CN114181972A - 适用于难治性血管新生性眼疾病基因治疗的慢病毒载体 - Google Patents

适用于难治性血管新生性眼疾病基因治疗的慢病毒载体 Download PDF

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CN114181972A
CN114181972A CN202111397423.8A CN202111397423A CN114181972A CN 114181972 A CN114181972 A CN 114181972A CN 202111397423 A CN202111397423 A CN 202111397423A CN 114181972 A CN114181972 A CN 114181972A
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凌思凯
汪啸渊
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Abstract

本发明公开了一种适用于难治性血管新生性眼疾病基因治疗的慢病毒载体；其包装质粒包括表达包膜蛋白的pMD.2G、表达REV蛋白的pRSV‑REV、整合酶突变失活的pMDlg/pRRE‑IN mut和表达VEGFA抗体基因的载体质粒。该载体质粒是将抗VEGFA基因替换质粒pCCL‑PGK‑eGFP中的eGFP基因构建而得；该抗VEGFA基因是在雷珠单抗的重链和轻链可变区分别加上人源IgG的Fc片段恒定区CH3，并对基因进行密码子优化而得。本发明提供的非整合型慢病毒载体感染细胞后，细胞可以长期稳定表达血管内皮生长因子VEGFA抗体，并且不会整合至人染色体组，避免了对基因组的影响。

Description

适用于难治性血管新生性眼疾病基因治疗的慢病毒载体
技术领域
本发明属于基因工程技术领域，涉及一种适用于难治性血管新生性眼疾病基因治疗的慢病毒载体；尤其涉及一种适用于难治性血管新生性眼疾病基因治疗的经改造的非整合型慢病毒载体***。
背景技术
难治性血管新生性眼疾病是一组以脉络膜或视网膜血管新生为病理特征的疾病，包括血管新生性年龄相关性黄斑变性(nAMD)，糖尿病视网膜病变相关黄斑水肿(DME)及视网膜静脉阻塞相关黄斑水肿(RVO-ME)等。由于新生血管壁的结构异常，导致血管的渗漏和出血，进而引发一系列的继发性病理改变，疾病常累及视功能最为密切的黄斑区，引起黄斑区视网膜神经上皮下或神经上皮层间积液，进而导致明显的视力下降，最终使中心视力丧失。经过治疗后，患者仍可能出现纤维化和(或)RPE层萎缩的情况，导致严重的永久性中枢视力丧失。所以，难治性血管新生性眼疾病成为世界难治性眼盲的重要原因(Wong et al2016)，也是世界卫生组织重点防盲眼病。
目前，难治性血管新生性眼疾病的发病机制还未完全阐明，但是血管内皮生长因子(VEGF-A)是公认的能促进脉络膜新生血管的重要因子。VEGF-A因子是一种高度特异性的促血管内皮细胞生长因子，具有促进血管通透性增加、细胞外基质变性、血管内皮细胞迁移、增殖和血管形成等作用。因此，现有治疗方法主要通过玻璃体腔注射VEGF-A因子的抗体药物，抑制VEGF-A因子，减弱其增生新的血管作用。抗血管内皮生长因子(Anti-VEGF)治疗是目前难治性血管新生性眼疾病的标准治疗方法。现有的药物主要包括雷珠单抗(Ranibizumab)，阿柏西普(Aflibercept)，康柏西普(Conbercept)。但目前现有的抗VEGF治疗方法通常需要较多的注射次数，根据患者情况每次注射相隔时间不确定，平均约4-8周需要进行一次注射。这对于患者和医生来说治疗负担较重。有些患者经历了抗VEGF治疗疲劳，选择停止治疗(Boulanger-Scemama et al 2015)，这可能会导致疾病复发和永久视力丧失(Essex et al 2016)。
近年来基因治疗技术逐步建立和完善。应用基因治疗的方法治疗难治性血管新生性眼疾病是实现一次注射，终身治疗目标的发展趋势。现在已有多家公司开展基因治疗难治性血管新生性眼疾病的药物研发，乃至进入临床试验阶段。其原理是通过视网膜下注射带有目标基因片段的病毒载体，可通过表达血管生成抑制因子，或增加RPE细胞分泌内源性抗VEGF抗体，长期有效控制眼内VEGF浓度在较低的水平，达到治疗血管新生性疾病的效果。
但是，临床上现有治疗方法存在以下问题：经济方面：抗体药物比较昂贵。治疗wAMD需要每4-8周就要进行一次抗体注射。除了药物本身带来的直接医疗费用，还有交通费、营养费等直接非医疗费用，甚至还有误工费等间接损失。副面影响方面：1)长期、频繁的玻璃体腔注射抗体药物引起眼部二次伤害；2)长期大量注射抗VEGF药物可能扩散全身，引起对机体其它器官或组织的VEGF抑制等问题。
发明内容
本发明的目的在于解决现有技术中治疗难治性血管新生性眼疾病安全性与有效性问题，提供一种适用于难治性血管新生性眼疾病基因治疗的慢病毒载体；具体是一种适用于难治性血管新生性眼疾病基因治疗的非整合型慢病毒载体。
本发明的目的是通过以下技术方案实现的：本发明提供了一种非整合型慢病毒载体，所述载体的包装质粒包括表达包膜蛋白的pMD.2G、表达REV蛋白的pRSV-REV、整合酶突变失活的pMDlg/pRRE-IN mut和载体质粒pCCL-PGK-eGFP。
整合酶突变失活的pMDlg/pRRE-IN mut，包括整合酶具有D64V的pMDlg/pRRE-D64V；也可以是其他位置突变，如N120L,W235E,Q148A,K264R，K264E,F185A,D116A,D64A,H12A，D64E，D116N。
本发明还提供一种前述非整合型慢病毒载体的制备方法，将质粒pMD.2G、pRSV-REV、pMDlg/pRRE-IN mut和载体质粒pCCL-PGK-eGFP共转染进宿主细胞；经过浓缩、纯化，得到非整合型慢病毒颗粒。
作为一个实施方案，所述宿主细胞包括HEK293T细胞。
本发明还提供了一种非整合型慢病毒载体***，所述载体***的包装质粒包括表达包膜蛋白的pMD.2G、表达REV蛋白的pRSV-REV、整合酶突变失活的pMDlg/pRRE-IN mut和表达VEGFA抗体基因的载体质粒。所述载体质粒可以为pCCL-PGK-antiVEGFA，是将抗VEGFA基因替换质粒pCCL-PGK-eGFP中的eGFP基因构建而得。
整合酶突变失活的pMDlg/pRRE-IN mut，包括整合酶具有D64V的pMDlg/pRRE-D64V；也可以是其他位置突变，如N120L,W235E,Q148A,K264R，K264E,F185A,D116A,D64A,H12A，D64E，D116N。
VEGFA抗体指的是可与VEGFA抗原结合的抗体，包括但不限于雷珠单抗、贝伐珠单抗、布洛赛珠单抗、康柏西普、阿柏西普等。
作为一个实施方案，整合酶的氨基酸序列如SEQ ID NO.2所示。
作为一个实施方案，编码所述整合酶的基因序列包括如SEQ ID NO.1所示序列。
作为一个实施方案，抗VEGFA基因是在雷珠单抗的重链和轻链可变区分别加上人源IgG的Fc片段恒定区CH3，并对基因进行密码子优化而得。抗VEGFA抗体是根据雷珠单抗和人源IgG改造而来，与VEGFA的结合力更强。本发明优化改造后的抗VEGFA抗体基因相对于未改造抗VEGFA抗体基因在人源细胞的蛋白表达水平上具有显著进步。
作为一个实施方案，抗VEGFA基因由重链和轻链组成，重链的氨基酸序列为SEQ IDNO.5；轻链的氨基酸序列为SEQ ID NO.7。
作为一个实施方案，所述重链的基因序列如SEQ ID NO.4所示；所述轻链的基因序列为SEQ ID NO.6所示。
作为一个实施方案，所述载体质粒pCCL-PGK-antiVEGFA的载体序列如SEQ IDNO.3所示。
本发明还提供一种非整合型慢病毒载体***的制备方法，将所述质粒pMD.2G、pRSV-REV、pMDlg/pRRE-IN mut(如pMDlg/pRRE-D64V)和载体质粒共转染进宿主细胞，浓缩、纯化，得到能表达抗VEGFA抗体的非整合型慢病毒***。
作为一个实施方案，所述宿主细胞包括HEK293T细胞。
本发明的非整合型慢病毒载体***可用于治疗难治性血管新生性眼疾病。包括血管新生性年龄相关性黄斑变性(nAMD)，糖尿病视网膜病变相关黄斑水肿(DME)及视网膜静脉阻塞相关黄斑水肿(RVO-ME)等。
因此，本发明还提供一种非整合型慢病毒载体***在制备治疗难治性血管新生性眼疾病的制剂中的用途。
在一些实施例中，提供一种非整合型慢病毒载体***在制备治疗血管新生性年龄相关性黄斑变性疾病、糖尿病视网膜病变相关黄斑水肿或视网膜静脉阻塞相关黄斑水肿等制剂中的用途。
与现有技术相比，本发明具有如下有益效果：
1、本发明利用非整合型的慢病毒载体递送，可经过视网膜下注射或脉络膜上腔注射到眼底后，更好地靶向RPE细胞，从源头中和VEGFA，以更少的病毒载量达到效果；
2、本发明提供的非整合型慢病毒载体，可以通过一次注射(脉络膜上腔注射)达到治疗效果；避免了多次注射带来的眼部创伤等医生和患者的治疗负担；
3、本发明提供的非整合型慢病毒载体感染细胞后，细胞可以长期稳定表达血管内皮生长因子VEGFA抗体，并且不会整合至人染色体组，减少了对基因组的影响；
4、可以通过剂量调控抗VEGFA抗体的表达量，使达到治疗效果的同时，减少抗VEGFA抗体的全身性扩散，避免抗VEGFA抗体过量给人体器官和组织带来负面影响。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为抗VEGFA抗体组成示意图；
图2为抗VEGFA抗体体外表达示意图；
图3为VEGFA抗体体内抑制新生血管增生示意图。
具体实施方式
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
本发明是将慢病毒载体改造为非整合型的慢病毒载体，用来递送改造和优化的抗VEGF抗体基因。具体的操作示例如下：
1、改造慢病毒载体的整合特性；
普通的慢病毒载体是具有基因整合的特性。第三代慢病毒的包装质粒是pMD.2G、pRSV-REV、pMDlg/pRRE和第三代慢病毒载体质粒pCCL-PGK-eGFP(质粒基因序列如SEQ IDNO.12所示)。在本发明中，将普通慢病毒整合酶的第64位氨基酸天冬氨酸(D)突变为缬氨酸(V)，整合酶失活，使慢病毒失去基因整合的特性。即将质粒pMDlg/pRRE中的整合酶基因做碱基突变，构建出质粒pMDlg/pRRE-D64V。在生产非整合型慢病毒时，将质粒pMD.2G、pRSV-REV、pMDlg/pRRE-D64V和载体质粒pCCL-PGK-eGFP共转染进HEK293T细胞。经过浓缩、纯化步骤得到非整合型慢病毒颗粒。
2、改造和优化抗VEGFA抗体的基因
本发明中，抗VEGFA抗体的构成(图1)是：雷珠单抗的重链和轻链可变区分别加上人源IgG的Fc片段恒定区CH3(重链的基因序列如SEQ ID NO.4所示，氨基酸序列如SEQ IDNO.5所示；轻链的基因序列如SEQ ID NO.6所示，氨基酸序列如SEQ ID NO.7所示)。并对基因进行适合在哺乳动物真核细胞表达的密码子优化。密码子优化是由测序公司的优化工具进行的，将基因优化为在真核细胞，尤其是人源的细胞中表达量增高的序列，而所表达的蛋白氨基酸序列不变。重链基因前有一段Ig k-chain leader(基因序列如SEQ ID NO.8所示，氨基酸序列如SEQ ID NO.9所示)。重链和轻链基因之间添加了P2A序列用于蛋白成熟时的分割(基因序列如SEQ ID NO.10所示，氨基酸序列如SEQ ID NO.11所示)，
3、慢病毒载体包装抗VEGFA抗体基因
将本发明中优化的抗VEGFA基因构建到慢病毒的载体质粒pCCL-PGK-eGFP里。构建时，将抗VEGFA基因替换原质粒中的eGFP基因。构建出质粒pCCL-PGK-antiVEGFA。将质粒pMD.2G、pRSV-REV、pMDlg/pRRE-D64V和载体质粒pCCL-PGK-antiVEGFA共转染进HEK293T细胞。经过浓缩、纯化步骤得到能表达抗VEGFA抗体的非整合型慢病毒颗粒，即BD311。BD311感染细胞后，载体基因通过反转录成环状DNA游离在细胞基因组外，表达抗VEGFA蛋白，达到治疗效果。
详见以下各实施例：
实施例1：构建pMDlg/pRRE-D64V质粒
1.合成的基因：合成带有D64V突变的整合酶的基因。
2.将合成的基因克隆到质粒pMDlg/pRRE中，替换原有的整合酶基因。克隆采用试剂
Figure BDA0003370411070000051
HiFi DNA Assembly进行一部分无缝克隆。
实施例2：构建表达VEGFA抗体的质粒pCCL-PGK-antiVEGFA
1.设计需要合成的基因：合成抗VEGFA抗体基因时在基因的5’端加入限制性酶切位点BamHI，和Kozak序列。在重链和轻链之间加入P2A序列用于蛋白成熟时分割重链和轻链。P2A的基因序列如SEQ ID NO.10所示，氨基酸序列如SEQ ID NO.11所示。在3’端加入限制性酶切位点XhoI。
2.根据所设计的基因序列合成基因。
3.用限制性内切酶BamHI/XhoI酶切合成的基因和载体pCCL-PGK-eGFP，经过DNA纯化后连接基因和载体，构建出质粒pCCL-PGK-antiVEGFA。
实施例3：生产非整合型表达抗VEGFA抗体的慢病毒颗粒-BD311
1.第一天，接种HEK293T细胞到15cm规格的细胞培养皿，细胞数量为1.3×107每皿。
2.第二天，细胞接种24小时后，密度接近85％。换新鲜的细胞完全培养基。1小时后转染。将质粒9.07μg pMD.2G、7.26μg pRSV-REV、31.46μg pMDlg/pRRE-D64V和31.46μg载体质粒pCCL-PGK-antiVEGFA共转染进HEK293T细胞。
本实施例应用钙磷转染法转染，方法步骤如下：
1)取15mL离心管，依次加无菌水和各种质粒配成总体积为1089μL的DNA MIX混匀；
2)在DNA MIX液面上缓慢滴加121μL 2.5M CaCl2溶液，轻轻吸取反复滴加混匀(约重复3次左右)；
3)滴加1210μL 2×HEBS溶液，混匀，静置；
4)5分钟内将液体滴加到细胞中，混匀，放入培养箱。
3.第三天，弃掉原来的培养基，换成新鲜的细胞完全培养基。
4.第四天，质粒转染48小时后收取粗病毒液，再加入新鲜的细胞完全培养基。
5.第五天，质粒转染72小时后收取粗病毒液，与前一天收的粗病毒液合并。加入全能核酸酶，37℃处理粗病毒液1小时。用0.45μm的滤膜的一次性过滤器将粗病毒液过滤到超滤管中，每管加入32mL粗病毒液,并在管底加6mL 20％的蔗糖PBS溶液，使蔗糖和粗病毒液界限分明。把超滤管放入超速离心机的适配器中，配平衡后，在Beckman&optima L-100XP超速离心机中以25000rpm,4℃的条件下离心2小时。离心结束后，拿出超离管，倒掉上清液，擦干内管壁，加入100μL PBS浸泡慢病毒颗粒4-18h。
6.浸泡完全后，用移液器轻柔地将慢病毒吹吸均匀后分装，储存在-80℃冰箱。
实施例4：BD311体外感染293T细胞表达抗VEGFA抗体
实验方法是：
1.第一天，将HEK293T细胞以4×104/孔的密度接种到48孔板，
2.第二天，接种细胞24h后感染BD311。其感染量是以慢病毒载体物理滴度p24的量为标准的。分别为100ng，200ng和400ng三个梯度。每个梯度3个复孔。
3.第三天，感染BD311 18h后换新鲜完全培养基，每孔500mL。
4.第四天，感染48h后收细胞上清液，用ELISA方法检测抗体表达量。
ELIAS的检测所用的空白包被蛋白是VEGF165，标准品是体外表达后纯化的抗VEGF抗体蛋白。
5.结果显示(图2)，抗VEGF抗体表达量随感染BD311量的增加而增多。表达量最高的孔达到56ug/mL。
实施例5：BD311体内抑制血管新生
采用激光诱导的小鼠黄斑变性模型验证BD311减缓脉络膜新生血管增长的效果。
具体操作如下：
1.视网膜下注射。选取C57BL/6J，雄性，6周龄小鼠进行体内实验。1.25％三溴乙醇麻醉小鼠后，在解剖镜下用微量注射器(Hamilton)进行视网膜下注射。将2uL(100ng p24)BD311注射液注射到小鼠右眼，2uL PBS经视网膜下注射到小鼠左眼作为对照。
2.激光诱导。视网膜下注射7天，1.25％三溴乙醇麻醉小鼠后，采用Laser System(IQ 532,IRIDEX)，120mW,50μm光斑和100ms曝光时间的条件下在视神经盘周围进行4处激光灼伤。在膜处产生气泡并未出血的激光灼伤代表造模成功。
3.检测脉络膜新生血管。
1)摘取眼球：1.25％三溴乙醇麻醉小鼠并脱颈后，小心摘取小鼠眼球。
2)固定眼球：用针头在虹膜边缘扎一个小孔，整个眼球置于4％的多聚甲醛溶液中室温固定1h。PBS清洗3次。
3)解剖眼球：在解剖显微镜下去除多余的***；沿赤道(虹膜边缘)呈圆形切除眼壁；取出角膜、晶状体和玻璃体；剩下视网膜、脉络膜和巩膜的碗状组织；沿边缘向视神经盘方向剪四条切口，使碗状组织被均匀分成四部分，展开铺平，将部分视网膜朝上，用镊子夹出视网膜，剩下RPE/脉络膜/巩膜的组织。
4)染色：5％的山羊血清4℃封闭和0.5％Triton X-100/PBS清洗样品后，用抗体Alexa Fluor 488Conjugates isolectin GS-B4染色，染色抗体稀释比例和方法按照说明书操作。
5)制片：将组织平铺在载玻片上，滴加荧光抗淬灭剂，盖上盖玻片，用封片剂封片。
6)数据采集和处理：用荧光显微镜采集图片。ImageJ软件计算脉络膜新生血管的面积。结果如图3。
以上对本发明的具体实施示例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，也不局限于上述VEGFA抗体；事实上，应用本发明所述非整合型慢病毒载体可用来递送抗体如雷珠单抗、贝伐珠单抗、布洛赛珠单抗、康柏西普、阿柏西普的基因，所表达的相应的抗体可以与VEGFA结合，阻碍VEGFA的促血管新生的作用，从而缓解黄斑变性疾病。本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。
序列表
<110> 上海本导基因技术有限公司
<120> 适用于难治性血管新生性眼疾病基因治疗的慢病毒载体
<130> DD16237
<141> 2021-09-27
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 867
<212> DNA
<213> Artificial Sequence
<400> 1
tttttagatg gaatcgataa ggctcaagaa gaacacgaaa agtaccactc taattggaga 60
gccatggcaa gtgattttaa cctgccacct gtagtagcaa aagaaatagt agccagctgt 120
gataaatgtc agctaaaagg agaagccatg catggacaag tagactgtag tccaggaata 180
tggcaactag tttgtacaca tctagaagga aaaattatcc tggtagcagt tcatgtagcc 240
agtggatata tagaagcaga agttattcca gcagagacag ggcaggaaac agcatatttt 300
ctcttaaaat tagcaggaag atggccagta aaaacaatac atacagacaa tggcagcaat 360
ttcaccagta ctacggttaa ggccgcctgt tggtgggcag ggatcaagca ggaatttggc 420
attccctaca atccccaaag ccaaggagta gtagaatcta tgaataatga attaaagaaa 480
attataggac aggtaagaga tcaggctgaa caccttaaga cagcagtaca aatggcagta 540
ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata 600
gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt 660
caaaattttc gggtttatta cagggacagc agagatccag tttggaaagg accagcaaag 720
ctcctctgga aaggtgaagg ggcagtagta atacaagata atagtgacat aaaagtagtg 780
ccaagaagaa aagcaaagat catcagggat tatggaaaac agatggcagg tgatgattgt 840
gtggcaagta gacaggatga ggattaa 867
<210> 2
<211> 288
<212> PRT
<213> Artificial Sequence
<400> 2
Phe Leu Asp Gly Ile Asp Lys Ala Gln Glu Glu His Glu Lys Tyr His
1 5 10 15
Ser Asn Trp Arg Ala Met Ala Ser Asp Phe Asn Leu Pro Pro Val Val
20 25 30
Ala Lys Glu Ile Val Ala Ser Cys Asp Lys Cys Gln Leu Lys Gly Glu
35 40 45
Ala Met His Gly Gln Val Asp Cys Ser Pro Gly Ile Trp Gln Leu Val
50 55 60
Cys Thr His Leu Glu Gly Lys Ile Ile Leu Val Ala Val His Val Ala
65 70 75 80
Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro Ala Glu Thr Gly Gln Glu
85 90 95
Thr Ala Tyr Phe Leu Leu Lys Leu Ala Gly Arg Trp Pro Val Lys Thr
100 105 110
Ile His Thr Asp Asn Gly Ser Asn Phe Thr Ser Thr Thr Val Lys Ala
115 120 125
Ala Cys Trp Trp Ala Gly Ile Lys Gln Glu Phe Gly Ile Pro Tyr Asn
130 135 140
Pro Gln Ser Gln Gly Val Val Glu Ser Met Asn Asn Glu Leu Lys Lys
145 150 155 160
Ile Ile Gly Gln Val Arg Asp Gln Ala Glu His Leu Lys Thr Ala Val
165 170 175
Gln Met Ala Val Phe Ile His Asn Phe Lys Arg Lys Gly Gly Ile Gly
180 185 190
Gly Tyr Ser Ala Gly Glu Arg Ile Val Asp Ile Ile Ala Thr Asp Ile
195 200 205
Gln Thr Lys Glu Leu Gln Lys Gln Ile Thr Lys Ile Gln Asn Phe Arg
210 215 220
Val Tyr Tyr Arg Asp Ser Arg Asp Pro Val Trp Lys Gly Pro Ala Lys
225 230 235 240
Leu Leu Trp Lys Gly Glu Gly Ala Val Val Ile Gln Asp Asn Ser Asp
245 250 255
Ile Lys Val Val Pro Arg Arg Lys Ala Lys Ile Ile Arg Asp Tyr Gly
260 265 270
Lys Gln Met Ala Gly Asp Asp Cys Val Ala Ser Arg Gln Asp Glu Asp
275 280 285
<210> 3
<211> 8623
<212> DNA
<213> Artificial Sequence
<400> 3
aagcttggcc attgcatacg ttgtatccat atcataatat gtacatttat attggctcat 60
gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 120
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 180
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 240
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 300
ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 360
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 420
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 480
acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 540
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 600
actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 660
gagctcgttt agtgaaccgg ggtctctctg gttagaccag atctgagcct gggagctctc 720
tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt 780
agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc 840
agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca 900
gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg 960
cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg 1020
tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta 1080
aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta 1140
gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg 1200
ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca 1260
gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta 1320
gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat 1380
cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa 1440
agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca 1500
gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg 1560
aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg 1620
tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca 1680
actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct 1740
aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc 1800
tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac 1860
ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga 1920
agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc 1980
aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat 2040
gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag 2100
agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc 2160
cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg 2220
attagtgaac ggatctcgac ggtatcggtt aacttttaaa agaaaagggg ggattggggg 2280
gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt 2340
acaaaaacaa attacaaaaa ttcaaaattt tatcgatcac gagactagcc tcgacgatgg 2400
tcgagtaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag 2460
ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca 2520
ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc 2580
ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg 2640
aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg 2700
taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc 2760
tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc 2820
gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt 2880
tctcctcttc ctcatctccg ggcctttcga cctctagcgg gatccaccgg tcgccaccat 2940
ggagaccgac accctgctgc tgtgggtgct gctgctgtgg gtgcccggca gcaccggaga 3000
ggtgcagctg gtggagagcg gcggcggact ggtgcagcct ggaggaagcc tgagactgag 3060
ttgcgccgcc agcggctatg attttaccca ctacggcatg aactgggtga gacaggcccc 3120
cggcaaaggg ctggagtggg tgggatggat caacacatat acaggagaac ccacctacgc 3180
tgctgatttc aaaaggagat tcaccttcag cctggacacc tccaagagca ccgcctacct 3240
gcagatgaac agcctgagag ccgaggacac cgccgtgtac tactgcgcca aataccccta 3300
ctactacgga acaagccact ggtacttcga cgtgtggggc cagggaaccc tggtgaccgt 3360
gagcagcgcc cagcccagag agccccaggt gtataccctg ccccctagcc gcgacgaact 3420
gaccaaaaac caggtgagcc tgagctgcgc tgtgaaaggc ttctacccct ctgacatcgc 3480
cgtggaatgg gagagcaacg gccagcctga gaacaactac aagaccactc cccccgtgct 3540
ggacagcgac ggcagcttct tcctggtgtc taaactgact gtggacaaga gcaggtggca 3600
gcagggcaac gtgttcagtt gcagcgtgat gcacgaggcc ctgcacaacc actataccca 3660
gaagtccctg tccctgagcc ccggaaaggg ctccggcgct accaacttca gcctgctgaa 3720
gcaggccggg gacgtggagg agaaccccgg accagacatc cagctgaccc agagccccag 3780
cagcctgtcc gccagcgtgg gagacagagt gaccatcaca tgcagcgcct cccaggacat 3840
cagcaactac ctgaactggt accagcagaa gcccggcaag gcccccaaag tgctgatcta 3900
cttcactagc agcctgcaca gcggcgtgcc cagcagattt tccggcagcg gctccggcac 3960
cgacttcacc ctgactatca gcagcctgca gcccgaggat ttcgccacct actactgcca 4020
gcagtactcc accgtgccct ggaccttcgg acagggcacc aaggtggaaa ttaaaagaca 4080
gcccagagaa ccccaggtgt acaccctgcc ccccagcaga gacgaactga caaagaacca 4140
ggtgtccctg tggtgcctgg tgaagggctt ttaccccagc gatatcgccg tggagtggga 4200
gagcaatggc cagcccgaga acaactataa aacaacacca cccgtgctgg atagcgacgg 4260
ctcctttttc ctgtattcta agctgaccgt ggacaagagt agatggcagc aggggaacgt 4320
gttcagctgc agcgtgatgc atgaggccct gcataaccac tatactcaga agagcctgag 4380
cctgagcccc gggaagtaaa gcggcctcga gggaattccg ataatcaacc tctggattac 4440
aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga 4500
tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc 4560
tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa 4620
cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc 4680
acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc 4740
atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc 4800
gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg 4860
attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct 4920
tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg 4980
agtcggatct ccctttgggc cgcctccccg catcgggaat tcgagctcgg tacctttaag 5040
accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact 5100
ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct 5160
ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa 5220
gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc 5280
tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagcatcta 5340
gctagaatta attccgtgta ttctatagtg tcacctaaat cgtatgtgta tgatacataa 5400
ggttatgtat taattgtagc cgcgttctaa cgacaatatg tacaagccta attgtgtagc 5460
atctggctta ctgaagcaga ccctatcatc tctctcgtaa actgccgtca gagtcggttt 5520
ggttggacga accttctgag tttctggtaa cgccgtcccg cacccggaaa tggtcagcga 5580
accaatcagc agggtcatcg ctagcctagg gacgtaccca attcgcccta tagtgagtcg 5640
tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 5700
acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 5760
gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc 5820
tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 5880
gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 5940
ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 6000
cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 6060
tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 6120
ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 6180
ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 6240
tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtggcacgta 6300
gaaagccagt ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg 6360
gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg 6420
atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc 6480
ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat 6540
ctgatggcgc aggggatcaa gctctgatca agagacagga tgaggatcgt ttcgcatgat 6600
tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta 6660
tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca 6720
ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcaaga 6780
cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga 6840
cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct 6900
cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg 6960
gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga 7020
gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca 7080
tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc ccgacggcga 7140
ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg 7200
cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc 7260
gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt 7320
gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga 7380
gttcttctga attattaact cgagctgtca gaccaagttt actcatatat actttagatt 7440
gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 7500
atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 7560
atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 7620
aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 7680
aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag 7740
ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 7800
ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 7860
tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 7920
ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 7980
acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 8040
gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 8100
cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 8160
aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 8220
atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga 8280
gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg 8340
gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc 8400
tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt 8460
tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt 8520
ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag 8580
cgcgcaatta accctcacta aagggaacaa aagctggagc tgc 8623
<210> 4
<211> 690
<212> DNA
<213> Artificial Sequence
<400> 4
gaggtgcagc tggtggagag cggcggcgga ctggtgcagc ctggaggaag cctgagactg 60
agttgcgccg ccagcggcta tgattttacc cactacggca tgaactgggt gagacaggcc 120
cccggcaaag ggctggagtg ggtgggatgg atcaacacat atacaggaga acccacctac 180
gctgctgatt tcaaaaggag attcaccttc agcctggaca cctccaagag caccgcctac 240
ctgcagatga acagcctgag agccgaggac accgccgtgt actactgcgc caaatacccc 300
tactactacg gaacaagcca ctggtacttc gacgtgtggg gccagggaac cctggtgacc 360
gtgagcagcg cccagcccag agagccccag gtgtataccc tgccccctag ccgcgacgaa 420
ctgaccaaaa accaggtgag cctgagctgc gctgtgaaag gcttctaccc ctctgacatc 480
gccgtggaat gggagagcaa cggccagcct gagaacaact acaagaccac tccccccgtg 540
ctggacagcg acggcagctt cttcctggtg tctaaactga ctgtggacaa gagcaggtgg 600
cagcagggca acgtgttcag ttgcagcgtg atgcacgagg ccctgcacaa ccactatacc 660
cagaagtccc tgtccctgag ccccggaaag 690
<210> 5
<211> 230
<212> PRT
<213> Artificial Sequence
<400> 5
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe
50 55 60
Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Gln Pro Arg Glu
115 120 125
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
130 135 140
Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile
145 150 155 160
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
165 170 175
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
180 185 190
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
195 200 205
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
210 215 220
Ser Leu Ser Pro Gly Lys
225 230
<210> 6
<211> 642
<212> DNA
<213> Artificial Sequence
<400> 6
gacatccagc tgacccagag ccccagcagc ctgtccgcca gcgtgggaga cagagtgacc 60
atcacatgca gcgcctccca ggacatcagc aactacctga actggtacca gcagaagccc 120
ggcaaggccc ccaaagtgct gatctacttc actagcagcc tgcacagcgg cgtgcccagc 180
agattttccg gcagcggctc cggcaccgac ttcaccctga ctatcagcag cctgcagccc 240
gaggatttcg ccacctacta ctgccagcag tactccaccg tgccctggac cttcggacag 300
ggcaccaagg tggaaattaa aagacagccc agagaacccc aggtgtacac cctgcccccc 360
agcagagacg aactgacaaa gaaccaggtg tccctgtggt gcctggtgaa gggcttttac 420
cccagcgata tcgccgtgga gtgggagagc aatggccagc ccgagaacaa ctataaaaca 480
acaccacccg tgctggatag cgacggctcc tttttcctgt attctaagct gaccgtggac 540
aagagtagat ggcagcaggg gaacgtgttc agctgcagcg tgatgcatga ggccctgcat 600
aaccactata ctcagaagag cctgagcctg agccccggga ag 642
<210> 7
<211> 214
<212> PRT
<213> Artificial Sequence
<400> 7
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile
35 40 45
Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Gln Pro Arg Glu
100 105 110
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
115 120 125
Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
130 135 140
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
145 150 155 160
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
165 170 175
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
180 185 190
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
195 200 205
Ser Leu Ser Pro Gly Lys
210
<210> 8
<211> 60
<212> DNA
<213> Artificial Sequence
<400> 8
atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgcccgg cagcaccgga 60
<210> 9
<211> 20
<212> PRT
<213> Artificial Sequence
<400> 9
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly
20
<210> 10
<211> 66
<212> DNA
<213> Artificial Sequence
<400> 10
ggctccggcg ctaccaactt cagcctgctg aagcaggccg gggacgtgga ggagaacccc 60
ggacca 66
<210> 11
<211> 22
<212> PRT
<213> Artificial Sequence
<400> 11
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
1 5 10 15
Glu Glu Asn Pro Gly Pro
20
<210> 12
<211> 7751
<212> DNA
<213> Artificial Sequence
<400> 12
aagcttggcc attgcatacg ttgtatccat atcataatat gtacatttat attggctcat 60
gtccaacatt accgccatgt tgacattgat tattgactag ttattaatag taatcaatta 120
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 180
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 240
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 300
ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 360
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 420
cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 480
acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 540
acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 600
actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 660
gagctcgttt agtgaaccgg ggtctctctg gttagaccag atctgagcct gggagctctc 720
tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt 780
agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc 840
agtgtggaaa atctctagca gtggcgcccg aacagggacc tgaaagcgaa agggaaacca 900
gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg 960
cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc 1020
gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg 1080
ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa 1140
cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga 1200
cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta 1260
gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac 1320
aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt 1380
cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt 1440
agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag 1500
agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag 1560
cactatgggc gcagcctcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat 1620
agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact 1680
cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa 1740
ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt 1800
gccttggaat gctagttgga gtaataaatc tctggaacag attggaatca cacgacctgg 1860
atggagtggg acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa 1920
tcgcaaaacc agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt 1980
ttgtggaatt ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata 2040
gtaggaggct tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt 2100
aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac 2160
aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta 2220
gtgaacggat ctcgacggta tcggttaact tttaaaagaa aaggggggat tggggggtac 2280
agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa 2340
aaacaaatta caaaaattca aaattttatc gatcacgaga ctagcctcga cgatggtcga 2400
gtaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc 2460
gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg 2520
taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct 2580
agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt 2640
agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg 2700
cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg 2760
aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag 2820
gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc 2880
ctcttcctca tctccgggcc tttcgacctc tagcgggatc caccggtcgc caccatggtg 2940
agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac 3000
gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag 3060
ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg 3120
accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac 3180
gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag 3240
gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac 3300
cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg 3360
gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc 3420
aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac 3480
taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg 3540
agcacccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg 3600
gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc 3660
ctcgagggaa ttccgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta 3720
ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc 3780
atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt 3840
ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg 3900
ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt 3960
tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct 4020
ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt 4080
cctttccatg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct 4140
acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc 4200
ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct 4260
ccccgcatcg ggaattcgag ctcggtacct ttaagaccaa tgacttacaa ggcagctgta 4320
gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga 4380
agacaagatc tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg 4440
gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg 4500
cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc 4560
ttttagtcag tgtggaaaat ctctagcagc atctagctag aattaattcc gtgtattcta 4620
tagtgtcacc taaatcgtat gtgtatgata cataaggtta tgtattaatt gtagccgcgt 4680
tctaacgaca atatgtacaa gcctaattgt gtagcatctg gcttactgaa gcagacccta 4740
tcatctctct cgtaaactgc cgtcagagtc ggtttggttg gacgaacctt ctgagtttct 4800
ggtaacgccg tcccgcaccc ggaaatggtc agcgaaccaa tcagcagggt catcgctagc 4860
ctaggctttt gcgtcgagac gtacccaatt cgccctatag tgagtcgtat tacgcgcgct 4920
cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc 4980
gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 5040
gcccttccca acagttgcgc agcctgaatg gcgaatggcg cgacgcgccc tgtagcggcg 5100
cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 5160
tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 5220
gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 5280
accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 5340
tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 5400
gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 5460
cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 5520
tattaacgtt tacaatttcc caggtggcac ttttcgggga aatgtgcgcg gaacccctat 5580
ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 5640
aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct 5700
tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 5760
agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 5820
cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt 5880
taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 5940
tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 6000
tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 6060
cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt 6120
gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 6180
cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 6240
actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 6300
ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 6360
tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 6420
tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 6480
acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 6540
ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 6600
ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 6660
ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct 6720
gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 6780
ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc 6840
aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc 6900
gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc 6960
gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg 7020
aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata 7080
cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta 7140
tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc 7200
ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg 7260
atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt 7320
cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt 7380
ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga 7440
gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc 7500
cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg 7560
cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca 7620
ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg 7680
aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa 7740
gctggagctg c 7751

Claims (10)

1.一种非整合型慢病毒载体***，其特征在于，所述载体***的包装质粒包括表达包膜蛋白的pMD.2G、表达REV蛋白的pRSV-REV、整合酶突变失活的pMDlg/pRRE-IN mut和表达VEGFA抗体基因的载体质粒。
2.根据权利要求1所述的非整合型慢病毒载体***，其特征在于，所述整合酶的氨基酸序列包括如SEQ ID NO.2所示序列。
3.根据权利要求1所述的非整合型慢病毒载体***，其特征在于，所述整合酶的基因序列包括如SEQ ID NO.1所示序列。
4.根据权利要求1所述的非整合型慢病毒载体***，其特征在于，所述VEGFA抗体基因是在雷珠单抗的重链和轻链可变区分别加上人源IgG的Fc片段恒定区CH3，并对基因进行密码子优化而得。
5.根据权利要求4所述的非整合型慢病毒载体***，其特征在于，所述VEGFA抗体基因由重链和轻链组成；重链的氨基酸序列为SEQ ID NO.5，轻链的氨基酸序列为SEQ IDNO.7。
6.根据权利要求4所述的非整合型慢病毒载体***，其特征在于，所述VEGFA抗体基因的重链的基因序列如SEQ ID NO.4所示，轻链的基因序列为SEQ ID NO.6所示。
7.根据权利要求1所述的非整合型慢病毒载体***，其特征在于，所述载体质粒为pCCL-PGK-antiVEGFA，载体序列如SEQ ID NO.3所示。
8.一种根据权利要求1-7中任一项所述的非整合型慢病毒载体***的制备方法，其特征在于，将所述质粒pMD.2G、pRSV-REV、整合酶突变失活的pMDlg/pRRE-IN mut和载体质粒共转染进宿主细胞，浓缩、纯化，得到能表达抗VEGFA抗体的非整合型慢病毒***。
9.一种根据权利要求1-7中任一项所述的非整合型慢病毒载体***在制备治疗难治性血管新生性眼疾病的制剂中的用途。
10.根据权利要求9所述的用途，其特征在于，所述制剂包括治疗血管新生性年龄相关性黄斑变性疾病、糖尿病视网膜病变相关黄斑水肿或视网膜静脉阻塞相关黄斑水肿的制剂。
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