CN115161294B - 新城疫疫苗株及其构建方法、禽类免疫识别方法、应用 - Google Patents

新城疫疫苗株及其构建方法、禽类免疫识别方法、应用 Download PDF

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CN115161294B
CN115161294B CN202210653186.5A CN202210653186A CN115161294B CN 115161294 B CN115161294 B CN 115161294B CN 202210653186 A CN202210653186 A CN 202210653186A CN 115161294 B CN115161294 B CN 115161294B
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孙敏华
宋家升
周洁
任涛
廖明
孙慧敏
董嘉文
韩翡
涂玉蓉
李林林
张俊勤
黄允真
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Guangdong Winsun Biopharmaceuticals Co ltd
Zhejiang Difu Runsi Biotechnology Co ltd
South China Agricultural University
Institute of Animal Health of Guangdong Academy of Agricultural Sciences
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Zhejiang Difu Runsi Biotechnology Co ltd
South China Agricultural University
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Abstract

本发明属于生物技术领域，公开了一种新城疫疫苗株，分类名为新城疫病毒(Newcastle disease virus)，保藏编号为：CCTCC No：V202235；保藏日期：2022年5月7日，保藏单位：武汉大学中国典型培养物保藏中心。该疫苗株突变缺失了HN蛋白的关键中和位点，通过病毒拯救获得了重组病毒rLa‑mGM。将重组病毒和LaSota疫苗免疫SPF鸡后，通过测定HI抗体能够确定是否免疫成功，通过间接ELISA方法能够实现野毒和疫苗毒的鉴别。同时，本发明还提供了该疫苗株的构建方法、禽类免疫识别方法、应用。

Description

新城疫疫苗株及其构建方法、禽类免疫识别方法、应用
技术领域
本发明涉及生物技术领域，具体为一种新城疫疫苗株及其构建方法、禽类免疫识别方法、应用。
背景技术
新城疫(Newcastle disease，ND)已被列入《广东省中长期动物疫病防治规划(2012-2020年)》，是必须控制和净化的传染病。现有的ND疫苗只有弱毒活疫苗和全病毒灭活疫苗，这些疫苗免疫后产生的抗体和野毒株感染产生的抗体几乎一致，导致疫苗免疫后无法区分疫苗免疫和野毒感染。当前疫苗只能控制该病的发生，却无法净化。
对比文件1(D1)：中国农业科学院兰州兽医研究所提出了一项专利申请CN201510230344.6公开了利用已建立的新城疫病毒反向遗传操作平台，将G7株的NP蛋白缺失18个氨基酸并将F蛋白裂解位点进行突变，通过筛选救获一株毒力高度致弱、病毒滴度高的基因VII型新城疫病毒标记疫苗株MG7-NPΔ18+Fmut，其微生物保藏编号为：CCTCC NO：V201505。该发明标记疫苗株在鸡胚中具有高生长滴度和低致病力的生物学特性且遗传稳定。免疫保护试验结果表明，该标记疫苗株免疫原性好，可诱导高水平的保护性抗体，对免疫鸡只实现完全保护，能用于防控目前流行的基因VII型新城疫病毒，并为鉴别疫苗免疫和野毒感染奠定基础。
D1是通过基因重组，将NP蛋白的某些氨基酸进行了缺失处理，并对F蛋白裂解位点进行突变，具体来说第112-117位的氨基酸序列突变为GRQGRL。
从理论上来说，该MG7-NPΔ18+Fmut可用494DEQARLNPVSAVFDNI509-BSA藕连蛋白反应，如果不反应，那么说明MG7-NPΔ18+Fmut没有F蛋白裂解位点突变。
D1的说明书表4记载：ICPI由1.6降低到0.49。
对比文件2(D2)：湖北省农业科学院畜牧兽医研究所提出了一项专利申请CN202011036432.X，公开了一种基因VII型新城疫标记疫苗株及其制备方法与应用。利用反向遗传操作技术，以血清2型禽副粘病毒Y2株的全基因组转录质粒为骨架，将其F和HN基因的胞外域分别替换为基因VII型新城疫病毒株的F和HN基因的胞外域，通过病毒拯救，获得重组嵌合病毒。该疫苗株已保藏于中国典型培养物保藏中心，保藏编号为CCTCC NO:202052。该嵌合病毒可以用于我国主要流行的基因VII新城疫的防控。同时，该嵌合病毒不含有新城疫病毒NP蛋白，其免疫鸡后不会产生针对新城疫病毒NP蛋白的抗体。以常规的新城疫病毒NP抗体ELISA检测方法对rY2-HB疫苗株免疫鸡群进行抗体检测，可实现特异检测临床野毒株产生的NP抗体，排除疫苗毒株产生的抗体干扰，有利于新城疫的监测与净化。
由此可见D2是让新的疫苗株作用于对象后，不产生针对新城疫病毒NP蛋白的抗体以实现可以通过NP抗体ELISA检测方法来检测。
对比文件3(D3)：中国农业大学提出了一项专利申请CN201610556238.1，公开了一种基因Ⅶ型NDV标记疫苗株aSG10-mHN，所述标记疫苗株为HN基因5’非编码区具有SEQ IDNO.1所示的核苷酸序列的致弱毒株aSG10。其构建的标记疫苗株毒力明显降低且遗传稳定。免疫保护试验结果表明，标记疫苗株对NDV具有良好的免疫保护效果，并能有效抑制排毒，可用于预防当前流行的基因Ⅶ型新城疫病毒。进一步，通过PCR方法能鉴别野毒感染动物与疫苗免疫动物，对ND的监测、诊断、控制和净化有重要意义，且有广泛的应用价值。
可见，D3是采用引物对进行检测。
对比文件4(D4)：浙江鼎持生物制品有限公司提出了一项专利申请CN202010441121.5，公开了一种新城疫嵌合病毒标记疫苗株及其构建方法和应用，属于新城疫嵌合疫苗株的拯救及应用领域。其将新城疫基因GⅦ型毒株的F蛋白裂解位点突变为弱毒株的裂解位点，该突变后的基因VII型新城疫毒株的F和HN基因与基因II型NDV La Sota株的NP、P、M、L构建全长嵌合cDNA序列，并在P和M之间的非编码区***18bp的核苷酸标记序列。转染细胞拯救获得了新城疫嵌合病毒NDV DC株。嵌合后的毒株含有基因VII型新城疫毒株的囊膜表面糖蛋白，并含有基因II型毒株的骨架，兼具新城疫基因VII型毒株的免疫原性和基因II型La Sota株的高繁殖和高安全特性。
D4在其说明书23段记载：通过测序和酶切鉴定的方法来进行毒株的鉴别，标记疫苗株为鉴别感染和疫苗免疫奠定了基础。
所以，D4也是采用特异性引物来进行免疫识别的。
综上所述，上述D1-D4，其技术方案和识别方法如下。
技术方案：
D1:NP蛋白氨基酸部分缺失、F蛋白裂解位点部分氨基酸突变；
D2：Y2株的转录质粒中F和HN基因的胞外域采用基因VII型新城疫病毒株的F和HN基因胞外域替换。
D3：HN基因ORF终止密码子后第6个核苷酸处***一段核苷酸序列；
D4：将VII型F基因序列和基因VII型的HN基因序列***到II型La Sota株的NP、P、M和L基因中，并在P和M之间***标记序列。
鉴别方法：
D1理论上可通过HI抗体结合494DEQARLNPVSAVFDNI509-BSA藕连蛋白鉴别；D3、D4通过引物对鉴别；D2通过NP抗体ELISA检测方法识别。
但是D1的毒性虽然有所减弱，但是无法达到我们想要的程度。
因此，亟待研发一种低毒、能够用相对简单的方法区分感染和免疫动物(Differentiation of infected from vaccinated animals，DIVA)的基因标记疫苗，为ND的净化提供物质保障。
发明内容
本发明的目的在于提供一种新城疫疫苗株及其构建方法、禽类免疫识别方法、应用，该疫苗株突变缺失了HN蛋白的关键中和位点，通过病毒拯救获得了重组病毒rLa-mGM。将重组病毒和LaSota疫苗免疫SPF鸡后，通过测定HI抗体能够确定是否免疫成功，通过间接ELISA方法能够实现野毒和疫苗毒的鉴别，同时本疫苗株的ICPI为0，属于传代稳定、低毒的疫苗株。
为实现上述目的，本发明提供如下技术方案：一种疫苗株，分类名为新城疫病毒(Newcastle disease virus)，保藏编号为：CCTCC No：V202235；保藏日期：2022年5月7日，保藏单位：武汉大学中国典型培养物保藏中心，保藏地点：中国.湖北.武汉，邮编：。
研究基因标记疫苗的关键就是对现有疫苗株进行基因改造，一方面使它和当前流行毒株的抗原性匹配度高；另外一方面使它具有独特的基因标记，能够通过血清学方法进行检测和鉴别。
LaSota疫苗株是当前使用最广泛的ND疫苗株，该疫苗株培养特性稳定、效价高、成本低，长期使用结果证实该疫苗株十分安全，效果理想。鉴于我国当前的流行毒株与LaSota疫苗株之间存在近20％的序列差异，同时非典型新城疫仍然时有发生，因此对现有LaSota疫苗株的主要抗原性相关基因F(Fusion protein)和HN(Hemagglutinin-neuraminidaseprotein)进行反向遗传改造是抗原性匹配的关键。
为此，本发明选取当前流行的基因VII型毒株GM株的F和HN基因，替换掉LaSota疫苗株的F和HN基因，使疫苗株的保护性抗原与野毒株一致。
同时，为了实现区分野毒感染和疫苗免疫动物，本研究选择HN基因作为基因标记分子。通过分析发现，HN蛋白存在两个主要抗原位点区域，340～347位存在H340、N342和E347共3个抗原位点；494～509位存在D494、E495、N508、I509共4个抗原位点，这两个区域均认为和抗原性关系非常密切，因此可作为理想的基因标记位点。通过反向遗传操作技术将HN蛋白494(D)、495(E)、508(N)和509(I)位氨基酸全部突变为丙氨酸(A)，F蛋白裂解位点序列由112RRQKRF117突变为112GRQGRL117。经质粒提取、转染BSR细胞、接种SPF鸡胚获得拯救毒株rLa-mGM株。经鉴定该毒株ICPI值为0，传代稳定，且HA效价为7log2。利用rLa-mGM株制备灭活疫苗，免疫10只SPF鸡，制备相应血清。同时取10只SPF鸡免疫LaSota灭活疫苗，另外10只为不免疫对照。14天后，取HZ株攻毒，结果疫苗免疫组均能保护。利用494DEQARLNPVSAVFDNI509-BSA藕连蛋白包被ELISA反应板，对rLa-mGM株、LaSota灭活疫苗免疫后血清进行检测，结果rLa-mGM株免疫后，不与494DEQARLNPVSAVFDNI509-BSA藕连蛋白反应，而现有LaSota疫苗免疫后能够与上述蛋白发生反应，这表明rLa-mGM株(保藏编号：CCTCC NO:V202235)是潜在的DIVA疫苗株，能够实现基因标记，可为ND的净化提供技术支撑。
同时，本发明还提供一种禽类免疫识别方法，将禽类血清与测试蛋白反应，根据反应结果，判断检测对象体内是否具有针对于如上所述疫苗株的抗体；
所述测试蛋白为494DEQARLNPVSAVFDNI509-BSA藕连蛋白；
同时，结合HI抗体检测判断禽类是否已经产生抗体；
通过上述方法的结合，可以获知禽类是否接种了本疫苗株制备的疫苗。
最后，本发明还提供一种如上所述的疫苗株的构建方法，用VII型毒株GM株的F和HN基因替换掉LaSota疫苗株的F和HN基因，得到疫苗株。
以及，该疫苗株在新城疫疫苗制备中的应用，同时还要求保护采用所述的疫苗株生产得到的生物制品。
与现有技术相比，本发明的有益效果是：
本发明以LaSota疫苗株为骨架，用基因VII型NDV强毒GM株F和HN基因替换LaSota疫苗株的F和HN基因，实现与当前流行株主要抗原的匹配。在F蛋白裂解位点突变为弱毒的基础上，进一步选择将HN蛋白的D494、E495、N508、I509抗原位点，并全部突变为中性氨基酸A。上述突变缺失了HN蛋白的关键中和位点，通过病毒拯救获得了重组病毒rLa-mGM。将重组病毒和LaSota疫苗免疫SPF鸡后，通过测定HI抗体能够确定是否免疫成功，通过间接ELISA方法能够实现野毒和疫苗毒的鉴别。这表明rLa-mGM株可作为疫苗候选株实现新城疫净化。
附图说明
图1为GM株F和HN基因致弱片段扩增结果；
图2为pBeloBAC11-LaSota骨架片段扩增结果；
图3为HN中和位点基因突变扩增结果；
图4为HN中和位点基因突变骨架片段扩增结果；
图5为rLa-mGM突变位点序列比对图；
图6为rLa-mGM株HN基因中和位点突变序列比对图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例
1材料与方法
1.1主要试剂
Figure BDA0003686660240000061
Max DNA Polymerase、HB101感受态细胞、DNA Marker、T4 DNALigase、Reverse Transcriptase M-MLV(RNase H-)、
Figure BDA0003686660240000062
GXL DNA Polymerase均购自Takara公司；
Figure BDA0003686660240000063
HiFi DNA Assembly Master Mix购自NEB公司；QIAGENPlasmid Plus Midi Kit购自QIAGEN公司；DMEM培养基、0.25％EDTA胰酶、胎牛血清均购自Gibco公司；Lipofectamine LTX DNA Transfection Reagents购自Invitrogen公司；
Figure BDA0003686660240000064
Viral RNA Kit购自Omega公司；
Figure BDA0003686660240000065
II One Step RT-PCR Kit(Dye Plus)购自诺唯赞医疗科技有限公司；常规试剂均为国产分析纯。494DEQARLNPVSAVFDNI509-BSA藕连多肽由生工生物工程(上海)股份有限公司合成。
1.2毒株、细胞与质粒
表达T7聚合酶的MVA/T7痘病毒、基因VII型HZ株；BSR-T7/5细胞、克隆载体pBeloBAC11、pBeloBAC11-LaSota全长cDNA克隆、辅助质粒pCI-NP、pCI-P、pCI-L均由本实验室保存。
备注：NP、P、L基因均在GenBank中有记载；辅助质粒pCI-NP、pCI-P、pCI-L的制备、鉴定可参考《新城疫病毒GM株反向遗传操作***的建立及重组疫苗的研制》，博士论文，孙敏华。
NP基因序列可见SEQ ID NO：11；
P基因序列可见SEQ ID NO：12；
L基因序列可见SEQ ID NO：13。
1.3鸡胚和鸡
25日龄SPF级白来航鸡和9日龄SPF鸡胚购自新兴大华农禽蛋有限公司。
1.4病毒
攻毒用基因VII型HZ株通过尿囊腔接种法在9日龄SPF鸡胚中繁殖。
1.5重组病毒的构建
以含GM株F和HN基因(SEQ ID NO：14)的质粒为模板，利用GM-F-F和GM-ZR-R引物扩增获得F蛋白裂解位点突的片段1；利用GM-ZR-F和GM-HN-R引物扩增获得F蛋白裂解位点突的片段2。同时以pBeloBAC11-LaSota全长cDNA克隆质粒(SEQ ID NO：15)为模板，利用ZT-L+GM-F和ZT-L-R引物扩增获得骨架基因片段3，利用同源重组试剂盒(
Figure BDA0003686660240000071
HiFi DNAAssembly)，将片段1、片段2和片段3连接，获得以LaSota株为骨架，嵌合GM株F基因突变和HN基因的重组cDNA克隆。利用HN2-F和L+GM-R15186引物扩增获得HN基因突变(对应D494A、E495A、N508A、I509A氨基酸突变)的片段4，利用L+GM-F15158和HN2-R引物扩增获得骨架片段5，利用同源重组试剂盒连接片段4和5，提取全长cDNA质粒，将突变后构建质粒的命名为pBeloBAC11-La-mGM(SEQ ID NO：16)。
表1 BAC-La-mGM构建引物
Figure BDA0003686660240000072
Figure BDA0003686660240000081
a：下划线表示突变核苷酸位点。
1.6重组病毒的拯救
利用QIAGEN质粒提取试剂盒提取pBeloBAC11-La-mGM、pCI-NP、pCI-P和pCI-L质粒，以4：2：1：1的比例依次加入全长质粒pBeloBAC11-La-mGM和辅助质粒pCI-NP、pCI-P、pCI-L，利用LTX试剂转染BSR-T7/5细胞。转染24h后，加入终浓度为1μg/μL的TPCK胰酶继续培养。转染60h后，冻融1次，将细胞混合物接种至9日龄SPF鸡胚尿囊腔，培养72h对尿囊液进行血凝活性(HA)检测，有HA效价的尿囊液即为拯救成功的重组病毒。
1.7重组病毒的遗传稳定性
将重组病毒在SPF鸡胚中连续传代15代，检测其遗传稳定性。将病毒尿囊液接种9日龄SPF鸡胚尿囊腔。37℃温箱孵育72h后，收获尿囊液，测定其HA效价。有HA效价的尿囊液进行传代，一直传代至第15代。提取第5、10和15代病毒尿囊液RNA，RT-PCR扩增测序确认突变序列的传代稳定性。
1.8MDT、EID50和ICPI测定
为了确定rLa-mGM的生物学特性，测定尿囊液的9日龄SPF鸡胚的MDT(平均死亡时间)、EID50(鸡胚半数感染量)和1日龄SPF鸡的ICPI(脑内致病性指数)。当MDT值大于120h、ICPI小于0.4方可作为疫苗候选株。
1.9活疫苗的免疫效果
为确定重组病毒的免疫效果，将30只25日龄SPF白来航鸡分为3组，每组各10只，分别标记脚号。第1组和第2组分别胸肌注射含106.0EID50的rLa-mGM株和LaSota株活疫苗，0.2mL/只。免疫后14天，采集血清进行HI检测以确定新城疫病毒抗体水平，同时每组用0.2mL含106.0EID50的基因型VII型NDV强毒HZ株滴鼻点眼攻毒，攻毒后第3、5、7天采集每只鸡的口咽及泄殖腔棉拭子，进行病毒分离。
1.10免疫血清的ELISA鉴别
取494DEQARLNPVSAVFDNI509-BSA多肽包被反应板，利用棋盘法确定最佳包被浓度和反应条件，建立检测血清抗体的ELISA方法。随后将rLa-mGM和LaSota疫苗株免疫后14天血清分析进行检测，分析两种疫苗株免疫后血清抗体效价是否存在差异。
2、结果与分析
2.1致弱突变株cDNA克隆的构建和鉴定
利用高保真酶扩增分段扩增获得F蛋白裂解位点核苷酸序列致弱突变的GM株的F和HN基因，获得与预期片段(366bp、3266bp)相符合的扩增产物(图1)。利用ZT-L+GM-F和ZT-L-R引物扩增LaSota骨架病毒核酸片段，获得与符合预期大小(18536bp)相符合的扩增产物(图2)。将上述三个片段回收后，经连接，PCR鉴定为阳性的，提质粒，经测序确认，F蛋白裂解位点突变的GM株F和HN基因克隆至骨架病毒中。采用同样的方法，HN基因4个中和位点突变的核酸片段替换到pBeloBAC11-LaSota骨架的相应区域(图3和4)，经测序确认，获得rLa-mGM质粒。
图1为GM株F和HN基因致弱片段扩增结果，其中：1-4：F基因突变片段1的PCR扩增产物(366bp)；5-8：F+HN基因突变片段2的PCR扩增产物(3266bp)；M：DL 5000
图2为pBeloBAC11-LaSota骨架片段扩增结果，其中，M：DL 15000；1-4：LaSota骨架基因扩增产物(18536bp)；
图3为HN中和位点基因突变扩增结果，其中，1-4：HN中和位点基因突变扩增条带；M：DL 15000；
图4为HN中和位点基因突变骨架片段扩增结果，其中，M：DL 15000；1-4：HN中和位点基因突变骨架片段扩增条带。
图5为rLa-mGM突变位点序列比对图，其中，A：rLa-mGM质粒F基因***位点序列比对；B：rLa-mGM质粒F蛋白裂解位点核苷酸序列比对；C：rLa-mGM质粒HN基因***位点序列比对；红色方框表示和GM株一致序列；蓝色方框表示和LaSota株一致序列。
2.2重组病毒的拯救
大量制备rLa-mGM质粒后，经转染BSR-T7/5细胞，将细胞混合物接种至9日龄SPF鸡胚尿囊腔，收获24～72h内所有死亡和存活的鸡胚尿囊液，经HA试验表明，其中的2枚鸡胚血凝效价分别为5log2和6log2。证实所拯救的病毒具有血凝活性，命名为rLa-mGM株。
2.3重组病毒的遗传稳定性
取拯救的重组病毒rLa-mGM株在SPF鸡胚中连续传代10代，其HA效价第5代和第10代分别为6log2、7log2。提取第10代病毒尿囊液RNA，RT-PCR扩增HN基因序列，经测序表明病毒HN基因突变位点遗传稳定(图6)。
图6为rLa-mGM株HN基因中和位点突变序列比对图，其中，红色方框表示和突变位点差异核苷酸。
2.4重组病毒MDT、EID50和ICPI测定
经测定，rLa-mGM株的MDT为160h，ICPI为0，EID50为10-8.3/0.2mL(表2)。该毒株符合NDV无毒株特征。
表2 rLa-mGM株部分生物学特性测定
Figure BDA0003686660240000111
需要说明的是：
对比毒株rLa-mGM1的结构是：通过PCR突变获得HN蛋白第340、342、347位丙氨酸突变，采用1.5和1.6中描述的方法进行构建和拯救，获得的重组病毒按照1.8中描述的方法进行测定。参照1.9和1.10中的方法进行免疫，结果340HNNTCPDE347-BSA多肽无法鉴别rLa-mGM1株和LaSota疫苗株免疫后的血清。
通过以上设计可以得到如下结论：
针对HN蛋白的基因修饰并不是任何修饰都能够达到低毒的效果，本发明不仅仅限于以上rLa-mGM1的重组实验，在其他的针对于HN蛋白基因的重组位点的实验中，我们发现在IPCI、EID50(0.2mL)无法达到一致的统一以使低毒的目的实现；
特别是，没有任何一个案例(除了本实施例)能够达到ICPI为0。
这是本案一个非常具有实用意义之处。
2.5重组活疫苗免疫效果的比较
为了确定rLa-mGM株能否产生足够的保护，将rLa-mGM株繁殖后以106.0EID50免疫SPF鸡。免疫后14天，其血清平均HI效价为4.8log2，比LaSota疫苗株的平均HI效价低4个滴度。攻毒后rLa-mGM和LaSota疫苗株免疫组所有鸡均存活，攻毒后第3天，rLa-mGM免疫组咽拭子排毒率为4/10，而LaSota免疫组为6/10。第5天时，rLa-mGM免疫组咽拭子排毒率为1/10，而LaSota免疫组为2/10(表3)。rLa-mGM免疫组尽管HI抗体不高，但保护效果良好。
表3活疫苗免疫组攻毒后的病毒分离率
Figure BDA0003686660240000121
a：O表示咽拭子；b：C表示泄殖腔拭子
2.6重组活疫苗免疫血清的ELISA鉴别
取LaSota株免疫的SPF鸡血清和SPF鸡阴性血清，以利用合成多肽建立间接ELISA方法，结果表明，当多肽包被量为0.1μg/孔，血清1:100稀释时，ELISA方法的P/N值最高(表4)，阴阳性临界值为0.6。利用上述方法对rLa-mGM和LaSota免疫血清进行ELISA检测，结果表明，未免疫空白组SPF鸡血清OD值均低于0.6；Lasota株免疫组均大于0.6，rLa-mGM免疫组血清均低于0.6，这表明rLa-mGM可作为疫苗候选株实现免疫鉴别。
表4合成多肽ELISA检测血清抗体OD450值
Figure BDA0003686660240000131
表5不同疫苗免疫组血清OD450值
Figure BDA0003686660240000132
三、结论
本发明以LaSota疫苗株为骨架，用基因VII型NDV强毒GM株F和HN基因替换LaSota疫苗株的F和HN基因，实现与当前流行株主要抗原的匹配。在F蛋白裂解位点突变为弱毒的基础上，进一步选择将HN蛋白的D494、E495、N508、I509抗原位点，并全部突变为中性氨基酸A。上述突变缺失了HN蛋白的关键中和位点，通过病毒拯救获得了重组病毒rLa-mGM。将重组病毒和LaSota疫苗免疫SPF鸡后，通过测定HI抗体能够确定是否免疫成功，通过间接ELISA方法能够实现野毒和疫苗毒的鉴别。这表明rLa-mGM株可作为疫苗候选株实现新城疫净化。
同时，本发明的疫苗株为毒力极度致弱疫苗株，其虽然HI抗体不高，但保护效果良好。
序列表
<110> 广东省农业科学院动物卫生研究所
浙江迪福润丝生物科技有限公司
华南农业大学
广东永顺生物制药股份有限公司
<120> 新城疫疫苗株及其构建方法、禽类免疫识别方法、应用
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<170> SIPOSequenceListing 1.0
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<210> 10
<211> 48
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 10
tgcggcgtca aatactgcag atacggggtt aagcctcgct tgtgcagc 48
<210> 11
<211> 1470
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 11
atgtcgtctg ttttcgacga atacgagcag ctcctcgctg ctcagacccg ccctaacgga 60
gctcatggag ggggagagaa agggagcact ttaaaagttg aggtcccagt atttacccta 120
aacagtgatg atccagagga tagatggaat tttgcggtat tctgtcttcg gattgctgtt 180
agcgaggatg ccaacaaacc actcaggcaa ggtgctctta tatccctctt atgctcccat 240
tctcaggtga tgagaaacca tgttgccctt gcagggaaac agaatgaggc cacactggct 300
gttcttgaga tcgatggttt tgctaacagt gtgccccagt tcaacaatag gagtggagtg 360
tccgaggaga gagcacagag attcatggta atcgcaggat ccctccctcg ggcatgcagc 420
aacggtactc cgtttgtcac agctggggtt gaagacgatg caccagaaga tatcactgac 480
actctggaaa gaatcctatc tatccaagtc caggtatggg tcacggtagc aaaggccatg 540
actgcatatg agacagcaga tgagtcagaa acaagaagaa taaataagta tatgcagcaa 600
ggtagagttc agaagaagta catccttcat cctgtatgca ggagtgcaat tcaactcaca 660
atcagacatt ctctggcagt ccgtattttc ctagttagtg agctcaagag gggccgtaat 720
acagcaggtg ggagctctac atattacaac ttggtcgggg atgtagactc atacatcaga 780
aacaccgggc ttactgcatt tttcctaacg ctcaaatatg gaatcaatac caagacgtca 840
gccctcgcac tcagcagcct cacaggtgat atccaaaaaa tgaaacagct catgcgttta 900
tatcggatga aaggtgaaaa tgcaccatac atgacattgt taggtgacag tgaccagatg 960
agctttgcac cagccgaata tgcacaactt tattcttttg ccatgggcat ggcatcggtc 1020
ttagataagg ggactggcaa gtaccaattc gccagggact ttatgagcac atcattctgg 1080
agacttggag tagagtatgc tcaggctcag ggaagtagca tcaatgagga catggctgct 1140
gagttaaaac taaccccggc agcaaggaga ggcctggcag ctgccgccca acgagtatct 1200
gaagaagtcg gcagcatgga cattcctact caacaagcgg gagtcctcac cgggctcagt 1260
gacgagggcc cccgaacttc acagggcgga tcaaacaagc cgcaagggca accagatgcc 1320
ggggatgggg agacccaatt cttggatttt atgagagcag tggcgaacag catgcgggaa 1380
gcgccaaatc ctgcacagag caccacccat ccagagcccc ccccaacccc tggggcgtcc 1440
caagacaacg acactgactg ggggtactga 1470
<210> 12
<211> 1188
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 12
atggccacct ttacagatgc ggagatagat gacatatttg agaccagtgg gactgtcatt 60
gacagcataa ttacggccca gggcaaatca gttgagaccg tcggaagaag cgcgatcccg 120
cagggcaaga ccaaagctct aagcatagca tgggagaagc acgggagtgt ccagccacac 180
gccagtcagg acgcccctga ccaacaagac agaacagaaa aacagccatc cacacctgag 240
caggcgactc cacacaacaa cccgccgatc acatccactg aaccgcctcc cactcaggcc 300
ccaagcgaga ccagtgacac acagctcaag accggagcaa gcaactccct tctgtccatg 360
ctcgacaaat tgagcaataa atcgtctaat gctaaaaagg gcccatggtc gggtccccaa 420
gaagggcatc accaacctcc ggcccaacaa cacgggaacc aaccgagcta tggaagcagc 480
cagggaagac cgcagcacca ggccaaggcc gtccctggaa accggggcat agacgagaac 540
acagcatatc atggacaatg gaaggagtca caaccatcag ctggtgcaac ccctcatgcg 600
ccccagtcag ggcagagcca agacaatact cctgtacctg tggatcgtgt ccagctacct 660
gccgactttg cgcaggcgat gatgtctatg atggaggcat tatcacagaa ggtaagtaaa 720
gttgatcatc agctggacct agtcttgaaa cagacatcct ctattcctat gatgcgatct 780
gaaatccaac agctcaagac atctgttgcg atcatggaag ctaacttagg catgatgaaa 840
attctggacc ctggttgtgc caacgtttca tccttaagtg atctccgggc agtagcccga 900
tcccacccag tcctagtttc aggccccgga gacccatctc cttacgtgac acaagggggt 960
gaaatgacgc tcaataaact ctcacaaccg gtgcagcacc cctctgaatt gattaagcct 1020
gccactgcaa gcgggcctga catgggagtg gagaaggaca ctgtccgcgc attaatcacc 1080
tcacgcccga tgcatccaag ctcctcggct aagctcctga gcgagctaga tgcagccagg 1140
tcaattgaag agatcaggaa gatcaaacgc cttgcgctga atggttga 1188
<210> 13
<211> 6615
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 13
atggcgggct ccggtcccga aagggcagag caccagatca tcctaccaga gtcacatcta 60
tcctccccat tggtcaagca caaattgcta tactactgga aattgactgg gctaccgctt 120
cctgatgaat gcgactttga tcatctcatt atcagcaggc aatggaagaa aatactggag 180
tcggccactc ctgacacaga gagaatgata aaactcgggc gggcagtgca ccagactctc 240
aaccacaatt ccaagataac cggagtgctc catcccaggt gtttagaaga actggctagt 300
attgaggtcc ctgattcaac taacaatttc cggaagattg aaaagaagat ccagattcac 360
aacacaaggt atggagacct gttcacaaag ctgtgcacgc aagttgagaa taaattgcta 420
ggatcatctc ggtctaataa tgtcccacga tcagaggaat tcagtagcat ccgtacagat 480
ccggcattct ggtttcactc aaaatggtcc agagccaagt tcgcgtggct ccatataaaa 540
caagtccaaa ggcatctgat tgtagcagca aggacaaggt ctgcagtcaa caagttagta 600
acattaagtc ataagatagg ccacgtcttt gttactcctg agcttgtcat tgtgacacat 660
acagatgaga acaaattcac atgcctcacc caggaacttg tattgatgta tgcggatatg 720
atggaaggca gggacatggt caatataata tcttctacag cagcacatct cagaaaccta 780
tccgagaaaa ttgatgatat tctgcggtta gtagatgctc tggcaaagga cttaggtaat 840
caagtctatg atgttgtagc attaatggag ggatttgcat acggtgccgt tcagctgctt 900
gagccatcag gtacatttgc aggagatttc tttgcattta acctacagga gctcaaagac 960
acgttaatcg aacttctccc aaataatata gcggaatcag taactcacgc tattgccact 1020
gtattctccg gcttagaaca gaatcaagca gctgagatgt tgtgcttact gcgtttgtgg 1080
ggccacccat tgcttgagtc tcgtagtgca gcaagagcag tcaggagcca gatgtgcgca 1140
ccaaagatgg tagacttcga tatgatcctc caggtattat ctttcttcaa aggaacaatc 1200
atcaatggat acagaaagaa gaactcaggt gtgtggtcgc gtgtcaaagt agatacaata 1260
tacggaaata tcattgggca gctacatgct gattcagcag agatctcaca tgatgtcatg 1320
ttgagggagt acaagagttt atctgctctt gaatttgagc catgtataga ttatgaccct 1380
gttaccaatc taagcatgtt cctaaaagac aaggcaatcg cacatcctag tgataactgg 1440
ctcgcctcat ttaggcggaa cctactctct gaggaccaga agaaacagat aaaagaggca 1500
acttcaacta accgcctcct gatagagttc ttagaatcaa atgattttga tccatataaa 1560
gaaatggaat acctgacaac cctcgagtac ctaagagatg acagtgtggc agtatcgtac 1620
tcactcaaag agaaaggggt gaaagtgaat gggcggattt ttgctaaatt aacaaagaaa 1680
ctaaggaact gccaggtaat ggcagaagga attctagctg accagattgc acctttcttt 1740
cagggaaatg gggtcattca agatagcata tccttgacaa agagtatgtt agcgatgagt 1800
caactgtcct ttaacagcaa taagaaacgt atcactggct gcaaagagag ggtttcctca 1860
gaccgcaatc atgatcctaa gagcaagaat cgtagaagag ttgccacttt tatcacgact 1920
gacctacaaa agtattgtct taactggaga tatcagacag tcaaactatt cgcccatgcc 1980
atcaatcagc tgatgggcct acctcatttc tttgagtgga ttcatcttag gctgatggac 2040
actacaatgt ttgtagggga tcctttcaat cctccaagtg acccgactga ctgtgatcta 2100
tcaagagtcc cgaatgatga tatatatatt gtcagtgcta gagggggcat tgagggactc 2160
tgccagaagc tatggacgat gatctcgatt gctgcaatcc aacttgctgc agcaagatct 2220
cattgtcgag ttgcctgcat ggtacaaggt gacaaccaag taatagctgt aacgagagag 2280
gtgagatcag acgattcccc ggatatggtg ttgacgcagt tgcatcaagc tagtgataat 2340
ttcttcaagg aattgattca tgtcaatcat ctgattggcc ataacctgaa ggatcgtgaa 2400
accattagat cagacacatt cttcatatac agcaaacgaa tattcaaaga tggaacaata 2460
ctcagtcagg tcctcaaaaa ttcatctaaa ttggtgctaa tatcaggcga ccttagcgag 2520
aacactgtaa tgtcttgtgc caacattgca tccactgtgg cacgactatg tgagaatggg 2580
cttcctaagg atttctgtta ctatttgaac tacctaatga gttgcgtgca gacatacttt 2640
gattcggagt tttctattac ccacagctcg caatcagatt ccaaccagtc ctggatcgag 2700
gatatctctt tcgtacactc atacgtgtta acccctgccc agctgggggg actgagcaac 2760
cttcaatact caaggctcta cacaaggaat attggtgacc cagggaccac tgcttttgca 2820
gaggtcaagc gactagaagc agtggggttg ctgagtccca gcatcatgac taacatctta 2880
accaggccac ctggcaatgg agactgggcc agcctatgca atgacccata ctcttttaat 2940
tttgagactg ttgcaagccc aaatattgtc ctcaagaaac atacacagaa agtcctattt 3000
gagacatgtt caaacccttt attatccggg gtacatacag aggacaatga ggcagaagag 3060
aaagcattgg ctgaattctt actcaatcaa gaagtgattc acccacgtgt cgcacatgct 3120
atcatggaag caagctctgt gggtaggaga aagcaaattc aagggcttgt tgacacaacg 3180
aacactgtga ttaagattgc actgactagg aggcccctcg gtatcaaaag actgatgcgg 3240
ataatcaatt actcgagcat gcatgcaatg ttgttcagag atgatatttt cttatccaat 3300
agatccaacc acccattagt ttcttctaat atgtgctcgc tgacgctagc agattatgcc 3360
cggaacagaa gctggtcacc cctgacaggg ggcaggaaaa tactgggtgt atccaacccc 3420
gataccatag aacttgtgga gggagagatt ctcagcgtca gtggagggtg cacaaaatgt 3480
gacagcggag atgagcagtt tacttggttc catcttccaa gcaatataga gctgactgat 3540
gacaccagca agaatccccc gatgagagtg ccatatctcg ggtcgaagac tcaagagagg 3600
agagccgcct cgcttgcgaa aatagcccac atgtcaccac atgtgaaagc agcactaagg 3660
gcatcatccg tgttaatctg ggcttatggg gacaatgaag tgaactggac tgctgctctt 3720
aatattgcaa ggtctcgatg caacataagc tcagagtatc ttcggctatt gtcacccctg 3780
cccacagctg ggaatctcca acatagattg gatgatggca taacccagat gacatttacc 3840
cctgcatctc tctacagagt gtcgccttac gttcacatat ccaacgattc tcaaaggcta 3900
ttcaccgaag aaggggtcaa agagggaaac gtggtttacc aacaaattat gctcttgggt 3960
ttatctctaa ttgaatcact cttcccaatg acaacaacca gaacatatga tgagatcaca 4020
ttacacctcc acagtaaatt tagctgctgt atccgagaag cgcctgttgc ggttcctttc 4080
gagctcttcg ggctggcacc ggaattaagg atggtaacct caaataagtt catgtatgat 4140
cccagtccta tatcagagag agattttgcg agacttgact tagctatctt caagagttat 4200
gagcttaatt tggaatcata ttccacgctg gagctaatga acattctttc aatatctagc 4260
gggaaattga ttggccaatc cgtggtttct tatgatgaag atacttctat aaagaatgat 4320
gctataatag tgtatgacaa cacacgaaat tggattagtg aggcgcagaa ctcagatgtg 4380
gtccgcctgt ttgagtatgc agcactcgaa gtgctccttg actgtgctta tcaactctac 4440
tatctgaggg taaggggtct aagcaacatt gtcctataca tgaatgactt atataagaac 4500
atgccaggga tcctactctc taatattgcg gccacgatat cccaccccct cattcactca 4560
aggttgaatg cagtaggtct aattaaccat gacgggtcac accagcttgc agatatagac 4620
ttcgtcgagg tgtctgcgaa attgttagtc tcttgcactc gacgcgtggt ctcaggctta 4680
tatgcaggga ataagtacga tctgctgttc ccatctgtct tagatgataa cctgaatgag 4740
aagatgcttc aactgatttc ccggttatgc tgtctgtaca cagtgctctt tgctacaaca 4800
agagaaatcc caaaaataag gggcctatcg gcagaagaga aatgctcaat actcactgag 4860
tatctactgt cagatgctgt aaaaccgttg cttaggcccg aacaagtgag ttctatcatg 4920
tctcccaaca taatcacgtt cccagccaat ctatattaca tgtctaggaa gagccttaat 4980
ttgatcagag aacgagagga cagagatact atcttgtcgt tgttgttccc tcaggaacca 5040
ctgcttgagc ttcgcccagt acgagacatt ggtgctcgag tgaaagaccc gtttacccga 5100
caacccgcat cattcataca agagctagat ctgagtgccc cagcaaggta cgacgcattt 5160
acactgagta aggtttgctt cgagcacaca ttaccgaacc caaaggaaga ttacctagta 5220
cggtacttgt tcagaggaat agggactgct ccatcttctt ggtataaggc atctcatctt 5280
ctatccgtac ctgaggtcag gtgtgcaaga catgggaact ccttatactt agcagaagga 5340
agcggagcca tcatgagtct tcttgaattg catataccac atgagactat ctattacaat 5400
acacttttct cgaatgagat gaaccctcca cagcgacatt tcggacctac accaacacag 5460
tttctgaact cggtcgttta taggaatcta caagcggaag tgccgtgtaa agatggatat 5520
gtccaggagt tctgcccatt atggagagag aatgcagaag aaagtgacct gacctcagat 5580
aaggcagttg gatatatcac atctgtggta ccctacaggt ctgtatcatt actacattgt 5640
gacattgaga ttcctccagg gtccagtcaa agcttattag atcaactggc tactaattta 5700
tccctgattg ccatgcattc tgtgagagag ggcggggtag tgatcatcaa agtactgtat 5760
gcaatggggt actacttcca tttactcatg aatttattca ctccatgttc cacgaaagga 5820
tacatactct ccaatggcta tgcctgtaga ggggatatgg agtgttacct gatattcgtt 5880
atgggctact taggcgggcc caccttcgtg cacgaagtgg taaggatggc aaaaactcta 5940
atacaacgac acggtacact tctatctaaa tcagatgaaa tcacattgac taagctattt 6000
acctcacagc agcgtcgtgt aacagatctc ctatccagcc ctttaccgaa gctaatgaag 6060
ctcttaagtg aaaatattga tgctgcacta attgaagccg ggggacagcc cgtccgtcca 6120
ttctgtgcag aaagtttggt gagcacacta acagatatga cccagacaac tcagatcatt 6180
gccagccaca ttgacacagt cattcggtct gtgatttaca tggaggctga gggtgacctc 6240
gccgacacag tgttcttatt tactccttac aatctatcca cagacggtaa aaagagaaca 6300
tcacttaagc agggcaccaa acagatcttg gaagtcacaa tactgggtct cagagccaaa 6360
gatatcaata aagtaggtga tgtaatcagc ttagtactca gaggtgcggt ttccttagag 6420
gatctcatcc cattaaggac atacctgaag cgcagtacct gccctaaata cctgaaagcg 6480
gtcctaggta ttactaaact caaagaaatg ttcacagata cctcgttact gtacttgact 6540
cgtgctcaac aaaaattcta catgaaaacc ataggtaatg ctgccaaggg atattacagt 6600
aataatgact cttaa 6615
<210> 14
<211> 18321
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 14
agctcggatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct 60
gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg 120
aaaggaggaa ctatatccgg atcgagatcc tctagagtcg acctgcaggc atgcaagctt 180
gtattctata gtgtcaccta aatcgtatgt gtatgataca taaggttatg tattaattgt 240
agccgcgttc taacgacaat atgtacaagc ctaattgtgt agcatctggc ttactgaagc 300
agaccctatc atctctctcg taaactgccg tcagagtcgg tttggttgga cgaaccttct 360
gagtttctgg taacgccgtt ccgcaccccg gaaatggtca gcgaaccaat cagcagggtc 420
atcgctagcc agatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt 480
gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc 540
gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg 600
ttgggcgcca tctccttgca ccattccttg cggcggcggt gctcaacggc ctcaacctac 660
tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgatat ggtgcactct 720
cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc 780
tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 840
ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa 900
gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac 960
gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 1020
acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 1080
aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 1140
attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 1200
tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 1260
gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 1320
cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 1380
tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 1440
agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 1500
tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 1560
tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 1620
tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 1680
acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 1740
accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 1800
tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 1860
cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 1920
tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 1980
actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 2040
tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 2100
cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 2160
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 2220
tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 2280
gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 2340
gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 2400
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 2460
acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 2520
agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 2580
cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 2640
tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 2700
gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 2760
ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 2820
ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 2880
cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 2940
ttaatgcagg gggatctcga tcccgcgaaa ttaatacgac tcactatatg ttaagcgtct 3000
gatgagtccg tgaggacgaa actataggaa aggaattcct atagtcacca aacagagaat 3060
ctgtgaggta cgataaaagg cgaggaagca atcgagatcg tacgggtaga aggtgtgaac 3120
cccgagcgcg aggccgaagc ttgaacccga gggaaccttc taccgatatg tcgtctgttt 3180
tcgacgaata cgagcagctc ctcgctgctc agacccgccc taacggagct catggagggg 3240
gagagaaagg gagcacttta aaagttgagg tcccagtatt taccctaaac agtgatgatc 3300
cagaggatag atggaatttt gcggtattct gtcttcggat tgctgttagc gaggatgcca 3360
acaaaccact caggcaaggt gctcttatat ccctcttatg ctcccattct caggtgatga 3420
gaaaccatgt tgcccttgca gggaaacaga atgaggccac actggctgtt cttgagatcg 3480
atggttttgc taacagtgtg ccccagttca acaataggag tggagtgtcc gaggagagag 3540
cacagagatt catggtaatc gcaggatccc tccctcgggc atgcagcaac ggtactccgt 3600
ttgtcacagc tggggttgaa gacgatgcac cagaagatat cactgacact ctggaaagaa 3660
tcctatctat ccaagtccag gtatgggtca cggtagcaaa ggccatgact gcatatgaga 3720
cagcagatga gtcagaaaca agaagaataa ataagtatat gcagcaaggt agagttcaga 3780
agaagtacat ccttcatcct gtatgcagga gtgcaattca actcacaatc agacattctc 3840
tggcagtccg tattttccta gttagtgagc tcaagagggg ccgtaataca gcaggtggga 3900
gctctacata ttacaacttg gtcggggatg tagactcata catcagaaac accgggctta 3960
ctgcattttt cctaacactc aaatatggaa tcaataccaa gacgtcagcc ctcgcactca 4020
gcagcctcac aggtgatatc caaaaaatga aacagctcat gcgtttatat cggatgaaag 4080
gtgaaaatgc accatacatg acattgttag gtgacagtga ccagatgagc tttgcaccag 4140
ccgaatatgc acaactttat tcttttgcca tgggcatggc atcggtctta gataagggga 4200
ctggcaagta ccaattcgcc agggacttta tgagcacatc attctggaga cttggagtag 4260
agtatgctca ggctcaggga agtagcatca atgaggacat ggctgctgag ttaaaactaa 4320
ccccggcagc aaggagaggc ctggcagctg ctgcccaacg agtatctgaa gaagtcggca 4380
gcatggacat tcctactcaa caagcgggag tcctcaccgg gctcagtgac gagggccccc 4440
gaacttcaca gggcggatca aacaagccgc aagggcaacc agatgccggg gatggggaga 4500
cccaattctt ggattttatg agagcagtgg cgaacagcat gcgggaagcg ccaaatcctg 4560
cacagagcac cacccatcca gagccccccc caacccctgg ggcgtcccaa gacaacgaca 4620
ctgactgggg gtactgatcg actacaccca gcctgccttc acaggatcac atcaaaccct 4680
ccgcccaaaa ccctcccaca ctccctgacc cacaaccctg cacgacccca cctacaaaag 4740
atccccccca ccctctcccc cactcccagc cgcacgatcc cacctacccg ggacagcaca 4800
ggcacagctc ggttagtcaa caatccgccc agagtccaag gtattagaaa aaaatacggg 4860
tagaagagag acatccagag accaggacgg gtcaccaagt tctctgttct cccttctacc 4920
tagtgaatta gggtgaagat ggccaccttt acagatgcgg agatagatga catatttgag 4980
accagtggga ctgtcattga cagcataatt acggcccagg gcaaatcagt tgagaccgtc 5040
ggaagaagcg cgatcccgca gggcaagacc aaagctctaa gcatagcatg ggagaagcac 5100
gggagtgtcc agccacacgc cagtcaggac gcccctgacc aacaagacag aacagaaaaa 5160
cagccatcca cacctgagca ggcgactcca cacaacaacc cgccgatcac atccactgaa 5220
ccgcctccca ctcaggccgc aagcgagacc agtgacacac agctcaagac cggagcaagc 5280
aactcccttc tgtccatgct cgacaaattg agcaataaat cgtctaatgc taaaaagggc 5340
ccatggtcgg gtccccaaga agggcatcac caacctccgg cccaacaaca cgggaaccaa 5400
ccgagctatg gaagcagcca gggaagaccg cagcaccagg ccaaggccgt ccctggaaac 5460
cggggcatag acgagaacac agcatatcat ggacaatgga aggagtcaca accatcagct 5520
ggtgcaaccc ctcatgcgcc ccagtcaggg cagagccaag acaatactcc tgtacctgtg 5580
gatcgtgtcc agctacctgc cgactttgcg caggcgatga tgtctatgat ggaggcatta 5640
tcacagaagg taagtaaagt tgatcatcag ctggacctag tcttgaaaca gacatcctct 5700
attcctatga tgcgatctga aatccaacag ctcaagacat ctgttgcgat catggaagct 5760
aacttaggca tgatgaaaat tctggaccct ggttgtgcca acgtttcatc cttaagtgat 5820
ctccgggcag tagcccgatc ccacccagtc ctagtttcag gccccggaga cccatctcct 5880
tacgtgacac aagggggtga aatgacgctc aataaactct cacaaccggt gcagcacccc 5940
tctgaattga ttaagcctgc cactgcaagc gggcctgaca tgggagtgga gaaggacact 6000
gtccgcgcat taatcacctc acgcccgatg catccaagct cctcggctaa gctcctgagc 6060
aagctagatg cagccaggtc aattgaagag atcaggaaga tcaaacgcct tgcgctgaat 6120
ggttgatggc catcacaact cataacaggc tcctgtcact tcagcgtcac acggaatccc 6180
ttgggggccc ccccttgcaa atccacgctt caacacccca tacaacagcc ctctctcacc 6240
ccccccaatc ccccgaatga tcgcacaact gcaaccaatc cagcagcatt agaaattaag 6300
aaaaaatacg ggtagaatca aagtgcctcg attgcaccaa aatggactca tccaggacaa 6360
tcgggctgta ctttgattct gccctccctt ccagcagcct gttagcattt ccgattgtct 6420
tacaagacac aggagacggg aagaagcaaa tcaccccaca atacaggatc cagcgtcttg 6480
attcgtggac agacagtaag gaagactcgg tattcatcac cacctacggg ttcatctttc 6540
aagttgggaa tgaagaagcc accgtcggtg tgatcaatga caatcccagg cacgagctac 6600
tatcttccgc aatgctctgc ttagggagtg tcccgaacga tggagatctt gttgagctgg 6660
cgagagcctg cctcaccatg gtggtcacct gcaagaagag tgcaactaac actgagagaa 6720
tagtcttctc agtagtgcag gcacctcggg tgctgcaaag ctgtatggtt gtgtcaaata 6780
ggtactcatc agtgaatgca gtgaagcatg tgaaggcgcc cgaaaagatc cctgggagcg 6840
gaaccctaga gtataaagtg aattttgtct ctttgactgt ggtgccgaga aaggatgtct 6900
acaggatccc aactgcagta ttgaaagtgt ctggctcaag cctgtacaat cttgcgctca 6960
atgtcactat tgatgtggac gtggatccga agagcccgtt agtcaaatcc ctttctaagt 7020
ccgatagcgg atactatgcg aatctttttc tgcatatcgg gcttatgtcc actgtagata 7080
agaagggaaa gaaagtgaca tttgacaaga tagaggaaaa gataaggaga ctcaatctat 7140
ctgttgggct cagtgatgtg ctcggaccct ctgtgcttgt gaaggcgaga ggtgcacgga 7200
ctaagctact tgctcctttt ttctctagca gtgggacagc ctgctaccct atagcaaatg 7260
cctctcccca ggttgccaag atactctgga gccagaccgc gcacctgcgg agcgtgaaag 7320
tcatcattca agccggcact cagcgtgctg tcgcagtgac cgccgatcat gaggtaacct 7380
ccactaagat agagaggagg cacgccattg ctaaatacaa tcctttcagg aaataagttg 7440
catccctaag actgcagttc atctgctttc ctgaatcacc atgacaccag ataatgatcc 7500
atctcgaccg cttatagtta gttcacctgt ctagcaaatt agaaaaaaca cgggtagaag 7560
agtctggatc ccgaccggca cattcaggtc acaatatggg cttcaaacct tctaccagga 7620
tcccagcacc tctaatgctg atcactcgga ttatgctgat attgagctgt atccgtctga 7680
caagctctct tgacggcagg ccccttgcag ctgcaggaat tgtagtaaca ggagataagg 7740
cagtcaatgt atacacctcg tctcagacag ggtcaatcat agtcaagttg ctcccgaata 7800
tgcccagaga taaggaggca tgtgcaaaag ccccattaga ggcatataac agaacactga 7860
ctactctgct cactcctctt ggcgactcca tccgcaagat ccaagggtct gtgtccatgt 7920
ctggaggaag gagacaaaaa cgctttatag gtgctgttat tggcagtgta gctcttgggg 7980
ttgcaacagc ggcccagata acagcagctg cggccctaat acaagccaaa cagaatgccg 8040
ccaacatcct ccggcttaag gagagcattg ccgcaaccaa tgaagctgtg catgaagtca 8100
ccgacggatt atcacaacta tcagtggcag ttgggaagat gcagcagttt gtcaatgacc 8160
agtttaataa tacggcgcga gaattggact gtataaaaat cacacaacag gtcggtgtag 8220
aactcaacct atacctaact gaattgacta cagtattcgg gccacagatc acctcccctg 8280
cattaactca gctgaccatc caggcacttt ataatttagc tggtggcaat atggattact 8340
tattaactaa gttaggtata ggaaacaatc aactcagctc attaattggt agcggcctga 8400
tcactggtta tcctatactg tatgactcac atactcaact cttgggcata caagtaaacc 8460
tgccctcagt cgggaactta aataatatgc gtgccaccta tttggagacc ttatctgtaa 8520
gtacaaccaa aggatatgcc tcagcacttg tcccgaaagt agtgacacaa gtcggttctg 8580
tgatagaaga gcttgacacc tcatactgta tagagtccga tctggattta tattgtacta 8640
gaatagtgac attccccatg tccccaggta tttattcctg tttgagcggc aacacatcag 8700
cttgcatgta ttcaaagact gaaggtgcac tcactgcgcc atatatggcc cttaaaggct 8760
cagttattgc caattgtaag ataacaacat gtagatgtac agaccctcct ggtatcatat 8820
cgcaaaatta tggagaagct gtatccctga tagatagaca ttcatgcaat gtcttatcat 8880
tagacggaat aactctgagg ctcaatgggg aatttgatgc aacttatcaa aagaacatct 8940
caatattaga ttctcaagtc atcgtgacag gcaatcttga tatatcaact gaacttggaa 9000
acgtcaacaa ttcaatcagc aatgccttgg ataggttggc agaaagcaac agcaagctag 9060
aaaaagtcga tgtcagacta actagcacat ctgctctcat tacctatatt gttctaactg 9120
tcatttctct aattttcggt gcacttagtc tggttttagc gtgttacctg atgtacaaac 9180
agaaggcaca acaaaagacc ttgctatggc ttgggaataa taccctcgat cagatgagag 9240
ccactacaag agcatgaatg cagataagag gtggatatat acccgacagc agcctgtgtg 9300
ccaattccga taacctgtca agtagaagac ttaagaaaaa attactggga acaagcaact 9360
aaagaacaat acacgggtag aacggtcaga ggagccaccc ttcaatcgag aattaggctt 9420
cacaacatcc gttctaccac atcaccagca acaagagtca atcatggacc gcgcggttaa 9480
cagagtcgtg ctggagaatg aggaaagaga agcaaagaac acatggcgcc tggttttccg 9540
ggtcgtagtc ttacttttaa tggtaatgac tctagctatc tccgcagctg ccctggcata 9600
cagcacgggg gccagtacgc cgcacgacct cgcaggcata tcgactgtga tctccaagac 9660
agaagataag attacgtctt tactcagttc aagtcaagat gtgatagata ggatatacaa 9720
gcaggtggct cttgaatccc cgctggcgct actaaacact gaatctataa ttatgaatgc 9780
aataacctct ctttcttatc aaattaacgg ggctgagaac aatagcgaat gtggcgcgcc 9840
tgttcatgac ccagattata tcggggggat aggcaaagaa ctcatagtgg acgacatcag 9900
tgatgtcaca tcattttatc cttctgcata tcaagaacac ttgaatttca tcccggcgcc 9960
tactacagga tcaggttgca ctcggatacc ctcatttgac atgagcacca cccattattg 10020
ttatactcac aatgtgatac tatccggttg cagagatcac tcacactcac atcaatactt 10080
agcacttggt gtgcttcgga catctgcaac agggagggta ttcttttcta ctctgcgctc 10140
catcaattta gatgacaccc aaaatcggaa gtcctgcagt gtgagtgcaa cccctctagg 10200
ttgtgatatg ctgtgctcta aggtcacagg gactgaagag gaggattaca agtcagttgc 10260
ccccacatca atggtgcacg gaaggctagg gtttgacggt caataccatg agaaggactt 10320
agacaccacg gtcttattta aggattgggt ggcaaattac ccgggagtgg gaggagggtc 10380
ttttattggc gaccgtgtat ggttcccagt ttacggaggg ctcaaaccca attcacccag 10440
tgacactgca caagaaggga aatatgtaat atacaagcgc cataacaaca catgccccga 10500
tgaacaagat taccaaattc ggatggctaa gtcttcatat aaacccgggc gatttggtgg 10560
aaagcgcata cagcaagcca tcctatccat caaagtgtca acatccctgg gtaaggaccc 10620
agtgctgact attccaccta atacaatcac actcatggga gccgaaggca gaatcctcac 10680
agtagggaca tctcatttct tgtaccaacg agggtcttca tatttctccc ctgccttatt 10740
atatcccatg acagtaaata acaaaacggc tacactccat agtccttaca cgtttaatgc 10800
tttcactcga ccaggtagtg tcccttgcca ggcatcagca agatgcccca actcatgcat 10860
cactggggtc tataccgatc catatccctt aatcttccat aggaatcata ctctacgagg 10920
ggtcttcggg acgatgcttg atgatgaaca agcgaggctt aaccccgtat ctgcagtatt 10980
tgacaacata tctcgcagtc gtgtcacccg ggtgagttca agcagcacca aggcagcata 11040
cacgacatcg acatgtttta aagttgtcaa gaccaataaa gcttattgtc ttagtatcgc 11100
agaaatatcc aataccctat tcggggaatt taggatcgtt cccttactag ttgagatcct 11160
caaggatgat agagtttaag aagctagact tagccgattg agccaatcat aggatggttg 11220
ggaagacgac accgcgccaa tcatctccca taatgcttag agtcaagctg aatattaaca 11280
taagccagga tcccatgttg ttgggcaacc acaatccgac aatgctaaca tgattattct 11340
gagtcccgcc cactgtcatt ttattaagaa aaaaaacaag aagcattgag atataaggga 11400
aaacaaccaa caagagagaa cacgggtagg acatggcggg ctccggtccc gaaagggcag 11460
agcaccagat catcctacca gagtcacatc tatcctcccc attggtcaag cacaaattgc 11520
tatactactg gaaattgact gggctaccgc ttcctgatga atgcgacttt gatcatctca 11580
ttatcagcag gcaatggaag aaaatactgg agtcggccac tcctgacaca gagagaatga 11640
taaaactcgg gcgggcagtg caccagactc tcaaccacaa ttccaagata accggagtgc 11700
tccatcccag gtgtttagaa gaactggcta gtattgaggt ccctgattca actaacaaat 11760
tccggaagat tgaaaagaag atccagattc acaacacaag gtatggagac ctgttcacaa 11820
agctgtgcac gcaagttgag aataaattgc taggatcatc tcggtctaat aatgtcccac 11880
gatcagagga attcagtagc atccgtacag atccggcatt ctggtttcac tcaaaatggt 11940
ccagagccaa gttcgcgtgg ctccatataa aacaagtcca aaggcatctg attgtagcag 12000
caaggacaag gtctgcagtc aacaagttag taacattaag tcataagata ggccacgtct 12060
ttgttactcc tgagcttgtc attgtgacac atacagatga gaacaaattc acatgcctca 12120
cccaggaact tgtattgatg tatgcggata tgatggaagg cagggacatg gtcaatataa 12180
tatcttctac agcagcacat ctcagaaacc tatccgagaa aattgatgat attctgcggt 12240
tagtagatgc tctggcaaag gacttaggta atcaagtcta tgatgttgta gcattaatgg 12300
agggatttgc atacggtgcc gttcagctgc ttgagccatc aggtacattt gcaggagatt 12360
tctttgcatt taacctacag gagctcaaag acacgttaat cgaacttctc ccaaataata 12420
tagcggaatc agtaactcac gctattgcca ctgtattctc cggcttagaa cagaatcaag 12480
cagctgagat gttgtgctta ctgcgtttgt ggggccaccc attgcttgag tctcgtagtg 12540
cagcaagagc agtcaggagc cagatgtgcg caccaaagat ggtagacttc gatatgatcc 12600
tccaggtatt atctttcttc aaaggaacaa tcatcaatgg atacagaaag aagaactcag 12660
gtgtgtggcc gcgtgtcaaa gtagatacaa tatacggaaa tatcattggg cagctacatg 12720
ctgattcagc agagatctca catgatgtca tgttgaggga gtacaagagt ttatctgctc 12780
ttgaatttga gccatgtata gattatgacc ctgttaccaa tctaagcatg ttcctaaaag 12840
acaaggcaat cgcacatcct agtgataact ggctcgcctc atttaggcgg aacctactct 12900
ctgaggacca gaagaaacag ataaaagagg caacttcaac taaccgcctc ctgatagagt 12960
tcttagaatc aaatgatttt gatccatata aagaaatgga atacctgaca accctcgagt 13020
acctaagaga tgacagtgtg gcagtatcgt actcactcaa agagaaagag gtgaaagtga 13080
atgggcggat ttttgctaaa ttaacaaaga aactaaggaa ctgccaggta atggcagaag 13140
gaattctagc tgaccagatt gcacctttct ttcagggaaa tggggtcatt caagatagca 13200
tatccttgac aaagagtatg ttagcgatga gtcaactgtc ctttaacagc aataagaaac 13260
gtatcactga ctgcaaagag agggtttcct caaaccgcaa tcatgatcct aagagcaaga 13320
atcgtagaag agttgccact tttatcacga ctgacctaca aaagtattgt cttaactgga 13380
gatatcagac agtcaaacta ttcgcccatg ccatcaatca gctgatgggc ctacctcatt 13440
tctttgagtg gattcatctt aggctgatgg acactacaat gtttgtaggg gatcctttca 13500
atcctccaag tgacccgact gactgtgatc tatcaagagt cccgaatgat gatatatata 13560
ttgtcagtgc tagagggggc attgagggac tctgccagaa gctatggacg atgatctcga 13620
ttgctgcaat ccaacttgct gcagcaagat ctcattgtcg agttgcctgc atggtacaag 13680
gtgacaacca agtaatagct gtaacgagag aggtgagatc agacgattcc ccggatatgg 13740
tgttgacgca gttgcatcaa gctagtgata atttcttcaa ggaattgatt catgtcaatc 13800
atctgattgg ccataacctg aaggatcgtg aaaccattag atcagacaca ttcttcatat 13860
acagcaaacg aatattcaaa gatggaacaa tactcagtca ggtcctcaaa aattcatcta 13920
aattggtgct aatatcaggc gaccttagcg agaacactgt aatgtcttgt gccaacattg 13980
catccactgt ggcacgacta tgtgagaatg ggcttcctaa ggatttctgt tactatttga 14040
actacctaat gagttgcgtg cagacatact ttgattcgga gttttctatt actcacagct 14100
cgcaatcaga ttccaaccag tcctggatcg aggatatctc tttcgtacac tcatacgtgt 14160
taacccctgc ccagctgggg ggactgagca accttcaata ctcaaggctc tacacaagga 14220
atattggtga cccagggacc actgcttttg cagaggtcaa gcgactagaa gcagtggggt 14280
tgctgagtcc cagcatcatg actaacatct taaccaggcc acctggcaat ggagactggg 14340
ccagcctatg caatgaccca tactctttta attttgagac tgttgcaagc ccaaatattg 14400
tcctcaagaa acatacacag aaagtcctat ttgagacatg ttcaaaccct ttattatccg 14460
gggtacatac agaggacaat gaggcagaag agaaagcatt ggctgaattc ttactcaatc 14520
aagaagtgat tcacccacgt gtcgcacatg ctatcatgga agcaagctct gtgggtagga 14580
gaaagcaaat tcaagggctt gttgacacaa cgaacactgt gattaagatt gcactgacta 14640
ggaggcccct cggtatcaaa agactgatgc ggataatcaa ttactcgagc atgcatgcaa 14700
tgttgttcag agatgatatt ttcttatcca atagatccaa ccacccatta gtttcttcta 14760
atatgtgctc gctgacgcta gcagattatg cccggaacag aagctggtca cccctgacag 14820
ggggcaggaa aatactgggt gtatccaacc ccgataccat agaacttgtg gagggagaga 14880
ttctcagcgt cagtggaggg tgtacaaaat gtgacagcgg agatgagcag tttacttggt 14940
tccatcttcc aagcaatata gagctgactg atgacaccag caagaatccc ccgatgagag 15000
tgccatatct cgggtcgaag actcaagaga ggagagccgc ctcgcttgcg aaaatagccc 15060
acatgtcacc acatgtgaaa gcagcactaa gggcatcatc cgtgttaatc tgggcttatg 15120
gggacaatga agtgaactgg actgctgctc ttaatattgc aaggtctcga tgcaacataa 15180
gctcagagta tcttcggcta ttgtcacccc tgcccacagc tgggaatctc caacatagat 15240
tggatgatgg cataacccag atgacattta cccctgcatc tctctacaga gtgtcgcctt 15300
acgttcacat atccaacgat tctcaaaggc tattcaccga agaaggggtc aaagagggaa 15360
acgtggttta ccaacaaatt atgctcttgg gtttatctct aattgaatca ctcttcccaa 15420
tgacaacaac cagaacatat gatgagatca cattacacct ccacagtaaa tttagctgct 15480
gtatccgaga agcgcctgtt gcggttcctt tcgagctctt cgggctggca ccggaattaa 15540
ggatggtaac ctcaaataag ttcatgtatg atcccagtcc tatatcagag agagattttg 15600
cgagacttga cttagctatc ttcaagagtt atgagcttaa tttggaatca tattccacgc 15660
tggagctaat gaacattctt tcaatatcta gcgggaaatt gattggccaa tccgtggttt 15720
cttatgatga agatacttct ataaagaatg atgctataat agtgtatgac aacacacgaa 15780
attggattag tgaggcgcag aactcagatg tggtccgcct gtttgagtat gcagcactcg 15840
aagtgctcct tgactgtgct tatcaactct actatctgag ggtaaggggt ctaaacaaca 15900
tcgtcctata catgaatgac ttatataaga acatgccagg gatcctactc tctaatattg 15960
cggccacgat atcccacccc ctcattcact caaggttgaa tgcagtaggt ctaattaacc 16020
atgacgggtc acaccagctt gcagatatag acttcgtcga ggtgtctgcg aaattgttag 16080
tctcttgcac tcgacgcgtg gtctcaggct tatatgcagg gaataagtac gatctgctgt 16140
tcccatctgt cttagatgat aacctgaatg agaagatgct tcaactgatt tcccggttat 16200
gctgtctgta cacagtgctc tttgctacaa caagagaaat cccaaaaata aggggcctat 16260
cggcagaaga gaaatgctca atactcactg agtatctact gtcagatgct gtaaaaccgt 16320
tgcttaggcc cgaacaagtg agttctatca tgtctcccaa cataatcacg ttcccagcca 16380
atctatatta catgtctagg aagagcctta atttgatcag agaacgagag gacagagata 16440
ctatcttgtc gttgttgttc cctcaggaac cactgcttga gcttcgccca gtacgagaca 16500
ttggtgctcg agtgaaagac ccgtttaccc gacaacccgc atcattcata caagagctag 16560
atctgagtgc cccagcaagg tacgacgcat ttacactgag taaggtttgc ttcgagcaca 16620
cattaccgaa cccaaaggaa gattacctag tacggtactt gttcagagga atagggactg 16680
cttcatcttc ttggtataag gcatctcatc ttctatccgt acctgaggtc aggtgtgcaa 16740
gacatgggaa ctccttatac ttagcagaag gaagcggagc catcatgagt cttcttgaat 16800
tgcatatacc acatgagact atctattaca atacactttt ctcgaatgag atgaaccctc 16860
cacagcgaca tttcggacct acaccaacac agtttctgaa ctcggtcgtt tataggaatc 16920
tacaagcgga agtgccgtgt aaagatggat atgtccagga gttctgccca ttatggagag 16980
agaatgcaga agaaagtgac ctgacctcag ataaggcagt tggatatatc acatctgtgg 17040
taccctacag gtctgtatca ttactacatt gtgacattga gattcctcca gggtccagtc 17100
aaagcttatt agatcaactg gccactaatt tatccctgat tgccatgcat tctgtgagag 17160
agggcggggt agtgatcatc aaagtactgt atgcaatggg gtactacttc catttactca 17220
tgaatttatt cactccatgt tccacgaaag gatacatact ctccaatggc tatgcctgta 17280
gaggggatat ggagtgttac ctgatattcg ttatgggcta cttaggcggg cccaccttcg 17340
tgcacgaagt ggtaaggatg gcaaaaactc taatacaacg acacggtaca cttctatcta 17400
aatcagatga aatcacattg actaagctat ttacctcaca gcagcgtcgt gtaacagatc 17460
tcctatccag ccctttaccg aagctaatga agctcttaag tgaaaatatt gatgctgcac 17520
taattgaagc cgggggacag cccgtccgtc cattctgtgc agaaagtttg gtgagcacac 17580
taacagatat gacccagaca actcagatca ttgccagcca cattgacaca gtcattcggt 17640
ctgtgattta catggaggct gagggtgacc tcgccgacac agtgttctta tttactcctt 17700
acaatctatc cacagacggt aaaaagagaa catcacttaa gcagtgcacc aaacagatct 17760
tggaagtcac aatactgggt ctcagagcca aagatatcaa taaagtaggt gatgtaatca 17820
gcttagtact cagaggtgcg gtttccttag aggatctcat cccattaagg acatacctga 17880
agcgcagtac ctgccctaaa tacctgaaag cggtcctagg tattactaaa ctcaaagaaa 17940
tgttcacaga tacctcgtta ctgtacttga ctcgtgctca acaaaaattc tacatgaaaa 18000
ccataggtaa tgctgccaag ggatattaca gtaataatga ctcttaaagg caatcgtaca 18060
ccaatcagtt atcttcttag ctgatgactc cctcactgac ttaattatac cagattagaa 18120
aaaagttaaa ttccgactct ttggaactcg tattcggatt cagttagtta acgttaagca 18180
aaaatgcgca aagtcgtccc taattatagt tatgtcattc accaaatctc tgtttggtgg 18240
gtcggcatgg catctccacc tcctcgcggt ccgacctggg catccgaagg aggacgtcgt 18300
ccactcggat ggctaaggga g 18321
<210> 15
<211> 22250
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 15
gcggccgcaa ggggttcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 60
cggcattaat acgactcact atagggacca aacagagaat ccgtaagtta cgataaaagg 120
cgaaggagca attgaagtcg cacgggtaga aggtgtgaat ctcgagtgcg agcccgaagc 180
acaaactcga gaaagccttc tgccaacatg tcttccgtat ttgatgagta cgaacagctc 240
ctcgcggctc agactcgccc caatggagct catggagggg gagaaaaagg gagtacctta 300
aaagtagacg tcccggtatt cactcttaac agtgatgacc cagaagatag atggagcttt 360
gtggtattct gcctccggat tgctgttagc gaagatgcca acaaaccact caggcaaggt 420
gctctcatat ctcttttatg ctcccactca caggtaatga ggaaccatgt tgcccttgca 480
gggaaacaga atgaagccac attggccgtg cttgagattg atggctttgc caacggcacg 540
ccccagttca acaataggag tggagtgtct gaagagagag cacagagatt tgcgatgata 600
gcaggatctc tccctcgggc atgcagcaac ggaaccccgt tcgtcacagc cggggccgaa 660
gatgatgcac cagaagacat caccgatacc ctggagagga tcctctctat ccaggctcaa 720
gtatgggtca cagtagcaaa agccatgact gcgtatgaga ctgcagatga gtcggaaaca 780
aggcgaatca ataagtatat gcagcaaggc agggtccaaa agaaatacat cctctacccc 840
gtatgcagga gcacaatcca actcacgatc agacagtctc ttgcagtccg catctttttg 900
gttagcgagc tcaagagagg ccgcaacacg gcaggtggta cctctactta ttataacctg 960
gtaggggacg tagactcata catcaggaat accgggctta ctgcattctt cttgacactc 1020
aagtacggaa tcaacaccaa gacatcagcc cttgcactta gtagcctctc aggcgacatc 1080
cagaagatga agcagctcat gcgtttgtat cggatgaaag gagataatgc gccgtacatg 1140
acattacttg gtgatagtga ccagatgagc tttgcgcctg ccgagtatgc acaactttac 1200
tcctttgcca tgggtatggc atcagtccta gataaaggta ctgggaaata ccaatttgcc 1260
agggacttta tgagcacatc attctggaga cttggagtag agtacgctca ggctcaggga 1320
agtagcatta acgaggatat ggctgccgag ctaaagctaa ccccagcagc aaggaggggc 1380
ctggcagctg ctgcccaacg ggtctccgag gagaccagca gcatagacat gcctactcaa 1440
caagtcggag tcctcactgg gcttagcgag ggggggtccc aagctctaca aggcggatcg 1500
aatagatcgc aagggcaacc agaagccggg gatggggaga cccaattcct ggatctgatg 1560
agagcggtag caaatagcat gagggaggcg ccaaactctg cacagggcac tccccaatcg 1620
gggcctcccc caactcctgg gccatcccaa gataacgaca ccgactgggg gtattgatgg 1680
acaaaaccca gcctgcttcc acaaaaacat cccaatgccc tcacccgtag tcgacccctc 1740
gatttgcggc tctatatgac cacaccctca aacaaacatc cccctctttc ctccctcccc 1800
ctgctgtaca actccgcacg ccctagatac cacaggcaca atgcggctca ctaacaatca 1860
aaacagagcc gagggaatta gaaaaaagta cgggtagaag agggatattc agagatcagg 1920
gcaagtctcc cgagtctctg ctctctcctc tacctgatag accaggacaa acatggccac 1980
ctttacagat gcagagatcg acgagctatt tgagacaagt ggaactgtca ttggcaacat 2040
aattacagcc cagggtaaac cagcagagac tgttggaagg agtgcaatcc cacaaggcaa 2100
gaccaaggtg ctgagcgcag catgggagaa gcatgggagc atccagccac cggccagtca 2160
agacaacccc gatcgacagg acagatctga caaacaacca tccacacccg agcaaacgac 2220
cccgcatgac agcccgccgg ccacatccgc cgaccagccc cccacccagg ccacagacga 2280
agccgtcgac acacagctca ggaccggagc aagcaactct ctgctgttga tgcttgacaa 2340
gctcagcaat aaatcgtcca atgctaaaaa gggcccatgg tcgagccccc aagaggggaa 2400
tcaccaacgt ccgactcaac agcaggggag tcaacccagt cgcggaaaca gtctggaaag 2460
accgcagaac caagtcaagg ccgcccctgg aaaccagggc acagacgtga acacagcata 2520
tcatggacaa tgggaggagt cacaactatc agctggtgca acccctcatg ctctccgatc 2580
aaggcagagc caagacaata cccttgtatc tgcggatcat gtccagccac ctgtagactt 2640
tgtgcaagcg atgatgtcta tgatggaggc gatatcacag agagtaagta aggttgacta 2700
tcagctagat cttgtcttga aacagacatc ctccatccct atgatgcggt ccgaaatcca 2760
acagctgaaa acatctgttg cagtcatgga agccaacttg ggaatgatga agattctgga 2820
tcccggttgt gccaacattt catctctgag tgatctacgg gcagttgccc gatctcaccc 2880
ggttttagtt tcaggccctg gagacccctc tccctatgtg acacaaggag gcgaaatggc 2940
acttaataaa ctttcgcaac cagtgccaca tccatctgaa ttgattaaac ccgccactgc 3000
atgcgggcct gatataggag tggaaaagga cactgtccgt gcattgatca tgtcacgccc 3060
aatgcacccg agttcttcag ccaagctcct aagcaagtta gatgcagccg ggtcgatcga 3120
ggaaatcagg aaaatcaagc gccttgctct aaatggctaa ttactactgc cacacgtagc 3180
gggtccctgt ccactcggca tcacacggaa tctgcaccga gttccccccc gcagacccaa 3240
ggtccaactc tccaagcggc aatcctctct cgcttcctca gccccactga atgatcgcgt 3300
aaccgtaatt aatctagcta catttaagat taagaaaaaa tacgggtaga attggagtgc 3360
cccaattgtg ccaagatgga ctcatctagg acaattgggc tgtactttga ttctgcccat 3420
tcttctagca acctgttagc atttccgatc gtcctacaag acacaggaga tgggaagaag 3480
caaatcgccc cgcaatatag gatccagcgc cttgacttgt ggactgatag taaggaggac 3540
tcagtattca tcaccaccta tggattcatc tttcaagttg ggaatgaaga agccactgtc 3600
ggcatgatcg atgataaacc caagcgcgag ttactttccg ctgcgatgct ctgcctagga 3660
agcgtcccaa ataccggaga ccttattgag ctggcaaggg cctgtctcac tatgatagtc 3720
acatgcaaga agagtgcaac taatactgag aggatggttt tctcagtagt gcaggcaccc 3780
caagtgctgc aaagctgtag ggttgtggca aacaaatact catcagtgaa tgcagtcaag 3840
cacgtgaaag cgccagagaa gattcccggg agtggaaccc tagaatacaa ggtgaacttt 3900
gtctccttga ctgtggtacc gaagaaggat gtctacaaga tcccagctgc agtattgaag 3960
gtttctggct cgagtctgta caatcttgcg ctcaatgtca ctattaatgt ggaggtagac 4020
ccgaggagtc ctttggttaa atctctgtct aagtctgaca gcggatacta tgctaacctc 4080
ttcttgcata ttggacttat gaccaccgta gataggaagg ggaagaaagt gacatttgac 4140
aagctggaaa agaaaataag gagccttgat ctatctgtcg ggctcagtga tgtgctcggg 4200
ccttccgtgt tggtaaaagc aagaggtgca cggactaagc ttttggcacc tttcttctct 4260
agcagtggga cagcctgcta tcccatagca aatgcttctc ctcaggtggc caagatactc 4320
tggagtcaaa ccgcgtgcct gcggagcgtt aaaatcatta tccaagcagg tacccaacgc 4380
gctgtcgcag tgaccgccga ccacgaggtt acctctacta agctggagaa ggggcacacc 4440
cttgccaaat acaatccttt taagaaataa gctgcgtctc tgagattgcg ctccgcccac 4500
tcacccagat catcatgaca caaaaaacta acctgtcttg attatttaca gttagtttac 4560
ctgtctatca agttagaaaa aacacgggta gaagattctg gatcccggtt ggcgccctcc 4620
aggtgcaaga tgggcttcaa accttctacc aggatcccag cacatctaat gctgatcact 4680
cggattatgc tgatattgag ctgtatccgt ctgacaagct ctcttgacgg caggcccctt 4740
gcagctgcag gaattgtagt aacaggagat aaggcagtca atgtatacac ctcgtctcag 4800
acagggtcaa tcatagtcaa gttgctcccg aatatgccca gagataagga ggcatgtgca 4860
aaagccccat tagaggcata taacagaaca ctgactactc tgctcactcc tcttggcgac 4920
tccatccgca agatccaagg gtctgtgtcc atgtctggag gagggagaca ggggcgcctt 4980
ataggtgctg ttattggcag tgtagctctt ggggttgcaa cagcggccca gataacagca 5040
gctgcggccc taatacaagc caaacagaat gccgccaaca tcctccggct taaggagagc 5100
attgccgcaa ccaatgaagc tgtgcatgaa gtcaccgacg gattatcaca actatcagtg 5160
gcagttggga agatgcagca gtttgtcaat gaccagttta ataatacggc gcgagaattg 5220
gactgtataa aaatcacaca acaggtcggt gtagaactca acctatacct aactgaattg 5280
actacagtat tcgggccaca gatcacctcc cctgcattaa ctcagctgac catccaggca 5340
ctttataatt tagctggtgg caatatggat tacttattaa ctaagttagg tataggaaac 5400
aatcaactca gctcattaat tggtagcggc ctgatcactg gttatcctat actgtatgac 5460
tcacatactc aactcttggg catacaagta aacctgccct cagtcgggaa cttaaataat 5520
atgcgtgcca cctatttgga gaccttatct gtaagtacaa ccaaaggata tgcctcagca 5580
cttgtcccga aagtagtgac acaagtcggt tctgtgatag aagagcttga cacctcatac 5640
tgtatagagt ccgatctgga tttatattgt actagaatag tgacattccc catgtcccca 5700
ggtatttatt cctgtttgag cggcaacaca tcagcttgca tgtattcaaa gactgaaggt 5760
gcactcactg cgccatatat ggcccttaaa ggctcagtta ttgccaattg taagataaca 5820
acatgtagat gtacagaccc tcctggtatc atatcgcaaa attatggaga agctgtatcc 5880
ctgatagata gacattcatg caatgtctta tcattagacg gaataactct gaggctcaat 5940
ggggaatttg atgcaactta tcaaaagaac atctcaatat tagattctca agtcatcgtg 6000
acaggcaatc ttgatatatc aactgaactt ggaaacgtca acaattcaat cagcaatgcc 6060
ttggataggt tggcagaaag caacagcaag ctagaaaaag tcgatgtcag actaactagc 6120
acatctgctc tcattaccta tattgttcta actgtcattt ctctaatttt cggtgcactt 6180
agtctggttt tagcgtgtta cctgatgtac aaacagaagg cacaacaaaa gaccttgcta 6240
tggcttggga ataataccct cgatcagatg agagccacta caagagcatg aatgcagata 6300
agaggtggat atatacccga cagcagcctg tgtgccaatt ccgataacct gtcaagtaga 6360
agacttaaga aaaaattact gggaacaagc aactaaagaa caatacacgg gtagaacggt 6420
cagaggagcc acccttcaat cgagaattag gcttcacaac atccgttcta ccacatcacc 6480
agcaacaaga gtcaatcatg gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa 6540
gagaagcaaa gaacacatgg cgcctggttt tccgggtcgt agtcttactt ttaatggtaa 6600
tgactctagc tatctccgca gctgccctgg catacagcac gggggccagt acgccgcacg 6660
acctcgcagg catatcgact gtgatctcca agacagaaga taagattacg tctttactca 6720
gttcaagtca agatgtgata gataggatat acaagcaggt ggctcttgaa tccccgctgg 6780
cgctactaaa cactgaatct ataattatga atgcaataac ctctctttct tatcaaatta 6840
acggggctga gaacaatagc gaatgtggcg cgcctgttca tgacccagat tatatcgggg 6900
ggataggcaa agaactcata gtggacgaca tcagtgatgt cacatcattt tatccttctg 6960
catatcaaga acacttgaat ttcatcccgg cgcctactac aggatccggt tgcactcgga 7020
taccctcatt tgacatgagc accacccatt attgttatac tcacaatgtg atactatccg 7080
gttgcagaga tcactcacac tcacatcaat acttagcact tggtgtgctt cggacatctg 7140
caacagggag ggtattcttt tctactctgc actccatcaa tttagatgac acccaaaatc 7200
ggaagtcctg cagtgtgagt gcaacccctc taggttgtga tatgctgtgc tctaaggtca 7260
cagggactga agaggaggat tacaagtcag ttgcccccac atcaatggtg cacggaaggc 7320
tagggtttga cggtcaatac catgagaagg acttagacac cacggtctta tttaaggatt 7380
gggtggcaaa ttacccggga gtgggaggag ggtcttttat tggcgaccgt gtatggttcc 7440
cagtttacgg agggctcaaa cccaattcac ccagtgacac tgcacaagaa gggaaatatg 7500
taatatacaa gcgccataac aacacatgcc ccgatgaaca agattaccaa attcggatgg 7560
ctaagtcttc atataaaccc gggcgatttg gtggaaagcg catacagcaa gccatcctat 7620
ccatcaaagt gtcaacatcc ctgggtaagg acccagtgct gactattcca cctaatacaa 7680
tcacactcat gggagccgaa ggcagaatcc tcacagtagg gacgtctcat ttcttgtacc 7740
aacgagggtc ttcatatttc tcccctgcct tattatatcc catgacagta aataacaaaa 7800
cggctacact ccatagtcct tacacgttta atgctttcac tcgaccaggt agtgtccctt 7860
gccaggcatc agcaagatgc cccaactcat gcatcactgg ggtctatacc gatccatatc 7920
ccttaatctt ccataggaat catactctac gaggggtctt cgggacgatg cttgatgatg 7980
aacaagcgag gcttaacccc gtatctgcag tatttgacaa catatctcgc agtcgtgtca 8040
cccgggtgag ttcaagcagc accaaggcag catacacgac atcgacatgt tttaaagttg 8100
tcaagaccaa taaagcttat tgtcttagta tcgcagaaat atccaatacc ctattcgggg 8160
aatttaggat cgttccctta ctagttgaga tcctcaagga tgatagagtt taattgagtc 8220
aattataaag gagttggaaa gatggcattg tatcacctat cttctgcgac atcaagaatc 8280
aaaccgaatg ccggcgcgtg ctcgaattcc atgttgccag ttgaccacaa tcagccagtg 8340
ctcatgcgat cagattaagc cttgtcaata gtctcttgat taagaaaaaa tgtaagtggc 8400
aatgagatac aaggcaaaac agctcatggt aaataatacg ggtaggacat ggcgagctcc 8460
ggtcctgaaa gggcagagca tcagattatc ctaccagagt cacacctgtc ttcaccattg 8520
gtcaagcaca aactactcta ttactggaaa ttaactgggc taccgcttcc tgatgaatgt 8580
gacttcgacc acctcattct cagccgacaa tggaaaaaaa tacttgaatc ggcctctcct 8640
gatactgaga gaatgataaa actcggaagg gcagtacacc aaactcttaa ccacaattcc 8700
agaataaccg gagtgctcca ccccaggtgt ttagaagaac tggctaatat tgaggtccca 8760
gattcaacca acaaatttcg gaagattgag aagaagatcc aaattcacaa cacgagatat 8820
ggagaactgt tcacaaggct gtgtacgcat atagagaaga aactgctggg gtcatcttgg 8880
tctaacaatg tcccccggtc agaggagttc agcagcattc gtacggatcc ggcattctgg 8940
tttcactcaa aatggtccac agccaagttt gcatggctcc atataaaaca gatccagagg 9000
catctgatgg tggcagctag gacaaggtct gcggccaaca aattggtgat gctaacccat 9060
aaggtaggcc aagtctttgt cactcctgaa cttgtcgttg tgacgcatac gaatgagaac 9120
aagttcacat gtcttaccca ggaacttgta ttgatgtatg cagatatgat ggagggcaga 9180
gatatggtca acataatatc aaccacggcg gtgcatctca gaagcttatc agagaaaatt 9240
gatgacattt tgcggttaat agacgctctg gcaaaagact tgggtaatca agtctacgat 9300
gttgtatcac taatggaggg atttgcatac ggagctgtcc agctactcga gccgtcaggt 9360
acatttgcag gagatttctt cgcattcaac ctgcaggagc ttaaagacat tctaattggc 9420
ctcctcccca atgatatagc agaatccgtg actcatgcaa tcgctactgt attctctggt 9480
ttagaacaga atcaagcagc tgagatgttg tgtctgttgc gtctgtgggg tcacccactg 9540
cttgagtccc gtattgcagc aaaggcagtc aggagccaaa tgtgcgcacc gaaaatggta 9600
gactttgata tgatccttca ggtactgtct ttcttcaagg gaacaatcat caacgggtac 9660
agaaagaaga atgcaggtgt gtggccgcga gtcaaagtgg atacaatata tgggaaggtc 9720
attgggcaac tacatgcaga ttcagcagag atttcacacg atatcatgtt gagagagtat 9780
aagagtttat ctgcacttga atttgagcca tgtatagaat atgaccctgt caccaacctg 9840
agcatgttcc taaaagacaa ggcaatcgca caccccaacg ataattggct tgcctcgttt 9900
aggcggaacc ttctctccga agaccagaag aaacatgtaa aagaagcaac ttcgactaat 9960
cgcctcttga tagagttttt agagtcaaat gattttgatc catataaaga gatggaatat 10020
ctgacgaccc ttgagtacct tagagatgac aatgtggcag tatcatactc gctcaaggag 10080
aaggaagtga aagttaatgg acggatcttc gctaagctga caaagaagtt aaggaactgt 10140
caggtgatgg cggaagggat cctagccgat cagattgcac ctttctttca gggaaatgga 10200
gtcattcagg atagcatatc cttgaccaag agtatgctag cgatgagtca actgtctttt 10260
aacagcaata agaaacgtat cactgactgt aaagaaagag tatcttcaaa ccgcaatcat 10320
gatccgaaaa gcaagaaccg tcggagagtt gcaaccttca taacaactga cctgcaaaag 10380
tactgtctta attggagata tcagacaatc aaattgttcg ctcatgccat caatcagttg 10440
atgggcctac ctcacttctt cgaatggatt cacctaagac tgatggacac tacgatgttc 10500
gtaggagacc ctttcaatcc tccaagtgac cctactgact gtgacctctc aagagtccct 10560
aatgatgaca tatatattgt cagtgccaga gggggtatcg aaggattatg ccagaagcta 10620
tggacaatga tctcaattgc tgcaatccaa cttgctgcag ctagatcgca ttgtcgtgtt 10680
gcctgtatgg tacagggtga taatcaagta atagcagtaa cgagagaggt aagatcagac 10740
gactctccgg agatggtgtt gacacagttg catcaagcca gtgataattt cttcaaggaa 10800
ttaattcatg tcaatcattt gattggccat aatttgaagg atcgtgaaac catcaggtca 10860
gacacattct tcatatacag caaacgaatc ttcaaagatg gagcaatcct cagtcaagtc 10920
ctcaaaaatt catctaaatt agtgctagtg tcaggtgatc tcagtgaaaa caccgtaatg 10980
tcctgtgcca acattgcctc tactgtagca cggctatgcg agaacgggct tcccaaagac 11040
ttctgttact atttaaacta tataatgagt tgtgtgcaga catactttga ctctgagttc 11100
tccatcacca acaattcgca ccccgatctt aatcagtcgt ggattgagga catctctttt 11160
gtgcactcat atgttctgac tcctgcccaa ttagggggac tgagtaacct tcaatactca 11220
aggctctaca ctagaaatat cggtgacccg gggactactg cttttgcaga gatcaagcga 11280
ctagaagcag tgggattact gagtcctaac attatgacta atatcttaac taggccgcct 11340
gggaatggag attgggccag tctgtgcaac gacccatact ctttcaattt tgagactgtt 11400
gcaagcccaa atattgttct taagaaacat acgcaaagag tcctatttga aacttgttca 11460
aatcccttat tgtctggagt gcacacagag gataatgagg cagaagagaa ggcattggct 11520
gaattcttgc ttaatcaaga ggtgattcat ccccgcgttg cgcatgccat catggaggca 11580
agctctgtag gtaggagaaa gcaaattcaa gggcttgttg acacaacaaa caccgtaatt 11640
aagattgcgc ttactaggag gccattaggc atcaagaggc tgatgcggat agtcaattat 11700
tctagcatgc atgcaatgct gtttggagac gatgtttttt cctccagtag atccaaccac 11760
cccttagtct cttctaatat gtgttctctg acactggcag actatgcacg gaatagaagc 11820
tggtcacctt tgacgggagg caggaaaata ctgggtgtat ctaatcctga tacgatagaa 11880
ctcgtagagg gtgagattct tagtgtaagc ggagggtgta caagatgtga cagcggagat 11940
gaacaattta cttggttcca tcttccaagc aatatagaat tgaccgatga caccagcaag 12000
aatcctccga tgagggtacc atatctcggg tcaaagacac aggagaggag agctgcctca 12060
cttgcaaaaa tagctcatat gtcgccacat gtaaaggctg ccctaagggc atcatccgtg 12120
ttgatctggg cttatgggga taatgaagta aattggactg ctgctcttac gattgcaaaa 12180
tctcggtgta atgtaaactt agagtatctt cggttactgt cccctttacc cacggctggg 12240
aatcttcaac atagactaga tgatggtata actcagatga cattcacccc tgcatctctc 12300
tacagggtgt caccttacat tcacatatcc aatgattctc aaaggctgtt cactgaagaa 12360
ggagtcaaag aggggaatgt ggtttaccaa cagatcatgc tcttgggttt atctctaatc 12420
gaatcgatct ttccaatgac aacaaccagg acatatgatg agatcacact gcacctacat 12480
agtaaattta gttgctgtat cagagaagca cctgttgcgg ttcctttcga gctacttggg 12540
gtggtaccgg aactgaggac agtgacctca aataagttta tgtatgatcc tagccctgta 12600
tcggagggag actttgcgag acttgactta gctaccttca agagttatga gcttaatctg 12660
gagtcatatc ccacgataga gctaatgaac attctttcaa tatccagcgg gaagttgatt 12720
ggccagtctg tggtttctta tgatgaagat acctccataa agaatgacgc cataatagtg 12780
tatgacaata cccgaaattg gatcagtgaa gctcagaatt cagatgtggt ccgcctattt 12840
gaatatgcag cacttgaagt gctcctcgac tgttcttacc aactctatta cctgagagta 12900
agaggcctag acaatattgt cttatatatg ggtgatttat acaagaatat gccaggaatt 12960
ctactttcca acattgcagc tacaatatct catcccgtca ttcattcaag gttacatgca 13020
gtgggcctgg tcaaccatga cggatcacac caacttgcag atacggattt tatcgaaatg 13080
tctgcaaaac tattagtatc ttgcacccga cgtgtgatct ccggcttata ttcaggaaat 13140
aagtatgatc tgctgttccc atctgtctta gatgataacc tgaatgagaa gatgcttcag 13200
ctgatatccc ggttatgctg tctgtacacg gtactctttg ctacaacaag agaaatcccg 13260
aaaataagag gcttaactgc agaagagaaa tgttcaatac tcactgagta tttactgtcg 13320
gatgctgtga aaccattact tagccccgat caagtgagct ctatcatgtc tcctaacata 13380
attacattcc cagctaatct gtactacatg tctcggaaga gcctcaattt gatcagggaa 13440
agggaggaca gggatactat cctggcgttg ttgttccccc aagagccatt attagagttc 13500
ccttctgtgc aagatattgg tgctcgagtg aaagatccat tcacccgaca acctgcggca 13560
tttttgcaag agttagattt gagtgctcca gcaaggtatg acgcattcac acttagtcag 13620
attcatcctg aactcacatc tccaaatccg gaggaagact acttagtacg atacttgttc 13680
agagggatag ggactgcatc ttcctcttgg tataaggcat ctcatctcct ttctgtaccc 13740
gaggtaagat gtgcaagaca cgggaactcc ttatacttag ctgaagggag cggagccatc 13800
atgagtcttc tcgaactgca tgtaccacat gaaactatct attacaatac gctcttttca 13860
aatgagatga accccccgca acgacatttc gggccgaccc caactcagtt tttgaattca 13920
gttgtttata ggaatctaca ggcggaggta acatgcaaag atggatttgt ccaagagttc 13980
cgtccattat ggagagaaaa tacagaggaa agtgacctga cctcagataa agtagtgggg 14040
tatattacat ctgcagtgcc ctacagatct gtatcattgc tgcattgtga cattgaaatt 14100
cctccagggt ccaatcaaag cttactagat caactagcta tcaatttatc tctgattgcc 14160
atgcattctg taagggaggg cggggtagta atcatcaaag tgttgtatgc aatgggatac 14220
tactttcatc tactcatgaa cttgtttgct ccgtgttcca caaaaggata tattctctct 14280
aatggttatg catgtcgagg agatatggag tgttacctgg tatttgtcat gggttacctg 14340
ggcgggccta catttgtaca tgaggtggtg aggatggcga aaactctggt gcagcggcac 14400
ggtacgcttt tgtctaaatc agatgagatc acactgacca ggttattcac ctcacagcgg 14460
cagcgtgtga cagacatcct atccagtcct ttaccaagat taataaagta cttgaggaag 14520
aatattgaca ctgcgctgat tgaagccggg ggacagcccg tccgtccatt ctgtgcggag 14580
agtctggtga gcacgctagc gaacataact cagataaccc agatcatcgc tagtcacatt 14640
gacacagtta tccggtctgt gatatatatg gaagctgagg gtgatctcgc tgacacagta 14700
tttctattta ccccttacaa tctctctact gacgggaaaa agaggacatc acttaaacag 14760
tgcacgaggc agatcctaga ggttacaata ctaggtctta gagtcgaaaa tctcaataaa 14820
ataggcgata taatcagcct agtgcttaaa ggcatgatct ccatggagga ccttatccca 14880
ctaaggacat acttgaagca tagtacctgc cctaaatatt tgaaggctgt cctaggtatt 14940
accaaactca aagaaatgtt tacagacact tctgtactgt acttgactcg tgctcaacaa 15000
aaattctaca tgaaaactat aggcaatgca gtcaaaggat attacagtaa ctgtgactct 15060
taacgaaaat cacatattaa taggctcctt ttttggccaa ttgtattctt gttgatttta 15120
tcatattatg ttagaaaaaa gttgaaccct gactccttag gactcgaatt cgaactcaaa 15180
taaatgtctt aaaaaaaggt tgcgcacaat tattcttgag tgtagtctcg tcattcacca 15240
aatctttgtt tggtggccgg catggtccca gcctcctcgc tggcgccggc tgggcaacat 15300
tccgagggga ccgtcccctc ggtaatggcg aatgggacct agcataaccc cttggggcct 15360
ctaaacgggt cttgaggggt tttttgccgt cgaccaattc tcatgtttga cagcttatca 15420
tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa ccaggcgttt 15480
aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact 15540
gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct 15600
gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa 15660
cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc 15720
agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt 15780
tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt 15840
ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag 15900
ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat 15960
gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt 16020
tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt 16080
gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg 16140
tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata 16200
actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta 16260
cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag 16320
ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcgcgat 16380
aagctcatgg agcggcgtaa ccgtcgcaca ggaaggacag agaaagcgcg gatctgggaa 16440
gtgacggaca gaacggtcag gacctggatt ggggaggcgg ttgccgccgc tgctgctgac 16500
ggtgtgacgt tctctgttcc ggtcacacca catacgttcc gccattccta tgcgatgcac 16560
atgctgtatg ccggtatacc gctgaaagtt ctgcaaagcc tgatgggaca taagtccatc 16620
agttcaacgg aagtctacac gaaggttttt gcgctggatg tggctgcccg gcaccgggtg 16680
cagtttgcga tgccggagtc tgatgcggtt gcgatgctga aacaattatc ctgagaataa 16740
atgccttggc ctttatatgg aaatgtggaa ctgagtggat atgctgtttt tgtctgttaa 16800
acagagaagc tggctgttat ccactgagaa gcgaacgaaa cagtcgggaa aatctcccat 16860
tatcgtagag atccgcatta ttaatctcag gagcctgtgt agcgtttata ggaagtagtg 16920
ttctgtcatg atgcctgcaa gcggtaacga aaacgatttg aatatgcctt caggaacaat 16980
agaaatcttc gtgcggtgtt acgttgaagt ggagcggatt atgtcagcaa tggacagaac 17040
aacctaatga acacagaacc atgatgtggt ctgtcctttt acagccagta gtgctcgccg 17100
cagtcgagcg acagggcgaa gccctcgagt gagcgaggaa gcaccaggga acagcactta 17160
tatattctgc ttacacacga tgcctgaaaa aacttccctt ggggttatcc acttatccac 17220
ggggatattt ttataattat tttttttata gtttttagat cttctttttt agagcgcctt 17280
gtaggccttt atccatgctg gttctagaga aggtgttgtg acaaattgcc ctttcagtgt 17340
gacaaatcac cctcaaatga cagtcctgtc tgtgacaaat tgcccttaac cctgtgacaa 17400
attgccctca gaagaagctg ttttttcaca aagttatccc tgcttattga ctctttttta 17460
tttagtgtga caatctaaaa acttgtcaca cttcacatgg atctgtcatg gcggaaacag 17520
cggttatcaa tcacaagaaa cgtaaaaata gcccgcgaat cgtccagtca aacgacctca 17580
ctgaggcggc atatagtctc tcccgggatc aaaaacgtat gctgtatctg ttcgttgacc 17640
agatcagaaa atctgatggc accctacagg aacatgacgg tatctgcgag atccatgttg 17700
ctaaatatgc tgaaatattc ggattgacct ctgcggaagc cagtaaggat atacggcagg 17760
cattgaagag tttcgcgggg aaggaagtgg ttttttatcg ccctgaagag gatgccggcg 17820
atgaaaaagg ctatgaatct tttccttggt ttatcaaacg tgcgcacagt ccatccagag 17880
ggctttacag tgtacatatc aacccatatc tcattccctt ctttatcggg ttacagaacc 17940
ggtttacgca gtttcggctt agtgaaacaa aagaaatcac caatccgtat gccatgcgtt 18000
tatacgaatc cctgtgtcag tatcgtaagc cggatggctc aggcatcgtc tctctgaaaa 18060
tcgactggat catagagcgt taccagctgc ctcaaagtta ccagcgtatg cctgacttcc 18120
gccgccgctt cctgcaggtc tgtgttaatg agatcaacag cagaactcca atgcgcctct 18180
catacattga gaaaaagaaa ggccgccaga cgactcatat cgtattttcc ttccgcgata 18240
tcacttccat gacgacagga tagtctgagg gttatctgtc acagatttga gggtggttcg 18300
tcacatttgt tctgacctac tgagggtaat ttgtcacagt tttgctgttt ccttcagcct 18360
gcatggattt tctcatactt tttgaactgt aatttttaag gaagccaaat ttgagggcag 18420
tttgtcacag ttgatttcct tctctttccc ttcgtcatgt gacctgatat cgggggttag 18480
ttcgtcatca ttgatgaggg ttgattatca cagtttatta ctctgaattg gctatccgcg 18540
tgtgtacctc tacctggagt ttttcccacg gtggatattt cttcttgcgc tgagcgtaag 18600
agctatctga cagaacagtt cttctttgct tcctcgccag ttcgctcgct atgctcggtt 18660
acacggctgc ggcgagcgct agtgataata agtgactgag gtatgtgctc ttcttatctc 18720
cttttgtagt gttgctctta ttttaaacaa ctttgcggtt ttttgatgac tttgcgattt 18780
tgttgttgct ttgcagtaaa ttgcaagatt taataaaaaa acgcaaagca atgattaaag 18840
gatgttcaga atgaaactca tggaaacact taaccagtgc ataaacgctg gtcatgaaat 18900
gacgaaggct atcgccattg cacagtttaa tgatgacagc ccggaagcga ggaaaataac 18960
ccggcgctgg agaataggtg aagcagcgga tttagttggg gtttcttctc aggctatcag 19020
agatgccgag aaagcagggc gactaccgca cccggatatg gaaattcgag gacgggttga 19080
gcaacgtgtt ggttatacaa ttgaacaaat taatcatatg cgtgatgtgt ttggtacgcg 19140
attgcgacgt gctgaagacg tatttccacc ggtgatcggg gttgctgccc ataaaggtgg 19200
cgtttacaaa acctcagttt ctgttcatct tgctcaggat ctggctctga aggggctacg 19260
tgttttgctc gtggaaggta acgaccccca gggaacagcc tcaatgtatc acggatgggt 19320
accagatctt catattcatg cagaagacac tctcctgcct ttctatcttg gggaaaagga 19380
cgatgtcact tatgcaataa agcccacttg ctggccgggg cttgacatta ttccttcctg 19440
tctggctctg caccgtattg aaactgagtt aatgggcaaa tttgatgaag gtaaactgcc 19500
caccgatcca cacctgatgc tccgactggc cattgaaact gttgctcatg actatgatgt 19560
catagttatt gacagcgcgc ctaacctggg tatcggcacg attaatgtcg tatgtgctgc 19620
tgatgtgctg attgttccca cgcctgctga gttgtttgac tacacctccg cactgcagtt 19680
tttcgatatg cttcgtgatc tgctcaagaa cgttgatctt aaagggttcg agcctgatgt 19740
acgtattttg cttaccaaat acagcaatag taatggctct cagtccccgt ggatggagga 19800
gcaaattcgg gatgcctggg gaagcatggt tctaaaaaat gttgtacgtg aaacggatga 19860
agttggtaaa ggtcagatcc ggatgagaac tgtttttgaa caggccattg atcaacgctc 19920
ttcaactggt gcctggagaa atgctctttc tatttgggaa cctgtctgca atgaaatttt 19980
cgatcgtctg attaaaccac gctgggagat tagataatga agcgtgcgcc tgttattcca 20040
aaacatacgc tcaatactca accggttgaa gatacttcgt tatcgacacc agctgccccg 20100
atggtggatt cgttaattgc gcgcgtagga gtaatggctc gcggtaatgc cattactttg 20160
cctgtatgtg gtcgggatgt gaagtttact cttgaagtgc tccggggtga tagtgttgag 20220
aagacctctc gggtatggtc aggtaatgaa cgtgaccagg agctgcttac tgaggacgca 20280
ctggatgatc tcatcccttc ttttctactg actggtcaac agacaccggc gttcggtcga 20340
agagtatctg gtgtcataga aattgccgat gggagtcgcc gtcgtaaagc tgctgcactt 20400
accgaaagtg attatcgtgt tctggttggc gagctggatg atgagcagat ggctgcatta 20460
tccagattgg gtaacgatta tcgcccaaca agtgcttatg aacgtggtca gcgttatgca 20520
agccgattgc agaatgaatt tgctggaaat atttctgcgc tggctgatgc ggaaaatatt 20580
tcacgtaaga ttattacccg ctgtatcaac accgccaaat tgcctaaatc agttgttgct 20640
cttttttctc accccggtga actatctgcc cggtcaggtg atgcacttca aaaagccttt 20700
acagataaag aggaattact taagcagcag gcatctaacc ttcatgagca gaaaaaagct 20760
ggggtgatat ttgaagctga agaagttatc actcttttaa cttctgtgct taaaacgtca 20820
tctgcatcaa gaactagttt aagctcacga catcagtttg ctcctggagc gacagtattg 20880
tataagggcg ataaaatggt gcttaacctg gacaggtctc gtgttccaac tgagtgtata 20940
gagaaaattg aggccattct taaggaactt gaaaagccag caccctgatg cgaccacgtt 21000
ttagtctacg tttatctgtc tttacttaat gtcctttgtt acaggccaga aagcataact 21060
ggcctgaata ttctctctgg gcccactgtt ccacttgtat cgtcggtctg ataatcagac 21120
tgggaccacg gtcccactcg tatcgtcggt ctgattatta gtctgggacc acggtcccac 21180
tcgtatcgtc ggtctgatta ttagtctggg accacggtcc cactcgtatc gtcggtctga 21240
taatcagact gggaccacgg tcccactcgt atcgtcggtc tgattattag tctgggacca 21300
tggtcccact cgtatcgtcg gtctgattat tagtctggga ccacggtccc actcgtatcg 21360
tcggtctgat tattagtctg gaaccacggt cccactcgta tcgtcggtct gattattagt 21420
ctgggaccac ggtcccactc gtatcgtcgg tctgattatt agtctgggac cacgatccca 21480
ctcgtgttgt cggtctgatt atcggtctgg gaccacggtc ccacttgtat tgtcgatcag 21540
actatcagcg tgagactacg attccatcaa tgcctgtcaa gggcaagtat tgacatgtcg 21600
tcgtaacctg tagaacggag taacctcggt gtgcggttgt atgcctgctg tggattgctg 21660
ctgtgtcctg cttatccaca acattttgcg cacggttatg tggacaaaat acctggttac 21720
ccaggccgtg ccggcacgtt aaccgggctg catccgatgc aagtgtgtcg ctgtcgacga 21780
gctcgcgagc tcggacatga ggttgccccg tattcagtgt cgctgatttg tattgtctga 21840
agttgttttt acgttaagtt gatgcagatc aattaatacg atacctgcgt cataattgat 21900
tatttgacgt ggtttgatgg cctccacgca cgttgtgata tgtagatgat aatcattatc 21960
actttacggg tcctttccgg tgatccgaca ggttacgggg cggcgacctc gcgggttttc 22020
gctatttatg aaaattttcc ggtttaaggc gtttccgttc ttcttcgtca taacttaatg 22080
tttttattta aaataccctc tgaaaagaaa ggaaacgaca ggtgctgaaa gcgagctttt 22140
tggcctctgt cgtttccttt ctctgttttt gtccgtggaa tgaacaatgg aagtccgagc 22200
tcatcgctaa taacttcgta tagcatacat tatacgaagt tatattcgat 22250
<210> 16
<211> 22250
<212> DNA
<213> 人工序列(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 16
gcggccgcaa ggggttcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 60
cggcattaat acgactcact atagggacca aacagagaat ccgtaagtta cgataaaagg 120
cgaaggagca attgaagtcg cacgggtaga aggtgtgaat ctcgagtgcg agcccgaagc 180
acaaactcga gaaagccttc tgccaacatg tcttccgtat ttgatgagta cgaacagctc 240
ctcgcggctc agactcgccc caatggagct catggagggg gagaaaaagg gagtacctta 300
aaagtagacg tcccggtatt cactcttaac agtgatgacc cagaagatag atggagcttt 360
gtggtattct gcctccggat tgctgttagc gaagatgcca acaaaccact caggcaaggt 420
gctctcatat ctcttttatg ctcccactca caggtaatga ggaaccatgt tgcccttgca 480
gggaaacaga atgaagccac attggccgtg cttgagattg atggctttgc caacggcacg 540
ccccagttca acaataggag tggagtgtct gaagagagag cacagagatt tgcgatgata 600
gcaggatctc tccctcgggc atgcagcaac ggaaccccgt tcgtcacagc cggggccgaa 660
gatgatgcac cagaagacat caccgatacc ctggagagga tcctctctat ccaggctcaa 720
gtatgggtca cagtagcaaa agccatgact gcgtatgaga ctgcagatga gtcggaaaca 780
aggcgaatca ataagtatat gcagcaaggc agggtccaaa agaaatacat cctctacccc 840
gtatgcagga gcacaatcca actcacgatc agacagtctc ttgcagtccg catctttttg 900
gttagcgagc tcaagagagg ccgcaacacg gcaggtggta cctctactta ttataacctg 960
gtaggggacg tagactcata catcaggaat accgggctta ctgcattctt cttgacactc 1020
aagtacggaa tcaacaccaa gacatcagcc cttgcactta gtagcctctc aggcgacatc 1080
cagaagatga agcagctcat gcgtttgtat cggatgaaag gagataatgc gccgtacatg 1140
acattacttg gtgatagtga ccagatgagc tttgcgcctg ccgagtatgc acaactttac 1200
tcctttgcca tgggtatggc atcagtccta gataaaggta ctgggaaata ccaatttgcc 1260
agggacttta tgagcacatc attctggaga cttggagtag agtacgctca ggctcaggga 1320
agtagcatta acgaggatat ggctgccgag ctaaagctaa ccccagcagc aaggaggggc 1380
ctggcagctg ctgcccaacg ggtctccgag gagaccagca gcatagacat gcctactcaa 1440
caagtcggag tcctcactgg gcttagcgag ggggggtccc aagctctaca aggcggatcg 1500
aatagatcgc aagggcaacc agaagccggg gatggggaga cccaattcct ggatctgatg 1560
agagcggtag caaatagcat gagggaggcg ccaaactctg cacagggcac tccccaatcg 1620
gggcctcccc caactcctgg gccatcccaa gataacgaca ccgactgggg gtattgatgg 1680
acaaaaccca gcctgcttcc acaaaaacat cccaatgccc tcacccgtag tcgacccctc 1740
gatttgcggc tctatatgac cacaccctca aacaaacatc cccctctttc ctccctcccc 1800
ctgctgtaca actccgcacg ccctagatac cacaggcaca atgcggctca ctaacaatca 1860
aaacagagcc gagggaatta gaaaaaagta cgggtagaag agggatattc agagatcagg 1920
gcaagtctcc cgagtctctg ctctctcctc tacctgatag accaggacaa acatggccac 1980
ctttacagat gcagagatcg acgagctatt tgagacaagt ggaactgtca ttggcaacat 2040
aattacagcc cagggtaaac cagcagagac tgttggaagg agtgcaatcc cacaaggcaa 2100
gaccaaggtg ctgagcgcag catgggagaa gcatgggagc atccagccac cggccagtca 2160
agacaacccc gatcgacagg acagatctga caaacaacca tccacacccg agcaaacgac 2220
cccgcatgac agcccgccgg ccacatccgc cgaccagccc cccacccagg ccacagacga 2280
agccgtcgac acacagctca ggaccggagc aagcaactct ctgctgttga tgcttgacaa 2340
gctcagcaat aaatcgtcca atgctaaaaa gggcccatgg tcgagccccc aagaggggaa 2400
tcaccaacgt ccgactcaac agcaggggag tcaacccagt cgcggaaaca gtctggaaag 2460
accgcagaac caagtcaagg ccgcccctgg aaaccagggc acagacgtga acacagcata 2520
tcatggacaa tgggaggagt cacaactatc agctggtgca acccctcatg ctctccgatc 2580
aaggcagagc caagacaata cccttgtatc tgcggatcat gtccagccac ctgtagactt 2640
tgtgcaagcg atgatgtcta tgatggaggc gatatcacag agagtaagta aggttgacta 2700
tcagctagat cttgtcttga aacagacatc ctccatccct atgatgcggt ccgaaatcca 2760
acagctgaaa acatctgttg cagtcatgga agccaacttg ggaatgatga agattctgga 2820
tcccggttgt gccaacattt catctctgag tgatctacgg gcagttgccc gatctcaccc 2880
ggttttagtt tcaggccctg gagacccctc tccctatgtg acacaaggag gcgaaatggc 2940
acttaataaa ctttcgcaac cagtgccaca tccatctgaa ttgattaaac ccgccactgc 3000
atgcgggcct gatataggag tggaaaagga cactgtccgt gcattgatca tgtcacgccc 3060
aatgcacccg agttcttcag ccaagctcct aagcaagtta gatgcagccg ggtcgatcga 3120
ggaaatcagg aaaatcaagc gccttgctct aaatggctaa ttactactgc cacacgtagc 3180
gggtccctgt ccactcggca tcacacggaa tctgcaccga gttccccccc gcagacccaa 3240
ggtccaactc tccaagcggc aatcctctct cgcttcctca gccccactga atgatcgcgt 3300
aaccgtaatt aatctagcta catttaagat taagaaaaaa tacgggtaga attggagtgc 3360
cccaattgtg ccaagatgga ctcatctagg acaattgggc tgtactttga ttctgcccat 3420
tcttctagca acctgttagc atttccgatc gtcctacaag acacaggaga tgggaagaag 3480
caaatcgccc cgcaatatag gatccagcgc cttgacttgt ggactgatag taaggaggac 3540
tcagtattca tcaccaccta tggattcatc tttcaagttg ggaatgaaga agccactgtc 3600
ggcatgatcg atgataaacc caagcgcgag ttactttccg ctgcgatgct ctgcctagga 3660
agcgtcccaa ataccggaga ccttattgag ctggcaaggg cctgtctcac tatgatagtc 3720
acatgcaaga agagtgcaac taatactgag aggatggttt tctcagtagt gcaggcaccc 3780
caagtgctgc aaagctgtag ggttgtggca aacaaatact catcagtgaa tgcagtcaag 3840
cacgtgaaag cgccagagaa gattcccggg agtggaaccc tagaatacaa ggtgaacttt 3900
gtctccttga ctgtggtacc gaagaaggat gtctacaaga tcccagctgc agtattgaag 3960
gtttctggct cgagtctgta caatcttgcg ctcaatgtca ctattaatgt ggaggtagac 4020
ccgaggagtc ctttggttaa atctctgtct aagtctgaca gcggatacta tgctaacctc 4080
ttcttgcata ttggacttat gaccaccgta gataggaagg ggaagaaagt gacatttgac 4140
aagctggaaa agaaaataag gagccttgat ctatctgtcg ggctcagtga tgtgctcggg 4200
ccttccgtgt tggtaaaagc aagaggtgca cggactaagc ttttggcacc tttcttctct 4260
agcagtggga cagcctgcta tcccatagca aatgcttctc ctcaggtggc caagatactc 4320
tggagtcaaa ccgcgtgcct gcggagcgtt aaaatcatta tccaagcagg tacccaacgc 4380
gctgtcgcag tgaccgccga ccacgaggtt acctctacta agctggagaa ggggcacacc 4440
cttgccaaat acaatccttt taagaaataa gctgcgtctc tgagattgcg ctccgcccac 4500
tcacccagat catcatgaca caaaaaacta acctgtcttg attatttaca gttagtttac 4560
ctgtctatca agttagaaaa aacacgggta gaagattctg gatcccggtt ggcgccctcc 4620
aggtgcaaga tgggcttcaa accttctacc aggatcccag cacatctaat gctgatcact 4680
cggattatgc tgatattgag ctgtatccgt ctgacaagct ctcttgacgg caggcccctt 4740
gcagctgcag gaattgtagt aacaggagat aaggcagtca atgtatacac ctcgtctcag 4800
acagggtcaa tcatagtcaa gttgctcccg aatatgccca gagataagga ggcatgtgca 4860
aaagccccat tagaggcata taacagaaca ctgactactc tgctcactcc tcttggcgac 4920
tccatccgca agatccaagg gtctgtgtcc atgtctggag gagggagaca ggggcgcctt 4980
ataggtgctg ttattggcag tgtagctctt ggggttgcaa cagcggccca gataacagca 5040
gctgcggccc taatacaagc caaacagaat gccgccaaca tcctccggct taaggagagc 5100
attgccgcaa ccaatgaagc tgtgcatgaa gtcaccgacg gattatcaca actatcagtg 5160
gcagttggga agatgcagca gtttgtcaat gaccagttta ataatacggc gcgagaattg 5220
gactgtataa aaatcacaca acaggtcggt gtagaactca acctatacct aactgaattg 5280
actacagtat tcgggccaca gatcacctcc cctgcattaa ctcagctgac catccaggca 5340
ctttataatt tagctggtgg caatatggat tacttattaa ctaagttagg tataggaaac 5400
aatcaactca gctcattaat tggtagcggc ctgatcactg gttatcctat actgtatgac 5460
tcacatactc aactcttggg catacaagta aacctgccct cagtcgggaa cttaaataat 5520
atgcgtgcca cctatttgga gaccttatct gtaagtacaa ccaaaggata tgcctcagca 5580
cttgtcccga aagtagtgac acaagtcggt tctgtgatag aagagcttga cacctcatac 5640
tgtatagagt ccgatctgga tttatattgt actagaatag tgacattccc catgtcccca 5700
ggtatttatt cctgtttgag cggcaacaca tcagcttgca tgtattcaaa gactgaaggt 5760
gcactcactg cgccatatat ggcccttaaa ggctcagtta ttgccaattg taagataaca 5820
acatgtagat gtacagaccc tcctggtatc atatcgcaaa attatggaga agctgtatcc 5880
ctgatagata gacattcatg caatgtctta tcattagacg gaataactct gaggctcaat 5940
ggggaatttg atgcaactta tcaaaagaac atctcaatat tagattctca agtcatcgtg 6000
acaggcaatc ttgatatatc aactgaactt ggaaacgtca acaattcaat cagcaatgcc 6060
ttggataggt tggcagaaag caacagcaag ctagaaaaag tcgatgtcag actaactagc 6120
acatctgctc tcattaccta tattgttcta actgtcattt ctctaatttt cggtgcactt 6180
agtctggttt tagcgtgtta cctgatgtac aaacagaagg cacaacaaaa gaccttgcta 6240
tggcttggga ataataccct cgatcagatg agagccacta caagagcatg aatgcagata 6300
agaggtggat atatacccga cagcagcctg tgtgccaatt ccgataacct gtcaagtaga 6360
agacttaaga aaaaattact gggaacaagc aactaaagaa caatacacgg gtagaacggt 6420
cagaggagcc acccttcaat cgagaattag gcttcacaac atccgttcta ccacatcacc 6480
agcaacaaga gtcaatcatg gaccgcgcgg ttaacagagt cgtgctggag aatgaggaaa 6540
gagaagcaaa gaacacatgg cgcctggttt tccgggtcgt agtcttactt ttaatggtaa 6600
tgactctagc tatctccgca gctgccctgg catacagcac gggggccagt acgccgcacg 6660
acctcgcagg catatcgact gtgatctcca agacagaaga taagattacg tctttactca 6720
gttcaagtca agatgtgata gataggatat acaagcaggt ggctcttgaa tccccgctgg 6780
cgctactaaa cactgaatct ataattatga atgcaataac ctctctttct tatcaaatta 6840
acggggctga gaacaatagc gaatgtggcg cgcctgttca tgacccagat tatatcgggg 6900
ggataggcaa agaactcata gtggacgaca tcagtgatgt cacatcattt tatccttctg 6960
catatcaaga acacttgaat ttcatcccgg cgcctactac aggatccggt tgcactcgga 7020
taccctcatt tgacatgagc accacccatt attgttatac tcacaatgtg atactatccg 7080
gttgcagaga tcactcacac tcacatcaat acttagcact tggtgtgctt cggacatctg 7140
caacagggag ggtattcttt tctactctgc actccatcaa tttagatgac acccaaaatc 7200
ggaagtcctg cagtgtgagt gcaacccctc taggttgtga tatgctgtgc tctaaggtca 7260
cagggactga agaggaggat tacaagtcag ttgcccccac atcaatggtg cacggaaggc 7320
tagggtttga cggtcaatac catgagaagg acttagacac cacggtctta tttaaggatt 7380
gggtggcaaa ttacccggga gtgggaggag ggtcttttat tggcgaccgt gtatggttcc 7440
cagtttacgg agggctcaaa cccaattcac ccagtgacac tgcacaagaa gggaaatatg 7500
taatatacaa gcgccataac aacacatgcc ccgatgaaca agattaccaa attcggatgg 7560
ctaagtcttc atataaaccc gggcgatttg gtggaaagcg catacagcaa gccatcctat 7620
ccatcaaagt gtcaacatcc ctgggtaagg acccagtgct gactattcca cctaatacaa 7680
tcacactcat gggagccgaa ggcagaatcc tcacagtagg gacgtctcat ttcttgtacc 7740
aacgagggtc ttcatatttc tcccctgcct tattatatcc catgacagta aataacaaaa 7800
cggctacact ccatagtcct tacacgttta atgctttcac tcgaccaggt agtgtccctt 7860
gccaggcatc agcaagatgc cccaactcat gcatcactgg ggtctatacc gatccatatc 7920
ccttaatctt ccataggaat catactctac gaggggtctt cgggacgatg cttgatgctg 7980
cacaagcgag gcttaacccc gtatctgcag tatttgacgc cgcatctcgc agtcgtgtca 8040
cccgggtgag ttcaagcagc accaaggcag catacacgac atcgacatgt tttaaagttg 8100
tcaagaccaa taaagcttat tgtcttagta tcgcagaaat atccaatacc ctattcgggg 8160
aatttaggat cgttccctta ctagttgaga tcctcaagga tgatagagtt taattgagtc 8220
aattataaag gagttggaaa gatggcattg tatcacctat cttctgcgac atcaagaatc 8280
aaaccgaatg ccggcgcgtg ctcgaattcc atgttgccag ttgaccacaa tcagccagtg 8340
ctcatgcgat cagattaagc cttgtcaata gtctcttgat taagaaaaaa tgtaagtggc 8400
aatgagatac aaggcaaaac agctcatggt aaataatacg ggtaggacat ggcgagctcc 8460
ggtcctgaaa gggcagagca tcagattatc ctaccagagt cacacctgtc ttcaccattg 8520
gtcaagcaca aactactcta ttactggaaa ttaactgggc taccgcttcc tgatgaatgt 8580
gacttcgacc acctcattct cagccgacaa tggaaaaaaa tacttgaatc ggcctctcct 8640
gatactgaga gaatgataaa actcggaagg gcagtacacc aaactcttaa ccacaattcc 8700
agaataaccg gagtgctcca ccccaggtgt ttagaagaac tggctaatat tgaggtccca 8760
gattcaacca acaaatttcg gaagattgag aagaagatcc aaattcacaa cacgagatat 8820
ggagaactgt tcacaaggct gtgtacgcat atagagaaga aactgctggg gtcatcttgg 8880
tctaacaatg tcccccggtc agaggagttc agcagcattc gtacggatcc ggcattctgg 8940
tttcactcaa aatggtccac agccaagttt gcatggctcc atataaaaca gatccagagg 9000
catctgatgg tggcagctag gacaaggtct gcggccaaca aattggtgat gctaacccat 9060
aaggtaggcc aagtctttgt cactcctgaa cttgtcgttg tgacgcatac gaatgagaac 9120
aagttcacat gtcttaccca ggaacttgta ttgatgtatg cagatatgat ggagggcaga 9180
gatatggtca acataatatc aaccacggcg gtgcatctca gaagcttatc agagaaaatt 9240
gatgacattt tgcggttaat agacgctctg gcaaaagact tgggtaatca agtctacgat 9300
gttgtatcac taatggaggg atttgcatac ggagctgtcc agctactcga gccgtcaggt 9360
acatttgcag gagatttctt cgcattcaac ctgcaggagc ttaaagacat tctaattggc 9420
ctcctcccca atgatatagc agaatccgtg actcatgcaa tcgctactgt attctctggt 9480
ttagaacaga atcaagcagc tgagatgttg tgtctgttgc gtctgtgggg tcacccactg 9540
cttgagtccc gtattgcagc aaaggcagtc aggagccaaa tgtgcgcacc gaaaatggta 9600
gactttgata tgatccttca ggtactgtct ttcttcaagg gaacaatcat caacgggtac 9660
agaaagaaga atgcaggtgt gtggccgcga gtcaaagtgg atacaatata tgggaaggtc 9720
attgggcaac tacatgcaga ttcagcagag atttcacacg atatcatgtt gagagagtat 9780
aagagtttat ctgcacttga atttgagcca tgtatagaat atgaccctgt caccaacctg 9840
agcatgttcc taaaagacaa ggcaatcgca caccccaacg ataattggct tgcctcgttt 9900
aggcggaacc ttctctccga agaccagaag aaacatgtaa aagaagcaac ttcgactaat 9960
cgcctcttga tagagttttt agagtcaaat gattttgatc catataaaga gatggaatat 10020
ctgacgaccc ttgagtacct tagagatgac aatgtggcag tatcatactc gctcaaggag 10080
aaggaagtga aagttaatgg acggatcttc gctaagctga caaagaagtt aaggaactgt 10140
caggtgatgg cggaagggat cctagccgat cagattgcac ctttctttca gggaaatgga 10200
gtcattcagg atagcatatc cttgaccaag agtatgctag cgatgagtca actgtctttt 10260
aacagcaata agaaacgtat cactgactgt aaagaaagag tatcttcaaa ccgcaatcat 10320
gatccgaaaa gcaagaaccg tcggagagtt gcaaccttca taacaactga cctgcaaaag 10380
tactgtctta attggagata tcagacaatc aaattgttcg ctcatgccat caatcagttg 10440
atgggcctac ctcacttctt cgaatggatt cacctaagac tgatggacac tacgatgttc 10500
gtaggagacc ctttcaatcc tccaagtgac cctactgact gtgacctctc aagagtccct 10560
aatgatgaca tatatattgt cagtgccaga gggggtatcg aaggattatg ccagaagcta 10620
tggacaatga tctcaattgc tgcaatccaa cttgctgcag ctagatcgca ttgtcgtgtt 10680
gcctgtatgg tacagggtga taatcaagta atagcagtaa cgagagaggt aagatcagac 10740
gactctccgg agatggtgtt gacacagttg catcaagcca gtgataattt cttcaaggaa 10800
ttaattcatg tcaatcattt gattggccat aatttgaagg atcgtgaaac catcaggtca 10860
gacacattct tcatatacag caaacgaatc ttcaaagatg gagcaatcct cagtcaagtc 10920
ctcaaaaatt catctaaatt agtgctagtg tcaggtgatc tcagtgaaaa caccgtaatg 10980
tcctgtgcca acattgcctc tactgtagca cggctatgcg agaacgggct tcccaaagac 11040
ttctgttact atttaaacta tataatgagt tgtgtgcaga catactttga ctctgagttc 11100
tccatcacca acaattcgca ccccgatctt aatcagtcgt ggattgagga catctctttt 11160
gtgcactcat atgttctgac tcctgcccaa ttagggggac tgagtaacct tcaatactca 11220
aggctctaca ctagaaatat cggtgacccg gggactactg cttttgcaga gatcaagcga 11280
ctagaagcag tgggattact gagtcctaac attatgacta atatcttaac taggccgcct 11340
gggaatggag attgggccag tctgtgcaac gacccatact ctttcaattt tgagactgtt 11400
gcaagcccaa atattgttct taagaaacat acgcaaagag tcctatttga aacttgttca 11460
aatcccttat tgtctggagt gcacacagag gataatgagg cagaagagaa ggcattggct 11520
gaattcttgc ttaatcaaga ggtgattcat ccccgcgttg cgcatgccat catggaggca 11580
agctctgtag gtaggagaaa gcaaattcaa gggcttgttg acacaacaaa caccgtaatt 11640
aagattgcgc ttactaggag gccattaggc atcaagaggc tgatgcggat agtcaattat 11700
tctagcatgc atgcaatgct gtttggagac gatgtttttt cctccagtag atccaaccac 11760
cccttagtct cttctaatat gtgttctctg acactggcag actatgcacg gaatagaagc 11820
tggtcacctt tgacgggagg caggaaaata ctgggtgtat ctaatcctga tacgatagaa 11880
ctcgtagagg gtgagattct tagtgtaagc ggagggtgta caagatgtga cagcggagat 11940
gaacaattta cttggttcca tcttccaagc aatatagaat tgaccgatga caccagcaag 12000
aatcctccga tgagggtacc atatctcggg tcaaagacac aggagaggag agctgcctca 12060
cttgcaaaaa tagctcatat gtcgccacat gtaaaggctg ccctaagggc atcatccgtg 12120
ttgatctggg cttatgggga taatgaagta aattggactg ctgctcttac gattgcaaaa 12180
tctcggtgta atgtaaactt agagtatctt cggttactgt cccctttacc cacggctggg 12240
aatcttcaac atagactaga tgatggtata actcagatga cattcacccc tgcatctctc 12300
tacagggtgt caccttacat tcacatatcc aatgattctc aaaggctgtt cactgaagaa 12360
ggagtcaaag aggggaatgt ggtttaccaa cagatcatgc tcttgggttt atctctaatc 12420
gaatcgatct ttccaatgac aacaaccagg acatatgatg agatcacact gcacctacat 12480
agtaaattta gttgctgtat cagagaagca cctgttgcgg ttcctttcga gctacttggg 12540
gtggtaccgg aactgaggac agtgacctca aataagttta tgtatgatcc tagccctgta 12600
tcggagggag actttgcgag acttgactta gctaccttca agagttatga gcttaatctg 12660
gagtcatatc ccacgataga gctaatgaac attctttcaa tatccagcgg gaagttgatt 12720
ggccagtctg tggtttctta tgatgaagat acctccataa agaatgacgc cataatagtg 12780
tatgacaata cccgaaattg gatcagtgaa gctcagaatt cagatgtggt ccgcctattt 12840
gaatatgcag cacttgaagt gctcctcgac tgttcttacc aactctatta cctgagagta 12900
agaggcctag acaatattgt cttatatatg ggtgatttat acaagaatat gccaggaatt 12960
ctactttcca acattgcagc tacaatatct catcccgtca ttcattcaag gttacatgca 13020
gtgggcctgg tcaaccatga cggatcacac caacttgcag atacggattt tatcgaaatg 13080
tctgcaaaac tattagtatc ttgcacccga cgtgtgatct ccggcttata ttcaggaaat 13140
aagtatgatc tgctgttccc atctgtctta gatgataacc tgaatgagaa gatgcttcag 13200
ctgatatccc ggttatgctg tctgtacacg gtactctttg ctacaacaag agaaatcccg 13260
aaaataagag gcttaactgc agaagagaaa tgttcaatac tcactgagta tttactgtcg 13320
gatgctgtga aaccattact tagccccgat caagtgagct ctatcatgtc tcctaacata 13380
attacattcc cagctaatct gtactacatg tctcggaaga gcctcaattt gatcagggaa 13440
agggaggaca gggatactat cctggcgttg ttgttccccc aagagccatt attagagttc 13500
ccttctgtgc aagatattgg tgctcgagtg aaagatccat tcacccgaca acctgcggca 13560
tttttgcaag agttagattt gagtgctcca gcaaggtatg acgcattcac acttagtcag 13620
attcatcctg aactcacatc tccaaatccg gaggaagact acttagtacg atacttgttc 13680
agagggatag ggactgcatc ttcctcttgg tataaggcat ctcatctcct ttctgtaccc 13740
gaggtaagat gtgcaagaca cgggaactcc ttatacttag ctgaagggag cggagccatc 13800
atgagtcttc tcgaactgca tgtaccacat gaaactatct attacaatac gctcttttca 13860
aatgagatga accccccgca acgacatttc gggccgaccc caactcagtt tttgaattca 13920
gttgtttata ggaatctaca ggcggaggta acatgcaaag atggatttgt ccaagagttc 13980
cgtccattat ggagagaaaa tacagaggaa agtgacctga cctcagataa agtagtgggg 14040
tatattacat ctgcagtgcc ctacagatct gtatcattgc tgcattgtga cattgaaatt 14100
cctccagggt ccaatcaaag cttactagat caactagcta tcaatttatc tctgattgcc 14160
atgcattctg taagggaggg cggggtagta atcatcaaag tgttgtatgc aatgggatac 14220
tactttcatc tactcatgaa cttgtttgct ccgtgttcca caaaaggata tattctctct 14280
aatggttatg catgtcgagg agatatggag tgttacctgg tatttgtcat gggttacctg 14340
ggcgggccta catttgtaca tgaggtggtg aggatggcga aaactctggt gcagcggcac 14400
ggtacgcttt tgtctaaatc agatgagatc acactgacca ggttattcac ctcacagcgg 14460
cagcgtgtga cagacatcct atccagtcct ttaccaagat taataaagta cttgaggaag 14520
aatattgaca ctgcgctgat tgaagccggg ggacagcccg tccgtccatt ctgtgcggag 14580
agtctggtga gcacgctagc gaacataact cagataaccc agatcatcgc tagtcacatt 14640
gacacagtta tccggtctgt gatatatatg gaagctgagg gtgatctcgc tgacacagta 14700
tttctattta ccccttacaa tctctctact gacgggaaaa agaggacatc acttaaacag 14760
tgcacgaggc agatcctaga ggttacaata ctaggtctta gagtcgaaaa tctcaataaa 14820
ataggcgata taatcagcct agtgcttaaa ggcatgatct ccatggagga ccttatccca 14880
ctaaggacat acttgaagca tagtacctgc cctaaatatt tgaaggctgt cctaggtatt 14940
accaaactca aagaaatgtt tacagacact tctgtactgt acttgactcg tgctcaacaa 15000
aaattctaca tgaaaactat aggcaatgca gtcaaaggat attacagtaa ctgtgactct 15060
taacgaaaat cacatattaa taggctcctt ttttggccaa ttgtattctt gttgatttta 15120
tcatattatg ttagaaaaaa gttgaaccct gactccttag gactcgaatt cgaactcaaa 15180
taaatgtctt aaaaaaaggt tgcgcacaat tattcttgag tgtagtctcg tcattcacca 15240
aatctttgtt tggtggccgg catggtccca gcctcctcgc tggcgccggc tgggcaacat 15300
tccgagggga ccgtcccctc ggtaatggcg aatgggacct agcataaccc cttggggcct 15360
ctaaacgggt cttgaggggt tttttgccgt cgaccaattc tcatgtttga cagcttatca 15420
tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcaa ccaggcgttt 15480
aagggcacca ataactgcct taaaaaaatt acgccccgcc ctgccactca tcgcagtact 15540
gttgtaattc attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct 15600
gaatcgccag cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa 15660
cgggggcgaa gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc 15720
agggattggc tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt 15780
tttcaccgta acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt 15840
ggtattcact ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag 15900
ggtgaacact atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat 15960
gagcattcat caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt 16020
tctttacggt ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt 16080
gagcaactga ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg 16140
tggtatatcc agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata 16200
actcaaaaaa tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta 16260
cgtgccgatc aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag 16320
ggacaccagg atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcgcgat 16380
aagctcatgg agcggcgtaa ccgtcgcaca ggaaggacag agaaagcgcg gatctgggaa 16440
gtgacggaca gaacggtcag gacctggatt ggggaggcgg ttgccgccgc tgctgctgac 16500
ggtgtgacgt tctctgttcc ggtcacacca catacgttcc gccattccta tgcgatgcac 16560
atgctgtatg ccggtatacc gctgaaagtt ctgcaaagcc tgatgggaca taagtccatc 16620
agttcaacgg aagtctacac gaaggttttt gcgctggatg tggctgcccg gcaccgggtg 16680
cagtttgcga tgccggagtc tgatgcggtt gcgatgctga aacaattatc ctgagaataa 16740
atgccttggc ctttatatgg aaatgtggaa ctgagtggat atgctgtttt tgtctgttaa 16800
acagagaagc tggctgttat ccactgagaa gcgaacgaaa cagtcgggaa aatctcccat 16860
tatcgtagag atccgcatta ttaatctcag gagcctgtgt agcgtttata ggaagtagtg 16920
ttctgtcatg atgcctgcaa gcggtaacga aaacgatttg aatatgcctt caggaacaat 16980
agaaatcttc gtgcggtgtt acgttgaagt ggagcggatt atgtcagcaa tggacagaac 17040
aacctaatga acacagaacc atgatgtggt ctgtcctttt acagccagta gtgctcgccg 17100
cagtcgagcg acagggcgaa gccctcgagt gagcgaggaa gcaccaggga acagcactta 17160
tatattctgc ttacacacga tgcctgaaaa aacttccctt ggggttatcc acttatccac 17220
ggggatattt ttataattat tttttttata gtttttagat cttctttttt agagcgcctt 17280
gtaggccttt atccatgctg gttctagaga aggtgttgtg acaaattgcc ctttcagtgt 17340
gacaaatcac cctcaaatga cagtcctgtc tgtgacaaat tgcccttaac cctgtgacaa 17400
attgccctca gaagaagctg ttttttcaca aagttatccc tgcttattga ctctttttta 17460
tttagtgtga caatctaaaa acttgtcaca cttcacatgg atctgtcatg gcggaaacag 17520
cggttatcaa tcacaagaaa cgtaaaaata gcccgcgaat cgtccagtca aacgacctca 17580
ctgaggcggc atatagtctc tcccgggatc aaaaacgtat gctgtatctg ttcgttgacc 17640
agatcagaaa atctgatggc accctacagg aacatgacgg tatctgcgag atccatgttg 17700
ctaaatatgc tgaaatattc ggattgacct ctgcggaagc cagtaaggat atacggcagg 17760
cattgaagag tttcgcgggg aaggaagtgg ttttttatcg ccctgaagag gatgccggcg 17820
atgaaaaagg ctatgaatct tttccttggt ttatcaaacg tgcgcacagt ccatccagag 17880
ggctttacag tgtacatatc aacccatatc tcattccctt ctttatcggg ttacagaacc 17940
ggtttacgca gtttcggctt agtgaaacaa aagaaatcac caatccgtat gccatgcgtt 18000
tatacgaatc cctgtgtcag tatcgtaagc cggatggctc aggcatcgtc tctctgaaaa 18060
tcgactggat catagagcgt taccagctgc ctcaaagtta ccagcgtatg cctgacttcc 18120
gccgccgctt cctgcaggtc tgtgttaatg agatcaacag cagaactcca atgcgcctct 18180
catacattga gaaaaagaaa ggccgccaga cgactcatat cgtattttcc ttccgcgata 18240
tcacttccat gacgacagga tagtctgagg gttatctgtc acagatttga gggtggttcg 18300
tcacatttgt tctgacctac tgagggtaat ttgtcacagt tttgctgttt ccttcagcct 18360
gcatggattt tctcatactt tttgaactgt aatttttaag gaagccaaat ttgagggcag 18420
tttgtcacag ttgatttcct tctctttccc ttcgtcatgt gacctgatat cgggggttag 18480
ttcgtcatca ttgatgaggg ttgattatca cagtttatta ctctgaattg gctatccgcg 18540
tgtgtacctc tacctggagt ttttcccacg gtggatattt cttcttgcgc tgagcgtaag 18600
agctatctga cagaacagtt cttctttgct tcctcgccag ttcgctcgct atgctcggtt 18660
acacggctgc ggcgagcgct agtgataata agtgactgag gtatgtgctc ttcttatctc 18720
cttttgtagt gttgctctta ttttaaacaa ctttgcggtt ttttgatgac tttgcgattt 18780
tgttgttgct ttgcagtaaa ttgcaagatt taataaaaaa acgcaaagca atgattaaag 18840
gatgttcaga atgaaactca tggaaacact taaccagtgc ataaacgctg gtcatgaaat 18900
gacgaaggct atcgccattg cacagtttaa tgatgacagc ccggaagcga ggaaaataac 18960
ccggcgctgg agaataggtg aagcagcgga tttagttggg gtttcttctc aggctatcag 19020
agatgccgag aaagcagggc gactaccgca cccggatatg gaaattcgag gacgggttga 19080
gcaacgtgtt ggttatacaa ttgaacaaat taatcatatg cgtgatgtgt ttggtacgcg 19140
attgcgacgt gctgaagacg tatttccacc ggtgatcggg gttgctgccc ataaaggtgg 19200
cgtttacaaa acctcagttt ctgttcatct tgctcaggat ctggctctga aggggctacg 19260
tgttttgctc gtggaaggta acgaccccca gggaacagcc tcaatgtatc acggatgggt 19320
accagatctt catattcatg cagaagacac tctcctgcct ttctatcttg gggaaaagga 19380
cgatgtcact tatgcaataa agcccacttg ctggccgggg cttgacatta ttccttcctg 19440
tctggctctg caccgtattg aaactgagtt aatgggcaaa tttgatgaag gtaaactgcc 19500
caccgatcca cacctgatgc tccgactggc cattgaaact gttgctcatg actatgatgt 19560
catagttatt gacagcgcgc ctaacctggg tatcggcacg attaatgtcg tatgtgctgc 19620
tgatgtgctg attgttccca cgcctgctga gttgtttgac tacacctccg cactgcagtt 19680
tttcgatatg cttcgtgatc tgctcaagaa cgttgatctt aaagggttcg agcctgatgt 19740
acgtattttg cttaccaaat acagcaatag taatggctct cagtccccgt ggatggagga 19800
gcaaattcgg gatgcctggg gaagcatggt tctaaaaaat gttgtacgtg aaacggatga 19860
agttggtaaa ggtcagatcc ggatgagaac tgtttttgaa caggccattg atcaacgctc 19920
ttcaactggt gcctggagaa atgctctttc tatttgggaa cctgtctgca atgaaatttt 19980
cgatcgtctg attaaaccac gctgggagat tagataatga agcgtgcgcc tgttattcca 20040
aaacatacgc tcaatactca accggttgaa gatacttcgt tatcgacacc agctgccccg 20100
atggtggatt cgttaattgc gcgcgtagga gtaatggctc gcggtaatgc cattactttg 20160
cctgtatgtg gtcgggatgt gaagtttact cttgaagtgc tccggggtga tagtgttgag 20220
aagacctctc gggtatggtc aggtaatgaa cgtgaccagg agctgcttac tgaggacgca 20280
ctggatgatc tcatcccttc ttttctactg actggtcaac agacaccggc gttcggtcga 20340
agagtatctg gtgtcataga aattgccgat gggagtcgcc gtcgtaaagc tgctgcactt 20400
accgaaagtg attatcgtgt tctggttggc gagctggatg atgagcagat ggctgcatta 20460
tccagattgg gtaacgatta tcgcccaaca agtgcttatg aacgtggtca gcgttatgca 20520
agccgattgc agaatgaatt tgctggaaat atttctgcgc tggctgatgc ggaaaatatt 20580
tcacgtaaga ttattacccg ctgtatcaac accgccaaat tgcctaaatc agttgttgct 20640
cttttttctc accccggtga actatctgcc cggtcaggtg atgcacttca aaaagccttt 20700
acagataaag aggaattact taagcagcag gcatctaacc ttcatgagca gaaaaaagct 20760
ggggtgatat ttgaagctga agaagttatc actcttttaa cttctgtgct taaaacgtca 20820
tctgcatcaa gaactagttt aagctcacga catcagtttg ctcctggagc gacagtattg 20880
tataagggcg ataaaatggt gcttaacctg gacaggtctc gtgttccaac tgagtgtata 20940
gagaaaattg aggccattct taaggaactt gaaaagccag caccctgatg cgaccacgtt 21000
ttagtctacg tttatctgtc tttacttaat gtcctttgtt acaggccaga aagcataact 21060
ggcctgaata ttctctctgg gcccactgtt ccacttgtat cgtcggtctg ataatcagac 21120
tgggaccacg gtcccactcg tatcgtcggt ctgattatta gtctgggacc acggtcccac 21180
tcgtatcgtc ggtctgatta ttagtctggg accacggtcc cactcgtatc gtcggtctga 21240
taatcagact gggaccacgg tcccactcgt atcgtcggtc tgattattag tctgggacca 21300
tggtcccact cgtatcgtcg gtctgattat tagtctggga ccacggtccc actcgtatcg 21360
tcggtctgat tattagtctg gaaccacggt cccactcgta tcgtcggtct gattattagt 21420
ctgggaccac ggtcccactc gtatcgtcgg tctgattatt agtctgggac cacgatccca 21480
ctcgtgttgt cggtctgatt atcggtctgg gaccacggtc ccacttgtat tgtcgatcag 21540
actatcagcg tgagactacg attccatcaa tgcctgtcaa gggcaagtat tgacatgtcg 21600
tcgtaacctg tagaacggag taacctcggt gtgcggttgt atgcctgctg tggattgctg 21660
ctgtgtcctg cttatccaca acattttgcg cacggttatg tggacaaaat acctggttac 21720
ccaggccgtg ccggcacgtt aaccgggctg catccgatgc aagtgtgtcg ctgtcgacga 21780
gctcgcgagc tcggacatga ggttgccccg tattcagtgt cgctgatttg tattgtctga 21840
agttgttttt acgttaagtt gatgcagatc aattaatacg atacctgcgt cataattgat 21900
tatttgacgt ggtttgatgg cctccacgca cgttgtgata tgtagatgat aatcattatc 21960
actttacggg tcctttccgg tgatccgaca ggttacgggg cggcgacctc gcgggttttc 22020
gctatttatg aaaattttcc ggtttaaggc gtttccgttc ttcttcgtca taacttaatg 22080
tttttattta aaataccctc tgaaaagaaa ggaaacgaca ggtgctgaaa gcgagctttt 22140
tggcctctgt cgtttccttt ctctgttttt gtccgtggaa tgaacaatgg aagtccgagc 22200
tcatcgctaa taacttcgta tagcatacat tatacgaagt tatattcgat 22250

Claims (6)

1.一种新城疫疫苗株，其特征在于，分类名为新城疫病毒(Newcastle disease virus)，保藏编号为：CCTCC No：V202235；保藏日期：2022年5月7日，保藏单位：武汉大学中国典型培养物保藏中心。
2.一种禽类免疫识别方法，其特征在于，将禽类血清与测试蛋白反应，根据反应结果，判断检测对象体内是否具有针对于如权利要求1所述疫苗株的抗体；
所述测试蛋白为494DEQARLNPVSAVFDNI509-BSA藕连蛋白。
3.一种如权利要求1所述的疫苗株的构建方法，其特征在于，用VII型毒株GM株的F和HN基因替换掉LaSota疫苗株的F和HN基因，得到疫苗株；
所述方法具体包括如下步骤：
步骤1：以含VII型毒株GM株F和HN基因的质粒为模板，利用引物GM-F-F和引物GM-ZR-R扩增获得F蛋白裂解位点突的片段1；利用引物GM-ZR-F和引物GM-HN-R扩增获得F蛋白裂解位点突的片段2；
步骤2：以pBeloBAC11-LaSota全长cDNA克隆质粒为模板，利用引物ZT-L+GM-F和引物ZT-L-R扩增获得骨架基因片段3；
步骤3：将片段1、片段2、片段3同源重组得到以LaSota株为骨架，嵌合GM株F基因突变和HN基因的重组cDNA克隆；
步骤4：利用引物HN2-F和引物L+GM-R15186扩增重组cDNA克隆获得HN基因突变的片段4，利用引物L+GM-F15158和引物HN2-R扩增重组cDNA克隆获得骨架片段5，利用同源重组试剂盒连接片段4和5，提取全长cDNA质粒，将突变后构建质粒的命名为pBeloBAC11-La-mGM；
步骤5：将全长质粒pBeloBAC11-La-mGM和辅助质粒对细胞转染并接种鸡胚，得到疫苗株；
所述引物GM-F-F、引物GM-ZR-R、引物GM-ZR-F、引物GM-HN-R、引物ZT-L+GM-F、引物ZT-L-R、引物HN2-F、引物L+GM-R15186、引物L+GM-F15158、引物HN2-R的核苷酸序列如SEQ IDNO：1～SEQ ID NO：10所示。
4.根据权利要求3所述的疫苗株的构建方法，其特征在于，所述步骤5具体为：以4：2：1：1的比例依次加入全长质粒pBeloBAC11-La-mGM和辅助质粒pCI-NP、pCI-P、pCI-L，利用LTX试剂转染BSR-T7/5细胞，转染24 h后，加入终浓度为1 μg/μL的TPCK胰酶继续培养，转染60h后，冻融1次，将细胞混合物接种至9日龄SPF鸡胚尿囊腔，培养72h对尿囊液进行血凝活性检测，有HA效价的尿囊液即为拯救成功的重组病毒即疫苗株。
5.如权利要求1所述的疫苗株在新城疫疫苗制备中的应用。
6.含如权利要求1所述的疫苗株的生物制品。
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