CN110724673B - 嗜外层视网膜的腺相关病毒病毒体及其应用 - Google Patents
嗜外层视网膜的腺相关病毒病毒体及其应用 Download PDFInfo
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Abstract
本发明公开了一种嗜外层视网膜的腺相关病毒病毒体及其应用。所述嗜外层视网膜的腺相关病毒病毒体具有氨基酸突变AAV9衣壳蛋白，所述嗜外层视网膜的腺相关病毒病毒体赋予增强的嗜外层视网膜细胞的感染性。其中相对于相应亲本AAV9衣壳蛋白，所述突变AAV9衣壳蛋白的氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸。所述药物组合物包含所述嗜外层视网膜的腺相关病毒病毒体和药学上可接受的赋形剂。本发明通过对编码AAV9病毒衣壳进行氨基酸定点突变，获得亲嗜外层视网膜的腺相关病毒突变体，适用于外源治疗基因转导外层视网膜细胞治疗视网膜病变。
Description
技术领域
本发明涉及一种重组AAV病毒体，具体涉及一种在选定的腺相关病毒 (AAV9) 序列中进行氨基酸突变以赋予其亲嗜外层视网膜的方法，以及在携带外源治疗基因转导外层视网膜细胞治疗视网膜病变中的应用，属于基因工程技术领域。
背景技术
视网膜疾病是导致失明的主要原因之一，常见的有黄斑变性、糖尿病视网膜病变、青光眼、遗传性视网膜病变等。其中，大部分病变由基因突变、蛋白不工作或过表达引起异常，进而导致视细胞死亡，最终致盲。通过基因治疗可以介导正确基因或敲除异常基因，恢复正常表达，进而起到恢复视功能作用。
腺相关病毒（Adeno-associated virus，AAV）属于微小病毒科依赖病毒属的一员，它是一个直径约为20~26 nm的二十面体，携带有4.6~4.8 kb的线性单链DNA基因组。目前已经从鸟类和许多哺乳动物包括人的体内分离到13种血清型及120多种突变型的AAV病毒。AAV以非致病性、低免疫原性、稳定表达目的基因等优点成为最有前景的病毒载体，被广泛应用于视网膜病变的基因治疗。AAV作为基因载体在基因治疗应用中有其他病毒载体或非病毒载体所不具备的优点：（1）既可以感染分裂细胞，也可以感染静止细胞；（2）AAV是目前所知的唯一能定点整合到人类基因组特定位点的基因载体，且整合位点安全可靠；最重要的是，虽然80-90%的人类呈AAV阳性，但没有报道AAV引起任何疾病。目前为止，全世界已有33项眼部疾病基因治疗研究进入临床试验，其中有23个临床试验项目是由AAV作为基因治疗载体。美国基因治疗药物公司Spark Therapeutics，用AAV载体携带RPE65基因治疗RPE65基因突变所导致的Leber先天性黑朦（LCA2）的药物，已获得美国FDA批准上市。
已证实AAV可以感染多种视网膜细胞，包括光感受器细胞、视网膜色素上皮细胞(RPE)、Müller细胞、视网膜神经节细胞和角膜内皮细胞。AAV载体进行视网膜疾病基因治疗的给药途径主要有玻璃体注射和视网膜下注射，分别主要靶向内层视网膜和外层视网膜。视网膜下注射会导致一系列并发症，加重机械损伤，导致视功能受损，并且手术操作困难，安全性低。玻璃体注射相对更安全、容易，但对RPE、光感受器等视网膜外层细胞转导率低，因此在治疗靶向RPE光感受器等外层视网膜的疾病中使用率有限。为了提高玻璃体注射的临床使用率，需要提高其对视网膜外层细胞转导效率。
但是，目前尚未有任何关于重组AAV病毒体高效靶向外层视网膜细胞，以用于提高视网膜外层细胞转导效率方面的报道。
发明内容
本发明的主要目的在于提供一种嗜外层视网膜的腺相关病毒病毒体，以克服现有技术中的不足。
本发明的另一目的还在于提供包含前述腺相关病毒病毒体的突变AAV9衣壳蛋白。
本发明的另一目的还在于提供药物组合物及其用于将基因产物递送至个体的外层视网膜细胞的药物中的用途。
为实现前述发明目的，本发明采用的技术方案包括：
本发明实施例提供了一种嗜外层视网膜的腺相关病毒病毒体，它具有氨基酸突变AAV9衣壳蛋白，所述嗜外层视网膜的腺相关病毒病毒体赋予增强的嗜外层视网膜细胞的感染性。
本发明实施例还提供了包含编码前述嗜外层视网膜的腺相关病毒病毒体的组合物，其包含：
a) 突变AAV9衣壳蛋白，其中相对于相应亲本AAV9衣壳蛋白，氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸；
b) 包含编码基因产物的核苷酸序列的异源核酸。
本发明实施例还提供了一种编码突变AAV9衣壳蛋白的核酸序列，其中相对于相应亲本AAV9衣壳蛋白氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸。
本发明实施例还提供了一种突变AAV9衣壳蛋白，其中相对于相应亲本AAV9衣壳蛋白氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸。
本发明实施例还提供了包含编码前述腺相关病毒病毒体的突变AAV9衣壳蛋白的核酸序列的重组载体。
本发明实施例还提供了一种药物组合物，包含前述的嗜外层视网膜的腺相关病毒病毒体和药学上可接受的赋形剂。
本发明实施例还提供了前述的嗜外层视网膜的腺相关病毒病毒体或药物组合物用于将基因产物递送至个体的外层视网膜细胞的药物中的用途。
本发明实施例还提供了一种应用于视网膜疾病治疗方法的产品，所述方法包括向有需要的个体施用有效量的、前述的嗜外层视网膜的腺相关病毒病毒体。
与现有技术相比，本发明的有益效果至少在于：
1）本发明通过对编码AAV9病毒衣壳进行氨基酸定点突变，获得亲嗜外层视网膜（包括RPE和感光细胞）的腺相关病毒突变体，适用于外源治疗基因转导外层视网膜细胞治疗视网膜病变；
2）本发明采用一种更安全的给药方式，将重组AAV病毒体直接注入玻璃体腔，病毒悬液随玻璃体扩散入视网膜。野生型AAV9病毒载体通过玻璃体腔注射对RPE细胞转导效率比较高，对AAV9病毒衣壳进行定点突变，开发一种对RPE和外核层亲和性更高的AAV9载体，对于视网膜病变的基因治疗具有重要的意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明中记载的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1A是包含AAV9衣壳蛋白核苷酸序列的质粒图谱。
图1B是包含突变AAV9衣壳蛋白核苷酸序列的质粒图谱。
图2A是本发明实施例1中rAAV9-tYF玻璃体注射2周后在小鼠视网膜内EGFP表达冰冻切片荧光成像示意图。
图2B是本发明实施例1中rAAV9-tYF玻璃体注射三个月后在小鼠视网膜内EGFP表达冰冻切片荧光成像示意图。
图3A和图3B是本发明实施例1中rAAV9-tYF感染小鼠眼球视网膜铺片共聚焦内层层扫和外层层扫荧光图。
具体实施方式
rAAV以非致病性、低免疫原性、稳定表达目的基因等优点成为最有前景的病毒载体，被广泛应用于视网膜病变的基因治疗。因此，鉴于现有技术的诸多缺陷，本案发明人经长期研究和大量实践，得以提出本发明的技术方案，其主要是通过对编码AAV9病毒衣壳进行氨基酸定点突变，获得亲嗜外层视网膜（包括RPE和感光细胞）的腺相关病毒突变体，适用于外源治疗基因转导外层视网膜细胞治疗视网膜病变。
以下对本发明提及的一些术语的定义作出解释：
“AAV”是腺相关病毒的缩写，并且可用于指病毒本身或其衍生物。除非另有需要时，所述术语包括亚型及自然存在和重组形式。缩写“rAAV”指重组腺相关病毒，也称为重组AAV 载体 (或“rAAV 载体”)。术语“AAV”包括 1 型 AAV(AAV-1)、2型 AAV(AAV-2)、3型 AAV(AAV-3)、4型AAV(AAV-4)、5型AAV(AAV-5)、6型 AAV(AAV-6)、7型 AAV(AAV-7)、8 型AAV(AAV-8)、9 型AAV(AAV-9)、禽AAV、牛AAV、犬AAV、马AAV、灵长类 AAV、非灵长类 AAV 和羊AAV。“灵长类AAV”指感染灵长类动物的AAV，“非灵长类 AAV”指感染非灵长类哺乳动物的AAV，“牛AAV”指感染牛哺乳动物的AAV等。
如本文所使用，“rAAV载体”指包含非 AAV 起源的多核苷酸序列(即，与AAV异源的多核苷酸)，通常是细胞遗传转化的目标序列的AAV载体。一般而言，异源多核苷酸两侧有至少一个，而通常有两个AAV反向末端重复序列 (ITR)。术语rAAV载体涵盖rAAV载体颗粒和rAAV载体质粒。rAAV载体可为单链(ssAAV)或自身互补(scAAV)。
“AAV 病毒”或“AAV 病毒颗粒”或“rAAV载体颗粒”指由至少一种AAV衣壳蛋白 (通常为野生型 AAV 的所有衣壳蛋白)和壳体化多核苷酸 rAAV 载体构成的病毒颗粒。如果所述颗粒包含异源多核苷酸 (即除野生型 AAV 基因组外的多核苷酸，例如递送至哺乳动物细胞的转基因)，通常将其称为“rAAV 载体颗粒”或简称为“rAAV 载体”。因此，rAAV颗粒的生成必定包括rAAV 载体的生成，因为在rAAV 颗粒中含有这种载体。
“包装”指导致AAV颗粒装配和壳体化的一系列胞内事件。
AAV的“辅助病毒”指允许哺乳动物细胞复制并包装 AAV( 例如野生型 AAV) 的病毒。在本领域中已知 AAV 的多种此类辅助病毒，包括腺病毒、疱疹病毒和痘病毒 (例如牛痘)。虽然 C 亚类的 5 型腺病毒最常用，但是腺病毒涵盖许多不同亚类。已知人、非人类哺乳动物和禽类来源的许多腺病毒并且可从贮藏所例如ATCC获得。疱疹家族的病毒包括(例如 ) 单纯疱疹病毒 (HSV) 和埃 - 巴二氏病毒 (Epstein-Barr viruses)(EBV) 以及巨细胞病毒 (CMV) 和假狂犬病病毒 (PRV) ；也可从贮藏所例如 ATCC 获得。
“分离的”质粒、核酸、载体、病毒、病毒体、宿主细胞或其它物质指没有在所述物质或类似物质自然存在或最初制备时也可能存在的至少一些其它组分的物质的制剂。因此，例如，可使用纯化计数制备分离物质以使其从源混合物中富集。可绝对地测量富集物，例如每体积溶液的颗粒数，或可相对于源混合物中存在的第二潜在干扰物质测量。逐渐多次分离本公开实施方案越来越多的富集物。
如本文所使用，术语“治疗”等指获得所需药理学和/或生理学作用。所述作用在完全或部分预防疾病或其症状方面可为预防性和/或在对疾病和/或可归因于所述疾病的副作用的部分或完全治愈方面可为治疗性。如本文所使用，“治疗”包括对哺乳动物( 特别是人类 )疾病的任何治疗，并且包括：(a)预防疾病在可能易患病或有患病风险但尚未诊断为患病的受试者中出现；(b)抑制疾病，即阻止其发展；和(c)缓解疾病，即引起疾病消退。
如下将结合附图对该技术方案、其实施过程及原理等作进一步的解释说明。
本发明实施例的一个方面提供的一种嗜外层视网膜的腺相关病毒病毒体，它具有氨基酸突变AAV9衣壳蛋白，所述嗜外层视网膜的腺相关病毒病毒体赋予增强的嗜外层视网膜细胞的感染性。
进一步地，其中相对于相应亲本AAV9衣壳蛋白，氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸。
进一步地，所述病毒体是嗜外层视网膜细胞的。
本发明实施例的另一个方面还提供了包含编码前述嗜外层视网膜的腺相关病毒病毒体的组合物，其包含：
a) 突变AAV9衣壳蛋白，其中相对于相应亲本AAV9衣壳蛋白，氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸；
b) 包含编码基因产物的核苷酸序列的异源核酸。
本发明实施例的另一个方面还提供了包含编码前述腺相关病毒病毒体的腺相关病毒病毒体的突变AAV9衣壳蛋白的核酸序列的重组载体。
本发明实施例的另一个方面还提供了包含编码突变AAV9衣壳蛋白的核酸序列，其中相对于相应亲本AAV9衣壳蛋白氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸。
其中，包含AAV9衣壳蛋白核苷酸序列的质粒图谱可参见图1A，包含突变AAV9衣壳蛋白核苷酸序列的质粒图谱可参见图1B。
其中，AAV9-衣壳蛋白的核苷酸序列如SEQ ID NO:1所示，所述编码突变AAV9衣壳蛋白的核酸序列如SEQ ID NO:2所示。
本发明实施例的另一个方面还提供了一种突变AAV9衣壳蛋白，其中相对于相应亲本AAV9衣壳蛋白，氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸。
其中，AAV9-衣壳蛋白的氨基酸序列如SEQ ID NO:3所示，所述突变AAV9衣壳蛋白的氨基酸序列如SEQ ID NO:4所示。
本发明实施例的另一个方面还提供了包含前述分离核酸的重组载体。
进一步地，所述重组载体为质粒。
在一些优选实施例中，本发明根据AAV包装必要条件构建三个质粒（分别包含AAV基因组、AAV突变体衣壳蛋白和复制蛋白）。上述三个质粒的骨架都来源于pFastbacdual质粒，即，将目的基因表达盒通过酶切连接入pFastbacdual质粒的多克隆位点（MCS）中。
本发明中涉及的第一个质粒，为编码AAV9突变体衣壳蛋白的质粒pFastbacdual-inCap9-tYF。通过定点突变，改变野生型AAV9 Cap蛋白第274、446和731位酪氨酸突变为苯丙氨酸。通过融合PCR方法进行定点突变，需进行两轮PCR反应，其中，第一轮PCR时，以pFastbacdual-inCap9质粒中的Cap基因作为模板，第一轮PCR涉及两个反应体系，其中第一段序列的3'引物和第二段序列的5'引物有一段互补区；然后将第一轮PCR的所有产物加到一个反应体系中作为模板，第一段序列的5'引物和最后一段序列的3'引物做引物，进行第二次扩增，形成融合序列。将第二轮PCR产物通过双酶切连入载体pFastbacdual-inCap9替换野生型Cap9的部分序列，获得pFastbacdual- inCap9-tYF。此质粒还包括在真核细胞中表达衣壳蛋白所需的其他元件（包括启动子、增强子、内含子、polyA序列等）。
本发明中涉及的第二个质粒，为AAV基因组质粒pFastbacdual-ITR-EGFP，包含AAV血清型2（AAV2）的两个末端倒置重复序列（ITR），还包含在真核细胞中表达的外源基因表达盒（包括启动子、增强子、内含子、polyA序列，以及外源基因表达框包括绿色荧光蛋白基因EGFP等）。
本发明中涉及的第三个质粒，为编码AAV复制蛋白（Rep）的质粒pFastbacdual-inrep，包括AAV2的Rep基因表达框，以及其他表达元件（包括启动子、内含子、polyA序列等）。
进一步地，本发明中涉及的AAV载体均在昆虫细胞中生产获得，具体包括以下步骤：
首先，将上述三个重组质粒pFastbacdual-inCap9-tYF/pFastbacdual-ITR-EGFP/pFastbacdual-inrep用常规方法，分别转化大肠杆菌DH10Bac感受态细胞中，通过两轮蓝白斑筛选，包含重组杆粒的菌落为白色，原始菌落为蓝色，挑选白色菌落扩增，提取重组杆粒Bacmid-inCap9-tYF / Bacmid-ITR-EGFP /Bacmid-inrep。
然后，用昆虫细胞转染试剂，分别将上述三种重组杆粒Bacmid-inCap9-tYF/Bacmid-ITR-EGFP /Bacmid-inrep转染昆虫细胞Sf9，4~5天后，收集细胞上清经0.22 μm过滤器过滤后，获得P1代重组杆状病毒Baculovirus-inCap9-tYF / Baculovirus-ITR-EGFP/Baculovirus-inrep；将P1代重组杆状病毒经过两次感染Sf9细胞扩增获得P3代病毒。采用噬菌斑法测定P3代杆状病毒的滴度，病毒滴度（pfu/mL）=1/稀释倍数×噬菌斑数×1/每孔接种体积。
本发明中涉及一种在昆虫细胞中生产AAV载体的方法，将P3代的三种重组杆状病毒（Baculovirus-inCap9-tYF / Baculovirus-ITR-EGFP /Baculovirus-inrep）共同感染Sf9细胞，包装获得rAAV9-tYF。以及用CsCl密度梯度离心的方法纯化浓缩出高浓度的重组AAV病毒的方法，荧光定量PCR检测rAAV9-tYF滴度的方法，SDS-PAGE检测rAAV9-tYF纯度的方法。
本发明实施例的另一个方面还提供了一种包含前述的分离核酸的经分离、遗传修饰的宿主细胞。
本发明实施例的另一个方面还提供了一种药物组合物，包含前述的嗜外层视网膜的腺相关病毒病毒体和药学上可接受的赋形剂。
进一步地，此类赋形剂、载体、稀释剂和缓冲液包括可施用而无异常毒性的任何药剂。药学上可接受的赋形剂包括但不限于液体，例如水、盐水、甘油和乙醇。其中可包括药学上可接受的盐，例如矿物酸盐例如盐酸盐、氢溴酸盐、磷酸盐、硫酸盐等 ；和有机酸的盐例如醋酸盐、丙酸盐、丙二酸盐、苯甲酸盐等。另外，在此类媒介物中可存在辅助物质，例如润湿剂或乳化剂、pH 缓冲物质等。在本领域中已知多种多样的药学上可接受的赋形剂而不需要在本文中详细讨论。在多种出版物中已经详尽地描述了药学上可接受的赋形剂，包括(例如)A.Gennaro(2000)"Remington:The Science and Practice of Pharmacy,"第20版，Lippincott,Williams,&Wilkins ；Pharmaceutical Dosage Forms and DrugDeliverySystems(1999)H.C.Ansel 等编辑，第7版，Lippincott,Williams,&Wilkins；和Handbookof Pharmaceutical Excipients(2000)A.H.Kibbe 等编辑，第3版，Amer.Pharmaceutical Assoc。
本发明实施例的另一个方面还提供了前述的嗜外层视网膜的腺相关病毒病毒体或药物组合物用于将基因产物递送至个体的外层视网膜细胞的药物中的用途。
本发明实施例的另一个方面还提供了一种应用于视网膜疾病治疗方法的产品，所述方法包括向有需要的个体施用有效量的、前述的嗜外层视网膜的腺相关病毒病毒体。
进一步地，所述产品的施用方式是通过眼内注射。
更进一步地，所述产品的施用方式是通过玻璃体内注射。
更进一步地，本发明中涉及的给药方式为玻璃体注射，具体步骤包括：4%水合氯醛0.01mL/g麻醉小鼠，双眼散瞳，使用玻璃酸钠保持眼表湿润，抗生素眼药水、表麻药术前滴眼。调整小鼠头位使眼球保持角膜缘水平位。使用31G 针头在角膜缘后1mm处穿刺，汉密尔顿 (Hamilton)33G注射器在穿刺处注射病毒1μL。针尖垂直进入，随后倾斜，缓慢推注，推针以后留针0.5-1min，迅速出针。
综上所述，本发明采用一种更安全的给药方式，将重组AAV病毒体直接注入玻璃体腔，病毒悬液随玻璃体扩散入视网膜。野生型AAV9病毒载体通过玻璃体腔注射对RPE细胞有比较高的转导效率而AAV9病毒衣壳进行定点突变，对RPE和外核层都具有更高的亲和性，对于视网膜病变的基因治疗具有重要的意义。
以下结合若干较佳实施例及附图对本发明的技术方案作进一步的解释说明，但其中的实验条件和设定参数不应视为对本发明基本技术方案的局限。并且本发明的保护范围不限于下述的实施例。
实施例1
本发明涉及一种嗜外层视网膜的AAV载体，具体构建方法包括如下步骤：
（1）重组质粒的制备：
A. pFastbacdual-inCap9-tYF的制备
通过融合PCR方法进行定点突变，需进行两轮PCR反应，其中，第一轮PCR时，以pFastbacdual-inCap9质粒中的Cap基因作为模板，第一轮PCR涉及两个反应体系，其中第一段序列的3'引物和第二段序列的5'引物有一段互补区；然后将第一轮PCR的所有产物加到一个反应体系中作为模板，第一段序列的5'引物和最后一段序列的3'引物做引物，进行第二次扩增，形成融合序列。将第二轮PCR产物通过切胶和DNA纯化试剂盒进行纯化后，用HindIII和NcoI酶切，载体pFastbacdual-inCap9也用HindIII和NcoI酶切，二者通过T4 DNALigase连接，转化大肠杆菌Top10感受态细胞，挑取单克隆菌落扩增，提取质粒经过酶切鉴定，获得pFastbacdual-Cap9-tYF。此质粒还包括在真核细胞中表达衣壳蛋白所需的其他元件，包括启动子、增强子、内含子、polyA序列等。
B. pFastbacdual-ITR-EGFP的制备
腺相关病毒载体pAAV-MCS用Ehe I和Psc I双酶切得到包括AAV2 ITRs和CMV启动子、CMV增强子、beta-globin内含子、hGH polyA序列，杆状病毒载体pFastBacdual用Nco I和Stu I双酶切获得质粒骨架，37℃水浴，酶切过夜。酶切产物全部上样，1%的琼脂糖凝胶电泳(90 V, 65 min)，紫外灯下切下目的条带（4905 bp），用 DNA 胶回收试剂盒纯化回收目的片段。用T4 DNA Ligase将 2046 bp目的片段插入到4905 bp载体片段中，二者的摩尔比为5:1，16℃ 连接过夜。将酶连产物转化Sure 2感受态细菌，挑取平板上单克隆菌落，37℃恒温振荡 (220 r/min) 培养16 h，随后挑取实验组平板上单菌落，在含氨苄的LB 培养基中摇培16 h（37℃, 220 r/min），抽提质粒，经过Sma I酶切来鉴定重组质粒，获得pFastBacdual-ITR质粒。以质粒pUC57-minivector-EGFP为模板，通过PCR扩增EGFP序列(726bp)。用BamH I和Xho I双酶切PCR产物和pFastBacdual-ITR载体，T4 DNA Ligase连接，酶连产物转化Sure 2感受态，获得pFastBacdual-ITR-EGFP。
C. pFastbacdual-inrep质粒的制备
以pAAV-in-RC为模板，PCR扩展rep基因及其他表达元件，BstZ17 I和 Sph I 双酶切PCR 回收产物，pFastBacdual 用 Sma I和Sph I双切，二者连接，获得pFastbacdual-inrep质粒。
（2）重组Bacmid的制备
将上一步的三个重组质粒分别制备重组Bacmid，具体方法如下：
1)冰上缓慢融化100 μL DH10Bac感受态。
2)加入50 ng质粒DNA，轻轻混匀。
3)冰上放置30 min，42℃热休克90 s，立即转入冰上放置2 min。
4)加入900 μL SOC培养基，37℃ 225 rpm摇4 h。
5)在含50 μg/mL 卡那霉素（Kan），7 μg/mL 庆大霉素（Gen），10 μg/mL 四环素（Tet）的预制的90 mm琼脂板中央滴加40 μL 2%（20 mg/mL）的5-溴-3-吲哚基-β-D-吡喃半乳糖苷（Blue-gal）和7 μL 20% (200 mg/mL) IPTG。使用无菌涂布器使之分散于培养板整个表面，于室温孵育直至全部液体消失。
6)用SOC培养基10倍梯度稀释细胞（10-1，10-2，10-3），每个梯度取100 μL涂LB平板。
7)37℃放置48 h，挑取10个白色克隆，蘸取到新的LB琼脂板（同上抗性）上，37℃过夜。挑取确认的白斑，接种到LB液体培养基中（含50 μg/mL卡那霉素（Kan），7 μg/mL庆大霉素（Gen），10 μg/mL四环素（Tet））。
8)4℃放置过夜，使蓝色在这一期间充分显色。
9)对于白斑可以进行PCR鉴定正确后可以转染Sf9。
10)使用OMEGA试剂盒抽提分离重组的杆粒DNA，实验方法参照试剂盒说明书，测量杆粒浓度后分装后冻于–20℃，避免反复冻融。
11) PCR鉴定Bacmid，所用引物分别为：上游引物 5’-CCC AGT CAC GAC GTT GTAAAA CG-3’，下游引物 5’-GCT CTA GAT TAC TTG TAC AGC TCG TCC AT-3’。
12) 取出鉴定正确的Bacmid菌种，按照1:300的比例接种于3 mL LB（Kan+，Gen+，Tet+）摇菌12 h。然后按照1:100的比例接种于150 mL LB（Kan+，Gen+，Tet+）摇菌16 h，参照大型/大量质粒提取试剂盒说明书大量抽提Bacmid，以备转染细胞制备杆状病毒。
（3）杆状病毒的制备
① Sf9细胞培养。Sf9细胞提前一天铺六孔板，50%的孔密度，使用完全培养基，95%存活度。将重组Bacmid DNA在70℃水浴里面温浴20 min，12000 g离心10 min取上清液。
② 细胞铺板。
确保细胞密度在1.5~2.5×106 细胞/mL时进行操作（培养基不含抗生素）。加2 mL无添加剂的基础（Grace）培养基（不含抗生素和血清）到6孔板中。接种8×105细胞/mL步骤1中的Sf9（未更换培养基和洗细胞），让细胞室温贴壁15 min。
③ 配制转染试剂。
a)混匀转染试剂Ⅱ，加入8 μL到92 μL无添加剂的基础培养基（不含抗生素和血清），涡旋混匀。
b)取5 μL杆粒DNA（500 ng/μL，保证杆粒的量为2~3 μg）到95 μL无添加剂的基础培养基（不含抗生素和血清），轻轻混匀。
c)把上述两溶液混匀，室温孵育30 min。
④ 滴加上述DNA-Lipid混合物到铺有细胞的孔中，27℃孵育细胞5 h。
⑤ 移去板中培养基，换2 mL 完全培养基。
⑥ 在27℃孵育72 h，观察病毒感染迹象。
分离P1：
证实细胞处于晚期感染阶段后（通常是转染后4~5天），每孔收集2 mL含病毒的培养基到无菌的15 mL离心管中，1000 g离心5 min去除细胞碎片。
上清液经0.22 μm过滤器过滤到无菌的15 mL离心管中，4℃避光保存。若想长期保存，分装冻于–80℃。
扩增病毒：
取MOI为0.05~0.1，10 mL悬浮培养的细胞，密度为2×106细胞/mL；或是6孔板中的细胞，密度为2×106 细胞/孔，计算所需的P1体积。
① Sf9细胞铺六孔板，2×106细胞/孔。室温放置1 h使其贴附，显微镜下观察。
② 每孔加入适量的P1，27℃培养48 h~72 h。
③ 每孔收集2 mL含病毒的培养基于无菌的15 mL离心管中，1000 g离心5 min。
④ 转移上清到无菌的15 mL离心管中，该病毒上清为P2。4℃避光保存，若想长期保存，分装冻于–80℃。
⑤ 可按上述方法扩增得到P3(通常得到的P1病毒滴度在1×106~1×107之间，P2滴度在1×107~1×108之间)。
用噬菌斑法测定病毒滴度。详细实验步骤如下：
① 2 mL/孔细胞（5×105 细胞/mL）种入6孔板，室温孵育1 h使其贴壁，孵育后镜检其贴壁程度。
② 将4%琼脂糖凝胶放入70℃水浴锅融解，2个基础培养基与一个100 mL无菌瓶放入40℃水浴锅预热。
③ 将杆状病毒用无血清基础培养基进行梯度稀释：10-1~10-8。
④ 弃6孔板内上清，快速加入稀释好的病毒，1 mL/孔（复孔）室温孵育1 h。
⑤ 配置上层琼脂，加20 mL高温灭活FBS到2个100 mL基础培养基， 2个25 mL基础培养基（含FBS）+12.5 mL 无菌水+12.5 mL 4%琼脂糖凝胶，至预热的100 mL无菌瓶，轻轻混匀，放入37℃水浴锅备用。
⑥ 弃6孔板内上清，快速加2 mL上层琼脂，以防菌层干燥，静置10~20 min使其凝固。将6孔板放入27℃培养箱，培养5天。
⑦ 配制1 mg/mL中性红溶液，在基础完全培养基中，无菌过滤。
⑧ 1.5 mL的上述溶液，16.5 mL 基础完全培养基，6 mL 4%的琼脂配制成中性红上层琼脂。
⑨ 在病毒感染4天后，加1 mL中性红上层琼脂。
⑩ 继续放到培养箱中，4~5天后即可观察噬菌斑，计数噬菌斑的数量，求得病毒滴度。
注：病毒滴度（pfu/mL）=1/稀释倍数×噬菌斑数×1/每孔接种体积
（4）rAAV载体的制备及纯化
用CsCl密度梯度离心的方法纯化浓缩出高浓度的重组AAV病毒的方法，荧光定量PCR检测rAAV9-tYF滴度的方法，SDS-PAGE检测rAAV9-tYF纯度的方法。
（5）小鼠玻璃体注射方法
具体操作步骤如下：
4%水合氯醛 0.01ml/g麻醉小鼠，双眼散瞳，使用玻璃酸钠保持眼表湿润，抗生素眼药水、表麻药术前滴眼。调整小鼠头位使眼球保持角膜缘水平位。使用31G 针头在角膜缘后1mm处穿刺，汉密尔顿 (Hamilton)33G注射器在穿刺处注射病毒1μl。针尖垂直进入，随后倾斜，缓慢推注，推针以后留针0.5-1min，迅速出针。
（6）冰冻切片与DAPI染色
小鼠断颈处死，取出眼球，浸入4%多聚甲醛(Paraformaldehyde, PFA)，4℃固定2h，角膜处开口，浸入4% PFA，4℃ 过夜。除尽PFA，20%蔗糖4℃脱水12h，30%蔗糖4℃脱水24h以上。取出30%蔗糖溶液浸泡的眼，夹住角膜部分，去除角膜、晶状体，用纸吸干蔗糖。镊子、纸、刀等预冷。滴入少量OCT，将眼垂直放置塑料盖内，去除气泡，放入切片机冷冻，保持冷冻后垂直树立，继续滴入OCT包埋住整个眼。在预冷的标本盘上滴一圈OCT，样品放在标本盘上，OCT固定整个样品，小刀修整大小。 标本盘嵌入标本头，调整刀架和防卷板，粗调修片，待切出组织后，精切14μm。切出后的切片贴在载玻片，装入切片架。0.01 mol/L PBS浸泡载玻片，通风处晾干。
拿出切片架，放入盛0.01mol/L PBS的修复盒内，擦干四周，用免疫组化笔圈住四周，油滴干后，0.01 mol/L PBS清洗。加入DAPI（1：1000 PBS），孵育20 min。0.01 mol/L PBS清洗3次。保持切片湿润，倒置荧光显微镜观察，结果如图2A和图2B所示，图2A示出了rAAV9-tYF玻璃体注射2周后在小鼠视网膜内EGFP表达冰冻切片荧光成像图，图2B示出了rAAV9-tYF玻璃体注射三个月后在小鼠视网膜内EGFP表达冰冻切片荧光成像图。切片荧光结果图显示，荧光表达主要位于外层视网膜细胞，并且三个月后荧光表达有所增加，说明了rAAV9-tYF玻璃体注射小鼠眼能够稳定高效地感染外层视网膜细胞。
共聚焦显微镜观察视网膜铺片：玻璃体注射病毒，2周后取眼球，4% PFA固定两小时，取出视网膜，于共聚焦显微镜下观察，结果如图3A和图3B所示，示出了rAAV9-tYF感染小鼠眼球视网膜铺片共聚焦荧光图。其中，图3A为视网膜内层层扫图，图3B为视网膜外层层扫图。从图中可以显示出，外层视网膜的荧光强度（图3B）显著高于内层视网膜（图3A）的荧光强度，与切片结果一致。
由以上实施例可知，本发明通过对编码AAV9病毒衣壳进行氨基酸定点突变，获得亲嗜外层视网膜的腺相关病毒突变体，适用于外源治疗基因转导外层视网膜细胞治疗视网膜病变。
本发明的各方面、实施例、特征及实例应视为在所有方面为说明性的且不打算限制本发明，本发明的范围仅由权利要求书界定。在不背离所主张的本发明的精神及范围的情况下，所属领域的技术人员将明了其它实施例、修改及使用。
在本发明案中标题及章节的使用不意味着限制本发明；每一章节可应用于本发明的任何方面、实施例或特征。
在本发明案通篇中，在将组合物描述为具有、包含或包括特定组份之处或者在将过程描述为具有、包含或包括特定过程步骤之处，预期本发明教示的组合物也基本上由所叙述组份组成或由所叙述组份组成，且本发明教示的过程也基本上由所叙述过程步骤组成或由所叙述过程步骤组组成。
应理解，各步骤的次序或执行特定动作的次序并非十分重要，只要本发明教示保持可操作即可。此外，可同时进行两个或两个以上步骤或动作。
尽管已参考说明性实施例描述了本发明，但所属领域的技术人员将理解，在不背离本发明的精神及范围的情况下可做出各种其它改变、省略及/ 或添加且可用实质等效物替代所述实施例的元件。另外，可在不背离本发明的范围的情况下做出许多修改以使特定情形或材料适应本发明的教示。因此，本文并不打算将本发明限制于用于执行本发明的所揭示特定实施例，而是打算使本发明将包含归属于所附权利要求书的范围内的所有实施例。
最后，还需要说明的是，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
序列表
<110> 上海爱尔眼科医院有限公司
<120> 嗜外层视网膜的腺相关病毒病毒体及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2211
<212> DNA
<213> 人工序列(人工序列)
<400> 1
atggctgccg atggttatct tccagattgg ctcgaggaca accttagtga aggaattcgc 60
gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 120
aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180
aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240
cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300
caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360
gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420
ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480
aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540
tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600
cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660
gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720
accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780
tccaacagca catctggagg atcttcaaat gacaacgcct acttcggcta cagcaccccc 840
tgggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 900
ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960
caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 1020
acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080
gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 1140
acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 1200
ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 1260
cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 1320
gaccaatact tgtactatct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 1380
ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 1440
ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 1500
tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 1560
ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 1620
ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 1680
accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 1740
gccacaaacc accagagtgc ccaagcacag gcgcagaccg gctgggttca aaaccaagga 1800
atacttccgg gtatggtttg gcaggacaga gatgtgtacc tgcaaggacc catttgggcc 1860
aaaattcctc acacggacgg caactttcac ccttctccgc tgatgggagg gtttggaatg 1920
aagcacccgc ctcctcagat cctcatcaaa aacacacctg tacctgcgga tcctccaacg 1980
gccttcaaca aggacaagct gaactctttc atcacccagt attctactgg ccaagtcagc 2040
gtggagatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagatccag 2100
tacacttcca actattacaa gtctaataat gttgaatttg ctgttaatac tgaaggtgta 2160
tatagtgaac cccgccccat tggcaccaga tacctgactc gtaatctgta a 2211
<210> 2
<211> 2211
<212> DNA
<213> 人工序列(人工序列)
<400> 2
atggctgccg atggttatct tccagattgg ctcgaggaca accttagtga aggaattcgc 60
gagtggtggg ctttgaaacc tggagcccct caacccaagg caaatcaaca acatcaagac 120
aacgctcgag gtcttgtgct tccgggttac aaataccttg gacccggcaa cggactcgac 180
aagggggagc cggtcaacgc agcagacgcg gcggccctcg agcacgacaa ggcctacgac 240
cagcagctca aggccggaga caacccgtac ctcaagtaca accacgccga cgccgagttc 300
caggagcggc tcaaagaaga tacgtctttt gggggcaacc tcgggcgagc agtcttccag 360
gccaaaaaga ggcttcttga acctcttggt ctggttgagg aagcggctaa gacggctcct 420
ggaaagaaga ggcctgtaga gcagtctcct caggaaccgg actcctccgc gggtattggc 480
aaatcgggtg cacagcccgc taaaaagaga ctcaatttcg gtcagactgg cgacacagag 540
tcagtcccag accctcaacc aatcggagaa cctcccgcag ccccctcagg tgtgggatct 600
cttacaatgg cttcaggtgg tggcgcacca gtggcagaca ataacgaagg tgccgatgga 660
gtgggtagtt cctcgggaaa ttggcattgc gattcccaat ggctggggga cagagtcatc 720
accaccagca cccgaacctg ggccctgccc acctacaaca atcacctcta caagcaaatc 780
tccaacagca catctggagg atcttcaaat gacaacgcct tcttcggcta cagcaccccc 840
tgggggtatt ttgacttcaa cagattccac tgccacttct caccacgtga ctggcagcga 900
ctcatcaaca acaactgggg attccggcct aagcgactca acttcaagct cttcaacatt 960
caggtcaaag aggttacgga caacaatgga gtcaagacca tcgccaataa ccttaccagc 1020
acggtccagg tcttcacgga ctcagactat cagctcccgt acgtgctcgg gtcggctcac 1080
gagggctgcc tcccgccgtt cccagcggac gttttcatga ttcctcagta cgggtatctg 1140
acgcttaatg atggaagcca ggccgtgggt cgttcgtcct tttactgcct ggaatatttc 1200
ccgtcgcaaa tgctaagaac gggtaacaac ttccagttca gctacgagtt tgagaacgta 1260
cctttccata gcagctacgc tcacagccaa agcctggacc gactaatgaa tccactcatc 1320
gaccaatact tgtactttct ctcaaagact attaacggtt ctggacagaa tcaacaaacg 1380
ctaaaattca gtgtggccgg acccagcaac atggctgtcc agggaagaaa ctacatacct 1440
ggacccagct accgacaaca acgtgtctca accactgtga ctcaaaacaa caacagcgaa 1500
tttgcttggc ctggagcttc ttcttgggct ctcaatggac gtaatagctt gatgaatcct 1560
ggacctgcta tggccagcca caaagaagga gaggaccgtt tctttccttt gtctggatct 1620
ttaatttttg gcaaacaagg aactggaaga gacaacgtgg atgcggacaa agtcatgata 1680
accaacgaag aagaaattaa aactactaac ccggtagcaa cggagtccta tggacaagtg 1740
gccacaaacc accagagtgc ccaagcacag gcgcagaccg gctgggttca aaaccaagga 1800
atacttccgg gtatggtttg gcaggacaga gatgtgtacc tgcaaggacc catttgggcc 1860
aaaattcctc acacggacgg caactttcac ccttctccgc tgatgggagg gtttggaatg 1920
aagcacccgc ctcctcagat cctcatcaaa aacacacctg tacctgcgga tcctccaacg 1980
gccttcaaca aggacaagct gaactctttc atcacccagt attctactgg ccaagtcagc 2040
gtggagatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagatccag 2100
tacacttcca actattacaa gtctaataat gttgaatttg ctgttaatac tgaaggtgta 2160
tatagtgaac cccgccccat tggcaccaga ttcctgactc gtaatctgta a 2211
<210> 3
<211> 736
<212> PRT
<213> 人工序列(人工序列)
<400> 3
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> 4
<211> 736
<212> PRT
<213> 人工序列(人工序列)
<400> 4
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 Phe 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 Phe 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 Phe Leu Thr Arg Asn Leu
725 730 735
Claims (3)
1.包含编码嗜外层视网膜的腺相关病毒病毒体的组合物，所述嗜外层视网膜的腺相关病毒病毒体具有氨基酸突变AAV9衣壳蛋白，所述嗜外层视网膜的腺相关病毒病毒体赋予增强的嗜外层视网膜细胞的感染性，其特征在于，所述组合物包含：
a) 突变AAV9衣壳蛋白，其中相对于相应亲本AAV9衣壳蛋白，氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸；
b) 包含编码基因产物的核苷酸序列的异源核酸。
2.一种编码突变AAV9衣壳蛋白的核酸，其中相对于相应亲本AAV9衣壳蛋白氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸，所述编码突变AAV9衣壳蛋白的核酸序列如SEQ ID NO:2所示。
3.一种突变AAV9衣壳蛋白，其中相对于相应亲本AAV9衣壳蛋白氨基酸序列中第274、446和731位酪氨酸突变为苯丙氨酸，所述突变AAV9衣壳蛋白的氨基酸序列如SEQ ID NO:4所示。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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