CN112779227B - 一株嵌合犬瘟热病毒株及其构建方法和应用 - Google Patents

一株嵌合犬瘟热病毒株及其构建方法和应用 Download PDF

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CN112779227B
CN112779227B CN202110100419.4A CN202110100419A CN112779227B CN 112779227 B CN112779227 B CN 112779227B CN 202110100419 A CN202110100419 A CN 202110100419A CN 112779227 B CN112779227 B CN 112779227B
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薛向红
闫喜军
卜研
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Abstract

本发明涉及疫苗技术领域，公开了一株嵌合犬瘟热病毒株及其构建方法和应用。本发明所述嵌合犬瘟热病毒株以狐源犬瘟热病毒强毒HBF‑1株为基础，将其H基因替换为犬瘟热病毒疫苗株Onderstepoort的H基因。本发明利用反向遗传操作系统，将狐源犬瘟热病毒强毒HBF‑1的H基因替换为疫苗毒Onderstepoort的H基因，通过病毒拯救获得了一株嵌合犬瘟热病毒株rHBF‑vacH，该病毒可在Vero‑dSlam中稳定传代，且病毒滴度稳定达到107 . 0TCID50/ml，超过亲本毒株的105 . 0TCID50/ml，未来应用于犬瘟热病疫苗开发将会具有极大优势。

Description

一株嵌合犬瘟热病毒株及其构建方法和应用
技术领域
本发明涉及疫苗技术领域，具体的说是涉及一株嵌合犬瘟热病毒株及其构建方法和应用。
背景技术
犬瘟热弱毒活疫苗的广泛应用已经半个多世纪，防控效果虽然不错，但近年来仍然出现了多起动物感染犬瘟热发生死亡的报道。疫苗产品用的犬瘟热病毒(Caninedistemper virus,CDV)毒株，是通过连续鸡胚和细胞系传代致弱获得的，其基因型属于美洲I型。我国犬瘟热病毒流行毒株主要是亚洲I型，并呈现多基因型流行特征。犬瘟热病毒疫苗毒株Onderstepoort的H蛋白C末端提早出现了终止密码子，共编码氨基酸604个。犬瘟热病毒强毒株HBF-1的H蛋白则编码607个氨基酸。
2010年Bevan Sawatsky和Veronika von Messling构建了将强毒株CDV5804的H基因替换为onderstepoort H基因的重组病毒5804P-HOL，发现此重组病毒完全减毒，其最高滴度与亲本强毒CDV5804的基本一致。如果能够获得稳定传代且保持较高病毒滴度的犬瘟热病毒株，未来应用于疫苗开发将会具有极大优势。
发明内容
有鉴于此，本发明的目的在于提供一株嵌合犬瘟热病毒株及其构建方法，使得所述嵌合犬瘟热病毒株能够稳定传代并具备较高的病毒滴度；
本发明的另外一个目的在于提供上述嵌合犬瘟热病毒株在制备疫苗中的应用。
为了实现上述目的，本发明提供如下技术方案：
一株嵌合犬瘟热病毒株，以狐源犬瘟热病毒强毒HBF-1株为基础，将其H基因替换为犬瘟热病毒疫苗株Onderstepoort的H基因。
为了方便犬瘟热病毒疫苗株Onderstepoort的H基因的替换，本发明首先通过狐源犬瘟热病毒强毒HBF-1株(以下简写为HBF-1或wt HBF-1)的反向遗传系统构建其重组狐源犬瘟热病毒强毒HBF-1株(以下简写为rHBF-1)，具体操作如下：
以HBF-1株总RNA为模板，用RT-PCR方法扩增获得表达HBF-1株N蛋白、P蛋白、M蛋白、F蛋白、H蛋白和L蛋白的序列，将这些序列顺次拼接、插入到真核表达载体中，获得HBF-1株的全长cDNA重组质粒，同时构建表达HBF-1株N、P、L蛋白的三个辅助质粒，与全长cDNA重组质粒一起转染细胞进行病毒拯救，获得rHBF-1株。
与HBF-1株比，rHBF-1株的基因组中会多出一个或多个酶切位点(根据选择的载体确定)，其他方面均与HBF-1相同。作为优选，所述表达载体为市售载体pcDNA3.1(质粒图谱见图1)或经由pcDNA3.1改造多克隆位点而获得的pcDNA3.2(质粒图谱见图2)；所述HBF-1株N、P、L蛋白的三个辅助质粒是指表达HBF-1株N、P、L蛋白的序列插入到载体质粒的多克隆位点形成的质粒；例如HBF-N插入位点为PmeI处；HBF-P插入位点为NotI处；HBF-L插入位点为PmeI处。
在本发明具体实施方式中，所述反向遗传系统构建过程如下：
以适应Vero-dSlam细胞的狐源犬瘟热病毒强毒HBF-1株为模板，构建犬瘟热病毒感染性cDNA克隆。获得病毒的全基因组序列后，用RT-PCR方法，分4个长片段(N+P基因序列、M+F基因序列、H基因序列和L基因序列)扩增犬瘟热病毒的全基因组，通过酶切连接和无缝克隆的有机结合，将4个长片段顺次拼接、插入到真核表达载体pcDNA3.2(由pcDNA3.1改造多克隆位点而获得)的多克隆酶切位点处(见图3)。经过酶切鉴定，确定获得狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒(pcDNA3.2-HBF-1)，同时构建表达犬瘟热病毒强毒HBF-1株N、P、L蛋白的三个辅助质粒(pcDNA3.1-HBF-N、pcDNA3.1-HBF-P和pcDNA3.1-HBF-L)。经酶切和序列测定鉴定，全长重组质粒pcDNA3.2-HBF-1序列与预期一致。将全长质粒pcDNA3.2-HBF-1 5μg和三个辅助质粒pcDNA3.1-HBF-N 1μg，pcDNA3.1-HBF-P 0.8μg和pcDNA3.1-HBF-L 0.5μg，转染试剂X-treme GENE HP DNA 22μL，共转染BSR细胞，3天后，将上清接种到Vero-dSlam细胞上，37℃5％CO2培养5-10天，观察到犬瘟热病毒引起的典型合胞体病变。经过RT-PCR、间接免疫荧光(IFA)，电镜观察鉴定，证实拯救出重组病毒rHBF-1株。
在本发明具体实施方式中，本发明以rHBF-1株为基础，将其H基因替换为犬瘟热病毒疫苗株Onderstepoort的H基因，获得嵌合病毒rHBF-1-vacH；将嵌合病毒rHBF-vacH感染Vero-dSlam细胞，观察到典型细胞病变情况，同时空白对照细胞无病变，用犬瘟热N蛋白单克隆抗体为一抗、FITC标记的羊抗鼠IgG为二抗，进行间接免疫荧光检测。结果显示，发生病变的细胞能与CDVN蛋白单克隆抗体发生特异性结合，发出绿色荧光，呈阳性信号，而空白对照细胞无荧光，为阴性。
嵌合病毒rHBF-1-vacH的上清液用于负染，在电子显微镜下观察。结果可见，病毒粒子外部有一层囊膜包裹，囊膜上含有纤突，是副黏病毒的形态特征。
此外，嵌合病毒rHBF-1-vacH可在Vero-dSlam细胞上稳定传代，嵌合病毒在第二代时有明显病毒滴度的增高。传到第九代时，病毒滴度最高可稳定达到107.0TCID50/ml，而亲本毒株rHBF-1和野生毒株wtHBF-1的病毒滴度稳定在105.0TCID50/ml。
基于上述嵌合病毒的优异性能，本发明提出了所述嵌合犬瘟热病毒株在制备犬瘟热病疫苗中的应用。
同时，本发明还提供一种构建嵌合犬瘟热病毒株的方法，该方法可按照两种不同方式进行，在第一种方式中可直接跳过扩增获得HBF-1株H基因的步骤，直接扩增获得Onderstepoort的H基因，然后构建HBF-vacH全长重组质粒，进而通过病毒拯救获得嵌合病毒；在第二种方式中，首先获得HBF-1株的全长cDNA重组质粒，然后通过酶切方式将HBF-1株的全长cDNA重组质粒上的H基因替换为Onderstepoort的H基因，构建出HBF-vacH全长重组质粒(图4)，进而通过病毒拯救获得嵌合病毒；
具体地，第一种方式方法如下：
步骤1、以狐源犬瘟热病毒强毒HBF-1株总RNA为模板，用RT-PCR方法扩增获得表达狐源犬瘟热病毒强毒HBF-1株N蛋白、P蛋白、M蛋白、F蛋白和L蛋白的序列；
以犬瘟热病毒疫苗株Onderstepoort总RNA为模板，用RT-PCR方法获得表达犬瘟热病毒疫苗株Onderstepoort的H蛋白的序列；
步骤2、将步骤1获得的表达N蛋白、P蛋白、M蛋白、F蛋白、H蛋白和L蛋白的序列顺次拼接、插入到真核表达载体中，获得替换为Onderstepoort H基因的HBF-vacH全长重组质粒；
步骤3、HBF-vacH全长重组质粒与表达狐源犬瘟热病毒强毒HBF-1株N、P、L蛋白的三个辅助质粒一起转染细胞进行病毒拯救，获得所述嵌合犬瘟热病毒株。
第二种方式方法如下：
步骤1、以狐源犬瘟热病毒强毒HBF-1株总RNA为模板，用RT-PCR方法扩增获得表达狐源犬瘟热病毒强毒HBF-1株N蛋白、P蛋白、M蛋白、F蛋白、H蛋白和L蛋白的序列；将这些序列顺次拼接、插入到真核表达载体中，获得狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒；
步骤2、以犬瘟热病毒疫苗株Onderstepoort总RNA为模板，用RT-PCR方法获得表达犬瘟热病毒疫苗株Onderstepoort的H蛋白的序列；通过酶切方式将狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒上的H基因替换为Onderstepoort的H基因，获得替换为Onderstepoort H基因的HBF-vacH全长重组质粒；
步骤3、HBF-vacH全长重组质粒与表达狐源犬瘟热病毒强毒HBF-1株N、P、L蛋白的三个辅助质粒一起转染细胞进行病毒拯救，获得所述嵌合犬瘟热病毒株。
本发明中的病毒拯救方法可参照常规方式进行，在本发明具体实施方式中将4个质粒共转染BSR细胞，然后将上清接种到Vero-dSlam细胞上培养，观察到犬瘟热病毒引起的典型合胞体病变后，获得病毒株。
由以上技术方案可知，本发明利用反向遗传操作系统，将狐源犬瘟热病毒强毒HBF-1的H基因替换为疫苗毒Onderstepoort的H基因，通过病毒拯救获得了一株嵌合犬瘟热病毒株rHBF-vacH，该病毒可在Vero-dSlam中稳定传代，且病毒滴度稳定达到107.0TCID50/ml，超过亲本毒株的105.0TCID50/ml，未来应用于犬瘟热病疫苗开发将会具有极大优势。
附图说明
图1所示为pcDNA3.1质粒图谱；
图2所示为pcDNA3.2质粒图谱；
图3所示为狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒(pcDNA3.2-HBF-1)的构建示意图；
图4所示为替换为Onderstepoort H基因的HBF-vacH全长重组质粒(pcDNA3.2-HBF-vacH)的构建示意图；
图5所示为本发明嵌合病毒rHBF-vacH的RT-PCR鉴定结果；1.wtHBF-1H基因PCR产物的Cpo I酶切结果；2.rHBF-1H基因PCR产物的Cpo I酶切结果；3.rHBF-vacHH基因PCR产物的Cpo I酶切结果；M.DL 2,000DNA marker；
图6所示为rHBF-vacH的免疫荧光鉴定结果；(A)rHBF-vacH在Vero-dSlam细胞上形成的合胞体样细胞病变(×40)；(B)阴性Vero-dSlam细胞(×40)；(C)rHBF-vacH在Vero-dSlam细胞上的免疫荧光染色(×40)；(D)阴性Vero-dSlam细胞免疫荧光染色(×40)；
图7所示为电镜下rHBF-vacH的粒子形态；
图8所示为rHBF-vacH在Vero-dSlam细胞的传代和病毒滴度。
具体实施方式
本发明实施例公开了一株嵌合犬瘟热病毒株及其构建方法和应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明内。本发明所述嵌合犬瘟热病毒株及其构建方法和应用已通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述嵌合犬瘟热病毒株及其构建方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明中所涉及的序列编号如下：
狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒(pcDNA3.2-HBF-1)序列如SEQID No.1所示，根据图3插入位点可确定狐源犬瘟热病毒强毒HBF-1株N蛋白、P蛋白、M蛋白、F蛋白、H蛋白和L蛋白编码序列；其中，N蛋白、P蛋白和L蛋白编码序列依次如SEQ ID No.2-4所示；
犬瘟热病毒疫苗株Onderstepoort的H蛋白编码序列(H基因)如SEQ ID No.5所示；
pcDNA3.1质粒全基因序列如SEQ ID No.6所示；
pcDNA3.2质粒全基因序列如SEQ ID No.7所示；
本发明所述狐源犬瘟热病毒强毒HBF-1株和犬瘟热病毒疫苗株Onderstepoort(可从疫苗中分离获得)均为本领域常用毒株，记载于各文献中，如狐源犬瘟热病毒HBF-1株全基因组序列测定及其分析[J].中国兽医学报,2020,40(03):510-516；
犬瘟热病毒弱毒和野毒的全基因组扩增及序列测定[J].中国兽医学报,2019,39(9):1710-1714；
本发明中所用载体、细胞等试验材料如未特殊说明均可以通过购买获得。
以下就本发明所提供的一株嵌合犬瘟热病毒株及其构建方法和应用做进一步说明。
实施例1：rHBF-1的构建
以适应Vero-dSlam细胞(表达Slam受体的非洲绿猴肾细胞)的狐源犬瘟热病毒强毒HBF-1株为模板，构建犬瘟热病毒感染性cDNA克隆。获得病毒的全基因组序列后，用RT-PCR方法，分4个长片段(N+P基因序列、M+F基因序列、H基因序列和L基因序列)扩增犬瘟热病毒的全基因组，通过酶切连接和无缝克隆的有机结合，将4个长片段顺次拼接、插入到真核表达载体pcDNA3.2的多克隆酶切位点处(见图3)。经过酶切鉴定，确定获得狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒(pcDNA3.2-HBF-1)。
同时构建表达犬瘟热病毒强毒HBF-1株N、P、L蛋白的三个辅助质粒(pcDNA3.1-HBF-N、pcDNA3.1-HBF-P和pcDNA3.1-HBF-L)。其中，HBF-N插入位点PmeI处；HBF-P插入位点NotI处；HBF-L插入位点PmeI处。
经酶切和序列测定鉴定，全长重组质粒pcDNA3.2-HBF-1序列与预期一致。将全长质粒pcDNA3.2-HBF-1 5μg和三个辅助质粒pcDNA3.1-HBF-N 1μg，pcDNA3.1-HBF-P 0.8μg和pcDNA3.1-HBF-L 0.5μg，转染试剂X-treme GENE HP DNA 22μL，共转染BSR细胞，3天后，将上清接种到Vero-dSlam细胞上，37℃5％CO2培养5-10天，观察到犬瘟热病毒引起的典型合胞体病变。经过RT-PCR、间接免疫荧光(IFA)，电镜观察鉴定，证实拯救出重组病毒rHBF-1株，与HBF-1比，rHBF-1的基因组中，在3370处存在Bsiw I酶切位点，而HBF-1并没有。
实施例2：嵌合病毒的构建
1、引物设计和合成
以犬瘟热病毒疫苗毒Onderstepoort为模板，设计特异性引物(见表1，序列表中如SEQ ID No.10-11所示)，用于扩增其H基因，引物送上海生工公司合成。
表1
Figure BDA0002914185320000061
Figure BDA0002914185320000071
注：下划线部分为PmeI酶切位点，加粗部分为PacI酶切位点；
2、替换疫苗毒H基因的全长重组质粒的构建
根据QIAGEN总RNA试剂盒的说明，提取Onderstepoort病毒悬液的总RNA。依据SuperScriptTMⅢFirst-Strand Synthesis SuperMix试剂盒的操作说明，对总RNA进行反转录，获得cDNA。使用引物HBF-ORF3-F/R(表1)，以Onderstepoort的反转录产物cDNA为模板，用高保真DNA聚合酶进行扩增，PCR产物经1％琼脂糖凝胶电泳鉴定，大小正确后凝胶回收。将回收纯化H基因目的片段连接至pEASY-Blunt，酶切鉴定正确质粒送至上海生工生物技术公司测序。测序正确的质粒pEASY-Blunt-HBF-ORF3与全长质粒pcCDV3.2-HBF-1，分别用限制酶Pme I、Pac I酶切，分别回收纯化目的与载体片段，使用T4连接酶连接，将阳性质粒送至上海生工测序，将测序正确的、替换为Onderstepoort H基因的全长重组质粒命名为pcDNA3.2-HBF-vacH(图4)。
3、嵌合病毒的拯救
于六孔板中，当BSR细胞愈合至50％-60％时，将全长重组质粒pcDNA3.2-HBF-vacH与辅助质粒pcDNA3.1-HBF-N、pcDNA3.1-HBF-P、pcDNA3.1-HBF-L分别按5μg、1μg、0.8μg、0.5μg的比例进行共转染，使用X-treme GENE HP DNA转染试剂22μL，具体转染方法参照说明操作。48h后，取细胞悬液大约300μL，添加至密度约为80％的Vero-dSlam细胞上，35℃5％CO2培养5-10天，观察CDV引起的典型合胞体病变(CPE)。待出现CPE后，冻融收集细胞及上清，并继续接种Vero-dSlam细胞，进行传代。将获得的重组病毒命名为rHBF-vacH。
4、rHBF-vacH的RT-PCR鉴定
利用RNA提取试剂盒，分别提取wtHBF-1、rHBF-1、rHBF-vacH病毒悬液的总RNA，反转录获得cDNA。利用引物F3-F：TTCCTTCAAAAGCGTTTAAACTGCAACAAATAGTGGCG和F3-R：GGATTAATTAACACGGTCATCATCCCTCAGTTCAATTGA扩增wtHBF-1和rHBF-1的H基因，用引物表1中HBF-ORF3-F/R扩增rHBF-vacH的H基因。将三个PCR产物分别回收纯化后，用限制酶Cpo I酶切。OnderstepoortH基因中有Cpo I酶切位点，而wtHBF-1、rHBF-1的H基因中无此识别位点。同时，对从rHBF-vacH扩增获得的H基因片段进行序列测定，确定其来源于Onderstepoort毒株。
电泳结果表明，wtHBF-1、rHBF-1的H基因PCR产物不能被限制酶Cpo I切开，酶切产物仍然为2000bp大小条带，而因为Onderstepoort疫苗株的H基因含有Cpo I酶切位点，因此，嵌合病毒rHBF-vacH的H基因目的条带被切分为两条带，分别是1400bp和600bp(如图5)。
5、rHBF-vacH的免疫荧光鉴定
将收获的rHBF-vacH病毒悬液，接种于Vero-dSlam细胞，待细胞出现CPE后，以4％多聚甲醛固定，一抗用抗CDV N蛋白的单克隆抗体，二抗用FITC标记羊抗鼠IgG，在荧光显微镜的蓝紫光下观察绿色荧光。
将嵌合病毒rHBF-vacH感染Vero-dSlam细胞，观察到典型细胞病变情况(图6A)，同时空白对照细胞无病变(图6B)，用犬瘟热N蛋白单克隆抗体为一抗、FITC标记的羊抗鼠IgG为二抗，进行间接免疫荧光检测。结果显示，发生病变的细胞能与CDVN蛋白单克隆抗体发生特异性结合，发出绿色荧光，呈阳性信号(图6C)，而空白对照细胞无荧光，为阴性(图6D)。
6、rHBF-vacH的形态鉴定
嵌合病毒rHBF-vacH的上清液用于负染，在电子显微镜下观察。结果可见，病毒粒子外部有一层囊膜包裹，囊膜上含有纤突，是副黏病毒的形态特征(图7)。
7、rHBF-vacH的传代特性
为确定嵌合病毒rHBF-vacH在体外是否稳定遗传，将rHBF-vacH在Vero-dSlam细胞上传代9次，取每一代病毒液300μL，测定其TCID50/ml，使用软件GrapPad Prism 6.0绘制病毒传代图谱。
图8所示，rHBF-vacH嵌合病毒可在Vero-dSlam细胞上稳定传代，嵌合病毒在第二代时有明显病毒滴度的增高。传到第九代时，病毒滴度最高可稳定达到107.0TCID50/ml，而亲本毒株rHBF-1和野生毒株wtHBF-1的病毒滴度经测定后稳定在105.0TCID50/ml。
以上所述只是用于理解本发明的方法及其核心思想，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利的保护范围。
序列表
<110> 中国农业科学院特产研究所
<120> 一株嵌合犬瘟热病毒株及其构建方法和应用
<130> MP2035036
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21170
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttgcggcc gctgttaagc gtctgatgag tccgtgagga cgaaactata ggaaaggaat 960
tcctatagtc accagacaaa gttggctaag gatagttaaa ttattggata ttttattaaa 1020
aacttagggt caatgatcct accttagaga acaaggtcag ggttcagacc taccagtatg 1080
gctagccttc tcaagagcct cacattgttc aagaggactc gggaccaacc cccacttgcc 1140
tcgggctccg gaggagcaat aagagggata aagcatgtca ttatagtcct aatcccgggt 1200
gattcaagca ttgttacaag gtctcgacta ctggatagac ttgttagatt ggtcggtgat 1260
ccggaaatca acggacctaa attaactggg attttaatca gtatcctctc cttgttcgtg 1320
gaatcccctg gacagttgat ccagaggatc atagacgacc ctgatgtaag catcaagtta 1380
gtagaggtaa tcccaagcat caactctggt tgtggtctta catttgcatc cagaggagca 1440
agtttggatt ctgaggcaga tgagttcttc aaaattgtag acgaagggtc gaaagctcaa 1500
ggacaattag gctggttgga gaataaggat attgtagaca tagaagttga tgatgctgag 1560
caattcaata tattgctagc ttccatcttg gcccaaattt ggatcctgct cgctaaagca 1620
gtgactgctc ctgatactgc agccgactcg gaaatgagga ggtggattaa gtatacccaa 1680
cagagacgtg tggtcgggga atttagaatg aacaaaatct ggcttgatat tgttagaaac 1740
aggattgctg aggacctatc tttgaggcga ttcatggtgg cactcatctt ggacatcaaa 1800
cgatccccag ggaacaagcc tagaattgct gaaatgattt gtgatataga taactacatt 1860
gtggaagctg gattagctag tttcatctta actatcaaat ttggcattga aactatgtat 1920
ccggctcttg ggttgcatga gttttccgga gagttaacaa ctattgaatc ccttatgatg 1980
ctatatcaac agatgggtga aacagcaccg tacatggtta ttctggaaaa ttctgttcag 2040
aacaaattta gtgcaggatc ctacccactg ctctggagtt atgctatggg agttggtgtt 2100
gaacttgaaa actccatggg agggttaaat ttcggtagat cctactttga tccggcctat 2160
ttcaggctcg ggcaagaaat ggtgagaaga tctgccggca aagtaagctc tgcacttgcc 2220
gccgagcttg gcatcaccaa ggaagaggct cagctagtgt cagaaatagc atccaagaca 2280
acggaggacc ggacgattcg cactgctggt cccaagcaat ctcaaatcac ttttctgcac 2340
tcagaaagat ccgaagtcac caatcaacaa cccccaacca tcaacaagag gtccgaaaac 2400
caaggaggag acaaataccc catccacgtc aatgatgaac ggtttccagg gtacacccct 2460
gatgtcaaca gctccgaatg gagtgaatca cgctatgata cccagactat tcaagatgat 2520
ggaaacgacg atgaccggaa atcgatggaa gcaatcgcca agatgagaat gcttactaag 2580
atgctcagtc aacctgggac cagtgaagag agttctcctg tctataatga tagagagcta 2640
ctcaattaaa tattcaagac cagtgttaca tcagtcacca attatccttc taaactcatt 2700
ataaaaaact taggacccag gtccaacaat cccgatcaac cattcatccg accaaccatt 2760
ctatctctaa atggcagaag agcaggccta tcatgtcaat aaagggctgg aatgcctcaa 2820
agccctcaga gagaatcctc ctgacattga ggagattcaa gaggtcagca gtatcagaga 2880
tcaaacccgc gacccaggcc aagagaatgg aaccgcaagc atgcaggaag aagaggtctc 2940
tcaggatctc gatgaatcac acgagccagc aaaaggatca aactatgtcg gccatgtact 3000
ccaaaataat ccgggatgtg gagagagcaa cactgcgctt gtggaggaag agcagcccgc 3060
taaagatgat gtccaaccag gacctgaaat acgatgttat catgtttatg atcacagtgg 3120
tgaagaggtt aagggaatcg aagatgctga cagtctcgtg gtacctgcag gcgctgtcag 3180
taatcgagga ttcgagagag gagaaggaag ccttgatgat agcactgagg attctggcga 3240
agattattcc gagggaaatg cttcatctaa ctggggatat tctttcggcc ttaaaccaga 3300
cagagcggct gatgtgagca tgctgatgga agaggaattg agtactctgc tcaagacaag 3360
cagaaatgtg gggattaaga aaagggatgg gatgactctg cagttcccac acagtcccga 3420
aggtaagaca gaggatccgg agtgtggatc cattaaaaag ggcacaggag agaggtcagc 3480
ctcacatgga atggggatag ttgctggatc gacaagtggt gcaacccaat ctgcactcaa 3540
gtcaactggg ggatcatcag ggccacgtgt gtctgcggag aatgtccgcc aacctgcaat 3600
gaatgcaaag atgacccaga aatgcaaacc cgagtctggt acgcaactcc ctgccaggac 3660
ctcaaatgag gctgaatctg acagtgagta tgacgatgag cttttttctg aaatacaaga 3720
aattcgatct gctatcacta agctaatgga agataatcaa gcaatacttt ctaaactgga 3780
taccctatta ctgcttaaag gagagactga ttcaattaag aaacaaatta gcaagcaaaa 3840
tattgctatt tccacgattg aggggcatct atcaagcatt atgatagcta tacctggttt 3900
tgggaaggac actggagacc ctacggcaaa tgtcgacatt aacccagagc tccgccctat 3960
aatagggagg gattcaggaa gagcactggc ggaagttctc aagcaacccg catcatcccg 4020
cggtaatcgg aaggacagtg gtatcgcctt gggttcaaaa ggtcaactat tgagagacct 4080
ccagctgaaa cctattgaca aagagtctag ttcggcaatc ggatacaaac cgaaggatac 4140
cgcaccttcc aaagctgtac ttgcatcatt gattagatca agcaaaattg atcaaagtca 4200
caaacacaac atgctggccc ttctcaaaaa tattaagggg gatgacaatc taaacgagtt 4260
ctaccagatg atcaagagta tcacacatgc ttaagctgta gcatttacta atctattaac 4320
aggctcaaaa ctgctttcac gtacgcttaa aagcaattat aaaaaactta ggacacaaga 4380
gcctaagtcc tctccacaaa aatgactgag gtgtacgact tcgatcagtc ttcgtgggac 4440
accaaaggtt ccttggcccc cattttgccc accacctacc ccgatggtag gctagtaccc 4500
caagtcagag tgatagatcc aggactcgga gatcgaaaag atgaatgttt catgtatatt 4560
tttctactgg gtataataga agacaacgat ggcctcggac ccccgattgg aagaacattt 4620
ggatcgctgc ctttaggtgt tgggcgcact acagccagac ctgaagaatt attgaaggaa 4680
gccaccttgt tggacattgt ggtaaggcga actgcaggtg tcaaagaaca actggtattt 4740
tacaataaca ccccattgca catcttaact ccgtggaaga aggtccttac gagcgggagt 4800
gtattcagtg ctaatcaagt ctgtaacgcg gtcaatttaa taccattgga catagcacag 4860
agatttaggg tggtatatat gagcatcact cgactatcag acgatggaag ttacagaatt 4920
cctcgcggga tgtttgaatt ccgctccagg aatgctttag catttaatat tttagtcacc 4980
attcaagttg agggagatgt ctgttcaagc cgagggaatt tgagcatgtt caaagatcac 5040
caagtgacat tcatggtgca tatcggcaac ttcagtcgta agaagaacca agcttactct 5100
gctgattact gtaaacttaa aattgaaaag atgggattag tgtttgctct aggagggata 5160
ggaggaacca gtcttcacat acgatgtact ggtaagatga gcaaggcttt gaatgcccag 5220
ctaggattca agaaaatcct gtgttacccg ctcatggaga tcaacgaaga tttgaatcga 5280
tttctatgga gattagagtg caaaatagta agaatccaag cagtcttgca accatcagtc 5340
ccacaagatt tcagaattta taatgatgtt atcatcagtg atgatcaggg tcttttcaaa 5400
attctctaaa tcattagttc atgaactaaa actcaaatgc cttggtggca ttgtccagga 5460
tcccttaatc ctctcaaaca aggattgagg ctacaagtgt caattgtctc ggtgttgctc 5520
ctgcatttta agcaagttct ataggtttct aaacttctca ttcgtgccta ctattctggt 5580
gactctgcaa tacgaagaca gctgaatcaa accaattcat gctcaagagt aggttgatca 5640
ttatcggacc aagaaatgta tggatgcttg gggttttgaa attcacctct aggaatctca 5700
cttaaccaat tatacctcca cgcgcttgcc tgatctcaag ctattactag tagtcctgtt 5760
tcacgaagtt ctgactgtct atctttctat caccaatcgt taataattaa tcaaaactta 5820
ggatccagga cgcagcaagc caacaggcca accaagtcca ccaatccgag gccgggcagg 5880
aaccccaaca aacagacaag ccccatgcac aacaaaatcc ccaaaaaatc caaacccctg 5940
ccacacaccc gacaagatcc cctccaacaa cacagcacca gatccaccga gaccaagacc 6000
tcccaaggac gatatagcat aacatcggct cagcgatcca cgtaccatag tcctcgaaca 6060
tcggacaggc ccgtccacta cataatgaac aggaccaggt cttgcaagca aactagccac 6120
agatcggata acatcccgcc tcacagagac cacgagggta tcatccatca cacaccagag 6180
agtgtcaccc aaggagcgag ttcctggttc aagaggtggc aatccaatgc aaccaatgca 6240
ggctctcaat gcacctggtt agtcctgtgg tgcatcggaa tagccagtct ctttctttgt 6300
tccaaggctc agatacattg gaataatttg tcaacgattg ggattatcgg aactgacagt 6360
gtccattata agatcatgac taggcccagt caccagtact tggtcataaa actaatgcct 6420
aatgtttcac ttatagataa ttgtaccaaa gcagaattag gtgagtatga gaaattatta 6480
aattcagtcc tggagccaat caaccaagct ttgactctaa tgaccaataa tgtgaagccc 6540
ctacagtcag tagggtcagg taggagacaa aggcgttttg caggagtggt gcttgcaggt 6600
gcagctttag gggtggccac agccgcacaa atcactgcag ggatagcttt acatcagtcc 6660
aacctcaatg ctcaagcaat ccaatctctg agaactagcc ttgaacagtc caacaaggct 6720
atagaagaaa ttagggaggc aacccaggaa accgtcattg ctgttcaggg agtccaggat 6780
tacgtcaata atgaactcgt ccctgctatg caacatatgt cgtgtgagtt agttgggcag 6840
agattagggt taaaactgct taggtattac accgagttgt tgtcaatatt tggcccgagt 6900
ttacgtgacc ctatttcagc cgagatatca attcaagcac tgagttatgc tcttggggga 6960
gaaattcata agatacttga gaagttgggg tattctggta atgatatgat tgcaattttg 7020
gagagtcggg ggataaaaac aaaaataact catgtcgatc tccccgggaa actcatcata 7080
ttaagtatct catacccaac tttatcagaa gtcaaggggg ttatagtcca cagactggaa 7140
gccgtttctt ataacatagg gtcacaggag tggtacacca ctgtcccgaa gtatgttgca 7200
actaatggtt acttaatatc aaactttgat gagtcatcct gtgtattcgt ctcagaatca 7260
gccatttgta gccagaactc tctgtacccc atgagcccga ttctacaaca atgcattagg 7320
ggcgatactt catcttgtgc tcggaccttg gtgtctggga ctatgggcaa caagtttatt 7380
ctgtcaaaag gtaatatcgt tgcaaattgt gcttctatac tatgtaagtg ttatagcaca 7440
agcacaatta tcaatcagag tcctgataag ttgctgacat ttattgcctc cgatacctgc 7500
ccactggttg aaatagatgg tgtaactatc caagttggag ggaggcaata ccctgatatg 7560
gtatacgaaa gcaaagttgc cttaggacct gctatatcac ttgagaggtt ggatgtaggt 7620
acaaatttag ggaacgccct taagaaactg gatgatgcta aagtactgat agactcctct 7680
aaccagatcc ttgagacagt taagcgctct tcctttaatt ttggcagtct cctcagcgtt 7740
cccatattaa tctgtacagc tctggctttg ttgttgctaa tttactgctg taaaagacgc 7800
taccgacaga cattcaagca taatactaag gtcgatccga catttaaacc tgatttgact 7860
ggaacttcga aatcatatgt aagatcactc tgaagcactc tgatcacaag tcttacctga 7920
ttgttaggct tgaaatccat aagtcccgcc taatttcctt caaaagcgtt taaactgcaa 7980
caaatagtgg cgaggactga ctccaattat tataattaaa gaaaacttag ggctcaggta 8040
gtccaacaat gctctcttac caagacaagg tgggtgcctt ctacaaggac aatgcaagag 8100
ctaattcatc caagctgtcc ccagtgaccg aagagcaagg gggacggaga ccaccctatt 8160
tgctgtttgt ccttctcatc ctactgattg gaatcctggc cttgcttgcc atcactggag 8220
ttcgatttca ccaagtatca actagcaata tggaatttag cagattgctg aaagaggata 8280
tggagaaatc agaggccgta catcaccaag tcatagatgt cttgacaccg ctcttcaaaa 8340
ttattggaga tgagattggg ttacggttgc cacaaaaact aaacgagatc aaacaattta 8400
tccttcaaaa gacaaacttc ttcaatccga acagggaatt cgacttccgc gatctccact 8460
ggtgcattaa cccacctagc aagatcaagg tgaattttac taattactgt gatacagttg 8520
gggtcaaaaa atctattgca tcggcagcaa atcccatcat tttatcagca ctctccgggg 8580
ccagaggtga catattcccg ccgtacagat gcagtggagc tactacttca gtaggcaggg 8640
tattccccct atccgtatca ttatccatgt ctttgatatc aagaacatca gagataatca 8700
atatgctaac cgctatctca gacggagtgt atggtaaaac ttatttgcta gtgcctgatt 8760
atattgaagg ggagttcgac tcacaaaaga ttcgagtctt tgagataggg tttatcaaac 8820
ggtggctgaa tgacatgcct ttactccaga caaccaacta tatggtcctc ccggaaactt 8880
ccaaagccaa ggtatgtact atagcagtgg gtgagctgac actagcttcc ttgtgtgtag 8940
atgagagcac cgtattgtta tatcatgaca gcaacggttc acaagatggt attctagtag 9000
tgacattggg aatatttggg gcaacaccta tggatcaagt tgaagaggtg atacctaccg 9060
ctcacccatc agtggagaga atacatataa caaatcaccg tgggttcata aaagactcaa 9120
tagtaacctg gatggtgcct gtattggtct ctgagaaaca agaggagcaa aaaaactgtc 9180
tggagtctgc ttgtcacaga aaatcctacc ctatgtgcaa ccaaacgtca tgggaaccct 9240
ttggaggagg acagttgcct tcttatgggc ggttgacatt acctctagat ccaagcattg 9300
accttcaact taacatatca tttacatatg gtccggttat actgaacgga gacggtatgg 9360
attattatga aagcccactt ttggactccg gatggcttac catacctcct aagaacggga 9420
cagtccttgg attgataaac aaagcaagta gaggagacca gttcactgtg accccccatg 9480
tgttgacatt tgcgcccagg gaatcaagtg gaaattgtta tttgcctatt caaacatccc 9540
agattatgga taaagatgtc cttactgagt ccaatttagt ggtgttacct acacagaatt 9600
ttagatatgt catagcaaca tatgatatat cccggggcga tcatgcaatt gtttattatg 9660
tttatgaccc aatccggacg atttcttata catacccatt tagactaact accaagggta 9720
gacctgattt cctaaggatt gaatgttttg tgtgggatga cgatttgtgg tgtcatcaat 9780
tttaccgatt cgaggctaac atcactaact ctacaaccag tgttgagaat ttagtccgta 9840
taagattctc atgtaaccgt tcaaaacctt gatggtatgc tgatacacat ctcaattgaa 9900
ctgagggatg atgaccgtgt taattaatcc cttaccgatg attgaattaa accatcccca 9960
gcattataaa aaaactaagg atccaggatc cttttagtca tggactctgt ttcagtgaac 10020
cagattttat accctgaggt ccatctagat agcccaattg tgaccaataa gctagtggct 10080
attttagagt atgcacgaat tagacataac tatcgactcc ttgacacaac gttagtgcgt 10140
aatatcaaag agagaatttc agaagggtta tcaaaccaga tgatcattaa ctgtatcgaa 10200
attgggagca ttgttaatca gaccttgtta tcttatccca aacacaacca tgtgatatat 10260
ccaaattgca acaaacttct gtttcatgca caggatcgag tcatctctct gaggttgaga 10320
aatatattca aaagaggaaa tagcatctat agtaaaataa cagacggggt caaaaaatgc 10380
ttaaacgata ttaatcttag tattggttta ggaggtgcat tggataagac tattggggcc 10440
aaagttgatg aagcaggcat aattatgcaa agctcacagt ggttcgaacc tttccttctg 10500
tggtttacaa ttaagacaga aatgagatca gtgattaaat cctctactca caactgtcgc 10560
aaacgaaggc agaatcctgt ctttgtaaga ggtgaatcat ttaatgtgtt agtgtctcgg 10620
gatcttgtat gtatcattga cctcaccagt cacaatgttt attacctaac atttgaaatg 10680
gtcctgatgt attgtgatgt gatagaaggg agattaatga ctgataccgc tatggcaatt 10740
gatcaccgtt actcaacttt gcatgtcaga atcaggtatc tttgggatct aattgatgga 10800
tttttcccgg acttaggaaa ttcgacctat caactggtag ctctgctgga gcctctttca 10860
ttggcttact tgcaattaaa agacatcacc ttctctctca ggggtgcttt tttgagtcac 10920
tgctttgctg aaatccagga gattttacag gacaatggct tctatactga agagacattc 10980
caaaccttaa cccaggctct agactttgtt ttcatcacag aggatataca tataacagga 11040
gagatctttt ccttttttag gagtttcggt cacccaagat tagaagcaat aacagcagca 11100
gaaaatgtac ggaaacacat gaatcaaccc aaagttgtct cctatgagac catgatgaag 11160
ggacacgcta ttttctgtgg gataatcatt aacggttatc gggatagaca tggaggaact 11220
tggcctccaa tggatcttcc tgtccatgca tctcctatca tcaggaatgc tcatgcctca 11280
ggagagggaa tcacctatag tcaatgtata gaaaattgga aatcctttgc aggaattcga 11340
tttaaatgct ttatgcccct ctgcctagac agtgatctga ccatgtattt gaaagataag 11400
gctttagcag cccttaaaaa agagtgggat tcagtgtacc caaaagaatt cctcaggtac 11460
aacccacctc gctccactga atctcggaga cttgttaatg tgtttctaga ggactctcag 11520
tttgaccctt ataatatgat tatgtacgtt atctcaggac aatatctaga cgatcctgac 11580
ttcaacctat catacagtct taaagagaaa gagattaaag aagtggggag gttattcgct 11640
aaaatgacat acaaaatgcg ggcctgtcaa gtcatagcag aaaacttaat atctaatgga 11700
attgggaagt acttcaagga caatgggatg gcaaaggatg aacacgatct cactaaagca 11760
ttgcacactc tggctgtgtc cggggttcct aaagacaaga aagactccca tcgcggcctc 11820
actaaccagt gtaagtctaa aaaactgaca ccttatcgag gagcccttca ctccgtctct 11880
tctccaagta gtagatatat ggacccaaac ccaaattttt gcaccagtag aagagaagac 11940
aatgacatag agatctatga gaccgtaagt gcatttataa ctacagatct caaaaagtac 12000
tgtctgaatt ggcgatatga gaccattagt atatttgctc akagattaaa tgaaatctac 12060
ggtctcccct catttttcca atggttgcac agaagattgg aacagtcgat cctatacgta 12120
agtgaccccc actgccctcc agatctcgat cgccatgtgg acttgaacac agtccctaac 12180
tctcaaatat tcatcaaata cccaatggga ggagtagagg gatattgtca aaagttatgg 12240
actattagca caatacctta tctgtacttg gcagcacatg aaagcggtgt caggattgca 12300
tcacttgttc aaggtgataa ccaaaccatt gctgtcacta aaagagttcc aagcacctgg 12360
tcatatgcct tgaagaagtc tgaagccagt cgagtcacca cagaatactt tatagcctta 12420
agacagagat tacatgatgt cggacatcat ttgaaagcaa atgaaacaat aatatcttcc 12480
cacttttttg tatactcaaa aggaatctat tatgatggga tgttaatttc gcaatccttg 12540
aaaagtatag ctaggtgcgt attttggtca gaaacaatag tggatgagac ccgagccgcc 12600
tgtagcaaca tttcaacaac attggcaaaa gccattgaga aagggtttga ccggtattta 12660
gcctatgcgc tgaatatttt aaaaatcatt caacaagtat taatttcatt aggattcact 12720
atcaattcag ctatgacgcg ggatgtgata gaaccccttt tacaagacca ctgtctcttg 12780
accaagatgg caattcttcc tgcacccata ggtggtctta attacctcaa tatgagtagg 12840
ctttttgtca ggaacatcgg ggatcccgtg acatcttcta ttgctgacct caaacgaatg 12900
atccgatcag gccttctcgg agtagagatt ttacatcaag tcatgaccca atacccaggt 12960
gactcttcgt acttagattg ggcaagtgac ccttattctg ctaatctgcc ctgtgtccag 13020
agcataaccc gactccttaa aaatattacg gccaggcatg tccttatcaa cagtccaaat 13080
cccatgctga aaggattgtt ccatgatgaa agtcaggatg aggatgaagc tttagctgct 13140
ttcttgatgg atagaaaaat tattatccca agagctgcac atgaaattct agataacacg 13200
atcactggtg cgagggaggc aattgctgga atgctagata ccacaaaggg gttgataaga 13260
gcaagcatga aaagaggagg gctaacccct agaataataa accgtttgtc aacttatgat 13320
tatgagcaat ttagggcagg tatcagacta ttgtcaggga agggacatga cccactcatc 13380
gatcaagact catgctctgt ccagttagcg agagcattaa ggaaccacat gtgggctaag 13440
ttggcgaagg gtcgtcctat ttatggtcta gaagtcccgg acatccttga atcaatgaag 13500
ggctatatga taagaagaca tgagtcttgt ttgctttgcg catcaggctc tcataactat 13560
ggttggtttt ttgtaccggc aaattgtcaa ttagatagta ttacagaggg aacatctgca 13620
ctgagggtac catacattgg gtccacaaca gaagaaagaa cagacatgaa attagcattc 13680
gtcaaatctc ctagtaggtc tctaaaatca gcagtgagaa tagcaactgt gtactcatgg 13740
gcctatggtg atgatgacga atcttggcaa gaggcttgga ctttggcaaa acagagagcg 13800
aacatctcac ttgaagaatt acggatgatt accccaattt ctacctctac taatctagct 13860
caccggctca gggacaagag tactcaagtc aaatactcag ggacctctct tatcagagta 13920
gcacgttatg caacaatctc taatgataat ctttcttttg tgatagctga caagaaagtg 13980
gacacgaact ttatttatca gcaaggtatg ctcctggggt tggggattct cgagcacttg 14040
tttagactgt cttcaaccac cggcgacacc aacaccgtac tgcatttaca tgttgaaaca 14100
gattgttgcg taatacccat gagcgaccat ccaagggtcc cagggctcag aaaggtcgtt 14160
ataccaagaa atatttgtac aaatcctttg atctatgaca gtaaccctat tattgagaaa 14220
gatgcagtca ggctttataa ccagagtcac aggaagcaca ttgtagagtt tgtcacatgg 14280
acaacagggc agctttatca tgtactagct aaatctactg ctatgtctat ggttgagatg 14340
attacaaagt ttgaaaaaga ccacctaaat gaagtctccg cgttaattgg cgatgacgat 14400
atcaatagct ttatcactga gtttcttctc gttgagccca ggttatttac tgtgtatcta 14460
ggtcaatgtg ctgcaatcaa ctggggcttt gaaattcatt atcaccgacc ttctggaaag 14520
tatcaaatgg gtgaattgtt gttttctttc ttgagtagaa tgagtaaagg agtcttcaaa 14580
attttaacca atgcattgag tcatcccaaa gtatatagac gattttggga cagtgggatg 14640
attgaacctg tacacggacc ctctcttgac tcccagaacc tacacataac tgtatgcaac 14700
ctgatctata actgttacat gatttaccta gaccttctgt taaatgatga gttagatgat 14760
ttctcattca ttttatgcga aagtgatgag gatatcatac ctgaaagatt tgacaacata 14820
caagctaggc acctatgcat cctgtctgac ctttattgta accctcgtga ttgtccccag 14880
attcgtgggt tgacaccaac acagaaatgt gctgtgttat cgaggtactt aaaatcaaaa 14940
gctctagagt cccatgttgg tctgacatgg aatgacaaac ctatcctgat agatcagtat 15000
tcatgttccc tgacatatct gagaagaggc tcaatcaagc agataagatt gagagtggac 15060
cccgggttca tcactgatgc tgttggatgc ttagaaaagc gacctctgag gaaaagtcct 15120
atctctaacg cctcagaatt aaaatcagaa tttgacccac cgaaagatga cctggttaaa 15180
ctcctgagtc agctatcaac aaggacacac aacttaccta ttacaggatt aggagtccga 15240
aactatgagg ttcactcatt cagaagaatt gggatcaatt caacggcatg ttacaaggca 15300
gttgaaatag tctctgttat taagaacgaa ttcacgtctg aagaacatgg attattccta 15360
ggagagggtt caggtgcaat gctgacagta tataaagagc tattgaggtt gtcaagatgt 15420
tattataaca gtggtgtgtc agcagaggct agaactggac aacgagagat ttcaccttac 15480
ccttctgagg tcagccttgt ggaacatcaa ttaggactcg ataaattggt gactgtgctt 15540
ttcaatggga gaccagaggt aacttgggtt gggagtgttg attgttacaa gtacatacta 15600
agtcagatat ctgctagcag tcttggattg attcactcgg atatcgagtc actacctgat 15660
aaagacataa ttgaaaaatt ggaagaactg tctgccatat tatcgatgac tttgatatta 15720
gggaaggtag ggtcagtgtt agtaatcaag atcatgcccg ctagtggcga ctgggttcaa 15780
ggatttattc tgtatgcact cccacatttt cttcgaagtt acataattta cccaagatac 15840
agcaattttg tgtcaacaga ggcctacctc gttttcactg gtcttagagc agggagacta 15900
gtcaatccgg aggggattaa acaacagatt ttgcgagtcg gtattcgaac ttcacccgga 15960
ttggtagggc acatcctttc atcaaagcag acagcatgtg tgcaatcttt gcatggacct 16020
ccatttcaag ctaaatcttt taatccttac ctccagggtt taacaagtat tgagaagatt 16080
ttgatcaatt gtgggcttac aattaacggt cttaaagtat gcaaaaacct gcttcaccat 16140
gatatctcgt caggcgagga agggctgaaa ggatctatca caatccttta tagggaactc 16200
gcacggttca aggataacca ccaattttca catggaatgt tccatgcata cccagtttta 16260
atcgcaagtc aggaaaggga gctcgtatcc accattgcaa ggaagtattg tggttatatt 16320
ttgctttact cgggagactt atacgaaatt accaggatag ttcgagacct gaaagccaac 16380
cacataattt ttgacttaca ccggaactta tttatggata acctatccag atctgaccgg 16440
tctctcatcc tgacgacaat cccaaaaagg aattggctct ttcaacttga gaccaaagag 16500
ataaaagagt ggttcaaact gttggggtat agtgcactga ttagaaacca ctgacgggtt 16560
ggtctggttc ctaaccctct gctattcatt gttattaaat ttaattttac gaaaaaaaac 16620
aacgattatt aataagttat catacccagc tttgtctggt gggtcggcat ggcatctcca 16680
cctcctcgcg gtccgacctg ggcatccgaa ggaggacgca cgtccactcg gatggctaag 16740
ggagggcgaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt 16800
ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta 16860
ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 16920
ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc 16980
ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca 17040
cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 17100
tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 17160
gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 17220
tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 17280
atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 17340
actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata 17400
agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 17460
cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca 17520
gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 17580
ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 17640
gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 17700
ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag 17760
ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg 17820
ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg 17880
attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 17940
tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 18000
caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 18060
gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 18120
gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 18180
ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 18240
cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 18300
gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 18360
catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 18420
gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 18480
cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 18540
gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 18600
gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 18660
gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 18720
catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 18780
tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 18840
accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 18900
tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 18960
tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat 19020
agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 19080
gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 19140
gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 19200
aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 19260
cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 19320
ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 19380
aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 19440
acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 19500
gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 19560
ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 19620
gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 19680
cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 19740
taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 19800
atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 19860
cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 19920
cttgatccgg caaacaaacc accgctggta gcggtttttt tgtttgcaag cagcagatta 19980
cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 20040
agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 20100
cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 20160
cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 20220
ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 20280
taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 20340
tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 20400
ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 20460
atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 20520
gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 20580
tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 20640
cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 20700
taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 20760
ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 20820
ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 20880
cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 20940
ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 21000
gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 21060
gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 21120
aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 21170
<210> 5
<211> 1572
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggctagcc ttctcaagag cctcacattg ttcaagagga ctcgggacca acccccactt 60
gcctcgggct ccggaggagc aataagaggg ataaagcatg tcattatagt cctaatcccg 120
ggtgattcaa gcattgttac aaggtctcga ctactggata gacttgttag attggtcggt 180
gatccggaaa tcaacggacc taaattaact gggattttaa tcagtatcct ctccttgttc 240
gtggaatccc ctggacagtt gatccagagg atcatagacg accctgatgt aagcatcaag 300
ttagtagagg taatcccaag catcaactct ggttgtggtc ttacatttgc atccagagga 360
gcaagtttgg attctgaggc agatgagttc ttcaaaattg tagacgaagg gtcgaaagct 420
caaggacaat taggctggtt ggagaataag gatattgtag acatagaagt tgatgatgct 480
gagcaattca atatattgct agcttccatc ttggcccaaa tttggatcct gctcgctaaa 540
gcagtgactg ctcctgatac tgcagccgac tcggaaatga ggaggtggat taagtatacc 600
caacagagac gtgtggtcgg ggaatttaga atgaacaaaa tctggcttga tattgttaga 660
aacaggattg ctgaggacct atctttgagg cgattcatgg tggcactcat cttggacatc 720
aaacgatccc cagggaacaa gcctagaatt gctgaaatga tttgtgatat agataactac 780
attgtggaag ctggattagc tagtttcatc ttaactatca aatttggcat tgaaactatg 840
tatccggctc ttgggttgca tgagttttcc ggagagttaa caactattga atcccttatg 900
atgctatatc aacagatggg tgaaacagca ccgtacatgg ttattctgga aaattctgtt 960
cagaacaaat ttagtgcagg atcctaccca ctgctctgga gttatgctat gggagttggt 1020
gttgaacttg aaaactccat gggagggtta aatttcggta gatcctactt tgatccggcc 1080
tatttcaggc tcgggcaaga aatggtgaga agatctgccg gcaaagtaag ctctgcactt 1140
gccgccgagc ttggcatcac caaggaagag gctcagctag tgtcagaaat agcatccaag 1200
acaacggagg accggacgat tcgcactgct ggtcccaagc aatctcaaat cacttttctg 1260
cactcagaaa gatccgaagt caccaatcaa caacccccaa ccatcaacaa gaggtccgaa 1320
aaccaaggag gagacaaata ccccatccac gtcaatgatg aacggtttcc agggtacacc 1380
cctgatgtca acagctccga atggagtgaa tcacgctatg atacccagac tattcaagat 1440
gatggaaacg acgatgaccg gaaatcgatg gaagcaatcg ccaagatgag aatgcttact 1500
aagatgctca gtcaacctgg gaccagtgaa gagagttctc ctgtctataa tgatagagag 1560
ctactcaatt aa 1572
<210> 6
<211> 1524
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atggcagaag agcaggccta tcatgtcaat aaagggctgg aatgcctcaa agccctcaga 60
gagaatcctc ctgacattga ggagattcaa gaggtcagca gtatcagaga tcaaacccgc 120
gacccaggcc aagagaatgg aaccgcaagc atgcaggaag aagaggtctc tcaggatctc 180
gatgaatcac acgagccagc aaaaggatca aactatgtcg gccatgtact ccaaaataat 240
ccgggatgtg gagagagcaa cactgcgctt gtggaggaag agcagcccgc taaagatgat 300
gtccaaccag gacctgaaat acgatgttat catgtttatg atcacagtgg tgaagaggtt 360
aagggaatcg aagatgctga cagtctcgtg gtacctgcag gcgctgtcag taatcgagga 420
ttcgagagag gagaaggaag ccttgatgat agcactgagg attctggcga agattattcc 480
gagggaaatg cttcatctaa ctggggatat tctttcggcc ttaaaccaga cagagcggct 540
gatgtgagca tgctgatgga agaggaattg agtactctgc tcaagacaag cagaaatgtg 600
gggattaaga aaagggatgg gatgactctg cagttcccac acagtcccga aggtaagaca 660
gaggatccgg agtgtggatc cattaaaaag ggcacaggag agaggtcagc ctcacatgga 720
atggggatag ttgctggatc gacaagtggt gcaacccaat ctgcactcaa gtcaactggg 780
ggatcatcag ggccaagtgt gtctgcggag aatgtccgcc aacctgcaat gaatgcaaag 840
atgacccaga aatgcaaacc cgagtctggt acgcaactcc ctgccaggac ctcaaatgag 900
gctgaatctg acagtgagta tgacgatgag cttttttctg aaatacaaga aattcgatct 960
gctatcacta agctaatgga agataatcaa gcaatacttt ctaaactgga taccctatta 1020
ctgcttaaag gagagactga ttcaattaag aaacaaatta gcaagcaaaa tattgctatt 1080
tccacgattg aggggcatct atcaagcatt atgatagcta tacctggttt tgggaaggac 1140
actggagacc ctacggcaaa tgtcgacatt aacccagagc tccgccctat aatagggagg 1200
gattcaggaa gagcactggc ggaagttctc aagcaacccg catcatcccg cggtaatcgg 1260
aaggacagtg gtatcgcctt gggttcaaaa ggtcaactat tgagagacct ccagctgaaa 1320
cctattgaca aagagtctag ttcggcaatc ggatacaaac cgaaggatac cgcaccttcc 1380
aaagctgtac ttgcatcatt gattagatca agcaaaattg atcaaagtca caaacacaac 1440
atgctggccc ttctcaaaaa tattaagggg gatgacaatc taaacgagtt ctaccagatg 1500
atcaagagta tcacacatgc ttaa 1524
<210> 7
<211> 6555
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggactctg tttcagtgaa ccagatttta taccctgagg tccatctaga tagcccaatt 60
gtgaccaata agctagtggc tattttagag tatgcacgaa ttagacataa ctatcgactc 120
cttgacacaa cgttagtgcg taatatcaaa gagagaattt cagaagggtt atcaaaccag 180
atgatcatta actgtatcga aattgggagc attgttaatc agaccttgtt atcttatccc 240
aaacacaacc atgtgatata tccaaattgc aacaaacttc tgtttcatgc acaggatcga 300
gtcatctctc tgaggttgag aaatatattc aaaagaggaa atagcatcta tagtaaaata 360
acagacgggg tcaaaaaatg cttaaacgat attaatctta gtattggttt aggaggtgca 420
ttggataaga ctattggggc caaagttgat gaagcaggca taattatgca aagctcacag 480
tggttcgaac ctttccttct gtggtttaca attaagacag aaatgagatc agtgattaaa 540
tcctctactc acaactgtcg caaacgaagg cagaatcctg tctttgtaag aggtgaatca 600
tttaatgtgt tagtgtctcg ggatcttgta tgtatcattg acctcaccag tcacaatgtt 660
tattacctaa catttgaaat ggtcctgatg tattgtgatg tgatagaagg gagattaatg 720
actgataccg ctatggcaat tgatcaccgt tactcaactt tgcatgtcag aatcaggtat 780
ctttgggatc taattgatgg atttttcccg gacttaggaa attcgaccta tcaactggta 840
gctctgctgg agcctctttc attggcttac ttgcaattaa aagacatcac cttctctctc 900
aggggtgctt ttttgagtca ctgctttgct gaaatccagg agattttaca ggacaatggc 960
ttctatactg aagagacatt ccaaacctta acccaggctc tagactttgt tttcatcaca 1020
gaggatatac atataacagg agagatcttt tcctttttta ggagtttcgg tcacccaaga 1080
ttagaagcaa taacagcagc agaaaatgta cggaaacaca tgaatcaacc caaagttgtc 1140
tcctatgaga ccatgatgaa gggacacgct attttctgtg ggataatcat taacggttat 1200
cgggatagac atggaggaac ttggcctcca atggatcttc ctgtccatgc atctcctatc 1260
atcaggaatg ctcatgcctc aggagaggga atcacctata gtcaatgtat agaaaattgg 1320
aaatcctttg caggaattcg atttaaatgc tttatgcccc tctgcctaga cagtgatctg 1380
accatgtatt tgaaagataa ggctttagca gcccttaaaa aagagtggga ttcagtgtac 1440
ccaaaagaat tcctcaggta caacccacct cgctccactg aatctcggag acttgttaat 1500
gtgtttctag aggactctca gtttgaccct tataatatga ttatgtacgt tatctcagga 1560
caatatctag acgatcctga cttcaaccta tcatacagtc ttaaagagaa agagattaaa 1620
gaagtgggga ggttattcgc taaaatgaca tacaaaatgc gggcctgtca agtcatagca 1680
gaaaacttaa tatctaatgg aattgggaag tacttcaagg acaatgggat ggcaaaggat 1740
gaacacgatc tcactaaagc attgcacact ctggctgtgt ccggggttcc taaagacaag 1800
aaagactccc atcgcggcct cactaaccag tgtaagtcta aaaaactgac accttatcga 1860
ggagcccttc actccgtctc ttctccaagt agtagatata tggacccaaa cccaaatttt 1920
tgcaccagta gaagagaaga caatgacata gagatctatg agaccgtaag tgcatttata 1980
actacagatc tcaaaaagta ctgtctgaat tggcgatatg agaccattag tatatttgct 2040
cagagattaa atgaaatcta cggtctcccc tcatttttcc aatggttgca cagaagattg 2100
gaacagtcga tcctatacgt aagtgacccc cactgccctc cagatctcga tcgccatgtg 2160
gacttgaaca cagtccctaa ctctcaaata ttcatcaaat acccaatggg aggagtagag 2220
ggatattgtc aaaagttatg gactattagc acaatacctt atctgtactt ggcagcacat 2280
gaaagcggtg tcaggattgc atcacttgtt caaggtgata accaaaccat tgctgtcact 2340
aaaagagttc caagcacctg gtcatatgcc ttgaagaagt ctgaagccag tcgagtcacc 2400
acagaatact ttatagcctt aagacagaga ttacatgatg tcggacatca tttgaaagca 2460
aatgaaacaa taatatcttc ccactttttt gtatactcaa aaggaatcta ttatgatggg 2520
atgttaattt cgcaatcctt gaaaagtata gctaggtgcg tattttggtc agaaacaata 2580
gtggatgaga cccgagccgc ctgtagcaac atttcaacaa cattggcaaa agccattgag 2640
aaagggtttg accggtattt agcctatgcg ctgaatattt taaaaatcat tcaacaagta 2700
ttaatttcat taggattcac tatcaattca gctatgacgc gggatgtgat agaacccctt 2760
ttacaagacc actgtctctt gaccaagatg gcaattcttc ctgcacccat aggtggtctt 2820
aattacctca atatgagtag gctttttgtc aggaacatcg gggatcccgt gacatcttct 2880
attgctgacc tcaaacgaat gatccgatca ggccttctcg gagtagagat tttacatcaa 2940
gtcatgaccc aatacccagg tgactcttcg tacttagatt gggcaagtga cccttattct 3000
gctaatctgc cctgtgtcca gagcataacc cgactcctta aaaatattac ggccaggcat 3060
gtccttatca acagtccaaa tcccatgctg aaaggattgt tccatgatga aagtcaggat 3120
gaggatgaag ctttagctgc tttcttgatg gatagaaaaa ttattatccc aagagctgca 3180
catgaaattc tagataacac gatcactggt gcgagggagg caattgctgg aatgctagat 3240
accacaaagg ggttgataag agcaagcatg aaaagaggag ggctaacccc tagaataata 3300
aaccgtttgt caacttatga ttatgagcaa tttagggcag gtatcagact attgtcaggg 3360
aagggacatg acccactcat cgatcaagac tcatgctctg tccagttagc gagagcatta 3420
aggaaccaca tgtgggctaa gttggcgaag ggtcgtccta tttatggtct agaagtcccg 3480
gacatccttg aatcaatgaa gggctatatg ataagaagac atgagtcttg tttgctttgc 3540
gcatcaggct ctcataacta tggttggttt tttgtaccgg caaattgtca attagatagt 3600
attacagagg gaacatctgc actgagggta ccatacattg ggtccacaac agaagaaaga 3660
acagacatga aattagcatt cgtcaaatct cctagtaggt ctctaaaatc agcagtgaga 3720
atagcaactg tgtactcatg ggcctatggt gatgatgacg aatcttggca agaggcttgg 3780
actttggcaa aacagagagc gaacatctca cttgaagaat tacggatgat taccccaatt 3840
tctacctcta ctaatctagc tcaccggctc agggacaaga gtactcaagt caaatactca 3900
gggacctctc ttatcagagt agcacgttat gcaacaatct ctaatgataa tctttctttt 3960
gtgatagctg acaagaaagt ggacacgaac tttatttatc agcaaggtat gctcctgggg 4020
ttggggattc tcgagcactt gtttagactg tcttcaacca ccggcgacac caacaccgta 4080
ctgcatttac atgttgaaac agattgttgc gtaataccca tgagcgacca tccaagggtc 4140
ccagggctca gaaaggtcgt tataccaaga aatatttgta caaatccttt gatctatgac 4200
agtaacccta ttattgagaa agatgcagtc aggctttata accagagtca caggaagcac 4260
attgtagagt ttgtcacatg gacaacaggg cagctttatc atgtactagc taaatctact 4320
gctatgtcta tggttgagat gattacaaag tttgaaaaag accacctaaa tgaagtctcc 4380
gcgttaattg gcgatgacga tatcaatagc tttatcactg agtttcttct cgttgagccc 4440
aggttattta ctgtgtatct aggtcaatgt gctgcaatca actggggctt tgaaattcat 4500
tatcaccgac cttctggaaa gtatcaaatg ggtgaattgt tgttttcttt cttgagtaga 4560
atgagtaaag gagtcttcaa aattttaacc aatgcattga gtcatcccaa agtatataga 4620
cgattttggg acagtgggat gattgaacct gtacacggac cctctcttga ctcccagaac 4680
ctacacataa ctgtatgcaa cctgatctat aactgttaca tgatttacct agaccttctg 4740
ttaaatgatg agttagatga tttctcattc attttatgcg aaagtgatga ggatatcata 4800
cctgaaagat ttgacaacat acaagctagg cacctatgca tcctgtctga cctttattgt 4860
aaccctcgtg attgtcccca gattcgtggg ttgacaccaa cacagaaatg tgctgtgtta 4920
tcgaggtact taaaatcaaa agctctagag tcccatgttg gtctgacatg gaatgacaaa 4980
cctatcctga tagatcagta ttcatgttcc ctgacatatc tgagaagagg ctcaatcaag 5040
cagataagat tgagagtgga ccccgggttc atcactgatg ctgttggatg cttagaaaag 5100
cgacctctga ggaaaagtcc tatctctaac gcctcagaat taaaatcaga atttgaccca 5160
ccgaaagatg acctggttaa actcctgagt cagctatcaa caaggacaca caacttacct 5220
attacaggat taggagtccg aaactatgag gttcactcat tcagaagaat tgggatcaat 5280
tcaacggcat gttacaaggc agttgaaata gtctctgtta ttaagaacga attcacgtct 5340
gaagaacatg gattattcct aggagagggt tcaggtgcaa tgctgacagt atataaagag 5400
ctattgaggt tgtcaagatg ttattataac agtggtgtgt cagcagaggc tagaactgga 5460
caacgagaga tttcacctta cccttctgag gtcagccttg tggaacatca attaggactc 5520
gataaattgg tgactgtgct tttcaatggg agaccagagg taacttgggt tgggagtgtt 5580
gattgttaca agtacatact aagtcagata tctgctagca gtcttggatt gattcactcg 5640
gatatcgagt cactacctga taaagacata attgaaaaat tggaagaact gtctgccata 5700
ttatcgatga ctttgatatt agggaaggta gggtcagtgt tagtaatcaa gatcatgccc 5760
gctagtggcg actgggttca aggatttatt ctgtatgcac tcccacattt tcttcgaagt 5820
tacataattt acccaagata cagcaatttt gtgtcaacag aggcctacct cgttttcact 5880
ggtcttagag cagggagact agtcaatccg gaggggatta aacaacagat tttgcgagtc 5940
ggtattcgaa cttcacccgg attggtaggg cacatccttt catcaaagca gacagcatgt 6000
gtgcaatctt tgcatggacc tccatttcaa gctaaatctt ttaatcctta cctccagggt 6060
ttaacaagta ttgagaagat tttgatcaat tgtgggctta caattaacgg tcttaaagta 6120
tgcaaaaacc tgcttcacca tgatatctcg tcaggcgagg aagggctgaa aggatctatc 6180
acaatccttt atagggaact cgcacggttc aaggataacc accaattttc acatggaatg 6240
ttccatgcat acccagtttt aatcgcaagt caggaaaggg agctcgtatc caccattgca 6300
aggaagtatt gtggttatat tttgctttac tcgggagact tatacgaaat taccaggata 6360
gttcgagacc tgaaagccaa ccacataatt tttgacttac accggaactt atttatggat 6420
aacctatcca gatctgaccg gtctctcatc ctgacgacaa tcccaaaaag gaattggctc 6480
tttcaacttg agaccaaaga gataaaagag tggttcaaac tgttggggta tagtgcactg 6540
attagaaacc actga 6555
<210> 2
<211> 1815
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgctcccct accaagacaa ggtgggtgcc ttctacaagg ataatgcaag agccaattca 60
accaagctgt ccttagtgac agaaggacat gggggcagga gaccacctta tttgttgttt 120
gtccttctca tcttattggt tggtatcctg gccttgcttg ctatcactgg agttcgattt 180
caccaagtat caactagtaa tatggaattt agcagattgc tgaaagagga tatggagaaa 240
tcagaggccg tacatcacca agtcatagat gtcttgacac cgctcttcaa gattattgga 300
gatgagattg ggttacggtt gccacaaaag ctaaacgaga tcaaacaatt tatccttcaa 360
aagacaaatt tcttcaatcc gaacagagaa ttcgacttcc gcgatctcca ctggtgcatt 420
aacccgccta gtacggtcaa ggtgaatttt actaattact gtgagtcaat tgggatcaga 480
aaagctattg catcggcagc aaatcctatc cttttatcag ccctatctgg gggcagaggt 540
gacatattcc caccacacag atgcagtgga gctactactt cagtaggcaa agttttcccc 600
ctatcagtct cattatccat gtctttgatc tcaagaacct cagaggtaat caatatgctg 660
accgctatct cagacggcgt gtatggcaaa acttacttgc tagtgcctga tgatatagaa 720
agagagttcg acactcgaga gattcgagtc tttgaaatag ggttcatcaa aaggtggctg 780
aatgacatgc cattactcca aacaaccaac tatatggtac tcccgaagaa ttccaaagcc 840
aaggtatgta ctatagcagt gggtgagttg acactggctt ccttgtgtgt agaagagagc 900
actgtattat tatatcatga cagcagtggt tcacaagatg gtattctagt agtgacactg 960
gggatatttt gggcaacacc tatggatcac attgaggaag tgatacctgt cgctcaccca 1020
tcaatgaaga aaatacatat aacaaaccac cgtggtttta taaaagattc aattgcaacc 1080
tggatggtgc ctgccctggc ctctgagaaa caagaagaac aaaaaggttg tctggagtca 1140
gcttgtcaaa gaaaaaccta ccccatgtgc aaccaagcgt catgggaacc cttcggagga 1200
agacagttgc catcttatgg gcggttgaca ttacctctag atgcaagtgt tgaccttcaa 1260
cttaacatat cgttcacata cggtccggtt atactgaatg gagatggtat ggattattat 1320
gaaagcccac ttttgaactc cggatggctt accattcccc ccaaagacgg aacaatctct 1380
ggattgataa acaaagcagg tagaggagac cagttcactg tactccccca tgtgttaaca 1440
tttgcgccca gggaatcaag tggaaattgt tatttaccta ttcaaacatc tcaaattaga 1500
gatagagatg tcctcattga gtccaatata gtggtgttgc ctacacagag tattagatat 1560
gtcatagcaa cgtatgacat atcacgaagt gatcatgcta ttgtttatta tgtttatgac 1620
ccaatccgga cgatttctta tacgcaccca tttagactaa ctaccaaggg tagacctgat 1680
ttcctaagga ttgaatgttt tgtgtgggat gacaatttgt ggtgtcacca attttacaga 1740
ttcgaggctg acatcgccaa ctctacaacc agtgttgaga atttagtccg tataagattc 1800
tcatgtaacc gttaa 1815
<210> 3
<211> 5428
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc 960
agatatccag cacagtggcg gccgctcgag tctagagggc ccgtttaaac ccgctgatca 1020
gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 1080
ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 1140
cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 1200
gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag 1260
gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta 1320
agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg 1380
cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 1440
gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 1500
aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt 1560
cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 1620
acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc 1680
tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg 1740
tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 1800
tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa 1860
gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta actccgccca 1920
tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt 1980
ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag 2040
gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg 2100
gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg 2160
caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa 2220
tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg 2280
tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt 2340
ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa 2400
gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc 2460
ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg 2520
ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg 2580
aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg 2640
aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg 2700
gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact 2760
gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg 2820
ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc 2880
ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct 2940
ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac 3000
cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat 3060
cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt ttattgcagc 3120
ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc 3180
actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtatacc 3240
gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg 3300
ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg 3360
tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc 3420
gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 3480
gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 3540
gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga 3600
taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 3660
cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg 3720
ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 3780
aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 3840
tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 3900
gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 3960
cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 4020
ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 4080
cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 4140
gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac 4200
cgctggtagc ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 4260
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 4320
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 4380
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 4440
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 4500
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 4560
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 4620
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 4680
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 4740
cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 4800
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 4860
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 4920
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 4980
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 5040
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 5100
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 5160
gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 5220
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 5280
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 5340
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 5400
atttccccga aaagtgccac ctgacgtc 5428
<210> 4
<211> 5420
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttgcggcc gccgtacggt ttaaacgtta attaagcacg gtccgacctg ggcatccgaa 960
ggaggacgca cgtccactcg gatggctaag ggagggcgaa acccgctgat cagcctcgac 1020
tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct 1080
ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct 1140
gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg 1200
ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag 1260
aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc 1320
gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 1380
tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 1440
tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 1500
tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 1560
gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 1620
ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt 1680
aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag 1740
ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 1800
aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 1860
agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 1920
ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 1980
gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 2040
ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 2100
caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct 2160
ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc 2220
tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc 2280
gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc 2340
acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg 2400
ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag 2460
aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc 2520
ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt 2580
cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc 2640
gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc 2700
tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg 2760
ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag 2820
cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg 2880
cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg 2940
aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct 3000
tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc 3060
gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg 3120
gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt 3180
ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct 3240
ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 3300
tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 3360
gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 3420
tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 3480
ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 3540
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 3600
gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 3660
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 3720
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 3780
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 3840
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 3900
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 3960
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 4020
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 4080
ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 4140
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 4200
gcggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 4260
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 4320
tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 4380
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 4440
gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 4500
tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac 4560
cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg 4620
ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 4680
gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 4740
caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac 4800
gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc 4860
ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 4920
tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact 4980
caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa 5040
tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 5100
cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 5160
ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa 5220
aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac 5280
tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 5340
gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 5400
gaaaagtgcc acctgacgtc 5420

Claims (4)

1.一株嵌合犬瘟热病毒株，其特征在于，以重组狐源犬瘟热病毒强毒HBF-1株为基础，将其H基因替换为如SEQ ID No.5所示的犬瘟热病毒疫苗株Onderstepoort的H基因；所述重组狐源犬瘟热病毒强毒HBF-1株是以狐源犬瘟热病毒强毒HBF-1株为模板通过反向遗传系统构建的重组狐源犬瘟热病毒强毒HBF-1株。
2.根据权利要求1所述嵌合犬瘟热病毒株，其特征在于，所述重组狐源犬瘟热病毒强毒HBF-1株的构建方法如下：
以狐源犬瘟热病毒强毒HBF-1株总RNA为模板，用RT-PCR方法扩增获得表达狐源犬瘟热病毒强毒HBF-1株N蛋白、P蛋白、M蛋白、F蛋白、H蛋白和L蛋白的序列，将这些序列顺次拼接、插入到真核表达载体中，获得狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒，同时构建表达狐源犬瘟热病毒强毒HBF-1株N、P、L蛋白的三个辅助质粒，与全长cDNA重组质粒一起转染细胞进行病毒拯救，获得重组狐源犬瘟热病毒强毒HBF-1株；
所述狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒的核酸序列如SEQ ID NO:1所示；
编码所述狐源犬瘟热病毒强毒HBF-1株N蛋白的核酸序列如SEQ ID NO:2所示；
编码所述狐源犬瘟热病毒强毒HBF-1株P蛋白的核酸序列如SEQ ID NO:3所示；
编码所述狐源犬瘟热病毒强毒HBF-1株L蛋白的核酸序列如SEQ ID NO:4所示。
3.权利要求1或2所述嵌合犬瘟热病毒株在制备犬瘟热病疫苗中的应用。
4.一种构建嵌合犬瘟热病毒株的方法，其特征在于，包括：
步骤1、以狐源犬瘟热病毒强毒HBF-1株总RNA为模板，用RT-PCR方法扩增获得表达狐源犬瘟热病毒强毒HBF-1株N蛋白、P蛋白、M蛋白、F蛋白、H蛋白和L蛋白的序列；
将这些序列顺次拼接、插入到真核表达载体中，获得狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒；
步骤2、以犬瘟热病毒疫苗株Onderstepoort总RNA为模板，用RT-PCR方法获得表达犬瘟热病毒疫苗株Onderstepoort的H蛋白的序列；通过酶切方式将狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒上的H基因替换为Onderstepoort的H基因，获得替换为Onderstepoort H基因的HBF-vacH全长重组质粒；
步骤3、HBF-vacH全长重组质粒与表达狐源犬瘟热病毒强毒HBF-1株N、P、L蛋白的三个辅助质粒一起转染细胞进行病毒拯救，获得所述嵌合犬瘟热病毒株；
所述狐源犬瘟热病毒强毒HBF-1株的全长cDNA重组质粒的核酸序列如SEQ ID NO:1所示；
编码所述狐源犬瘟热病毒强毒HBF-1株N蛋白的核酸序列如SEQ ID NO:2所示；
编码所述狐源犬瘟热病毒强毒HBF-1株P蛋白的核酸序列如SEQ ID NO:3所示；
编码所述狐源犬瘟热病毒强毒HBF-1株L蛋白的核酸序列如SEQ ID NO:4所示。
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