来鉴定沿着序列的具有某个阈值下方的计算的后验概率的所有位置。选择了0.3的阈值,这意味着将具有大于0.3的计算的后验概率的任何氨基酸包括在文库的合成中。将这些残基选择为文库中感兴趣的变体。
为了最终确定(finalize)序列,必须编码附加效用工具(additional utility)来选择密码子。编写了脚本以得出与另一个AAV序列(AVVRh10,其具有与Anc80支架序列约92%的序列同一性)密码子类似的密码子,并应用新算法取代密码子,在那里基于密码子取代矩阵有序列错配。新算法显示如下:
给定:氨基酸序列,Pt,具有相应的核苷酸序列,NT,其中Nt编码Pt;和蛋白质序列,Pi,其中Pi展现出与Pt的强同源性。
使用Needleman-Wunsch使用用于评分的Blosum62表比对Pi与Pt。通过步进(stepping)通过蛋白质比对使用来自Nt的相应密码子产生新的核苷酸序列,Ni。
其中Pt中的氨基酸与Pi中的氨基酸完全匹配,
来自密码子-PAM矩阵(Codon-PAM matrix)(Schneider et al.,2005,BMCBioinform.,6:134))的“最好得分”密码子,在那里有取代,
缺口,在那里与Pt中的氨基酸比对的Pi中存在缺口,和
在Nt中最经常出现的核苷酸(编码给定氨基酸),在那里与Pt中的缺口比对的Pi中存在氨基酸。
此外,还产生两个单核苷酸变化以消除装配激活蛋白(assembly-activatingprotein)(AAP)的转录,所述装配激活蛋白在野生型AAV中的AAV壳体基因内的框外编码。因为AAP(当代或祖先)的编码不是本重构建的一部分,所以通过在cap序列中产生同义突变消除AAP的表达,并且在病毒产生期间以反式提供AAP序列。
实施例2:祖先AAV VP1序列的表达
进行实验以确定预测的祖先AAV壳体序列是否可用于制备病毒载体。
克隆了许多预测的祖先AAV壳体序列。将祖先壳体文库转移到rep-cap表达质粒,使得能够在瞬时转染中形成病毒颗粒。为了维持VP1,VP2和VP3的适当的表达水平和剪接,通过切割位于rep编码序列中的cap的5’的HindIII和在cap终止密码子和多聚腺苷酸化信号之间工程化改造的SpeI,克隆了文库cap基因。因此,为了将祖先壳体克隆到更常规的“REP/CAP”构建体中,用HindIII和SpeI消化传代质粒,凝胶纯化,并连接到经相似消化的rep/cap质粒中。
表达的多肽分别在10%SDS凝胶上解析。如图6中所示,壳体多肽是适当表达的,并从多个祖先AAV序列(Anc80L44,Anc80L27和Anc80L65)以及从当代AAV序列,AAV2/8剪接成VP1,VP2和VP3。
实施例3:病毒滴定
通过瞬时共转染编码病毒颗粒装配需要的所有元件的质粒在HEK293细胞中产生了AAV。简言之,培养HEK293细胞至90%汇合,并用以下转染所述HEK293细胞:(a)编码侧翼是AAV2ITR的萤光素酶转基因(通过CMV启动子表达)的病毒基因组质粒,(b)编码本文所公开的AAV2rep和合成的壳体蛋白的AAV包装质粒,(c)AAV2-AAP表达壳体,以及(d)AAV包装和装配需要的腺病毒辅助基因。细胞在37℃培养2天,收获并收集细胞和培养基。
通过连续3次冻融循环裂解细胞培养基悬液。之后,通过离心澄清裂解物,并在进行彻底的DNA消化的条件下用酶(本文用BenzonaseTM)处理,以消化存在于病毒颗粒外的任何DNA。稀释AAV制备物以落入对照DNA模板的线性测量范围内,在这种情况下,与载体基因组相比具有相同的TaqManTM引物和探针结合序列的线性化质粒。用退火至选择的病毒载体基因组的引物和探针进行TaqManTM PCR。基于TaqManTM测量以每毫升(ml)中的基因组拷贝(GC)计算滴度,如下面的表2中显示。
表2
滴度(GC/ml) | 小规模#1 | 小规模#2 |
AAV2/2 | 1.12x109 | 1.99x109 |
AAV2/8 | 4.17x1010 | 5.91x1010 |
Anc80L27 | 8.01x108 | 1.74x109 |
Anc80L44 | 1.52x109 | 1.43x109 |
Anc80L65 | 1.42x109 | 2.05x109 |
无壳体对照 | 5.23x105 | 7.25x105 |
在祖先重构建的AAV壳体颗粒上的小规模载体生产结果表明类似于AAV2,但相对于AAV8减少(这两者都是基于当代AAV的载体制备物)的产率。
实施例4:体外病毒转导
进行体外病毒转导以评价含有预测的祖先AAV序列的病毒感染细胞的能力。
在使用Anc80序列文库进行高通量载体生产后,用每一病毒载体转导HEK293细胞种。除了Anc80序列之外,每个病毒载体含有萤光素酶转基因。在将萤光素底物加入到经转导的细胞或细胞裂解物中之后,通过在96孔平板读数器中定量生物发光测量萤光素酶。在定量后,产生了在四个级联96孔板中的萤光素酶表达的热图(排除在每个板中的对照栏)。由于与高通量载体量生产过程相关联的大量***,缺失,和转换,许多载体是非功能性的。对于本文的目的,仅进一步评估了在此测定中(即,能够转导HEK293细胞并表达转基因)的功能性病毒。
用两个当代AAV载体(AAV2/2和AAV2/8)和三个预测的祖先AAV载体(Anc80L27,Anc80L44,和Anc80L65)以每细胞1×104个基因组拷贝(GC)的相等的多重感染(MOI)转导HEK293细胞。每个载体含有萤光素酶编码转基因或eGFP编码转基因。60小时后使用AMGEvosFl光学显微镜的GFP通道对细胞成像。图7显示在体外转导之后的萤光素酶表达。每一祖先AAV病毒表明HEK293细胞的有效转导。
实施例5-体内视网膜转导
进行视网膜转导以确定祖先AAV载体是否能够在体内靶向鼠视网膜细胞。
用2×108基因组拷贝(GC)的三种不同的祖先AAV(Anc80L27,Anc80L44,和Anc80L65)和当代AAV(AAV2/8)转导鼠眼,它们全部包括eGFP编码转基因。对于转导,经由用注射装置递送载体推注(vector bolus),通过产生在光感受器和视网膜色素上皮层之间的空间以外科手术将每个AAV载体递送到视网膜下方。将载体推注留在视网膜下间隙,并且随着时间的推移解决视网膜分离。通过用TropicamideTM对瞳孔散大后的动物视网膜进行底部照相术非侵入性监测GFP表达。所有呈现的视网膜表明将祖先AAV不同程度成功靶向视网膜。
还进行视网膜组织学,并在荧光显微术下可视化以鉴定经转导的细胞类型。在用如上所述的Anc80L65祖先AAV载体转导的鼠视网膜上进行组织学。Anc80L65介导的eGFP表达在外核层(ONL),内节(IS),和视网膜色素上皮(RPE)中是明显的,指示祖先Anc80L65载体靶向鼠光感受器和视网膜色素上皮细胞。
实施例6:中和抗体测定
进行中和抗体测定,以评估祖先AAV病毒是否比当代AAV病毒对抗体中和更具抗性。中和抗体测定测量相比于不存在抗体的情况下的对照,中和感染达50%或以上的抗体浓度(或实验样品包含抗体浓度的滴度)。
2倍连续稀释血清样品或IVIG储备溶液(200mg/ml),并且未稀释和稀释的样品与104MOI的祖先AAV病毒,Anc80L65和当代AAV病毒,AAV2/8,在在37℃下共温育约30分钟。每个病毒包括萤光素酶基因。然后将混合载体和抗体样品转导到HEK293细胞中。对于这些实验,所用的抗体样品是静脉内免疫球蛋白(IVIG),从超过1000名血液供体(市售,例如,GammagardTM(Baxter Healthcare;Deerfield,IL)或GamunexTM(Grifols;Los Angeles,CA))的血浆中提取的汇集的IgG。转导起始后48小时,通过生物发光测定细胞以检测萤光素酶。中和抗体滴度通过鉴定达到50%或以上的中和(样品转导/在不存在样品的情况下的对照病毒的转导)的样品的稀释度确定的。
如图8所示,为将祖先AAV病毒Anc80L65的转导效率降低至50%的无IVIG对照(虚线)以下,相比于当代AAV病毒AAV2/8,需要IVIG的显著更高的浓度。这些结果表明了与当代AAV病毒相比,祖先AAV病毒对IVIG的中和的更高的抗性。
实施例7:Anc80的表征
根据本文中描述的方法,获得了最可能的Anc80序列(如通过后验概率确定的),并且称为Anc80L1(SEQ ID NO:35显示Anc80L1壳体的核酸序列并且SEQ ID NO:36显示Anc80L1VP1多肽的氨基酸序列)。还使用本文描述的序列通过商业公司合成Anc80概率文库并亚克隆到表达载体中。
在联合测定中克隆评估Anc80文库的载体产率和感染力。在筛选中,进一步表征了Anc80L65(SEQ ID NO:23)以及其他多种变体。
在序列差异方面比较了Anc80文库和Anc80L65(图9;从对角线向上%,以下的氨基酸差异#)。使用NCBI-BLAST,与Anc80L65最接近的公开可获得的序列是rh10(GenBank登记号AAO88201.1)。
图10显示了Anc80L65产生与AAV2相当的载体产率(图A),在透射电镜法(Transmission Electroscopy,TEM)下产生病毒颗粒(B图),并且基于变性条件下的SDSPAGE(C图)和使用AAV壳体抗体B1进行的Western印迹法(图D)在生物化学上产生AAV cap和VP1,2和3蛋白质。这些实验在下面的段落中更详细地描述。
简要地,小规模生产了含有报道构建体的AAV2/8,AAV2/2,AAV2/Anc80L27,AAV2/Anc80L44,和AAV2/Anc80L65载体,所述报告构建体由CMV启动子控制下的eGFP和萤火虫萤光素酶构成。然后通过qPCR获得病毒的这些小规模制备物的滴度。基于这些实验,发现Anc80L27,Anc80L44和Anc80L65载体产生与AAV2的病毒水平相当的病毒水平(图10A)。
为了确认组装成合适大小和构象的完整病毒样颗粒的Anc80L65壳体蛋白,使用透射电子显微术(TEM)获得显微照片。将大规模的纯化的Anc80-L065制备物装载到聚乙烯醇缩甲醛(polyvinyl formal)涂覆的铜网格上,然后用乙酸双氧铀(uranylacetate)染色。显微照片揭示了具有20至25nm之间直径的完整的,六角形的颗粒(图10B)。
为了确定是否适当地加工了(即剪接和表达)合成的祖先壳体基因,在变性条件下将AAV2/8,AAV2/2,和AAV2/Anc80L65载体的大规模纯化的制备物装载到SDS-PAGE凝胶(1E10GC/孔)。对于每一种载体制备物,代表病毒壳体蛋白VP1,VP2和VP3的条带清楚地存在(图10C)。用AAV壳体抗体B1进行的Western印迹法进一步证实这些条带代表预测的蛋白质(图10D)。
此外,图11显示相对于AAV2和/或AAV8对照,使用GFP作为读出(图A)或萤光素酶(图B),Anc80L65以MOI 10E4GC/细胞在HEK293细胞上体外感染哺乳动物组织和细胞。在IV注射编码核LacZ转基因的指定AAV后(顶行,图C),在直接肌内(IM)注射编码GFP的指定AAV后(中间行,图C),和在视网膜下注射编码GFP的指定AAV后(底行,图C),Anc80L65也有效靶向肝。这些实验在下面的段落中更详细地描述。
为获得祖先病毒体的感染性的相对测量,产生含有双顺反子报道构建体的AAV2/2,AAV2/8,AAV2/Anc80L65,AAV2/Anc80L44,AAV2/Anc80L27,AAV2/Anc80L121,AAV2/Anc80L122,AAV2/Anc80L123,AAV2/Anc80L124,和AAV2/Anc80L125的粗制制备物,所述双顺反子报道构建体包括在CMV启动子控制下的eGFP和萤火虫萤光素酶。然后以1E4GC/细胞的MOI(通过如上所述的qPCR获得的滴度)用每一载体转导以HEK293细胞汇合的96孔板。48小时后,荧光显微术确认在经转导的细胞中GFP的的存在(图11A)。然后测定细胞的萤光素酶的存在(图11B),这确定了用Anc80衍生的载体转导的细胞中萤光素酶的表达是在用AAV8转导的细胞(较低水平的转导)和在用AAV2转导的细胞(较高水平的转导)的表达之间。
为了评估在体内情况下基因转移的相对效率,得到AAV2/2,AAV2/8和AAV2/Anc80L65的纯化的高滴度的制备物。在全身麻醉后通过IP注射将3.9E10GC的每个载体(其壳体化在TBG启动子控制下的编码核LacZ的转基因)注射到C57BL/6小鼠(每个条件3只小鼠)中。注射后28天,将小鼠处死并收集组织。通过标准组织学技术对肝切片,并针对β-半乳糖苷酶染色。然后在显微镜下对切片成像,并且代表性图像显示于图11C,顶行。
然后获得相同血清型的载体,其含有pCASI启动子控制下的编码eGFP和hA1AT的双顺反子转基因。为了评估Anc80L65转导鼠骨骼肌的能力,在全身麻醉后将1E10GC的每一载体注射到C57BL/6小鼠的骨骼肌中(每种条件5只小鼠)。注射后28天,处死小鼠,冰冻切片组织,并且用荧光共焦显微术评估eGFP的存在(蓝色是DAPI,绿色是EGFP)。代表性的图像显示于图11C,中间行。这些实验表明,Anc80L65载体能够通过肌肉内注射转导鼠骨骼肌。
获得了相同血清型的载体,此次壳体化在CMV启动子控制下的仅编码eGFP转基因的构建体。在全身麻醉后将2E9颗粒经视网膜下(sub-retinally)注射到C57BL/6小鼠中。注射后28天,处死小鼠,收集眼睛,冰冻切片,并且用荧光共焦显微镜评估eGFP的存在(蓝色是DAPI,绿色是EGFP)。代表性的图像显示于图11C,底行。这些实验表明,Anc80L65载体能够以与AAV8载体相当的水平转导鼠视网膜。
图12显示实验结果,其中相对于现存的AAV病毒载体评估祖先病毒载体的血清阳性率。使用体外中和抗体测定,显示Anc80L65展现出对使用IVIG(约10,000个体的药用汇合血清)中和的增加的抗性(图12A)。另外,在须盒图中,来自比利时个体的血清(n=100;图12C)或波士顿个体的血清(n=102;图12B)显示相对于AAV2,8对中和Anc80L65的降低的敏感性(或增加的抗性),和相对于rh32.33(一种具有已知最小血清阳性率但有限用作基因治疗载体的不同AVV载体)的类似的血清阳性率。从食蟹猴获得的血清展示出相比于AAV2,8和rh32.33的类似的增加的抗性(图12D)。这些实验在下面段落中更详细地描述。
简言之,获得了AAV2/8,AAV2/2,AAV2/rh32.33和AAV2/Anc80L65病毒载体的纯化的高滴度制备物,所述病毒载体壳体化包括在CMV启动子控制下的eGFP和萤火虫萤光素酶双顺反子转基因。然后将这些载体与两倍连续稀释的IVIG(10mg,5mg,2.5mg等)一起温育或不与IVIG温育(每种条件1E9GC)。温育后,以每孔1E4MOI(每孔一个稀释度)将载体用于转导HEK293细胞。48小时后,通过发光测定法测定萤光素酶的相对量。相对于无血清对照的转导显示于图12A中。使用来自比利时个体的血清(n=100;图12C)或波士顿个体的血清(n=102;图12B)进行了相似的实验。对于每个血清样品,中和滴度报告为相对于无血清对照,给定的载体减少了50%的稀释度。在图12B和12C中以盒须图报告了中和滴度,其确定了Anc80L65载体在这两个人群(比利时或波士顿人群)中,相比于AAV2和AAV8具有较小的流行性,接近Rh32.33的水平(具有已知的最小血清阳性率的不同AAV载体)。以相同的方式评估了从食蟹猕猴获得的血清(图12D),其中发现相比于AAV2,AAV8和Rh.32.33载体,Anc80L65具有明显更小的血清阳性率。
实施例8:其他祖先AAV壳体的产生
然后通过商业实验室(Gen9)合成最有可能的祖先AAV壳体序列(如通过后验概率确定)并以线性dsDNA提供。然后将这些氨基酸序列与现存AAV的氨基酸序列相比较以确定它们不同的程度(图13)。每一祖先VP1蛋白与选择的代表性现存AAV的VP1蛋白相差3.6%-9.3%(图13A),而祖先VP3蛋白相差4.2%-9.4%(图13B)。将这些壳体各自通过限制酶消化(HindIII&SpeI)和T4连接亚克隆到AAV生产质粒(pAAVector2/空)。这些克隆通过限制性消化和Sanger测序来确认,然后产生了中等规模的质粒DNA的制备物。
然后将这些质粒中的每一个用于产生AAV载体,其含有编码eGFP和萤火虫萤光素酶两者的报告基因。如前所述,小规模一式三份产生这些载体。然后通过qPCR滴定病毒的粗制备物并发现相对于AAV8产生2.71%至183.1%的病毒颗粒(图14和15)。然后,将这些滴度用于设立滴度对照实验以评估相对感染性。Anc126由于其显著降低的产生而没有滴度对照,因此,关于Anc126的感染性的数据不能与实验中其他病毒的感染性准确相比。以1.9E3GC/细胞的多重感染(MOI)将其它载体用于转导HEK293细胞。
转导后60小时,通过荧光显微术评估细胞的GFP表达。除了阴性对照外,在每一种条件下检测eGFP阳性细胞(图16)。这表明预测,合成并克隆的每一种祖先序列能够产生活的,感染性的病毒颗粒。为了获得感染性的相对水平的想法,也在相同的细胞上进行萤光素酶测定法。结果指示相对于AAV8,每一个祖先载体能够转导28.3%至850.8%的HEK293细胞(图17和18)。应该注意的是将Anc126排除在相对转导的分析之外,因为它没有滴度对照。
总之,合成了八种新的祖先AAV壳体基因并与AAV8,AAV2和前面描述的Anc80L65载体一起用于产生功能性病毒载体。在体外评估了产生和感染,那些发现的总结显示于图19。
实施例9:载体免疫预防
在有载体(vectored)的免疫预防中,将基因治疗载体(如AAV)用于递送编码针对传染原的广谱中和抗体的转基因。参见例如Balazs et al.(2013,Nat.Biotechnol.,31:647-52);Limberis et al.(2013,Sci.Transl.Med.,5:187ra72);Balazs et al.(2012,Nature,481:81-4);及Deal et al.(2014,PNAS USA,111:12528-32)。这种处理的一个优点是,宿主在它们自己的细胞中产生抗体,这意味着单次施用具有赋予针对病原体(etiologic agent)的终生保护的潜力。
实施例10:药物递送载体
(兰尼单抗(ranibizumab))和阿瓦斯丁(贝伐单抗)均是基于针对血管内皮生长因子A(VEGF-A)的相同的人源化小鼠单克隆抗体的抗血管生成剂。虽然贝伐单抗是完整的抗体,而兰尼单抗是片段(Fab),它们通过相同机制-通过拮抗VEGF发挥作用以治疗湿性年龄相关性黄斑变性。参见例如Mao等人(2011,Hum.Gene Ther.,22:1525-35);Xie et al.(2014,Gynecol.Oncol.,doi:10.1016/j.ygyno.2014.07.105);和Watanabe et al.(2010,Gene Ther.,17:1042-51)。由于这两种分子是蛋白质,它们可以通过DNA编码,并在用含有转基因的载体转导的细胞中产生,并且足够小从而可以被包装到AAV载体中。
其他实施方案
应理解虽然本文中已经结合多个不同方面描述了方法和物质组合物,但是前述多个方面的描述旨在说明而不是限制方法和物质组合物的范围。其他方面,优点和修改均落入所附权利要求书的范围之内。
本文公开的是方法和组合物,它们可用于公开的方法和组合物的产物,可结合公开的方法和组合物的产物使用,可用于制备公开的方法和组合物的产物或者是公开的方法和组合物的产物。在本文中公开了这些和其他物质,并且应当理解公开了这些方法和组合物的组合,亚组,相互作用,组等。即,虽然可能没有明确地公开具体提到这些组合物和方法的每个不同个体和集体组合以及替换,但每一个是本文中特别考虑并且描述的。例如,如果公开并讨论了特定的物质组合物或特定方法并且讨论了多种组合物和方法,那么除非有明确相反的指示,明确涵盖组合物和方法的每一个组合以及替换。同样,也明确涵盖和公开了这些的任何亚组或组合。
附件A
SEQ ID NO:1:Anc80多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAX1KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMX2AGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGX3STNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKX4LNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFX5FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRX6LQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTX7NQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITX8EEEIKTTNPVATEX9YGTVATNLQSX10NTAPATGTVNSQGALPGMVWQX11RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL
X1=K/R;X2=A/S;X3=A/G;X4=R/K;X5=E/Q;X6=T/E;X7=A/T;X8=S/N;X9=Q/E;X10=S/A;X11=N/D
SEQ ID NO:2:Anc80 DNA
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCAATCACCCCAGGAACCAGACTCCTCTTCGGGCATCGGCAAGAAAGGCCAGCAGCCCGCGXXX1AAGAGACTCAACTTTGGGCAGACAGGCGACTCAGAGTCAGTGCCCGACCCTCAACCACTCGGAGAACCCCCCGCAGCCCCCTCTGGTGTGGGATCTAATACAATGXXX2GCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAACGCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTCCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAGCCAATCGGGAXXX3AGCACCAACGACAACACCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACT6CCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGXXX4CTCAACTTCAAGCTCTTCAACATCCAGGTCAAGGAGGTCACGACGAATGATGGCACCACGACCATCGCCAATAACCTTACCAGCACGGTTCAGGTCTTTACGGACTCGGAATACCAGCTCCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGGTACCTGACTCTGAACAATGGCAGTCAGGCCGTGGGCCGTTCCTCCTTCTACTGCCTGGAGTACTTTCCTTCTCAAATGCTGAGAACGGGCAACAACTTTXXX5TTCAGCTACACGTTTGAGGACGTGCCTTTTCACAGCAGCTACGCGCACAGCCAAAGCCTGGACCGGCTGATGAACCCCCTCATCGACCAGTACCTGTACTACCTGTCTCGGACTCAGACCACGAGTGGTACCGCAGGAAATCGGXXX6TTGCAATTTTCTCAGGCCGGGCCTAGTAGCATGGCGAATCAGGCCAAAAACTGGCTACCCGGGCCCTGCTACCGGCAGCAACGCGTCTCCAAGACAXXX7AATCAAAATAACAACAGCAACTTTGCCTGGACCGGTGCCACCAAGTATCATCTGAATGGCAGAGACTCTCTGGTAAATCCCGGTCCCGCTATGGCAACCCACAAGGACGACGAAGACAAATTTTTTCCGATGAGCGGAGTCTTAATATTTGGGAAACAGGGAGCTGGAAATAGCAACGTGGACCTTGACAACGTTATGATAACCXXX8GAGGAAGAAATTAAAACCACCAACCCAGTGGCCACAGAAXXX9TACGGCACGGTGGCCACTAACCTGCAATCGXXX10AACACCGCTCCTGCTACAGGGACCGTCAACAGTCAAGGAGCCTTACCTGGCATGGTCTGGCAGXXX11CGGGACGTGTACCTGCAGGGTCCTATCTGGGCCAAGATTCCTCACACGGACGGACACTTTCATCCCTCGCCGCTGATGGGAGGCTTTGGACTGAAACACCCGCCTCCTCAGATCCTGATTAAGAATACACCTGTTCCCGCGAATCCTCCAACTACCTTCAGTCCAGCTAAGTTTGCGTCGTTCATCACGCAGTACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAACCCAGAGATTCAATACACTTCCAACTACAACAAATCTACAAATGTGGACTTTGCTGTTGACACAAATGGCGTTTATTCTGAGCCTCGCCCCATCGGCACCCGTTACCTCACCCGTAATCTG
XXX1=AAG/AAA;XXX2=GCA/AGC;XXX3=GCA/GGC;XXX4=AGA/AAG;XXX5=GAG/GAG;XXX6=ACG/GAG;XXX7=GCG/ACC;XXX8=AGT/AAC;XXX9=CAG/GAG;XXX10=TCA/GCC;XXX11=AAC/GAC
SEQ ID NO:3:Anc81多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSX1GIGKKGQQPAX2KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISX3X4QSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKX5LNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFX6FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTAGNX7X8LQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFPSSGVLIFGKQGAGNX9NVDX10X11NVMITX12EEEIKTTNPVATEEYGX13VATNLQSX14NTAPQTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFX15PAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTEGVYSEPRPIGTRYLTRNL
X1=T/S;X2=K/R;X3=N/S;X4=S/H;X5=R/K;X6=E/Q;X7=R/Q;X8=T/E;X9=D/S;X10=L/Y;X11=D/S;X12=S/N;X13=V/I;X14=A/S;X15=S/T
SEQ ID NO:4:Anc81 DNA
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCAATCACCCCAGGAACCAGACTCCTCTXXX1GGCATCGGCAAGAAAGGCCAGCAGCCCGCGXXX2AAGAGACTCAACTTTGGGCAGACTGGCGACTCAGAGTCAGTGCCCGACCCTCAACCACTCGGAGAACCCCCCGCAGCCCCCTCTGGTGTGGGATCTAATACAATGGCTGCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTCCCCACCTACAACAACCACCTCTACAAGCAAATCTCCXXX3XXX4CAATCGGGAGGAAGCACCAACGACAACACCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGXXX5CTCAACTTCAAGCTCTTCAACATCCAGGTCAAGGAGGTCACGACGAATGATGGCACCACGACCATCGCCAATAACCTTACCAGCACGGTTCAGGTCTTTACGGACTCGGAATACCAGCTCCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGGTACCTGACTCTGAACAATGGCAGTCAGGCCGTGGGCCGTTCCTCCTTCTACTGCCTGGAGTACTTTCCTTCTCAAATGCTGAGAACGGGCAACAACTTTXXX6TTCAGCTACACGTTTGAGGACGTGCCTTTTCACAGCAGCTACGCGCACAGCCAAAGCCTGGACCGGCTGATGAACCCCCTCATCGACCAGTACCTGTACTACCTGTCTCGGACTCAGACCACGGGAGGTACCGCAGGAAATXXX7XXX8TTGCAATTTTCTCAGGCCGGGCCTAGTAGCATGGCGAATCAGGCCAAAAACTGGCTACCCGGGCCCTGCTACCGGCAGCAACGCGTCTCCAAGACAACGAATCAAAATAACAACAGCAACTTTGCCTGGACCGGTGCCACCAAGTATCATCTGAATGGCAGAGACTCTCTGGTAAATCCCGGTGTCGCTATGGCAACCCACAAGGACGACGAAGACCGATTTTTTCCGTCCAGCGGAGTCTTAATATTTGGGAAACAGGGAGCTGGAAATXXX9AACGTGGACXXX10XXX11AACGTTATGATAACCXXX12GAGGAAGAAATTAAAACCACCAACCCAGTGGCCACAGAAGAGTACGGCXXX13GTGGCCACTAACCTGCAATCGXXX14AACACCGCTCCTCAAACAGGGACCGTCAACAGTCAAGGAGCCTTACCTGGCATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCTATCTGGGCCAAGATTCCTCACACGGACGGAAACTTTCATCCCTCGCCGCTGATGGGAGGCTTTGGACTGAAACACCCGCCTCCTCAGATCCTGATTAAGAATACACCTGTTCCCGCGAATCCTCCAACTACCTTCXXX15CCAGCTAAGTTTGCGTCGTTCATCACGCAGTACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAACCCAGAGATTCAATACACTTCCAACTACAACAAATCTACAAATGTGGACTTTGCTGTTGACACAGAAGGCGTTTATTCTGAGCCTCGCCCCATCGGCACCCGTTACCTCACCCGTAATCTG
XXXI=ACG/AGC;XXX2=AAA/AAG;XXX3=AAC/AGT;XXX4=AGC/CAC;XXX5=AGA/AAG;XXX6=GAG/CAG;XXX7=CGG/CAG;XXX8=ACG/GAG;XXX9=GAC/AGC;XXX10=CTT/TAC;XXXI1=GAC/AGC;XXX12=AGT/AAC;XXX13=GTG/ATC;XXX14=GCA/AGC;XXX15=AGT/ACC
SEQ ID NO:5:Anc82多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQREPDSSXIGIGKKGQQPAX2KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTAGTQTLQFSQAGPSSMANQAKNWLPGPCYRQQRVSTTTNQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFPSSGVLIFGKQGAGNDNVDYSNVMITX3EEEIKTTNPVATEEYGVVATNLQSANTAPQTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL
X1=T/S;X2=K/R;X3=S/N
SEQ ID NO:6:Anc82 DNA
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAATCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCAGTCACCACAGCGTGAGCCCGACTCCTCCXXXIGGCATCGGCAAGAAAGGCCAGCAGCCCGCCXXX2AAGAGACTCAATTTCGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCTCAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGATCTAATACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGTGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGGACCTCGGGAGGCAGCACCAACGACAACACCTACTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAACTTCAAGCTCTTCAACATCCAGGTCAAAGAGGTCACGACGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCGTCCAGGTGTTTACGGACTCGGAATACCAGCTGCCGTACGTCCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGCTACCTGACTCTCAACAACGGTAGTCAGGCCGTGGGACGTTCCTCCTTCTACTGCCTGGAGTACTTCCCCTCTCAGATGCTGAGAACGGGCAACAACTTTCAATTCAGCTACACTTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGTTTGGACAGGCTGATGAATCCTCTCATCGACCAGTACCTGTACTACCTGTCAAGAACCCAGACTACGGGAGGCACAGCGGGAACCCAGACGTTGCAGTTTTCTCAGGCCGGGCCTAGCAGCATGGCGAATCAGGCCAAAAACTGGCTGCCTGGACCCTGCTACAGACAGCAGCGCGTCTCCACGACAACGAATCAAAACAACAACAGCAACTTTGCCTGGACTGGTGCCACCAAGTATCATCTGAACGGCAGAGACTCTCTGGTGAATCCGGGCGTCGCCATGGCAACCCACAAGGACGACGAGGACCGCTTCTTCCCATCCAGCGGCGTCCTCATATTTGGCAAGCAGGGAGCTGGAAATGACAACGTGGACTATAGCAACGTGATGATAACCXXX3GAGGAAGAAATCAAGACCACCAACCCCGTGGCCACAGAAGAGTATGGCGTGGTGGCTACTAACCTACAGTCGGCAAACACCGCTCCTCAAACGGGGACCGTCAACAGCCAGGGAGCCTTACCTGGCATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCTATTTGGGCCAAGATTCCTCACACAGATGGCAACTTTCACCCGTCTCCTTTAATGGGCGGCTTTGGACTTAAACATCCGCCTCCTCAGATCCTCATCAAAAACACTCCTGTTCCTGCGGATCCTCCAACAACGTTCAACCAGGCCAAGCTGAATTCTTTCATCACGCAGTACAGCACCGGACAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAGCGCTGGAACCCAGAGATTCAGTATACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTTAATACTGAGGGTGTTTACTCTGAGCCTCGCCCCATTGGCACTCGTTACCTCACCCGTAATCTG
XXX1=ACG/AGC;XXX2=AAA/AGA;
SEQ ID NO:7:Anc83多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQREPDSSX1GIGKKGQQPAX2KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLX3FKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFX4FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTAGTQTLQFSQAGPSX5MANQAKNWLPGPCYRQQRVSTTTSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEX7RFFPSSGX7LIFGKQGAGKDNVDYSNVMLTSEEEIKTTNPVATEEYGVVADNLQQQNTAPQX8GTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL
X1=T/S;X2=R/K;X3=N/S;X4=Q/E;X5=N/T/S;X6=D/E;X7=I/V;X8=I/V
SEQ ID NO:8:Anc83 DNA
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAATCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCAGTCACCACAGCGTGAGCCCGACTCCTCCXXX1GGCATCGGCAAGAAAGGCCAGCAGCCCGCCXXX2AAGAGACTCAATTTCGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCTCAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGATCTAATACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGTGCCGACGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGGACCTCGGGAGGCAGCACCAACGACAACACCTACTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCXXX3TTCAAGCTCTTCAACATCCAGGTCAAAGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCATCCAGGTGTTTACGGACTCGGAATACCAGCTGCCGTACGTCCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGCTACCTGACTCTCAACAACGGTAGTCAGGCCGTGGGACGTTCCTCCTTCTACTGCCTGGAGTACTTCCCCTCTCAGATGCTGAGAACGGGCAACAACTTTXXX4TTCAGCTACACTTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGTTTGGACAGGCTGATGAATCCTCTCATCGACCAGTACCTGTACTACCTGTCAAGAACCCAGACTACGGGAGGCACAGCGGGAACCCAGACGTTGCAGTTTTCTCAGGCCGGGCCTAGCXXX5ATGGCGAATCAGGCCAAAAACTGGCTGCCTGGACCCTGCTACAGACAGCAGCGCGTCTCCACGACAACGTCGCAAAACAACAACAGCAACTTTGCCTGGACTGGTGCCACCAAGTATCATCTGAACGGCAGAGACTCTCTGGTGAATCCGGGCGTCGCCATGGCAACCCACAAGGACGACGAGXXX6CGCTTCTTCCCATCCAGCGGCXXX7CTCATATTTGGCAAGCAGGGAGCTGGAAAAGACAACGTGGACTATAGCAACGTGATGCTAACCAGCGAGGAAGAAATCAAGACCACCAACCCCGTGGCCACAGAAGAGTATGGCGTGGTGGCTGATAACCTACAGCAGCAAAACACCGCTCCTCAAXXX8GGGACCGTCAACAGCCAGGGAGCCTTACCTGGCATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCTATTTGGGCCAAGATTCCTCACACAGATGGCAACTTTCACCCGTCTCCTTTAATGGGCGGCTTTGGACTTAAACATCCGCCTCCTCAGATCCTCATCAAAAACACTCCTGTTCCTGCGGATCCTCCAACAACGTTCAACCAGGCCAAGCTGAATTCTTTCATCACGCAGTACAGCACCGGACAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAGCGCTGGAACCCAGAGATTCAGTATACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTTAATACTGAGGGTGTTTACTCTGAGCCTCGCCCCATTGGCACTCGTTACCTCACCCGTAATCTG
XXX1=ACG/AGC;XXX2=AGA/AAG;XXX3=AAC/AGC;XXX4=CAA/GAA; XXX7=ATC/GTC;XXX8=ATA/GTA
SEQ ID NO:9:Anc84多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAX1KRLNFGQTGDSESVPDPQPIGEPPAAPSGVGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLX2FKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPSNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEX3RFFPSSGX4LMFGKQGAGKDNVDYSNVMLTSEEEIKTTNPVATEQYGVVADNLQQQNTAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGVYSEPRPIGTRYLTRNL
X1=R/K;X2=N/S;X3=D/E;X4=I/V
SEQ ID NO:10:Anc84 DNA
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAATCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCCGTCACCACAGCGTTCCCCCGACTCCTCCACGGGCATCGGCAAGAAAGGCCAGCAGCCCGCCXXX1AAGAGACTCAATTTCGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCTCAACCTATCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGATCTGGTACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGTGCCGACGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGGACCTCGGGAGGCAGCACCAACGACAACACCTACTTTGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCXXX2TTCAAGCTCTTCAACATCCAGGTCAAAGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCATCCAGGTGTTTACGGACTCGGAATACCAGCTGCCGTACGTCCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGCTACCTGACTCTCAACAACGGTAGTCAGGCCGTGGGACGTTCCTCCTTCTACTGCCTGGAGTACTTCCCCTCTCAGATGCTGAGAACGGGCAACAACTTTGAGTTCAGCTACACTTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGTTTGGACAGGCTGATGAATCCTCTCATCGACCAGTACCTGTACTACCTGTCAAGAACCCAGTCTACGGGAGGCACAGCGGGAACCCAGCAGTTGCTGTTTTCTCAGGCCGGGCCTAGCAACATGTCGGCTCAGGCCAAAAACTGGCTGCCTGGACCCTGCTACAGACAGCAGCGCGTCTCCACGACACTGTCGCAAAACAACAACAGCAACTTTGCCTGGACTGGTGCCACCAAGTATCATCTGAACGGCAGAGACTCTCTGGTGAATCCGGGCGTCGCCATGGCAACCCACAAGGACGACGAGXXX3CGCTTCTTCCCATCCAGCGGCXXX4CTCATGTTTGGCAAGCAGGGAGCTGGAAAAGACAACGTGGACTATAGCAACGTGATGCTAACCAGCGAGGAAGAAATCAAGACCACCAACCCCGTGGCCACAGAACAGTATGGCGTGGTGGCTGATAACCTACAGCAGCAAAACACCGCTCCTATTGTGGGGGCCGTCAACAGCCAGGGAGCCTTACCTGGCATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCTATTTGGGCCAAGATTCCTCACACAGATGGCAACTTTCACCCGTCTCCTTTAATGGGCGGCTTTGGACTTAAACATCCGCCTCCTCAGATCCTCATCAAAAACACTCCTGTTCCTGCGGATCCTCCAACAACGTTCAACCAGGCCAAGCTGAATTCTTTCATCACGCAGTACAGCACCGGACAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAGCGCTGGAACCCAGAGATTCAGTATACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTTAATACTGAGGGTGTTTACTCTGAGCCTCGCCCCATTGGCACTCGTTACCTCACCCGTAATCTG
XXX2=AAC/AGC;XXX3=GAC/GAG;XXX4=ATC/GTC
SEQ ID NO:11:Anc94多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGXXRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPX1NMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNTAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFSQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL
X1=S/N
SEQ ID NO:12:Anc94 DNA
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCCATCACCCCAGCGTTCTCCAGACTCCTCTACGGGCATCGGCAAGAAAGGCCAGCAGCCCGCGAAAAAGAGACTCAACTTTGGGCAGACTGGCGACTCAGAGTCAGTGCCCGACCCTCAACCAATCGGAGAACCCCCCGCAGGCCCCTCTGGTCTGGGATCTGGTACAATGGCTGCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAGTTCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTCCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGGACTTCGGGAGGAAGCACCAACGACAACACCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAACTTCAAGCTCTTCAACATCCAGGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTTACCAGCACGATTCAGGTCTTTACGGACTCGGAATACCAGCTCCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGGTACCTGACTCTGAACAATGGCAGTCAGGCCGTGGGCCGTTCCTCCTTCTACTGCCTGGAGTACTTTCCTTCTCAAATGCTGAGAACGGGCAACAACTTTGAGTTCAGCTACACGTTTGAGGACGTGCCTTTTCACAGCAGCTACGCGCACAGCCAAAGCCTGGACCGGCTGATGAACCCCCTCATCGACCAGTACCTGTACTACCTGTCTCGGACTCAGTCCACGGGAGGTACCGCAGGAACTCAGCAGTTGCTATTTTCTCAGGCCGGGCCTXXXAACATGTCGGCTCAGGCCAAAAACTGGCTACCCGGGCCCTGCTACCGGCAGCAACGCGTCTCCACGACACTGTCGCAAAATAACAACAGCAACTTTGCCTGGACCGGTGCCACCAAGTATCATCTGAATGGCAGAGACTCTCTGGTAAATCCCGGTGTCGCTATGGCAACCCACAAGGACGACGAAGAGCGATTTTTTCCGTCCAGCGGAGTCTTAATGTTTGGGAAACAGGGAGCTGGAAAAGACAACGTGGACTATAGCAGCGTTATGCTAACCAGTGAGGAAGAAATTAAAACCACCAACCCAGTGGCCACAGAACAGTACGGCGTGGTGGCCGATAACCTGCAACAGCAAAACACCGCTCCTATTGTAGGGGCCGTCAACAGTCAAGGAGCCTTACCTGGCATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCTATCTGGGCCAAGATTCCTCACACGGACGGAAACTTTCATCCCTCGCCGCTGATGGGAGGCTTTGGACTGAAACACCCGCCTCCTCAGATCCTGATTAAGAATACACCTGTTCCCGCGGATCCTCCAACTACCTTCAGTCAAGCTAAGCTGGCGTCGTTCATCACGCAGTACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAACCCAGAGATTCAATACACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTTAACACAGAAGGCACTTATTCTGAGCCTCGCCCCATCGGCACCCGTTACCTCACCCGTAATCTG
XXX1=AGT/AAT
SEQ ID NO:13:Anc113多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEX1SPQRSPDSSTGIGKKGQQPAX2KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSAGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLX3FKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQSVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSTTGGTAGNRELQFX4QAGPSTMAEQAKNWLPGPCYRQQRVSKTLDQNNNSNFAWTGATKYHLNGRNSLVNPGVAMATHKDDEDRFFPSSGVLIFGKTGAANKTTLENVLMTX5EEEIKTTNPVATEEYGX6VSSNLQSX7NTAPQTQTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPEVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYDKSTNVDFAVDSEGVYSEPRPIGTRYLTRNL
X4=Y/H;
SEQ ID NO:14:Anc113 DNA
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCATTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGXXX1TCACCTCAGCGTTCCCCCGACTCCTCCACGGGCATCGGCAAGAAAGGCCAGCAGCCCGCCXXX2AAGAGACTCAATTTCGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCTCAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGATCTGGTACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGTGCCGACGGAGTGGGTAATGCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATTACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCCAGTCAAAGTGCAGGTAGTACCAACGACAACACCTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAAGCTGXXX3TTCAAGCTCTTCAACATCCAGGTCAAGGAGGTCACGACGAATGACGGCGTTACGACCATCGCTAATAACCTTACCAGCACGGTTCAGGTATTCTCGGACTCGGAATACCAGCTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGCTACCTGACTCTCAACAATGGCAGTCAGTCTGTGGGACGTTCCTCCTTCTACTGCCTGGAGTACTTCCCCTCTCAGATGCTGAGAACGGGCAACAACTTTGAGTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCACACAGCCAGAGCCTGGACCGGCTGATGAATCCCCTCATCGACCAGTACTTGTACTACCTGGCCAGAACACAGAGTACCACAGGAGGCACAGCTGGCAATCGGGAACTGCAGTTTXXX4CAGGCCGGGCCTTCAACTATGGCCGAACAAGCCAAGAATTGGTTACCTGGACCTTGCTACCGGCAACAAAGAGTCTCCAAAACGCTGGATCAAAACAACAACAGCAACTTTGCTTGGACTGGTGCCACCAAATATCACCTGAACGGCAGAAACTCGTTGGTTAATCCCGGCGTCGCCATGGCAACTCACAAGGACGACGAGGACCGCTTTTTCCCATCCAGCGGAGTCCTGATTTTTGGAAAAACTGGAGCAGCTAACAAAACTACATTGGAAAATGTGTTAATGACAXXX5GAAGAAGAAATTAAAACTACTAATCCTGTAGCCACGGAAGAATACGGGXXX6GTCAGCAGCAACTTACAATCGXXX7AATACTGCACCCCAGACACAAACTGTCAACAGCCAGGGAGCCTTACCTGGCATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCCATCTGGGCCAAGATTCCTCACACGGATGGCAACTTTCACCCGTCTCCTTTGATGGGCGGCTTTGGACTTAAACATCCGCCTCCTCAGATCCTGATCAAGAACACTCCCGTTCCCGCTAATCCTCCGGAGGTGTTTACTCCTGCCAAGTTTGCTTCGTTCATCACACAGTACAGCACCGGACAAGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATTCAGTACACCTCCAACTATGATAAGTCGACTAATGTGGACTTTGCCGTTGACAGCGAGGGTGTTTACTCTGAGCCTCGCCCTATTGGCACTCGTTACCTCACCCGTAATCTG
XXX1=CCG/CAG;XXX2=AAA/AGA;XXX3=CGG/AAC;XXX4=TAC/CAC;XXX5=AAT/AGT;XXX6=GTA/ATA;XXX7=GCT/TCT
SEQ ID NO:15:Anc126多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKX1GQQPAX2KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMASGGGAPMADNNEGADGVGNX3SGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKX4LNFXLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFX5FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLX6RTQTTSGTAQNRELX7FSQAGPSSMX8NQAKNWLPGPCYRQQRVSKTANDNNNSNFAWTGATKYHLNGRDSLVNPGPAMASHKDDEDKFFPMSGVLIFGKQGAGASNVDLDNVMITDEEEIKTTNPVATEQYGTVATNLQSSNTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSX9NVDFTVDTNGVYSEPRPIGTRYLTRNL
X1=S/T;X2=K/R;X3=A/S;X4=R/K;X5=T/Q;X6=S/N;X7=Q/L;X8=A/S;X9=A/T
SEQ ID NO:16:Anc126 DNA
ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGATGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGAGGGTTCTCGAACCTCTTGGTCTGGTTGAGGAAGGTGCTAAGACGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCTCGGGCATTGGCAAGXXXIGGCCAGCAGCCCGCTXXX2AAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCACAACCTCTCGGAGAACCTCCAGCAGCCCCCTCTGGTGTGGGATCTAATACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATXXX3TCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACATGGGCCTTGCCCACCTATAACAACCACCTCTACAAGCAAATCTCCAGTCAATCAGGGGCCAGCAACGACAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGATTTCAACAGATTCCACTGCCATTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAATTGGGGATTCCGGCCCAAGXXX4CTCAACTTCAAGCTCTTCAACATCCAAGTCAAGGAGGTCACGACGAATGATGGCACCACGACCATCGCTAATAACCTTACCAGCACGGTTCAAGTCTTCACGGACTCGGAGTACCAGTTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGGACGTGTTCATGATTCCGCAGTACGGCTACCTAACGCTCAACAATGGCAGCCAGGCAGTGGGACGGTCATCCTTTTACTGCCTGGAATATTTCCCATCGCAGATGCTGAGAACGGGCAATAACTTTXXX5TTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACCAGTACCTGTATTACCTGXXX6AGAACTCAGACTACGTCCGGAACTGCCCAAAACAGGGAGTTGXXX7TTTAGCCAGGCGGGTCCATCTAGCATGXXX8AATCAGGCCAAAAACTGGCTACCTGGACCCTGTTACCGGCAGCAGCGCGTTTCTAAAACAGCAAATGACAACAACAACAGCAACTTTGCCTGGACTGGTGCTACAAAATATCACCTTAATGGGCGTGATTCTTTAGTCAACCCTGGCCCTGCTATGGCCTCACACAAAGACGACGAAGACAAGTTCTTTCCCATGAGCGGTGTCTTGATTTTTGGAAAGCAGGGCGCCGGAGCTTCAAACGTTGATTTGGACAATGTCATGATCACAGACGAAGAGGAAATCAAAACCACTAACCCCGTGGCCACCGAACAATATGGGACTGTGGCAACCAATCTCCAGAGCAGCAACACAGCCCCTGCGACCGGAACTGTGAATTCTCAGGGAGCCTTACCTGGAATGGTGTGGCAAGACAGAGACGTATACCTGCAGGGTCCTATTTGGGCCAAAATTCCTCACACGGATGGACACTTTCACCCGTCTCCTCTCATGGGCGGCTTTGGACTTAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACGCCTGTTCCTGCGAATCCTCCGACAACGTTTTCGCCTGCAAAGTTTGCTTCATTCATCACCCAGTATTCCACAGGACAAGTGAGCGTGGAGATTGAATGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCCGAAATACAGTATACATCTAACTATAATAAATCTXXX9AACGTTGATTTCACTGTGGACACCAATGGAGTTTATAGTGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGTAACCTG
XXX1=TCA/ACA;XXX2=AAA/AGA;XXX3=GCC/TCC;XXX4=AGA/AAA;XXX5=ACC/CAG;XXX6=AGC/AAC;XXX7=CAG/CTG;XXX8=GCT/TCT;XXX9=GCC/ACC
SEQ ID NO:17:Anc127多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPQPXANQQHQDDX1RGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSSGIGKSGQQPAX2KRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKX3LNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFX4FSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLX5RTQTTSGTTQQSRLX6FSQAGPSSMX7QQAX8NWLPGPCYRQQRVSKTANDNNNSNFAWTX9ATKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGX10LIFGKQGTGASNVDLDNVMITDEEEIRTTNPVATEQYGTVATNLQSSNTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL
X1=G/S;X2=R/K;X3=K/R;X4=T/Q;X5=S/R;X6=Q/L;X7=A/S;X8=K/R;X9=G/A;X10=V/N
SEQ ID NO:18:Anc12 DNA
ATGGCTGCTGACGGTTATCTTCCAGATTGGCTCGAGGACAACCTTTCTGAAGGCATTCGTGAGTGGTGGGATCTGAAACCTGGAGCCCCTCAACCCAAAGCGAACCAACAACACCAGGACGACXXX1CGGGGTCTTGTGCTTCCGGGTTACAAATACCTCGGACCCTTTAACGGACTCGACAAAGGAGAGCCGGTCAACGAGGCGGACGCGGCAGCCCTCGAACACGACAAAGCTTACGACCAGCAGCTCAAGGCCGGTGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTTCAGGAGCGTCTTCAAGAAGATACGTCTTTTGGGGGCAACCTTGGCAGAGCAGTCTTCCAGGCCAAAAAGAGGGTCCTTGAGCCTCTTGGTCTGGTTGAGGAAGCAGCTAAAACGGCTCCTGGAAAGAAGAGGCCTGTAGAACAGTCTCCTCAGGAACCGGACTCATCATCTGGTATTGGCAAATCGGGCCAACAGCCTGCCXXX2AAAAGACTAAATTTCGGTCAGACTGGAGACTCAGAGTCAGTCCCAGACCCTCAACCTCTCGGAGAACCACCAGCAGCCCCCTCAGGTGTGGGATCTAATACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAGGGTGCCGATGGAGTGGGTAATTCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCAGAACCTGGGCCCTGCCCACTTACAACAACCATCTCTACAAGCAAATCTCCAGCCAATCAGGAGCTTCAAACGACAACCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATTAACAACAACTGGGGATTCCGGCCCAAGXXX3CTCAACTTCAAGCTCTTCAACATCCAAGTTAAAGAGGTCACGCAGAACGATGGCACGACGACTATTGCCAATAACCTTACCAGCACGGTTCAAGTGTTTACGGACTCGGAGTATCAGCTCCCGTACGTGCTCGGGTCGGCGCACCAAGGCTGTCTCCCGCCGTTTCCAGCGGACGTCTTCATGATCCCTCAGTATGGATACCTCACCCTGAACAACGGAAGTCAAGCGGTGGGACGCTCATCCTTTTACTGCCTGGAGTACTTCCCTTCGCAGATGCTAAGGACTGGAAATAACTTCXXX4TTCAGCTATACCTTCGAGGATGTACCTTTTCACAGCAGCTACGCTCACAGCCAGAGTTTGGATCGCTTGATGAATCCTCTTATTGATCAGTATCTGTACTACCTGXXXSAGAACGCAAACAACCTCTGGAACAACCCAACAATCACGGCTGXXX6TTTAGCCAGGCTGGGCCTTCGTCTATGXXX7CAGCAGGCCXXX8AATTGGCTACCTGGGCCCTGCTACCGGCAACAGAGAGTTTCAAAGACTGCTAACGACAACAACAACAGTAACTTTGCTTGGACAXXX9GCCACCAAATATCATCTCAATGGCCGCGACTCGCTGGTGAATCCAGGACCAGCTATGGCCAGTCACAAGGACGATGAAGAAAAATTTTTCCCTATGCACGGCXXX10CTAATATTTGGCAAACAAGGGACAGGGGCAAGTAACGTAGATTTAGATAATGTAATGATTACGGATGAAGAAGAGATTCGTACCACCAATCCTGTGGCAACAGAGCAGTATGGAACTGTGGCAACTAACTTGCAGAGCTCAAATACAGCTCCCGCGACTGGAACTGTCAATAGTCAGGGGGCCTTACCTGGCATGGTGTGGCAAGATCGTGACGTGTACCTTCAAGGACCTATCTGGGCAAAGATTCCTCACACGGATGGACACTTTCATCCTTCTCCTCTGATGGGAGGCTTTGGACTGAAACATCCGCCTCCTCAAATCTTGATCAAAAATACTCCGGTACCGGCAAATCCTCCGACGACTTTCAGCCCGGCCAAGTTTGCTTCATTTATCACTCAGTACTCCACTGGACAGGTCAGCGTGGAAATTGAGTGGGAGCTACAGAAAGAAAACAGCAAACGTTGGAATCCAGAGATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTAGACACTAATGGTGTTTATAGTGAACCTCGCCCTATTGGAACCCGGTATCTCACACGAAACTTG
XXX2=AGA/AAA;XXX3=AAA/AGA;XXX4=ACA/CAG;XXX5=AGC/AGA;XXX6=CAA/CTC;XXX7=GCT/TCT;XXX8=AAA/AGA;XXX9=GGG/GCG;XXX10=GTT/GAC
SEQ ID NO:19:L0027多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTANQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITNEEEIKTTNPVATEQYGTVATNLQSANTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL
SEQ ID NO:20:L0059多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRELQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITNEEEIKTTNPVATEEYGTVATNLQSANTAPATGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL
SEQ ID NO:21:L0060多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRELQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITSEEEIKTTNPVATEEYGTVATNLQSSNTAPATGTVNSQGALPGMVWQERDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL
SEQ ID NO:22:L0062多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPW.GYFDFNRFHCHFSPRDWQRLtNNNWGFRPKKLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRELQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITSEEEIKTTNPVATEEYGTVATNLQSANTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL
SEQ ID NO:23:L0065多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKAVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITNEEEIKTTNPVATEEYGTVATNLQSANTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL
SEQ ID NO:24:L0033多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTANQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITSEEEIKTTNPVATEQYGTVATNLQSSNTAPATGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTAYLTRNL
SEQ ID NO:25:L0036多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRTLQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTANQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITSEEEIKTTNPVATEEYGTVATNLQSSNTAPATGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL
SEQ ID NO:26:L0044多肽
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGSNTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSQSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLNFKLFNIQVKEVTTNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTSGTAGNRELQFSQAGPSSMANQAKNWLPGPCYRQQRVSKTTNQNNNSNFAWTGATKYHLNGRDSLVNPGPAMATHKDDEDKFFPMSGVLIFGKQGAGNSNVDLDNVMITNEEEIKTTNPVATEQYGTVATNLQSANTAPATGTVNSQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSTNVDFAVDTNGVYSEPRPIGTRYLTRNL
SEQ ID NO:27:AAV8 VP1多肽
SEQ ID NO:28:AAV9 VP1多肽
SEO ID NO:29:AAV6 VP1多肽
SEQ ID NO:30:AAV1 VP1多肽
SEQ ID NO:31:AAV2 VP1多肽
SEQ ID NO:32:AAV3 VP1多肽
SEQ ID NO:33:AAV3B VP1多肽
SEQ ID NO:34:AAV7 VP1多肽
Claims (37)
1.腺伴随病毒(AAV)壳体多肽,其具有选自由以下组成的组的氨基酸序列:SEQ ID NO:1、3、5、7、9、11、13、15和17。
2.权利要求1的AAV壳体多肽,其中所述AAV壳体多肽或包含所述AAV壳体多肽的病毒颗粒比AAV2壳体多肽或包含AAV2壳体多肽的病毒颗粒展现出更低的血清阳性率,并且其中所述AAV壳体多肽或包含所述AAV壳体多肽的病毒颗粒比AAV8壳体多肽或包含AAV8壳体多肽的病毒颗粒展现出相同或更低的血清阳性率(seroprevalence)。
3.权利要求1的AAV壳体多肽,其中所述AAV壳体多肽或包含所述AAV壳体多肽的病毒颗粒比AAV2壳体多肽或包含AAV2壳体多肽的病毒颗粒在更小程度上被人血清中和,并且其中所述AAV壳体多肽或包含所述AAV壳体多肽的病毒颗粒与AAV8壳体多肽或包含AAV8壳体多肽的病毒颗粒在相似或更小程度上被人血清中和。
4.权利要求1-3中任一项的AAV壳体多肽,其中所述AAV壳体多肽是纯化的。
5.权利要求1的AAV壳体多肽,其由选自由以下组成的组的核酸序列编码:SEQ ID NO:2、4、6、8、10、12、14、16和18。
6.核酸分子,其编码具有选自由以下组成的组的核酸序列的腺伴随病毒(AAV)壳体多肽:SEQ ID NO:2、4、6、8、10、12、14、16和18。
7.包含权利要求6的核酸分子的载体。
8.包含权利要求7的载体的宿主细胞。
9.纯化的病毒颗粒,其包含权利要求1-5中任一项的AAV壳体多肽。
10.权利要求9的纯化的病毒颗粒,其进一步包含转基因。
11.腺伴随病毒(AAV)壳体多肽,其与选自由以下组成的组的氨基酸序列具有至少95%的序列同一性:SEQ ID NO:19、20、21、22、23、24、25和26。
12.权利要求11的AAV壳体多肽,其中所述AAV壳体多肽或包含所述AAV壳体多肽的病毒颗粒比AAV2壳体多肽或包含AAV2壳体多肽的病毒颗粒展现出更低的血清阳性率,并且其中所述AAV壳体多肽或包含所述AAV壳体多肽的病毒颗粒比AAV8壳体多肽或包含AAV8壳体多肽的病毒颗粒展现出相同或更低的血清阳性率。
13.权利要求11的AAV壳体多肽,其中所述AAV壳体多肽或包含所述AAV壳体多肽的病毒颗粒比AAV2壳体多肽或包含AAV2壳体多肽的病毒颗粒在更小程度上被人血清中和,并且其中所述AAV壳体多肽或包含所述AAV壳体多肽的病毒颗粒与AAV8壳体多肽或包含AAV8壳体多肽的病毒颗粒在相似或更小程度上被人血清中和。
14.权利要求11-13中任一项的AAV壳体多肽,其中所述AAV壳体多肽是纯化的。
15.权利要求11-14中任一项的AAV壳体多肽,其中所述多肽与选自由以下组成的组的氨基酸序列具有至少99%的序列同一性:SEQ ID NO:19、20、21、22、23、24、25和26。
16.权利要求11-14中任一项的AAV壳体多肽,其中所述多肽与选自由以下组成的组的氨基酸序列具有100%的序列同一性:SEQ ID NO:19、20、21、22、23、24、25和26。
17.病毒颗粒,其包含权利要求11-16中任一项的AAV壳体多肽中的至少一种。
18.权利要求17的病毒颗粒,其进一步包含转基因。
19.用转基因进行基因转移或疫苗接种的方法,所述方法包括
将权利要求10或权利要求18的病毒颗粒施用于需要基因转移或免疫接种的受试者,其中所述病毒颗粒比AAV2病毒颗粒展现出更小的血清阳性率。
20.权利要求19的方法,其中所述病毒颗粒与AAV8病毒颗粒展现出大致相同或更小的血清阳性率。
21.权利要求19的方法,其中所述病毒颗粒比AAV2病毒颗粒在更小程度上被人血清中和,并且其中所述AAV病毒颗粒比AAV8病毒颗粒在相似或更小程度上被人血清中和。
22.对受试者接种疫苗的方法,所述方法包括
将可操作连接于权利要求1或权利要求11的AAV壳体多肽的靶抗原施用于需要疫苗接种的受试者,其中所述AAV壳体多肽比AAV2壳体多肽展现出更小的血清阳性率。
23.权利要求22的方法,其中所述AAV壳体多肽与AAV8壳体多肽展现出大致相同或更小的血清阳性率。
24.权利要求22的方法,其中所述AAV壳体多肽比AAV2壳体多肽在更小程度上被人血清中和,并且所述AAV壳体多肽比AAV8壳体多肽在相似或更小程度上被人血清中和。
25.预测祖先病毒或其部分的序列的计算机方法(in silico method),所述方法包括:
提供来自多个当代病毒或其部分的核苷酸或氨基酸序列;
使用多重序列比对(MSA)算法比对所述序列;
对进化建模以获得所述多个当代病毒或其部分的预测的祖先***发生;
在所述预测的祖先***发生的***发生节点处估计在所述序列的每个位置上的特定核苷酸或氨基酸残基的进化概率,并且
基于估计的在每个位置上的概率预测祖先病毒或其部分的序列。
26.权利要求25的方法,其中使用计算机处理器进行全部步骤。
27.权利要求25或权利要求26的方法,其中所述MSA算法使用***发生信息来预测所述比对中的缺口是否是缺失或***的结果。
28.权利要求27的方法,其中所述MSA算法是概率比对套件(Probabilistic AlignmentKit)(PRANK)。
29.权利要求25至28中任一项的方法,其中使用Aikake信息标准(Aikake InformationCriterion)(AIC)选择用于对进化建模的模型。
30.权利要求25至28中任一项的方法,其中使用JTT模型及伽马分布模型(Gammadistribution model)(“+G”)以及πi的频率计算(“+F”)获得所述预测的祖先***发生。
31.权利要求25的方法,其中使用JTT+G+F模型进行对进化建模的步骤。
32.权利要求25至31中任一项的方法,其进一步包括基于所述预测的序列合成所述祖先病毒或其部分。
33.权利要求32的方法,其进一步包括将所述祖先病毒或其部分组装成祖先病毒颗粒。
34.权利要求33的方法,其进一步包括对所述祖先病毒颗粒筛选下列至少一项:(1)复制;(b)基因转移性质;(c)受体结合;或(d)血清阳性率。
35.权利要求34的方法,其中所述祖先病毒颗粒比从所述多个当代病毒或其部分中的至少一种组装的病毒颗粒展现出更小的血清阳性率。
36.权利要求34的方法,其中所述祖先病毒颗粒比所述多个当代病毒或其部分中的至少一种组装的病毒颗粒在更小程度上被人血清中和。
37.权利要求25的方法,其中所述多个当代病毒或其部分属于选自由以下组成的组的家族:腺病毒(adenovirus)(AV),人免疫缺陷病毒(human immunodeficiency virus)(HIV),逆转录病毒(lentivirus),慢病毒(lentivirus),单纯疱疹病毒(herpes simplexvirus)(HSV),痘苗病毒(vaccinia virus),痘病毒(pox virus),流感病毒(influenzavirus),呼吸道合胞病毒(respiratory syncytial virus),副流感病毒(parainfluenzavirus)和泡沫病毒(foamy virus)。
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US11236402B2 (en) | 2011-04-22 | 2022-02-01 | The Regents Of The University Of California | Adeno-associated virus virions with variant capsid |
US11136557B2 (en) | 2013-05-31 | 2021-10-05 | The Regents Of The University Of California | Adeno-associated virus variants and methods of use thereof |
US11634691B2 (en) | 2013-05-31 | 2023-04-25 | The Regents Of The University Of California | Compositions and methods of treatment |
CN107532177A (zh) * | 2015-03-24 | 2018-01-02 | 加利福尼亚大学董事会 | 腺相关病毒变体及其使用方法 |
US10883117B2 (en) | 2015-03-24 | 2021-01-05 | The Regents Of The University Of California | Adeno-associated virus variants and methods of use thereof |
US11554180B2 (en) | 2016-07-29 | 2023-01-17 | The Regents Of The University Of California | Adeno-associated virus virions with variant capsid and methods of use thereof |
US11565001B2 (en) | 2016-07-29 | 2023-01-31 | The Regents Of The University Of California | Adeno-associated virus virions with variant capsid and methods of use thereof |
US11565000B2 (en) | 2016-07-29 | 2023-01-31 | The Regents Of The University Of California | Adeno-associated virus virions with variant capsid and methods of use thereof |
CN110831611A (zh) * | 2017-05-10 | 2020-02-21 | 马萨诸塞眼科耳科诊所 | 用于修饰病毒的组装活化蛋白(aap)依赖性的方法和组合物 |
US11680249B2 (en) | 2017-08-28 | 2023-06-20 | The Regents Of The University Of California | Adeno-associated virus capsid variants and methods of use thereof |
CN112368391A (zh) * | 2018-05-11 | 2021-02-12 | 马萨诸塞眼科耳科诊所 | 腺相关病毒的肝脏特异性嗜性 |
US11987801B2 (en) | 2018-05-11 | 2024-05-21 | Massachusetts Eye And Ear Infirmary | Altering tissue tropism of adeno-associated viruses |
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