CN114164159B - 一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗及其制备方法与应用 - Google Patents

一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗及其制备方法与应用 Download PDF

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CN114164159B
CN114164159B CN202111351867.8A CN202111351867A CN114164159B CN 114164159 B CN114164159 B CN 114164159B CN 202111351867 A CN202111351867 A CN 202111351867A CN 114164159 B CN114164159 B CN 114164159B
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t3ss
salmonicida
dbff01
edwardsiella tarda
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胡胜标
严惠娟
方琪婷
黄熙殷
王芳
刘四庆
何思美
李琛
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Hunan Normal University
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Abstract

本发明公开了一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗及其制备方法与应用，该二联疫苗为工程菌株DBFF01‑T3SS‑GAPDH。该二联疫苗采用DBFF01为出发菌，通过在DBFF01中异源合成来自发光杆菌的T3SS和异源表达迟缓爱德华氏菌中的抗原GAPDH，利用T3SS递送抗原GAPDH至靶细胞，使宿主获得抗迟缓爱德华氏菌感染的能力，而低毒力出发菌株DBFF01则使保护宿主不受杀鲑气单胞菌的的感染。该二联疫苗可用于制备抗鱼类病原菌感染药物，具有使用方便等优点，降低了免疫成本，减少动物应激反应。

Description

一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗 及其制备方法与应用
技术领域
本发明涉及水产养殖动物病害防治技术领域，特别涉及一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗以及制备方法与应用。
背景技术
水产养殖在我国农业结构中占比重大，水产是国民餐桌上重要的蛋白质来源之一，但随着养殖规模的扩大而来的，加上养殖环境不断恶化、养殖品种抗逆性衰退，最终导致水产养殖动物病害频繁发生，造成的直接经济损失。
杀鲑气单胞菌（Aeromonas salmonicida，A. salmonicida）和迟缓爱德华氏菌（Edwardsiella tarda，E. tarda）是水产养殖中常见病原菌，在世界范围内频繁爆发，给渔业带来巨大经济损失。在渔业病害防治过程中，主要手段是采用药物治疗和疫苗免疫。药物治疗涉及抗菌类药物的使用，而这类化学药物的使用不仅带来环境污染，还会造成水产品药物残留和细菌的耐药性，破坏生态环境平衡，对人类健康形成潜在危害，因而在世界范围内受到很大的限制。疫苗免疫在特异性、安全性和保护效果方面优于抗生素等药物治疗，被普遍接受和采用。为了坚持可持续发展，让消费者吃的放心，渔业疫苗的研制与开发成为各国研究者的研究热点。
Ш型分泌系统（Type III secretion system，T3SS）是一种广泛存在于革兰氏阴性细菌的分泌系统，可将细菌产生的效应因子传递给直接接触的真核细胞。近年来，T3SS被用作抗原传递工具在疫苗开发中得到应用。目前，用于防治杀鲑气单胞菌、迟缓爱德华氏菌的疫苗主要是利用鱼类病原菌表面抗原作为疫苗，一般是将该抗原异源表达后纯化收集该蛋白质，然后做注射免疫。该方法不仅疫苗制备过程繁琐，而且至少需要两次免疫才可达到免疫一次以防治两种甚至更多鱼类病原菌感染的目的，使得疫苗的接种过程费时费力，大大增加了疫苗的制备工序和成本，增大了医护人员的劳动强度和水产动物接种疫苗后的应激反应。
发明内容
本发明要解决的技术问题是：克服现有技术的不足，提供一种制备简单且接种方便的具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗以及制备方法与应用。
本发明解决其技术问题所采用的技术方案是：一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗，所述二联疫苗为工程菌株DBFF01-T3SS-GAPDH，其拉丁文名称为Aeromonas salmonicida DBFF01-T3SS-GAPDH，该菌株已保藏于中国典型培养物保藏中心，保藏地址：中国，武汉，武汉大学，保藏编号：CCTCC NO：M 2021557，保藏日期：2021年05月19日。
进一步，所述DBFF01-T3SS-GAPDH菌株的T3SS编码基因来源于P. luminescensTT01，其GAPDH编码基因来源于迟缓爱德华氏菌。
进一步，所述T3SS的核苷酸序列如SEQ ID NO.1所示。
进一步，所述P. luminescens TT01菌株中的III型效应因子LopT的N端信号肽编码基因与迟缓爱德华氏菌的GAPDH编码基因融合，并构建了该融合基因与LopT蛋白的伴侣蛋白SlcT编码基因的共表达载体LopT-GAPDH-3FALG-SlcT。
进一步，所述LopT-GAPDH-3FALG-SlcT的核苷酸序列如SEQ ID NO.2所示，其编码的蛋白质的氨基酸序列如SEQ ID NO.3所示。
进一步，所述二联疫苗的接种方式为浸泡免疫。
本发明还提供一种如上所述具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗的制备方法，包括以下步骤：
1）通过三亲本结合转移将P. luminescens TT01菌株的T3SS插入杀鲑气单胞菌DBFF01的基因组上，获得重组菌株DBFF01-T3SS；
2）构建pBBR1-Cm-GAPDH原核表达质粒；
3）将pBBR1-Cm-GAPDH原核表达质粒电击转化至E. coli MFDpir中，获得接合转移供体菌株MFDpir/pBBR1-Cm-GAPDH；
4）通过双亲本接合转移将pBBR1-Cm-GAPDH原核表达质粒转入重组菌株DBFF01-T3SS中，获得菌株DBFF01-T3SS-GAPDH。
本发明采用一株无Ш型分泌系统（Type III secretion system，T3SS）、低毒力的杀鲑气单胞菌DBFF01为出发菌，通过在DBFF01中异源合成来自发光杆菌（Photorhabdus luminescens，P. luminescens）的T3SS和异源表达迟缓爱德华氏菌（Edwardsiella tarda， E. tarda）中的抗原—甘油醛-3-磷酸脱氢酶（Glyceraldehyde3-phosphatedehydrogenase，GAPDH），制备二联疫苗。利用T3SS递送抗原GAPDH至靶细胞，使宿主获得抗迟缓爱德华氏菌感染的能力，而低毒力出发菌株DBFF01则使保护宿主不受杀鲑气单胞菌的的感染。
进一步，所述步骤2）的具体操作步骤为：采用PCR扩增方法扩增LopT-GAPDH-3FLAG-SlcT的基因片段，并克隆到表达质粒pBBR1MCS2-Tac-mCherry-Cm，获得重组质粒pBBR1-Cm-GAPDH。
本发明还提供了一种如上所述具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗的应用，所述二联疫苗用于制备抗鱼类病原菌感染药物。
本发明还提供了一种如上所述具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗的应用，所述二联疫苗应用于水产养殖鱼类的疫苗接种。
本发明一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗以及制备方法与应用的有益效果：
（1）本发明的二联疫苗为全菌疫苗，可直接使用工程菌株DBFF01-T3SS-GAPDH浸泡免疫，大大降低了疫苗制备过程的经济成本；
（2）本发明的二联疫苗采用低毒力的杀鲑气单胞菌DBFF01为出发菌株，能对多种亚型的杀鲑气单胞菌起到免疫防治作用，避免了一种疫苗只能免疫某种特定亚种甚至特定菌株的尴尬；
（3）本发明的二联疫苗可以同时防御杀鲑气单胞菌和迟缓爱德华氏菌对鱼类的攻击，具有减少免疫次数、使用方便等优点，简化了疫苗制备工序，降低了免疫接种劳动强度和免疫成本，减少了降低动物因疫苗免疫产生的应激反应，更经济环保。
附图说明
图1—为T3SS转入DBFF01示意图；
图2—为DBFF01-T3SS的构建及鉴定图；
图3—为构建并验证pBBR1-Cm-GAPDH原核表达质粒的凝胶电泳图；
图4-为构建pBBR1-Cm-GAPDH原核表达质粒的示意图；
图5-为pBBR1-Cm-GAPDH原核表达质粒转入重组菌株DBFF01-T3SS示意图；
图6-为红色荧光质粒pBBR1-Cm-GAPDH标记DBFF01与DBFF01-T3SS示意图。
具体实施方式
以下结合附图及实施例对本发明作进一步说明，但这些具体实施方案不以任何方式限制本发明的保护范围。
本发明涉及的微生物：
杀鲑气单胞菌（Aeromonas salmonicida）DBFF01由本课题组刘志鹏从草鱼肠道中筛选，保藏于中国典型培养物保藏中心，其分类命名为杀鲑气单胞菌DBFF01，拉丁文名称为Aeromonas salmonicida DBFF01，保藏地址：中国，武汉，武汉大学，保藏编号为CCTCC NO:M 2018282，保藏日期：2018年05月16日；
P. luminescens TT01和攻毒菌株杀鲑气单胞菌JF2267由University of BernInstitute of Veterinary Bacteriology惠赠，且均为现有技术中的菌株，如：P. luminescens TT01在“Jallouli W, Boukedi H, Sellami S, Frikha F, Abdelkefi-Mesrati L, Tounsi S. Combinatorial effect of Photorhabdus luminescens TT01and Bacillus thuringiensis Vip3Aa16 toxin against Agrotis segetum. Toxicon.2018 Feb;142:52-57. doi: 10.1016/j.toxicon.2017.12.054. Epub 2018 Jan 2.PMID: 29305079.”中有记载，杀鲑气单胞菌Aeromonas salmonicida JF2267在“Burr SE,Wahli T, Segner H, Pugovkin D, Frey J. Association of Type III secretiongenes with virulence of Aeromonas salmonicida subsp. salmonicida. Dis AquatOrgan. 2003 Dec 3;57(1-2):167-71. doi: 10.3354/dao057167. PMID: 14735937.” 中有记载；
迟缓爱德华氏菌、HGB283 、MFDpir/pEVS107-T3SS均为现有技术中的菌株，如：迟缓爱德华氏菌在“李杰,莫照兰,李贵阳,肖鹏. 迟缓爱德华氏菌弱毒株的构建及其免疫效果[A]. 海南大学、国家大宗淡水鱼类产业技术体系病害防控功能研究室、中国水产学会鱼病专业委员会、中国科学院水生生物研究所.中国水产学会鱼病专业委员会2013年学术研讨会论文摘要汇编[C].海南大学、国家大宗淡水鱼类产业技术体系病害防控功能研究室、中国水产学会鱼病专业委员会、 中国科学院水生生物研究所:海南大学海洋学院,2013:1.”中均有记载；HGB283 、MFDpir/pEVS107-T3SS在“侯小庆. 昆虫病原线虫共生菌中Ⅲ型分泌系统的功能研究[D].湖南师范大学,2020.”有记载。
实施例1
本发明的一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗为工程菌株DBFF01-T3SS-GAPDH本身，其拉丁文名称为Aeromonas salmonicida DBFF01-T3SS-GAPDH，该菌株已保藏于中国典型培养物保藏中心，保藏地址：中国，武汉，武汉大学，保藏编号：CCTCC NO：M 2021557，保藏日期：2021年05月19日，所述DBFF01-T3SS-GAPDH菌株的T3SS编码基因来源于P. luminescens TT01，其GAPDH编码基因来源于迟缓爱德华氏菌，所述T3SS的核苷酸序列如SEQ ID NO.1所示，所述P. luminescens TT01菌株中的III型效应因子LopT的N端信号肽编码基因与迟缓爱德华氏菌的GAPDH编码基因融合，并构建了该融合基因与LopT蛋白的伴侣蛋白SlcT编码基因的共表达载体LopT-GAPDH-3FALG-SlcT，所述LopT-GAPDH-3FALG-SlcT的核苷酸序列如SEQ ID NO.2所示，其编码的蛋白质的氨基酸序列如SEQID NO.3所示。
实施例2
本发明的一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗的制备方法，包括以下步骤：
（1）通过三亲本结合转移将P. luminescens TT01菌株的T3SS插入杀鲑气单胞菌DBFF01的基因组上，获得重组菌株DBFF01-T3SS，具体操作如下：
挑取供体菌E.Coli MFDpir/pEVS107-T3SS单克隆接种于1.4 mL含有Kan、Ery、DAP的LB液体培养基中，于37 ℃振荡器上培养过夜。挑取辅助菌HGB283单克隆接种于1.4mL含Amp的LB液体培养基中，于30 ℃振荡器上培养过夜（HGB283:含Tn7转座子，将转入杀鲑气单胞菌胞质中的质粒转座部分定点插入杀鲑气单胞菌基因组上）。挑取受体菌DBFF01单克隆接种于1.4 mL含Amp的LB液体培养基中，30 ℃振荡器上培养过夜。按1%的接种量，取50μL 上述过夜培养的实验菌转接至到5 mL新鲜培养基，分别加入相应的抗生素，全部于28℃摇床中培养3 h-3.5 h。
实验分为实验组和对照组。实验组：移取500 μL供体菌、500 μL辅助菌和900 μL受体菌于2 mL EP管中，混匀后10000 rpm离心2min收集菌体，用LB液体培养基洗涤沉淀，重复离心——洗涤一遍后，留20 μL培养基重悬菌体。对照组：取500 μL供体菌、900 μL受体菌和500 μL LB液体培养基于2mL EP管中，其余步骤与实验组一致。将20 μL实验组和对照组菌重悬菌液分别点在含有DAP的新鲜LB平板上，放置于30 ℃培养箱中过夜培养。
次日将LB板上的菌体刮下重悬与1 mL LB液体中，取100 μL涂布于含200 μg/mLErm的LB平板上，置于30 ℃培养箱中培养，不超过14 h，然后将平板置于室温中2-5天。挑选在实验平板上生长而在对照平板上不生长的单克隆菌落，分离纯化2-3 次，在纯化平板上挑取单克隆进行筛选鉴定，获得重组菌株DBFF01-T3SS。
通过接合转移将来源于Photorhabdus luminescens TT01的T3SS合成基因簇转移到杀鲑气单胞菌DBFF01中，该T3SS转入DBFF01的示意图如图所示，通过PCR扩增T3SS基因簇前、中、后三段相应条带进行检测，目的条带大小应分别为1010 bp、876 bp、793 bp。
以接合子基因组为模板，以T3SS-1-F/T3SS-1-R、T3SS-2-F/T3SS-2-R、T3SS-3-F/T3SS-3-R（表1所示）为引物进行 PCR 扩增，通过琼脂糖凝胶电泳检测PCR条带，条带大小与预期一致（如图2-A所示）。普通光学显微镜观察菌落形态（图2-C所示），使用16S rDNA引物PCR 扩增接合子的16S rDNA（图2-B所示），PCR产物进行测序分析，结果显示为杀鲑气单胞菌，表明T3SS基因簇已经成功转入至DBFF01染色体上，携带T3SS基因簇的重组菌株DBFF01-T3SS构建成功。
图2中2-A为PCR 扩增 T3SS 基因簇片段；M: DL2000; 1-4以T3SS-1-F/T3SS-1-R为引物，1阴性对照，2-3接合子，4阳性对照；5-8以T3SS-2-F/T3SS-2-R为引物，5阴性对照，6-7接合子，8阳性对照；9-12：以T3SS-3-F/T3SS-3-R为引物，9阴性对照，10-11接合子，12阳性对照；2-B为PCR扩增重组菌株DBFF01-T3SS的16S rDNA；M：DL2000；1-3为三个接合子；2-C为普通光学显微镜观察菌株形态。
（2）构建pBBR1-Cm-GAPDH原核表达质粒，具体操作如下：
将P. luminescens TT01菌株中的III型效应因子LopT的N端信号肽编码基因与迟缓爱德华氏菌的GAPDH编码基因融合，并构建了该融合基因与LopT蛋白的伴侣蛋白SlcT编码基因的共表达载体LopT-GAPDH-3FALG-SlcT；
以pUC57-GAPDH质粒为模板，以pBBR1-GAPDH-F（5’-ATGCCTCGAGACGATGCCATCAACGCCTC-3’）和pBBR1-GAPDH-R（5’-ATGCGATATCTCATTATTCTGCAAATTCAG-3’）为引物，采用高保真Taq酶扩增LopT-GAPDH-3FLAG-SlcT，并在其两端加上XhoⅠ和EcoRⅤ酶切位点，扩增反应的条件为95℃ 5 min，（95℃ 30s，58℃ 30 s，72℃ 2.5 min）共30个循环，72 ℃ 10 min。使用1 %琼脂糖凝胶电泳检测PCR扩增产物，其凝胶电泳图如图3-A所示，确认扩增正确后利用SanPrep 柱式 PCR 产物纯化试剂盒进行纯化。
纯化后的PCR基因片段和质粒pBBR1MCS2-Tac-mCherry-Cm分别经XhoⅠ和EcoRⅤ在37℃酶切4h（质粒/PCR基因片段46 μL，10×Buffer 10 μL，超纯水40 μL，XhoⅠ酶2 μL，EcoRⅤ酶2 μL，总体积100 μL）。使用SanPrep 柱式胶回试剂盒回收目的片段，于-20℃储存备用。
经双酶切后的目的片段LopT-GAPDH--3FLAG-SlcT和质粒pBBR1MCS2-Tac-mCherry-Cm 回收后16 ℃酶连过夜（目的片段7 μL，载体1 μL，T4连接酶1 μL，2x Buffer 1μL，总体积10 μL），酶连产物热激转化转入E. coli DH5α。
随机挑取重组筛选板上的重组子，分别接种于装有1.4 ML含Cm的LB液体培养基中，37 ℃培养。SanPrep柱式小型质粒抽提试剂盒提取重组质粒，使用EcoRⅤ和XhoⅠ酶切验证重组质粒（酶切体系同上），酶切2 h后进行琼脂糖凝胶电泳检测，该凝胶电泳图如图3-B所示，酶切片段与目的片段大小一致，结果正确。酶切验证的转化子pBBR1-Cm-GAPDH送至上海生物工程股份有限公司进行双向测序鉴定，获得原核表达质粒pBBR1-Cm-GAPDH，构建原核表达质粒pBBR1-GAPDH-Cm示意图如图4所示，重组质粒经测序比对，表明重组质粒上的目的片段基因序列与原始序列一致，证明原核表达质粒 pBBR1-GAPDH-FLAG 构建成功。
图3中3-A为PCR扩增LopT-GAPDH-3FLAG-SlcT；M: DL5000；1-4: PCR产物；3-B为双酶切检测重组质粒pBBR1-GAPDH-FLAG ；M: DL5000；1-4: 重组子经 XhoⅠ和EcoRⅤ双酶切。
（3）原核表达质粒质粒pBBR1-Cm-GAPDH电击转化至E. coli MFDpir菌株，获得接合转移供体菌株MFDpir/pBBR1-Cm-GAPDH。
（4）通过双亲本接合转移将pBBR1-Cm-GAPDH原核表达质粒转入重组菌株DBFF01-T3SS中，获得菌株DBFF01-T3SS-GAPDH，具体操作如下：
挑取供体菌MFDpir/pBBR1-Cm-GAPDH单克隆，接种于1.4 mL含有Cm、DAP的LB 液体培养基中，于37℃振荡器上培养过夜。次日按2%的接种量转接，加入相应抗生素及营养补充剂，37℃培养3 h。
挑取DBFF01-T3SS（Ery、Amp）和DBFF01（Amp）单克隆，分别接种于1.4 mL 含相应抗生素的LB液体培养基中，30 ℃振荡器培养过夜。次日按2%的接种量转接，30℃培养3 h。
取500 μL供体菌和900 μL受体菌于2 mL EP管中，混匀后10000 rpm 离心2 min收集菌体，用LB液体培养基洗涤沉淀，重复离心——洗涤后，留20 μL培养基重悬菌体。20 μL重悬菌液点在含有DAP的新鲜LB平板上，30 ℃过夜培养。次日将LB板上的菌体刮下重悬于1mL LB液体中，取100 μL涂布于含50 μg/mL Cm 的LB平板上，置于30 ℃培养箱中培养不超过14 h，然后将平板取出置于室温中2-5天。次日将LB板上的菌体刮下重悬于1 mL LB液体中，取100 μL涂布于含 50 μg/mL Cm 的 LB平板上， 置于30 ℃培养箱中培养不超过14 h，然后将平板取出置于室温中2-5天。挑选筛选平板上生长而对照平板上不生长的单克隆，分离纯化2-3 次，在纯化平板上挑取单克隆进行筛选鉴定，获得菌株DBFF01-T3SS-GAPDH，原核表达质粒pBBR1-GAPDH-Cm转入重组菌株DBFF01-T3SS示意图如图5所示，pBBR1-Cm-GAPDH转入重组菌株DBFF01-T3SS时红色荧光原核表达质粒pBBR1-Cm-GAPDH标记DBFF01与DBFF01-T3SS的示意图如图6所示，图6中白点为红色荧光。
对照平板是指对照组涂布的平板，实验组如上段所示，对照组取1400 μL受体菌于2 mL EP管中，其余步骤均相同。
实施例3 工程菌株DBFF01-T3SS-GAPDH免疫后攻毒处理
取 9个鱼缸，分为A、B、C三组，在每个鱼缸中添加20 L的经48h曝气的自来水，饲养20条合方鲫，水温控制在22 ℃，喂食普通鱼饲料，驯养7天。每组都有甲乙丙三个鱼缸，甲鱼缸中加入108 CFU/mL的DBFF01-T3SS-GAPDH，乙鱼缸中加入108 CFU/mL的DBFF01-GAPDH，丙鱼缸不做处理，浸泡免疫7天后，再次加入同样浓度工程菌株，强化免疫。
在浸泡免疫15天后，对三个组的甲鱼缸中每条鱼都腹腔注射0.2 mL的1×107 CFU/mL杀鲑气单胞菌JF2267菌液，在乙鱼缸中的每条条鱼都腹腔注射 0.2 mL的和1×107 CFU/mL迟缓爱德华氏菌菌液，丙鱼缸中的鱼注射0.2 mLPBS，连续观察7天，统计合方鲫死亡率（如表2所示）。
由表2可知，本发明中，通过使用108 CFU/mL 免疫工程菌株DBFF01-T3SS-GAPDH浸泡免疫鲫鱼，注射0.2 mL 107CFU/mL的典型杀鲑气单胞菌（JF2267）和攻毒迟缓爱德华氏菌（Et）进行攻毒，结果发现经工程菌DBFF01-T3SS-GAPDH免疫的合方鲫，注射杀鲑气单胞菌的合方鲫死亡率与经DBFF01-GAPDH免疫的合方鲫一致（50%），明显低于对照组（100%），本发明的二联疫苗的杀鲑气单胞菌攻毒保护率为50%；经工程菌DBFF01-T3SS-GAPDH免疫的合方鲫，注射迟缓爱德华氏菌后死亡率为70%，低于对照组（100%），而经过DBFF01-GAPDH免疫后的合方鲫，攻毒迟缓爱德华氏菌的鲫鱼死亡率与对照组相同，达到100%，本发明的二联疫苗的迟缓爱德华氏菌攻毒保护率为30%。表明工程菌株DBFF01-T3SS-GAPDH作为疫苗具有良好的免疫原性，可作为候选商业疫苗，该二联疫苗可以降低鱼类受杀鲑气单胞菌和迟缓爱德华氏菌感染后的死亡率。
以上所述，仅是本发明的较佳实施例而已，并非对本发明作任何形式上的限制，虽然本发明已以较佳实施例揭露如上，然而并非用以限定本发明，任何熟悉本专业的技术人员，在不脱离本发明技术方案范围内，当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。
序列表
<110> 湖南师范大学
<120> 一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗及其制备方法与应用
<141> 2021-06-21
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40809
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccggaatgta tttattacat cagcaaaaag aacaataaaa aatatatgtg atatttttct 60
atcatcataa aactaccgaa tctttttcga gccgcctcac aataattgaa taacctcatt 120
gttttattgt tccactcaac tcattttatc agcctgagtt aatgaataag gagataacct 180
catggtggaa caacaatatt tcattggtgt tgatgttggc tccgccagtg tccgtactgc 240
ggtttttgac caatgcggca aacggcatgc attctccgct cgccctatcc agcaatttca 300
tccaaaagct gattttgttg aacaatcttc tgctgacatc tgggcgcaag tatgtgcaac 360
agtaaaagaa gctgttgcac tggcaaatat taatcctatt gatgttaaat caatcggctt 420
cgatgccacc tgttcactcg ttgcggtagc agcagaggga tgctctcttt ctgtggccga 480
aaatggcaat cctgaacacg atatcatcat gtggatggat catcgtgcca taaaagaagc 540
catcacgata aatcaaacca atgaccctgc actctgctat gtaggcggtg aaatcagccc 600
tgaaatggaa ctaccgaaaa tcttatggtt aaaaaactac tttcctcaac gttatcagga 660
tatatggcgt ttctttgatt tagctgattt tctggtgtgg aaagccactg gcgctgatgt 720
agccagtacc tgcacgctta cttgcaaatg gaactatctg gctcatcaaa aacagttcag 780
cgaaaaacta ctctatgatg tggggttaga agatttaaca aataaagttc catctaccat 840
cattgaactt ggcgaaaaag caggttacct gactgctgag gtggctaaag attttgggct 900
gcacacagga atcatcgtcg ccggtggtat tattgatgct catgccggcg gattggcact 960
gactggtgcc tatccagagg gcagcttggt tattatcagc ggtacctcaa actgccacat 1020
gattgtcagt cctcatcctg tcatggttcc gggggtttgg ggcccctatt tcggcgcaat 1080
gctacccggt tactggctca acgaaggtgg acaaagcgcc gccggcgcat tagttgaatg 1140
gtcaattcgt caacacaata gctggccgga gctggcagcc gaagccgagg aatctggtca 1200
tcattactac cagctactga atgaagcggt tacacagtta gaaaaacaag agatataccc 1260
gaccgcacag cttcatattt taagtgacca tcatggtaat cgttcaccaa gagcaaaccc 1320
taatgccaag ggaatggtaa gcgggctgac gctggcaaat ggtcgtgatg atctggcccg 1380
tcattacttg gcaacactgc aatctatcgc ctatggtacc cgccatatta ttgatgcatt 1440
agtagaagca ggacaccaga taaacagact ggttatgtgt ggtggcgcaa ctaaaaaccc 1500
tctatggtta cgtgaatatg ccaatgccac tgggcgagag atccatctca ctcaggaaga 1560
ggatgctgtc aatcttggct ctgctttgct aggtgccgtt gcttgcggtt cctttgcaaa 1620
cttcgctcaa gcagcaaaat cgatggtacg tgacggcaac attatcaaac cggaaaaaga 1680
gacattttta tttcatcaag ccaaatatca ggtctatctg caaatgtacc gagatcaaca 1740
gcaatataac gaaatcatgc aaaccggtaa ttagcccagt gagtacagcc aacaaatagg 1800
caatttgaaa ggtaacgttc atcgctttct ggcgagaagc gtagcaaaat ttaattttat 1860
tctattacca ttttcatctg ttttatgaag atgaccaggg ttttcattat atttaatgat 1920
ttcccagtca gaataataat tctccaattc acctggcatg aggaaacttt tgaatggcaa 1980
taatgaatat ggtgcaaaat ctgtttcaat cgggcaaaca atcagattaa taccaccttt 2040
attcgtctgt ttttgcatat tgtaaataat atcggtgatt ttttcccgtt gtagaaacat 2100
cagtaccaca gttgaaataa ttaaatcata tttttcggtg atattgtgag agttgatatc 2160
ataaacagcc gtacggatat ttttaaattg ctgtttttct ttaataacat taattgtgtt 2220
aatacattgc tgactaatat ccacggctgt aacgtcataa cctctttctg ccagatagaa 2280
agtattccgg ccatgaccgg cccccagatc taatgtctta caaggagaaa taatatttgc 2340
caaatatata atttcagagt gaggtaatga caaattattt tgtttataaa acagatattg 2400
tggttcacat aaaaaagaga gctggcattt aatatcatca cttacccatt taattttatg 2460
ccaaatctgt ggctgaataa tgggaggctg cttttgtaca ttaaaatatg cagtttcttc 2520
ctgatcacca tcgaaaatca caaattccag ttcaccttca aagatctcaa gacaagcata 2580
agtcccctct tttgtgttat gcctttcagt gaacatttca ggaagggtat ttttctgcca 2640
gatgggtatt cgtttataac aaatgaggtc actcatagaa ggttccttga ccataaatta 2700
catttaaaat gcatcttata atgacattct acaactgtaa agctataaaa tcatcagaag 2760
aaaagtctct gggtttcacc atagctatgc ttgtaagata aaaggaaaaa tgatccactc 2820
tccgttaaat tgaacgaact gactcactat taagaaacat aggaaagaaa ataagcgctc 2880
attgataaaa tttctccggc cttataagcc ggagaaaagg cgatcaatcc ttataactgt 2940
attgataaca tccgctagaa tcataataga gaccagactt cttctctacc gcgtttagca 3000
acacaccttt aatatgaatg ccattttgtc taaaccggcg aatagtcaca ttaacttctt 3060
taacccttgt tttatgataa cgtgcaatta acaatgaagt tcctgcatat ctcccaataa 3120
tcgcagcatc agtgacagcc aaaataggag gagtatctac caagacataa tcataatgct 3180
cttctgccca agataataat tctttcagtc tattatgcat aagtaattct gacgggtttg 3240
gcggaatagc acctcggttg ataaaatcaa gctcagtaag atacgttctg gtaataatcc 3300
cttttaattg atgttgacct gccaaatatt cagaaagccc cagagattga ggactttcca 3360
atactttatg taaacggcct cttctcatat ctgcatcgat taaaagcacg cgtttattac 3420
ttaatgccaa tacagcggca agatttactg aaataaaaga ctttcctaat tcaggagcag 3480
aacctgaaag aagaataatt ttatttgcag attccatcat tgaaaaatgc aaggttgttc 3540
taaggctacg caatgcttca atagctaaat cagtaggatc ttgaatagct aataaccgca 3600
atttctgacg atgaattcgc tttttctgtt gttggtcaag tttgatctga gtctcagaga 3660
gtggaatagt ggcgtaaacg gaaacaccta tctcttccaa ttgttcggga ctttcgattc 3720
ctttacgaaa catgactcgt atgcaaatca acccagcaga caatataaat cccaatacca 3780
tcatcgatgc aataatcagt gacttcttcg gttttaccgc tgaactttct acaatagctt 3840
catcaataat acgcacatta cccactgtcc ccgctttcac gatattcaat tcttgctgtt 3900
tattgagtaa ctgcacataa atttcttggt caacttgaac atctctggtc aaacgtaaaa 3960
tttcctgctg tgttttaggt aaccgtttga ttgactgatt cagctccttt ttggtatcaa 4020
ccaaagtttt acgtttatca agcaacgcaa cataagcggg atgttgtttg gtatacaatt 4080
gctgtaattc agcttctttg aacgtcaatt cattaatctg tttctcaatt tggaccaatg 4140
tctccaaagc cgattttgct tcaagtgata aatcgataga ttcattcgct tgacgatatt 4200
gattaagcaa ctcttcagaa cgattgagct caattttaac tttaggcaaa tgatttttta 4260
agaaattaag actactttca gcctcttccg tctttcttgt cacattttgt tgcaagtaat 4320
ttttaccaat actatccagt atcaatttta ttcttgcacg atcatcacct tctatttcaa 4380
gcaagataat accggtatta ttaccttttt cttttacccg caaagcctgt tgcagcccgc 4440
gaataacacc caattgcccc cgtttaacca acgtgaaact ttgccctgga gaagctgaaa 4500
tatttgagat aagcaaattt atgccatcgg tatcaaatac tttgtttact tgcccgtgga 4560
aaacctgatc gtcaaaaaac aaccgataat tttctgcatc gtcaacatat atttttatcc 4620
cttcattaat atattcaggt gatattgtaa aatgtttaat aacaatatta ggctttggca 4680
taccacgaag acgggcaatg cccttaccta tcacaggaaa atagtccgct gtaacctgaa 4740
catccaaatt tagatccttt accgtctttc ctaatatcat tcgggaagtt aacaactcca 4800
attctgccga tgtatctgac ggttttccag aaagcatatc cgacatgtcg ccgattaaag 4860
gtaatgcact acttctcttt tcaacctgca ataaagcatt tgccttgtat atcggagtcg 4920
caatcaagga ataacaaagg ccaataacgg tgaacaatag ggtgattcca ccaatcaccc 4980
agcgataatc gaataaggca ctaagaagac ggcctaaatc aatttcatca ctatccgtat 5040
gagtgtgact tgatttatct gatgtagtca taaaaagcct ataattactt tattaataag 5100
cagcaaaccc ataatataaa aatacttata agttataatt tccctaacca actattagta 5160
ctcttaaata ataactgata cacatattca taaacctcga tatcagactt ataaggatcg 5220
gggatgacca tttcatttaa ccagtgacct aaaagcattg ttttccccct tgcccctaga 5280
tttatcctat ttacagcctc aatatgtttt ttttccataa ccaagatcaa acttgactgc 5340
tggcataatt cactggttag ttgacgcgct atatgccctt ctaaggataa tccatgtcta 5400
gccgcaaccc tacttgagaa ttcatttgca ggtttaccaa ttaaagccga tattcccgca 5460
gaataaatct ctttctgagg aaatagttgc cgcaataaac gctctcccgt tggcgatcgg 5520
cagatattac ccacacaaac aatcagaata gaattgaaca ttaattcggc cacttacgaa 5580
tccacttagt tgtttcagtc aggctattaa tgcttgatat cgttggcacc aactgattga 5640
taacccgatt ccagcgaaca actggcgcac tggtaacata gacaatatca taaggttgca 5700
aattgaactc agttcccagg actaacgccg tcgcatcctt agcattgagc tgataaatat 5760
tggctatttt ccccttatta tcaggtacat tacgcagcga acggataaca aagatgccat 5820
tagcatcact tgataattga tctatacctt cagcattacc caatgcttcg gtcaacgtca 5880
tcccactgcg atccatacgt agagtggttt gttttctgac ttcccccata acaaacactt 5940
tcaaatcatc attacgagga atgtataaga tatcgccaga atagagcaga cgattttgct 6000
tcagatcacc attttgcatc aaatcttgga gagaaataat cgtttctttt cccttatgag 6060
tcaaaatgat cctcctccaa tcagcgtttt cggttaagcc gccagcatgg ttaatcgcat 6120
caataatggt taaaggaacg ttagtgatag gttgttgagc agatcgggca acttcacctg 6180
ttacataggc tttttgtgaa cgaaaagcag caatattgac atcaacttga ggtgattcga 6240
tgtacttcga taacttatca gtaatcatcg tacgcacctg agtgaccgtt tttcctttca 6300
catgaagttt accaacatag ggataaaaaa tcgtcccatc agaatgcacc caattacccg 6360
tatcactagc actacgatat tgtcctgccg gtattgtcaa ttcaggatga tcccaaacag 6420
taaccataat gacatccccc acaccaatac ggtattcata ctgttgtaat tctttatcta 6480
actcaggatt aggacgtgca atagcagaat tcacccgtaa attttcaatt aattttggtg 6540
ttaccggata aacgtttacc aaattattaa tatcatagtt actatcatca tcattgatta 6600
tttctttatt ataaacagta agaccactcc caggcataac agtacaacca cctaatagca 6660
tagaaccgga caatgccaga actataatta attccttaat attcatcaga aatttatcct 6720
taaaaaataa taaacaaaaa agaatacaga aaatagtatt aattataatc cccctcacaa 6780
aactagatta taatataata aaatataata taatataaag attttgctta agggccgcaa 6840
gattccaggc tactggtcat atcacttggt aaaaacaata gaaatcaact taatagcttt 6900
gaaaaattca gattattaaa taatagtagt agtaaattca ttaattattt tattatttaa 6960
tatatcttat aagatattat tacaaacaga aagtaagagc agacctatca aataaaagac 7020
agtgaggata tttatcagaa tttatcaaga ccaagataca tgctctcgat gacattgtga 7080
agaaaacaga tacctactta catcaaatcc gtgttcatat catcgacttt ggtgcttgaa 7140
atggtgacaa aaaataaaaa aatagacaat caattcatca aacaactcca attataaaac 7200
cctaagttta atcatattgt aaataatagc tcggtagtat cggatatttt tagattgttt 7260
taactttaaa gatcaccact ttgtactttt cacgtttatc aactaccatt agcagttgtt 7320
ttatatcgca gtagaagatc tgaaaattgg acatattcca caaaaacttt atcaactggg 7380
attaccatga aaaaatctac tatactaaga atcgccaggc ccactgacaa tttaactcaa 7440
attgctgaaa tgtaccgccg aggtttagga tttattctcc tggagcaatt cgaaaatcat 7500
gaaaattttg atggcatcat tctcgggcat ccaaatcatc tctggcatct cgaatttacg 7560
catcatcgtg gcacaacagt aggaagatcc cccacacagg acaatttatt ggccttctat 7620
ataccagacc attcagaatg gttagatcag atatcattaa tggaaaatgc tggctttatt 7680
gcagtcccct cttacaaccc atattgggat attgaaggta aaacatttga agatattgat 7740
ggttatcggg ttgtattaca aaacacagta ttccttgctt gattattaat cgatcttaag 7800
catacttgcc ctaataacca acccacacct acgttgtttt acattacgca ataacattat 7860
caaaactcag ctattgacac tgagttttga tatattcttg caagtatttc gtttgctgtt 7920
cgttctcaac aatcattctt cggagatcga aataatcttg ttcagttgct gggttaagtc 7980
gtggggtgtc tgcatggccc acgctgccgg aggagccggc ttggccttga tgacaggtgg 8040
ctgcgatgcg caaccgacga tgaccagcgg caacatcatt gcgcagaaca tcaatttcag 8100
atttggcatt agcaagttcc tttgtatgct gattatccag tttagacagg agccttattc 8160
gctcctgctg attaacgatt tcattctgct gctccagcaa ctcactctgt agcttaactg 8220
tcgtatcaag ctgtttggta tacttaccgt aatagtgata agccagaagc gaggcaagcg 8280
ccagagcaat aacagtataa ccacgagagt taaacttcat aatatcgctc tacaaaagtg 8340
aaaatgcttt atcaatgacc tcattgctat aaggttgact accattttcc atggtaataa 8400
tggacttaat aagctctgtc ataaatactt tattaaatac atcaacaacc tgatcgcaag 8460
tcactcccgt acctttacat acgtggttaa tataggcatc agtattattt tcattagtag 8520
gagcccatcg tgaaatgatc tctttcactg ttttaaggcc atatttccga ttgtaattat 8580
gcaaaatttt aatcatcgcc cgaagaccat attcaggacc aacaaactga cagaaagatt 8640
tatcagtacg ctgtgatgca ggcatcagac cttgccagtc atcaccccaa cgaatattac 8700
ctgggttatt atttcgaata cctctgctca taattcactc ctattgttta ttacctgttt 8760
tgttattaac aacttttctt aagatctgac caaaatagtc agtgcccaga taaccaataa 8820
tgacactgct gatgtaagcc aaatccgtgc tcataccaaa aaaattcaac gcatcacgga 8880
taaaccaagc aatcatggca cacataatag aatccataat tgtcgtccaa aatttaccac 8940
cgttatatcg acccctgaga taagccattg cggcagccag cgccgcacct ataccttgtt 9000
ctttgactga cagcagccat aaccatagct gcatccagat gtcaggctgt ttatccatgt 9060
atttcatcct tcactcccac tcacataaat gggaatccat aggctgccgt gtagccttaa 9120
ttttgagtta ataggaatac tgcgaaatca gtagaataag aaaaggccac acatagcgca 9180
gccttaaatt ttataaggtg attacttaca tttaatataa taccaagctc atcaacaaat 9240
tctgcgggtg tatcattatg tgtaagggaa tgataataat taacaaatag atatatatcc 9300
tatggatttc aagatggatc acggcggcaa gggagcgaat ccccgggagc atagataact 9360
atgtgaccgg ggtgagtgaa tgcagccaac aaagaggcaa cttgaaagat gacgggtata 9420
gagcattgta gtcactcatc catgaacctc cttaatatta agactaaatt acaacgtata 9480
ataaaccata gttcatggta attatgcaca cctggtaata aatctctcaa agcaatttgg 9540
ttaataaaac gtttagcaag caattttttt aagcttggat cgtgtatctt ttttctatac 9600
aaaataaaaa aactaccaac ctctcgaatt gcatgacgac taagagatct ttttaacgat 9660
ttctcacctt taatttcttt tgagatttta ataattgaat taaccacagt aatataacta 9720
tcagctcgat gatataattt tttactttct cttgagcgag ttattgattg caaattatgt 9780
cgataacaat acaattgctc ctcacaaaag ccgattcgat tcgctataag tgacaaatta 9840
attgtccata agatatcttc atgaaagata ttttccataa aactcatttg gttatttata 9900
attaattctt ttttaataag ttgtaaccaa acaaaatgtg gccattctct acattcaacg 9960
caatatttta tccactcttt acctgttaat actctaccat aaggttgtct acgtgttatt 10020
cttctatttc cttgtgagaa tggatctttt gtaaaactaa ctccattgcc aattaacaca 10080
tctaatctct gttcaatagc gtgatcaagc cattttctta atatttgtgg ttttagccaa 10140
tcatcactat caacaaataa aatccactca cctctcgcat attttatgcc ataatttcta 10200
gctattgaaa caccacgatt ttcttgatta aagatccgta ttctattatc ttttctttca 10260
tattctttaa tgacttctaa gctattatct gttgaaccat cattaataat aatgacttca 10320
aaattaacat tttcttgtgc aaaaattgaa tctaacaaat aggttatatt attctcatca 10380
ttaaaaacag ggataataat agtaatatca aactgaaaca tctaccatcc ttttaatgag 10440
ttagttgttt ttgataaaaa taccattttc ataataaaaa tgcccctttt ccccattgct 10500
attcacatac catacaatgt tttaagtatc agttatagat gggattgaaa atttctacca 10560
aaatagtaaa tcattaagta cgataatgat aaaaatatgc attgggaatt taggtattac 10620
tatctcctgt aaagatagta acagtatcaa aaatctattg gaacagttta aataaatcgg 10680
taaatgtagc agataaaatt aaaaatgata ttaatttaaa atggcttatg taatctatat 10740
taaaaataca attaaatact tttaggatga aaaagtaaaa tataatcaag cataagaaat 10800
agaaagaaat aacagaagag gaagtgaaaa atattaacta tctttaccaa tcccaataat 10860
atgacagacg tgaggtttat cataccctaa ttggcaaata atattattat ataccctatg 10920
gatttcaaga tgcatcgcga cggcaaggga gttaatcccc gggagcatag gtaactatgt 10980
gaccggggtg agcgagtgca gccaacaaag aggcaacttg aaagatgacg ggtataaaca 11040
ctattgtgaa tattatctct atctaattta taattaaatt atttaagata atataagagg 11100
aactattcaa ctaattgtaa aataagcgat taaaatatta atgcattatc ctatagatat 11160
ttaacattga aaatatatta aattataccg gaaaataaaa tattacatga aattattagg 11220
tataaagcca tgtgatttaa atagcattta aaaatttaaa agacaatatt atcccttatt 11280
tattaatgcg aaaatattta gcattctgga taacatggca agggagacaa ttgcactaca 11340
attctttttc ctgcaatgat gcgttgttgg cttttcgcct gcactgcccg tttcccatcc 11400
caacgtagac gagctcttaa aaataactca ccatgagaaa actgcggatt caaccgcata 11460
gaaaaagtga cgggtagcat atccaaacga taatgcatct cacctactgt caacactttt 11520
ccttcaacca ccgtcaacaa tgacacctca acgtttgccg ttctgggtag agaggcccgc 11580
atggtcacat ccccctcaat cagacgtgat gcttcatgct caatagcctc actaacacaa 11640
ccagtcatca gcaaacacaa caagaccaac cctattttgg tcatgttagg ctgatcctac 11700
gttgtgacaa cggagtatgc tgcccatcca acaagcgctg tagtgttgtc tcatgttcaa 11760
gccccatggc aagacctcta ctcagcatcg tagaaagcgg ctcatgaacg ttttgtggta 11820
tataccctga cgtaacccga cgaccacaac aaaggctacc attttcatgg ggaacatttg 11880
ttaaatgaat gggtaacgaa ttctcaattt tcatactcgc attcccaaag attcgttctg 11940
ttgagagcca atcggaagtt gagcttgcaa taattcctgc caaaactcag catgatccaa 12000
catacttgaa agactctctt caaaaacagc ctgattcagc cgacttgctg acatttgccg 12060
ataaagctgc accatcagcg ttgactcatc aaaaccaaag tgaagatcaa actgatacca 12120
atgacgatta aagctggcta attgacggaa aatcccctct ccgaccgcag tgatatcagc 12180
gacatctatc tgtaaaacga gctggtcacg ttcaggaatt aagatcaaag agaggtgaag 12240
gtgctcatca aaacagagag cagccacacc ttcatgattt aaagctaagt gcggcaagcc 12300
gaatttagca gcaaattcag ccaaaacttg attgacgatg gctatcacat ccatgggtta 12360
ctcctactag agagggcaat cagaccaact tgaattacca agtagtcaaa caaacgggtt 12420
acccccgcag aaaagggact cagtttacta ccatcgcctt ttacgcctct gtcactcaag 12480
ggcaattctt ttaataaaaa ggtcacccca gtgattatca aaaaccaaac accggaatat 12540
aggaaaatgc tttttttcct taacacattc tgcctgttaa ggactgaaat cttaatttga 12600
tattttcgtt tttctatacc tttggtggtg ttagtggatt ttaaaaacga gatcaaataa 12660
cggtatccat tgataagagc gcacctctcg gtacgcccaa gcttattata taacactgat 12720
ctattttacc agcattacac cacgccagtc gccgcgcgca atgcctgaac atgattctga 12780
tagagttgtt gcaataattg caaaacatct ttcatgaaat tattattcaa tgagatttgg 12840
tcttcactct tctgtttttc atgttgggca atagatgaac ccacttcatg ttccttggct 12900
ttagcctgag aaagtccctg ttctacctgc acagaattat tggacatacc ctctaaagat 12960
ttcatcactg aggttaataa ctgctgattg ctgttacgac tttcaaaaac caaggtaaga 13020
gatttcaaag tgctcttgtc agcatcttgt gctacattcc atactttacc aattgctgcc 13080
cgatccgcat cgctactctt gcccaactgt tgcaacttag actcgattaa tttgttacga 13140
ccagcaatat tgctttccaa cgtcttctgc tgcttaaccg cctgactgtt tttaacggaa 13200
gaaaaactac ctatcagccc tgaaccaagc gtcattaagc ctgaaaccac tgccatggca 13260
atcatcaatt tggcaccatg tcgcatctca tccacttgtg atttttgagc atcaatggac 13320
gctttattct cagtgtcacg ctgcaaaata cccagttcac gcatcctctt tgcaatctca 13380
aaaagtagcg acatcatatc cagcgtgtta tttaattctt gtccgacatt tcctaaacga 13440
ctgggatcga ggccctgttt cggtacaatc aattcaacct tatccgtgag cctcgttttt 13500
tccggctgtt gaatagcagc ttcatgtgct gtcggtgtcg acacaaagtt gtcagttcca 13560
acttcaacac cattactgga agtaacccct tgactactgg cgttatcatt aattttcatc 13620
tttttattcc ctaaatttcc tgtggtcggc tggcaaggtt atttatcatg tctccccggg 13680
cattaagcat ctggaaaatc atttccatca cattttggaa agactctacc aggtgggaca 13740
actcctcttt gagcctgtcc ataacagctt ggaatgcggt caattcgcta cgtgaaagtt 13800
gcacgtcagc ttgtcggtta gccgctttac tctggaaggc cgcattggta gtttgagcag 13860
cgccattagt aacatcaact cccgaatcgc ccacctgcat agccactctc aagccccgag 13920
acgtggtatt tgctgctgta cccgcggctt ttgcagaaaa ttccgctgcc tttactgcca 13980
tcttagccgt catctcagcc actttactgc ccattttagc gcccattttc gcaatcaaac 14040
cggcagcaga accaccgaaa ctgactgccg ccgccgccac cgccatcgta atctcaatcg 14100
ctgttatgac tggccctagc catttcatgg tttccttgct gattaaacca tcttgagctg 14160
cttgttgtag tccctgagag actaccccca tcactcctcc gacaatcagc aaagcaccgg 14220
ctaccgcacc aacaccggta gcaaccataa tcgcgccgat cacaattgaa gtaattgcac 14280
taatccagcc aaagatctta gaaaacaatc cagatttctg cgcctctttg gcagaatttt 14340
cagcttcttt aatcttctgc tggttctctt ccattttctg caagctttgc tgacgtgctc 14400
gctggatatc ctggattttg agactcgtct gaatacgatc aagcttgctg gtcagctcgc 14460
ccagattaat ctctatttcc tcaagtgagt gcaacaccag agaggtcaac cacgaagttg 14520
ctgtcagctt tgctgtcagt tcctttatca gacctgttgc ctgcttagcc tcttgcataa 14580
ccgatttaag cctggccatt tgttcttgat cagggtgagt attcaccacc gtcaatggcg 14640
ctggcaatga tacccctcgt gacttagcgg catcagaaga caacgagcta cccgcctggc 14700
tgggcgtaac gtgctccgcg cttttcgaag gtgtagttgc gaggatttca ccaaccggag 14760
aactattgac caccggttga ggaattttag attcactaac ttgaaccatt ttcctgagtc 14820
ctttttattg taatagcttc taacattgca ctggaactgg cagccagagc ggcctgttcc 14880
ggcgaagctg ccgcaagttg ttgagccgag tagaagccac tctcagcccc atcaagttct 14940
cctaattgca ggagacattc ggcagcatga aatggaaaac ggggctcatt aatatcaagc 15000
atcgctccgt agctatagct ttgaactgcc tgctccagtt gccccattgc ctgacggcat 15060
gcccccaatc caaggaagaa tcgggaatca tagtgatcca acatacacaa agcctggaat 15120
atcttatgtg catcctgcca tttgcctgac tgatactggt taaacgctaa tgagtaaagt 15180
tgctccaacg tatcactaga aatatccttc aacatggcaa gagtgccacc atcatgaaga 15240
aaagattcca gctcttgtgc atattggtca gaagtggttg atgtttcttg atgctccata 15300
atcattcctt aaattgcgcc gagaatatta cgtataatgc tgtcatattt ttgaataaaa 15360
cggtttaacg cctcaatagc cgcgttataa cgggaactag cctcatttaa acgtgttgtt 15420
ttttcctgga cagtattgtt tagtggactt acacgatcgt taactgaagt cgaaaagttg 15480
gctaggcgat tattttcctt atcatattcg taagaattct ccagacctgc catagctcca 15540
ctctgtttat tagaactttc caaaaatgtc ttaatggaga tttcagaacc agaagatatc 15600
ttccttagca gcttatattc agcacttttc tcaaaagtgg gctgatcgct aaaaccatac 15660
ttcttatggt ccagcaaatt gaaagagtta tcatcaatct taagctcccc attagccgct 15720
aatttcgcat taatctccga ttgaataaca ctgtaaattt ttaactctgc ggtcaatgtc 15780
gccagttcat cacgcaactc aagtcgtttt ttatcatgac ctgacataac cgaaacaaaa 15840
atatcgatta catcatcatc aagacgatcc ggtgttaaat taaaatgaac cgcagccaaa 15900
aaatctttta atgtccacgt ttttaatgtt gagctattca agaaagattc aagattatta 15960
attccatttt ttatttggct atctaagtga ccaccctcga caaccgcgtc ggctggcagg 16020
aaataggcaa tcagtttttt caagagctcc ttattttcag taacaacact agaatcagat 16080
gtaatcttga tccccttatc aaaaataaga ttaaccaatc tatcaagctc cgaagatcca 16140
gaaccttgta gttgagctag cgaaaccttt tctagatctg ccaagaaaag ctgtgggtca 16200
ttttgatatg gcctaatttc caccttcaaa ctccttatac aataatgcca cgcatacgag 16260
gtgggcgacg aagacgcttt ccctcttcca gagagggagg ttggttgctt cgtaagcgat 16320
taacttctgt cagcaactct tgctcagcat tctgaaccag agtaacgtca gtatgaccaa 16380
acagttctgt acccacattg gggttgatgc caagagactg ccacatctcc tgcaatagtt 16440
gcagtctgtg atcactgtct ttgatggctg cttcagcagc ttgcagtact ttggcgttag 16500
gttcctcttg catccatgac tcctataata aagagagata ttgattcttc ctgtcaacgt 16560
tggacttttt tttgttgttt tctcatttga tgtagcgata taacgcaact ggcttctaac 16620
tcgaaccagc caggcaccac ctcatttgcg cctgtccagc gatggtaaaa gcgggccaaa 16680
cgttcatcat taaggggaga gtcaaccaaa gtattaactt gaccataaag ccggctaccc 16740
ggcccaagca tggccatcgt tgctgttgcc aacagataaa gaggcgcaaa tggattcgca 16800
agccctcgac gctgccgggt acggcgtaac atcacgatcc aaactcgcgg cgcatcataa 16860
cgccaggcaa tttcacagcc ttcataccac acacgccagc caagagggat tgggctattc 16920
cccatacagt gaggctgtac tgaataacct tgctcctcaa accaaggggt gagcggatca 16980
cggttcatag acacactcta ccaagcggtt ggatgttgat ctgttgggtc aactcttgat 17040
acgaaagcac tggcaagccg tggtgatcac cttcaattaa cttacgaaca taacggcgaa 17100
tatccattga tacgatcaac accggtttat tctgcatctg tgttaaatca cccacggttt 17160
tcttcatctg ttcaaggaaa ctttgcgtca ccgctgggtc cagcgccaag tagctacccg 17220
cgctcgtttg gcgaatacct ccccggatct gctcctctac ctgttgatcc aacagataag 17280
caggcaagat attgttgcca ttagcatact tgtaacagat gtaacgcttg agactactgc 17340
gaatatattc cgttaattgc accacatctt tttccttctg cccccacacc accatagctt 17400
cgagaatcgt acgtgtatta cgaatagaga tgtcctcgcc gaccaaccgc tgcaatatct 17460
cagtcatccg ctgcaatgga attatacgca tagcctcctt gaccaattcc ccataactcc 17520
cttccatctg ctccagaaga taacgggttt cctgcacgcc aataaaatct tcggcatact 17580
cccgcaacac atgtgacaaa tgccaggtga tcacctgaga catggataaa gccgccagcc 17640
cggatttcac caaacgttcc tgatgtgctt ccgctaccca caaggtaggc tggttaggta 17700
atagaggttc cccttcttca taagggatag ccagtaattc caactgactg actggttcct 17760
gcaccagtaa atgcgctgag cgcagccgac cacgggcaac gggcacctct tgtagctgga 17820
tcaggtattc tccttccttc atcccttcat taaagcgcaa atggataccc ggaaatggta 17880
cgcccagatc caaatagagg gcgcgtcgta cccgtattaa ttcatcattc agtgaaatag 17940
cttccagctc agcctgtaat cccgcatcca catcaataag taaaggaacc gtcatagcaa 18000
attcctcttt ctcactcagt tttccagctt tggccttact gccgctcttg gctttagatt 18060
tggccgccgg tgcgcctgct ccttgggcta acaggctagg aagatccgct tgttggctgg 18120
cgttagcttg gcgctgacgc tggaacagca aatagccacc acccgccacc accagtgcaa 18180
gtattaagaa agttaatgta gggaatccag gaataaggcc aaataccacc aataaaacac 18240
cgccaattaa tagtgccttt ggttgagcaa cgacctgatc accaatctca ctacctagat 18300
cagaggcgtc ttccgacgaa actcgggtaa caatgatgcc tgccgtaatc gcaatcaata 18360
gggcaggaac ctgagagacc ataccatcac caacagtcaa aatggcatag agctgcaatg 18420
catcggacgc tgacattcct ttttgggtga cgcctatggt tatcccgccc aaaatgttga 18480
caaagatgat gagaagactc gcaatcgcat cacccttaac aaatttcatc gcaccatcca 18540
ttgaaccaaa catctggctc tctttttcga taatgctacg cctctccctc gcttcattga 18600
cgtcaataac accagcacgc atatctccgt cgatactcat ctgcttaccc ggcatggcat 18660
caagggaaaa acgagcacta acctcagcta cgcgctccga acctttggtt atgacgataa 18720
attgtacaat agtgatgatc agaaagatca ctatcccgac tatcaggttg ccaccgacaa 18780
cgaaattacc aaaggtgtag actattttac cggcatctgc ttgcagtagg atcatccggg 18840
tggtactaac ggaaagcgcc aaacgaaata gcgtggtcac cagcaagacc gcaggaaacg 18900
cggaaaactg taaaggggaa ttgatataga ccgccatcat catgagcaca acagagatgg 18960
tcatattaat ggcgatcaaa atgtccagca ggataggcgg aagcggcaaa accatcatga 19020
agaccacggc tagcaacaat attgccaaca tgatgtcttt acgttcacca atcagccgca 19080
ataactcaaa cccacggttc ataatagagc cccttgagcg agataacgct gataagcact 19140
gcgtgcttct tccagattcc cttcctgttg acaagcacgg ctaacacaga gccagagggc 19200
ggcgctttgt tcaccctgtt cttgtaagag agtgcaatgt tcttttgcca tgctcccctt 19260
ttgcagttta agtaatgaaa ctaccaaggc acgacgccct tctttatgtt caggatgcaa 19320
agccagtaat gcatctaata aaatacgcgc tcggtcagga tgaccgtatt gaagttgaag 19380
ccagccgagc agcaacagag cgctctgctg tttagcactg agagtcatgg tcataccttg 19440
tgcaacaggt gcagagccat ctgcaacaac tgttcatctt gattcatgtt ttccaacagg 19500
gagacagcat gctgtaccct gatgtcatta ccttgttgca aaattgtacg cagaccacta 19560
accgcctgac gaaaatcatg tggacgcaac agagcgtgaa acccatcttg cgggcaggca 19620
aaagccaaca aagcctgatc agctttgttg gccggataaa gacgtgcggc gtgagtctca 19680
acggattgac catcgggcaa taacgcgtag cgatcgggta aactaacctc tatctcttcc 19740
cgactgatcg ccgttagttg ctcaattcct atatgggcgg cagcgatttt gctcacgaag 19800
cgagcacctc acgctgtact cttagcaaag aacgaaatgc ctgatgcagc aagggcaagg 19860
ttatatgtcg ttcatctagt gtcacaaaca ataacagacg atcttctccc agccagccac 19920
aacgcaaggg cagttcgggt ccctgaccac tgaaagtgag tagcatcgct ttcaccattg 19980
cctcttttcc ctgatgccag ggaatttcac gacccagcca aagtgttaat gcgccaccat 20040
agcgctcaat ttgcaacgta ccagaatact caaaatcaat ttggatcaag gggctagaag 20100
catcagacat ctccataccc aaatcctgac aaaactggag taactgcggc tctatccagt 20160
tcatgatgct ccctctcccg accacaattc gtcttcttct ctctcaattg ctgcatccag 20220
cgccagttga cacgcattca gcaggttttg gcgctgctct tcatcactga acacttccac 20280
cggaatcaag cgaattaatc gcttcaactc ccggtagaaa tgaatttgag tttggctatc 20340
ctgcaatccc aaagcatgac cgagtatttc aacatctcgc acattggccc aacgtttctc 20400
aaccagagaa acgacatccc ccataaggtc agaaggcccg tatgccatga ttttccccct 20460
ctaccacgct tttccataac tgttcaactt gaaccgataa actgttaagc aaacggagtt 20520
tatgcatgtc actcatcaca cgttccaact taataggatc aagttgccgc tgttggctgt 20580
ccagatcagc actcaacgct ttcatgatga atcctgttac ttccatcagg gaactactct 20640
tgaaattgga ttgaatatcc cgccaggcag ccgacaaccc ttgataatcc atcacggcat 20700
cacgatagat attgcgtaat gcttgcaagt tgccaacccc atttctagct gctgccgtgg 20760
catcaggcgt aatgcgggct cccaactcga tagcaattcc ctgctcttgc gctaaacgtg 20820
ataattcttg ttcaaccaat gtcaatatgg catgtgcctc tggcctgcct gccagcgcat 20880
cacgggcctg acttagcgca taaaattgct cactcacctc tccagagaaa ctttccagat 20940
aagcctgtag ttgagcaata ttagtcaact gagcactatt taagtgagag atcaatgcct 21000
tgactttttg ttggcgctct aaatctggca ccttctgcaa atattcaccc accagtgcct 21060
caatttctcg caccctggca tgtccatcgc taactttacg tttggaaagt ggcatatcct 21120
tgcgttccga aaatgcaaaa gtcatctctt cggccgcatc agcaaaagac tgggatgctg 21180
ataccagcac gactttctct ccctgaaact gacctacctt agcctgttcc tgttgtgcgg 21240
taaccccggc acccagttct gataaagtta tcgaatgaag atgattctgg ataatatcca 21300
tagtctaccc tgtcatatca gagttaataa tgaacaaatt ataaaggtac cgtcccagtg 21360
cctatgccaa aatcagtcgt gaatttttag caaaaaccgc cgtaactcaa ggaagagccg 21420
attgctctga tataaaacgt atatacccta tggatttcaa gatggatcgc ggcggcaagg 21480
gagcgaatcc ccgggagcat agataactat gtgaccgggg tgagtgagtg cagccaacaa 21540
agaggcaact tgaaagataa cgggtataaa acatgagcaa cacactgtga ataaaatagg 21600
aaatctcacc gataaagcag agaacagcca ggtaaactcc aaccattttc taaaaactcc 21660
cgactcaggt tgcaatccct tcctcctccc cttctttata gtattcgtag cagaggttat 21720
ataaacgatc atgaagcttt cccttgacca tattcccggc aaaatgcgcc acgccattaa 21780
tgaatgccgg ctaattcaga ttcgtggccg ggttactcag gtcacgggta cactccttaa 21840
agccgttatt cctggtgtgc ggataggcga actttgtcac ttacgcaatc ctgacaacac 21900
actgtcgctt ttggcggagg tgattggatt ccaacaacat caagccttgt tgactccact 21960
tggtgaaatg tttggtatct cttcaaatac tgaagtaagc ccaaccggag ccatgcacca 22020
agttggcgtg ggtgattatc tgttagggca agtccttgat gggctaggta atccattttc 22080
cggcggccaa ttgccggaac cacaagcttg gtatcccgtt tatcgggatg cgccggctcc 22140
gatgagtcgt aaacggattg aacacccgct ttcactcggt gtacgcgcta ttgatggctt 22200
actcacttgt ggcgaaggtc aacgtatggg gatttttgcc gcagcgggtg gcggaaaaag 22260
taccctgcta tctacgctga ttcgtagcgc cgaagttgat gttaccgtgc tggcgctcat 22320
cggtgaacgt ggccgtgaag tacgggaatt tatcgaatca gatctcggtg aagagggact 22380
aaaacgttcc gtgctggtgg tagcgacctc agatcgcccg gcaatggagc gagctaaagc 22440
agggtttgtc gccacatcaa tcgctgaata ttttcgtgat caaggcaaac gtgtactcct 22500
actgatggat tcagtgaccc gttttgcccg agcgcaacga gaaataggtt tagctgccgg 22560
cgagccgcca acccgacgag gttatccccc atcagtgttt gccgctttgc cacggctaat 22620
ggaacgtgct ggtcaatccg acaaaggttc gattaccgcc ctctataccg tcttggtgga 22680
aggcgatgac atgacagagc ctgtggcaga cgagactcgt tctatccttg atggtcacat 22740
tattttgtca cgcaaactgg cggcggctaa ccactatcca gctatcgatg tgttacgttc 22800
cgccagcagg gtgatgaatc aaattatcac tcctgagcat caggctcagg ccggcctgtt 22860
gcgcaaatgg ctagctaaat atgaagaagt tgagcttttg ttgcagattg gagagtacca 22920
aaaagggcaa gatcccgttg ctgacaatgc cattgcacac attgaagcaa tacgtaactg 22980
gttacgccaa ggcacccatg agccaagtga tctaccgcaa accttagctc aattacagca 23040
gataacaaaa taatgattag ccgtttacaa cgcataaaag ccttgcgagt cgaacgcgct 23100
gaaaaacatt tttccttgca acaaatgaag ctgcaaacgg cccgtcaaca ccatcagcag 23160
gctttgcaaa ccttgcaaga ttactgtcaa tggcgaatag aagaagaaca acggcttttt 23220
gcattgtgtc agggccaacc tattgaccgc aaaggtcttg aacgctggca acaacaagtt 23280
gccctgttac gcgaaaatga agcgcaattg gaaaagcaag tggctgaaat gactgaaaaa 23340
gtggagctgg aactccgcca attaaaagag tgtcaacgtg ttctgcacca cacacgccag 23400
cagcaagaaa aatttaatga attgggtcgt cagcaacaag aggcgatccg ggcccaaggt 23460
gaataccagg aagagctgga acaagaagag ttccatcgcc aggaaagagt gtaaaacaga 23520
agagtatgaa cccactgaca gctatcggca gaaaagatgt ctcctccctc ccaacgtcgt 23580
cacccgcaga ggcagacgca gatttgcaac gccgtttcga gcaagctatc acctctcatg 23640
ccgccaacac gcgccagtca accacacggc agacggaaca accgttaacc aatacaacgg 23700
caaaccatga accagcggat caacatgcca cacatgactc agatgaggag cttccactat 23760
tcgactctcc ccaaccggca attgatgatc tgcgcaagca agccttacca ttgacaggcc 23820
atgacatgcc tgcaccacat caaaatcaga aaaaaaactt atcggccccc gctcaccgcc 23880
ttaatgagat cgctaacgag ataaaggaat cgctgtcagc acccggagtc ccaacacgtc 23940
agtcaaacct gaatgccttg tctgattctg acctaaagcc ggttgatatc acggtaactt 24000
ctaatcctga cacagcaatg atgcagacaa cacctggccg atccccatct cagccagaca 24060
cacaacacag cgccattcct caccaacaga cagacccgac gttacctata cctgcccgca 24120
atgccaacga aatcaaccct gcggaaggaa aaacacgaga tgaaatcgaa acatcccatt 24180
tgttggtatt aaaaccaaaa gagtccgact ctcaatcctc tgactctggt ggatcagacg 24240
gaaaaacacc tttcttccca caaacccaat tcctgccggg ggagcgcatc ctcgccacga 24300
tgcaagcaac ttcaaccata tctccccttt tggaagtgtt gatcgacaaa ctgagtgtgg 24360
agatcagtat cgaactaacc caacagccgc gtccggcgac attacatctg acacttccta 24420
acttgggagc acttgagatc caactcacca gcgagcatgg caaacttcaa atagaaattt 24480
tagcgaatcc cgccgctcaa caactcctga aacaagcacg ttttgaactg attgaacgtt 24540
tacagcttct ttacccaaca caaactgtcg aactctccct tccgccccaa acggatagtg 24600
aacatggatc acgtcaacgt cgcagcgtat atgaagaatg gaaaaaagat gcatgaatag 24660
cctgacctta tccaaagttt cattggaaga attaacgctg catcaggccc tgagtcgcca 24720
tcagcaacaa tttagttggg ataatagcca tctcacccta gatgtgatct ctcctcccca 24780
aacgttagac aaggtactga tcgcccattg gcagggacaa actttttcct tttactgcca 24840
tgcagccgag ctagcgctgt ggctcgcacc tgatctacaa caagcggacc tcacatcact 24900
ccctcaagat ctgctgttag cactactgga atatcagagc aaaatgctac ccactctgtc 24960
atggtcagca ctgagtacca cgtcagaacg ccgccctttg tcagcttgct tacgactccg 25020
ccttgaacgg ccaggggcca cattgccgct ctggttacca gaaccatccc ccttaattgc 25080
catattgcca gaacgtcaac ctagagaatg cctgcctatt ccattacgac tttctctgca 25140
atggggagca ataatcctga ctttagatgt gtttcgtact ctggaatcag gggatgtgct 25200
gctactgccc ccacagcaac aaccagatga ccctttatta gcgtatctgg aaggacgccc 25260
ttgggcctac tttaaatccc atgaccataa attggagtta atcactatgc acacactctc 25320
tcctgacagt tctgacaaca ctctgccagt aacagatcta aacgatctgg aggttcatgt 25380
cagtttcgaa gtgggccgtc agaccttgga cctacagaca ctcaccagcc tgcaacccgg 25440
ctcattactc gatcttggtg ttccttcaga cggtgcagtt cgtattcttg tcaatcaaaa 25500
atgcctgggt tcagggcggt tggtggatat tgagggacgt ctgggagtaa gagtcgaaca 25560
tctctccgtg gagaaacaat catgatccaa ctaccggacg aattagatct tatcatcggt 25620
ctggctttgc tggctctgct gccctttatt gccgtgatgg caacctcttt tctcaaactg 25680
gcggtggtat tttccttact acgtaacgcg ttgggcgtac agcagatccc acccaacatg 25740
gctatgtacg gtttggcaat tatcctcact atctatgtga tggctccggt gggtttcgcc 25800
actcaagatt acctgcgtca aaatgaagtt tcattcagca atgcgcagtc agtagataaa 25860
tttgtggaag agggcctgac accttaccga aattttctca aacaacatat taaacccagt 25920
gaacgcactt tctttatcga tagtacccga caaatttggc caagccagta tgccgatcgg 25980
ctagaaccag acagcctatt gatactgtta cctgccttta ccgtcagtga attaacccgg 26040
gcttttgaaa ttgggtttct tctttatcta ccatttattg caatcgatct cattatttcc 26100
aacattctgc tggcaatggg gatgatgatg gtttctccaa tgactatctc acttcctttc 26160
aaattactgt tatttgtcct gcttgatggc tggacacgcc tgacacatgg ccttgttatc 26220
agttatggag attgaatatg agtaatgctg atattttaca tttcactagc caatctctct 26280
ggttagtgtt aatcctttcc ctgccaccgg tactcatggc tgcggcagtg ggaacattgg 26340
tctcgttgat ccaagcactc acccaaatac aagaacaaac attgggtttt gttatcaaat 26400
taattgcagt cattatcacc ctgttcgcta ccgctacctg gcttggcaac gaactctaca 26460
gcttcgccaa tatgaccttt cttaaggtgc cacagatcaa atgaccctac ctgagctcca 26520
gcagcaaata cttgcttata ccctgttgtt accacgcatc atgagttgct ttgtgatatt 26580
tcctgtgctc agcaaacaga tgctaggtgg tgggttaatc cgcaacggcg tggcctgctc 26640
gctggccctt tatgcttatc caacagtagc aggcagccag ttgcccacat taagtagctt 26700
ggaattgacg ctgttgctcg gtaaagaagt cctgcttggt ttgctcattg gctttatcgc 26760
ttctatccca ttttgggcga tggaagccac cggttttatt atagataacc aacggggagc 26820
aacaatggct tcagtgctca accctgccct gggaagccag acctccccaa caggtctgtt 26880
attgactcaa acgctgatca cactcttttt ttccggcggc gcttttctta gcctgctcgg 26940
agccctattt cagagttata acagttggcc ggtaacacag tttttcccca aaattaccaa 27000
ccagtggctc cactttttct atggtcagtt tagctatcta ctacagctct gcgctttaat 27060
ggcagcccct ttgctaatcg ccatgtttct ggcagagttc gggctggcac tgattagccg 27120
tttcgctcct tctctcaacg ttttcgtatt ggcaatgccg atcaaaagcg caattgccag 27180
cctgttactg gttatctata tccagttatt gatgcatcat gcctatgata aggtgctgtt 27240
aatgctctcc ccagtacgca tgttaatccc tattctggag gcaccatgag cggagaaaag 27300
actgaaaaac ctactcccaa gaaacttaga gacgcccgga agaaaggcca agtaaccaaa 27360
agtaacgaag ttgtctccac ctcgttaatc ttggggctaa tcggcatgat aatggtcatg 27420
tctgaatact atttggagca tctgagtaaa ctgatgctga tcccggctaa tctgctagag 27480
aaaccctttc cccaagcctt caaccatgtg gttgaaaact tgatgcaaga gctggtctac 27540
ctctgcctgc caattctaag cgtttccgct ttgctatcgc tggtatcaca ctttgctcaa 27600
tatggttttc tacttagtgg acactccatc aaacctgata ttaaaaagat caaccctgtt 27660
gaaggtgcta agagaatctt ctctatcaag agtctggtgg aattcatcaa atcgatactt 27720
aaagtaggac tgctctgcac cttggtttgg attactctgg caggcaatct acaagcacta 27780
ctgcggctgc cggagtgtgg aacaagatgt attgttcctg tactcggcat aatgttaacc 27840
cagctcatga ccgtatgcgg catcgggctt atcgtgattt ctattgccga ttatgctttt 27900
gaacactacc aacatattaa acagcttaga atgagtaaag acgagatcaa acgggagtat 27960
aaagagagtg aaggcagccc agagatcaaa agcaaacgac gccaatttca ccaagaatta 28020
caatcaagca atatgcgcgc tagcgtcaag aattcatcag ttatcgtcgc caacccaact 28080
cacattgcgg taggtatccg ttataagaaa ggagagacac cgttacccct tattacactc 28140
aagttcacca atgcgcaggc cctacaagtt cggcgtattg ccgaagagga aggtattccg 28200
gtgttacaac gcattccgct ggcccgagca ctttatcaag atggcctgat agaccaatat 28260
attcctgctg atctcatcca agctaccgca gaagttttgc gctggttaga acagtggcaa 28320
gatcgcccgc cggagccgta atctgtcctc aaacagcact ttttgacagt caaactgcat 28380
atatgaactg agtattggca tttaacatac cttgccaatg ctcaattgca acaaacgaaa 28440
tacaatttat atttgtctct taagcgttac gatttaatta cataataaca acaaatttaa 28500
tataaattaa aattactgct aataacctaa cattgcttaa acaatattat aataaacaat 28560
ccaattaggt taggtgagtt ttctgttttt atcgattgat caaatagctg aaaccagatt 28620
gaatttatta ttaagtgatt tatattacac gtagctattt ggatttcact ttgcgctgac 28680
ttctacacct gttaattcgt taatattact ctagctatca ttcattacaa ttactaacgt 28740
attatgaatc aataatagat tgcctgaata aaaggagata actatcaata tcagtcaaga 28800
caaaagactc ggtttatata cccgttatct ttcaagttgc ctctttgttg gctgcactca 28860
ctcaccccgg tcacatcgtt ctctatgctc ccggggattc gctcccttgc cgccgcgata 28920
catcttgaaa tccatagggt ataactaagg gtgaaaaatt tacaggcaat cgttatcgtg 28980
caaaaaactg atcgcttttc actcttcgtt tataaagcgt ttataaaacg tgagaataat 29040
acagtattcg gcccaaagaa cattataggc ccactagcaa aaatcatttt aattaaaaga 29100
tgctatagtt aatcatcagg ttaaatacgt aaaattattc ttcactgtct attccaggca 29160
gcgaaaccaa aaaaatatct attaataata atctgatttg tatttttaag acataacacc 29220
aacagatcca ggacaataga atgaacggca tcacaacatc agggaaggga atggatatct 29280
cacgactcca acgtagcatg cctgccttta acgttataga gcacccccaa gaagggatat 29340
atatcctgct cgaaggtgaa atgacatggc aggacagtac caatacctac gatatctccc 29400
acaacgagct gctttttgtt cgtcgtggca gttatgcagc aaaaacgcgt agcgatactt 29460
gcaaactact ctggttgcct ctgagtacga ctttcctcca cgggtttctg caacgctttg 29520
gtactctgct cagtgaagtc gagcgacaca acatgtcaac acccgaatta attgcattta 29580
cctcatcccc tctgttatct caaagtataa atgggctggt cgatctattg gctcatgatt 29640
accccacagc gttgggtcaa ctcagaacag aagagttgtt attgttatta gcatttggag 29700
aacaaggtac tttgctcatg tcagtattac gacaattgag caatcgccaa gtggaacgtt 29760
tacagagttt tatggagaat cactacctta aggagtggaa actcagtgaa ttcgctaaag 29820
aatttggtat gggattaacc accttcaagg agctgttccg ttcggtatac ggaatatccc 29880
cacgcgcttg gatcagtgaa cgccgcattc tttatgcaca tcagctacta ctcaatagcg 29940
agatgagtat tgttgatatc tcgatggaag caggattttc cagccaatcc tactttactc 30000
aaagctatcg gagacgcttt aattgcacgc ccagtcgagc cagatatggc aaagagtaac 30060
gattggctaa aataatctga cgttttttac aagttaaccg ttggcctgac agataaaata 30120
aggccatccc caatttaatc gacaagagtg actagcagca agatgagcat agcatgagcc 30180
agcaagacca taatcagact cgaaccagca tgtttcttgg ccgcaagatc tctgtcacgc 30240
agtcgggaat tccacgccgg gatcaattac tcggaaaatc atcggctatt cagtatcaga 30300
atagagacat tattaatagc caacagttta ttttattaca gcgtttgcta cccagacatc 30360
tgctggaaag cttgatgaag tccgtctggt ttcgtagacg gttaaattgc gggcaagctt 30420
tgcggcgtga tgacttacaa caactcatca gagcggcggc agaactggaa tgtgactgga 30480
ataaggtact gggtgaccgc ataaacctag ctgacagaca tcttttacag cactgggtat 30540
tacagccgct atttgactgg tggctccgtt tgttggagcc agagatagac ttctggttca 30600
ctgaatttga tcagctacaa atccaagaac ggcaactgca tgctaaagcg catttttggc 30660
agcaagccga aagcgtctcg tctcaatgcc gggaacaaca acataaagag gttacatctt 30720
tgcaaacagc gcttaatcaa cgcaagcaac tattagaaaa acggctgttg acaacggaaa 30780
ctcaggcgcg cgaagcatgg cccaactggt ttactgggct agatgccctc caaactggcg 30840
gcgacctaac cgcttttatg ccggttcccg aagcactgtc atcctgttgg gcgtggttga 30900
ctactttaga gcatgacacc gaggcggcta ctcacttgca aaaatggctc tgtgcccgtt 30960
tcctgtgcct gccacaggac aattttcact ggcaatcgac gacctaatta ttagaggaaa 31020
catgcaaact ctactcaatc atttggccag tcgtttaggc cagaagcctt ttgtttccga 31080
tagacaaggg cattaccatt tgcacattga taactacaac ctgttttttc gccaacaggg 31140
aactgaactt gtgttatcca gcccgctaag ctggcgccat aacccaaaag atccgtcagg 31200
cgctgaatta ttgaaaacac tattgcaaca agttacgcgc tggggacgcc atgctcccca 31260
tgcattgacc ttagatgaat cagagaatct tattttggaa gcacgcctta accttgatag 31320
tttggatgtt caaatactgg aacagatgct aactctgcac gtcgatctac tggaacaagt 31380
catggcttta ttgtcggaaa cccaatctgc ccctcaatgg aaacaggcaa tatggcaacc 31440
atgaaaccat ttcaaaagct atgccgttcg ctactgttgt cgttgagtat cagcggtgtg 31500
atgctcccct ctgcttatag ccaagatctg gattggtttc ctgcgcctta tagctatctg 31560
gcaaaaggag aaagcttgcg ggatgtactg gttaatttcg gtgccaacta tgaagcatca 31620
gtggttgtca gcgacaaggt gaccgatcaa gttaacggcc attttgagca ggacactcct 31680
caggaattcc tgcaacattt atcctctctc tataatctgg tctggtacta cgacgggagc 31740
gtgctttatg tattcaaaaa cagtgaaata caatcgcgcc tgcttaaact tgaacgaacc 31800
aacgcaacgg agttacagca agccctaaaa cgttccggta tttgggagcc acgctttggt 31860
tggcgtcctg atccaagcaa ccaactggtt tacgtatcag gccctcctcg ttatcttgaa 31920
ctggttgatc agactacatc cgcactggaa cagcagtcat tgctgcgtag cgaaaaaacg 31980
ggtgccttga cagtggaaat tttcccgctc aaatacgcct cagtggtcga tcgcaagatc 32040
cagtatcgtg ataccagtat tgaggcaccg ggcatagcca ccattctctc ccgcctgctc 32100
agtgatacca atgtaactgc tgttactggt gaagcatcat ctccttctat gggagaaagc 32160
tactcaagcc gtgcagtcgt acaagcagaa ccctccctaa atgccatcat tgtacgggat 32220
aacccggaaa ggatgtccat gtacgcccat ttaatcaacg cactggataa accctccgcc 32280
cggattgaag ttgccctttc catcgtcgac atcgacgcaa acaacctttc agaactggga 32340
gttgactggc aagtcggcat caacaccggg cctcgccata tcattgatat cacaacgtct 32400
gccagggaaa aaacagagat caaagaaaaa ggtgaaagca tcggttcagc cgcattaggt 32460
agcctggttg atcgccgtgg tctggattac ctgctcgcca aaatcacctt actagagagt 32520
aaaggaagcg ctcaggtgat ttcccgccca acgcttttaa cgcaggagaa tacgcaagcc 32580
gtcattgaca acaatgaaac ctactacgtg aaagttaatg gcgaacgggt tgccgagctt 32640
aaaagcctta cctatggcac tatgctacgc atgatcccca gagtcatcca agtaggcgat 32700
tcttctgaaa tcagtttaga cctgcatatt gaggatggta accagaaacc taatagctca 32760
ggacaggatg gcatcccgac catcagccga accgtcgtcg ataccgttgc cagagttgga 32820
catgggcaaa gtctgctgat tggtggcatc taccgcgacg aaatgagtca aataacacgc 32880
aaggtgccat ttttaggcga tatcccctat ctgggtgtct tgtttcgcaa taccagtgag 32940
caagttcggc gatcagtacg gttattcatt attgagccac gtctgattga tagtgggata 33000
gcccattatc tggctcttgg taacggacaa gatctacgcc ctggattact ggcaacacag 33060
gatatctcta atcagagctt atcgttgggc aaagttctag ggagtgcaca atgccaacca 33120
ttatcctcag ctcgtcaaat acaagaaaca ctacgacaag caggtaaaag ctcattttta 33180
gactcatgcc gaatgggtaa cacttacgga tggcgagtca ttgaacagca atgttcactg 33240
aaagaaacct ggtgtgttcg tgtacaaaat aaggcgaata gatgagttgg aaaattcgct 33300
tttaccgcgg actgaataaa agtattgagg tcaatttagc tgaggggcgt ttagccatcg 33360
gctcagaccc cctacaggca gatatcgttc tggtggatga agatgttgct cctgtgcacc 33420
tgatcttaga agtcgaacca gactcaattc gcctgttgga atgggccggg gaagctgcac 33480
caagccagaa tggcgaaccg ctgtcaccgg gtgcgacatt gcaagcgctg gcccgtcagg 33540
aagttggccc gttattgtgg gctttttgtg accagcaaca tacattccca agccaactag 33600
ctgaacccgt ggtcacacca cagcgccctg cccggcaaaa tcgcagccgg gtagcaggcg 33660
gaatgttaat actttgtctg gtactggcac tcggtcttct ggcgctactt ggacatgaag 33720
ggtggcaaag aaaccacatg tccaatggca tagccgagca agaactacgc cgttttctca 33780
gtagtgattc agcctatcgc caagtgagcg tacaaatgat gcccaatcac gggccgccgt 33840
tgctcaaagg ttatgtggct ctcaaccacc aacggctggc cctgcaacag tatctcgata 33900
cccactctct cagttatcgg ctggagctac gtactatgga agagttacgt cagggggtag 33960
atttcatttt acaaaaatta ggctatgccc agatccaaag tagcgatggt aaacaacccg 34020
gttggatccg ccttataggc gaagtcactg ataccagcca gcaatggaac aatatcgagt 34080
cactactcaa acgtgatgtt cctggtttgc ttggcataga aaaccagacg cggttcaccg 34140
gtaaccacct caagcgactg gatatgttat tggctaaaca tgatttacct gacacgctga 34200
cctacaagga taaaaatgat cgtatcgagt tcataggtca actggatgaa caccaaacgc 34260
ataacttcta ccgcttacag caggtattta agcaagaatt cggcaaccag ccggcactgg 34320
tattactgaa taataagcag ctcacccgca atgatgagct caatttcgac gtccgggccg 34380
tttctctcgg gcgtgtgccc tatgtggtac tcaaaaacaa cttgcgttac ccggttggag 34440
caaccacaga aaacggttta ataatcgagg ccatccgtca agatgcgatt gttattacca 34500
agggtaagca acaatttatc gtcaaactta acaggagccc agcttcatga tgacatccct 34560
ggaagcacga ctgtttggtg ctgatccctc ttatgtcaac gaaatacaac atcgcctttc 34620
tcaggcgctg aacgatatca aacaacgcct gagatgtggc ggttcggtac aagaatacca 34680
acaatggcaa ctggaggctg atgctatcca agctgcaatc acaatactta acacaacaga 34740
aggagaccat catggcccaa atttttagtt ccgcaacaac agtgaataca ttcgataagg 34800
ttgctgacca gttgaaagag ccagctaacg cagccaacaa agatgttgat gaggcaatca 34860
cggccctcaa aggcgggcct gataacccag ctttgctagc tgatttacaa cacaagatta 34920
acaaatggtc ggtcatctac aacatcaact ctaccatcat ccgggccatg aaagacctga 34980
tgcagggtat attacagaag atctaatcat gacacgttca cttaaagggc tactggcaga 35040
tattgctctg gttggaagcg gccatcactg ccatgacgaa gccaatatta ttgccgactg 35100
gctgatgtca ggcgaagaag agcaagaagc tgcaaacctg attcggctct cttctctaac 35160
gaaccaaggc aaatatcaac aggctctgga ttttgggcgg gaccttccct ggccaagttt 35220
ggaaccttgg ttagcattgt gcgagtggcg tttgggtctg gcatcagcct tggagcagag 35280
actgacgcga atggctggca gtgacgatcc acaattgctc tccttcgtcg acggcatgcg 35340
ggaacaactg gctcatgaat aggattgaag ggctatccta taacaccgtg tcgcaaccaa 35400
cggtacagcc ttcggtcagt tccaagcacc agcacagctt tgagcaagca ctggaaacca 35460
cgcccgatca gcaaatgttg aaagagcgcc agcaacgctg gcttcaggga gaaccgctgg 35520
aaaacgtttt aggcgactta gatccagcga cacaacgcaa agtgatatgg cagtggtatc 35580
aggctttatc gtctgataaa cagccctccc aacgtgctca gttacaggcc aagctgattg 35640
ccccggtaca agagcgctta tggtcgcagt ttggtggctt ggcaggcaac atcaaaccac 35700
cactggatat gccagaactt agaaagattg ttcgtgaatt cgcccccaca ggtcgacaac 35760
aggagacggt tcttcttgag gtactcggcg aaattaagcc tattcctggc aatgagtacc 35820
tgagtgacct tatccgacaa gagttaaaaa cgctgatccc ccgtaacggt atggtggata 35880
acctgatacg aaactcacac aaacccgatt tggaagaata atggagcagc cacaacacca 35940
tgaaacaagg acagacaacc taaataaact gcacacacct caaatttgga ggaactatgg 36000
atctaccaca gataagcgag acactgatca ccagcctgga tgaacttaat acacatcagg 36060
ccagtgccga aaatattgcg agttttgaac acgctatgtc caatcaagat caggggattg 36120
aaaacaatct gatcaatgaa ttgggtcagc ttagacagca acttactcag gcgaaacaag 36180
atttggaaac acagttagcg gtctctggta gtgatcctaa cagcctgatg caaatgcagt 36240
ggtctctgat gcgaatcacc ctgcaagaag agctgattgc caaaacggta ggacgactca 36300
atcaaaacat tgaaacccta atgaaagccc agtgaggagc tgtgaagaaa cactatataa 36360
tctgcgttat gctcgctgtc ctgatgctaa ctggctgtaa aatcgaactc tataccgatg 36420
tgagccaaaa agaaggcaac gaaatgttag ccttgctcag ggaagcgggc atttcctctg 36480
ataagcagcc agacaaagat ggcaatatca agctgttagt cgaagagtct gatgtggcac 36540
aagctgttga agtgctcaaa cgtaaaggat atccacggga gaacttctct tcgttacaag 36600
atgtcttccc caaggacggc cttatctcct caccaataga ggagcgggca cggctcaact 36660
ttgccaaagc acaggagatt gcccgaacac tttccgagat cgatggcgtg ctcgtcgccc 36720
gggtacacgt tgttttaccc gaggagcaag accgactggg caaaaagcta tcgccagcct 36780
cttcctcggt gtttattaaa cacgcagcgg acgtgcaact cgacacttac atccctcaaa 36840
tcaagcaact ggttaataac agcattgaag ggttgagtta tgatcggatc agtgtcgtat 36900
tagtgcccgc cgcgggcgtg cgtcaggtac cgttggcacc acgctactcc actctgttct 36960
ctatccaagt gacggaagag tcacagggac ggttgattgg gctgttggtg ctattattag 37020
ccctgctatt tatcagcaat ctggcacaat tcctgtggca ccgcagcagg atacaataat 37080
ggtaacagca ttaacgccct atcagtttcg gttttgccct gccagctata ttcattctga 37140
tcatctatcc ccagaatggc ttaccgtctt atcttctcta cctgagtggc gacactctcc 37200
caggcttaat ggactactgc taacccaatt tgatctaaat gtggattatg aattaccaac 37260
tggattggga aacatcgccc tgttagagca atcgtgctta gaacaacttc ttacctggct 37320
aggtgcttta cttcatgggc aagctatccg ccattgtctg atggcgacag aacttcgcca 37380
cttgcatgac tcactaggaa aagagggaca tcgcttctgc ttaaagtatc tcgacatcat 37440
tatcggcaac tggccgactg gctggcagcg ctcgttacca ccagaaatca atgcaaacta 37500
ttttcgtacc agtgccttac agttctggct aacggcgatg gagtctcctc ctattgattt 37560
cgctaagcgc ctgagcctgc ggcttccctc ttatgaaaac ctcgcagcct ggcctgtcag 37620
ccaggcagaa cgaccgttgg cacaagcatt gtgtctcaaa ttggctaagc aagtgaacac 37680
agaatgttac catttattaa aataacgaca gggcatcttc aactgtctcc tgaactccag 37740
atactgcgta aagcggacta tcagacctgc ctcagtgcca aatctctgct tgaggctgcc 37800
cggctccagg cacaagaaat tgaacgtgac gcgcaggcag tgtatgagca acaaaaagag 37860
ttaggttggc aagcaggcat tgatgctgcc cgcgcagaac aagctaacct gattcatcaa 37920
acccagttgc aatgccaaca atattaccgg caggtcgagc aacagatgag caatgtagtc 37980
ttgcaggctg tacgtaaaat cctcaagaat tatgaccaag tttccctcac cttacaggta 38040
gtgcgtgagg ccctgtcatt agtcagtaac caaaaacagg ttatcctgcg cgttaatcct 38100
gaacaggccg caacagttcg cgaacaaata tcacgggtac acaaagattt ccctgaaatc 38160
ggctatttag agatcactgc tgatgagcgt cttgatcaag ggggctgtat tttagaaaca 38220
gaagtcggga tcattgatgc cagtctggat agccaattag aagcactgat gtcagccatc 38280
aataaccaat ggcaaagttg acgatgccat cacgcctcaa agagataacc agttttcgac 38340
ggaaagtgaa aatttcttct gccgttatta atattctcta tttcgacaaa acattatgat 38400
tcactctact taatatccaa acatctatct caaaaaacca cttaaggagt gaccattgtc 38460
tcccatctca agtattaatc accaaagaat atttcctacc cagcagctcg ataccacagc 38520
cgttaatcaa ccacagggtg aactgtcggg taaatccctg aaagtctctt cccccagacc 38580
ggggttgtta gagcgcctct cagcggcgat tcaacgcaat ctaccgggcc actccatgct 38640
cgatcgccaa ctcactactg atggcaagaa gaaccaagag agccgcttct cgttcagcat 38700
gataaaagac agaatcgttc actttgcggt gagcaccacg ttagggagtg ttcgaaattc 38760
tgcgagtaag cacggcggcg aaatcacctt caagttcgcc cagaccaagg ggacctttct 38820
ggatcaaatc atgaaacata gtgacaccgc cggcggggtt tgtgaatcca tatcagctca 38880
ttggataagc gctcatgcca aaggggaaag tgtcttcgat caactctatg tgggcggcca 38940
aaaaggacag tttcatatcg actctctggt ttccattaag cagttacaga tggatggtat 39000
agcccaagat gtcgatcaag acactatgac cgaatcttgg cttagcgaaa atggcatcca 39060
accacgcatg aaaacgataa cctatcaaag cgccaacggg cccattaatt acaaacgtcc 39120
aatcgaagtc aacggtcaga ccggcagtaa tggcacatca gatctactaa acgctattct 39180
agacacaggc gataaaggtt ctagctataa aaagattagt ttttcaggac agatggccgg 39240
gcatgccgta gccgcttatg tagatgacca gaagggtgtt actttctttg accctaactt 39300
tggcgagttc cactttccaa acaaagaatc attctctaat tggttctctc aagatttctg 39360
gtccaaatcc atgtacaaca aggaaatcgg tcttgggcaa catttccaag tcctcaacta 39420
cgagcctaaa acacaatgac acacgactat caccaactta tagaagccct gttctctaaa 39480
ctcggggtac ctgcatcgcc tactgaggat ggtatctaca ctctcgaagt tgacgggcgt 39540
tttcaagtat tgattggctg ttatcacgac aactggctac agttgttctg cgaattaggg 39600
tccgaaatca gtcatgccaa caatcttttt gggcaacatt ggccggctca tgttcaaggt 39660
gatttggatg gacaagcaat tttgtggtcg caacgccctt tgggcgaggt agacgtagcc 39720
gagctacaag cttggttaga acgctttatt gatgatattg agcagcgcct gacacctaac 39780
acaacccaac agcctaaaaa tccgccgcct tccttggtgc gggtttgatc gctaaataca 39840
aaccactacc agaatataag ttcaatggga gcttcaatat ttagttccct ttaaactgcc 39900
aaggcaaaat aatcaagcaa agagccaagc aaaatgctac tgcctccatg caaaaaaccc 39960
gcaactaagc gggtttttac ttattaacaa ctttaactac tcaggatatt accttcatcg 40020
ctatcgtaaa gttgctaata gacttaatca gttaaattaa gataatacga taacatttac 40080
cgcagatggg cctttagcgc cattctcaat agagaatgag acttcctgac cttcttccaa 40140
tgttttgaaa ttattactct gaattgcaga ataatgaacg aaaacatctt tgctaccatc 40200
ttttggggaa ataaagccaa aacctttatc gttgttaaac cattttactg aaccagtcat 40260
tgaactagac atagaatttt cctttaaatt tattaagttg ccataaggcg ttatgaggtc 40320
tgtttttcag ttttacttat gggtactaat tagaaggaat tcgcaatgaa gtggtatcag 40380
ggataacgct aaatggtgaa ctgctttaat tttaatactt ttcattaata cttttcataa 40440
ataggtctgt acttccaaac cagtgacgct attaaatcac attgattggt cattagcaaa 40500
ttttaatttc atttattttt ttctattagt gttctcattt atcctaaatt tcggctttat 40560
ccatattgca tggctttacc atagccataa ctagctaatt atgaaaggtt tttatccaca 40620
agccctattt gccgagatta cggcactgag tttgaattta ccattcctcc ctagcaccct 40680
aatcagacta caacccgtaa cctaaaatta atgaaaataa agctaacatc tagtctgtaa 40740
ataaccatcg cagttcacga gctgtgatac cttaggatac gggaaaaacc gagtaccgtt 40800
atcgacagc 40809
<210> 2
<211> 2351
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
actagtacga tgccatcacg cctcaaagag ataaccagtt ttcgacggaa agtgaaaatt 60
tcttctgccg ttattaatat tctctatttc gacaaaacat tatgattcac tctacttaat 120
atccaaacat ctatctcaaa aaaccactta aggagtgacc attgtctccc atctcaagta 180
ttaatcacca aagaatattt cctacccagc agctcgatac cacagccgtt aatcaaccac 240
agggtgaact gtcgggtaaa tccctgaaag tctcttcccc cagaccgggg ttgttagagc 300
gcctctcagc ggcgattcaa cgcaatctac cgggccactc catgctcgat cgccaactca 360
ctactgatgg caagaagaac caagagagcc gcttctcgtt cagcatgata aaagacagaa 420
tcgttcactt tgcggtgagc accacgttag ggagtgttcg aaattctgcg agtaagcacg 480
gcggcgaaat catgactatc aaagtaggta ttaacggttt tggccgtatc ggtcgtttcg 540
tattccgtgc agcgcaagag cgcaacgaca tcgaagtagt aggtattaac gaccttatcg 600
atgtagatta catggcatac atgcttaagt atgactcaac tcacggccgt ttcaacggta 660
ctgttgaagt tgaaggcggt aacctaatcg ttaacggtaa aactgtacgt gttactgcag 720
agcgcaaccc tgaagaccta aaatgggacg aaatcggtgt tgacgttgta gctgaagcaa 780
ctggtctttt cctaactgac gagactgcac gtaagcacat cactgctggc gcgaaaaaag 840
ttgtactaac tggtccttct aaagacgcta ctccaatgtt cgttaacggc gtaaacttcg 900
acacttacgc tggtcaagac atcgtttcta acgcttcttg tactactaac tgtctagcgc 960
ctatcgctaa agttcttaac gacaagttcg gtatcgaatc tggtcttatg actacagttc 1020
acgcaactac agcaactcaa aaaactgtag atggtccttc tgctaaagac tggcgcggtg 1080
gtcgtggtgc ttctcaaaac atcatcccat cttcaactgg tgctgctaaa gctgtaggcg 1140
ttgtacttcc agaagtaaac ggcaaactaa ctggtatggc tttccgcgta ccaactgcta 1200
acgtttctgt agttgaccta actgttaacc tagttaacgg tgcttcttac gaagacatct 1260
gtaaagcgat gaaagaagct tctgaaggcg aactaaaagg cgttctaggc tacactgaag 1320
atgcagttgt atctcaagac ttcatcggcg aagtttgcac ttcagtattc gatgctaaag 1380
ctggtatcgc gctaactgac aaattcgtta aggttgtatc ttggtacgac aacgaaatcg 1440
gttactcaaa caaagttcta gacctaatcg ctcacatctc taaggattac aaggatgacg 1500
acgataaggg agattacaag gatgacgacg ataagatcga ttacaaggat gacgacgata 1560
aggggcaaca tttccaagtc ctcaactacg agcctaaaac acaatgacac acgactatca 1620
ccaacttata gaagccctgt tctctaaact cggggtacct gcatcgccta ctgaggatgg 1680
tatctacact ctcgaagttg acgggcgttt tcaagtattg attggctgtt atcacgacaa 1740
ctggctacag ttgttctgcg aattagggtc cgaaatcagt catgccaaca atctttttgg 1800
gcaacattgg ccggctcatg ttcaaggtga tttggatgga caagcaattt tgtggtcgca 1860
acgccctttg ggcgaggtag acgtagccga gctacaagct tggttagaac gctttattga 1920
tgatattgag cagcgcctga cacctaacac aacccaacag cctaaaaatc cgccgccttc 1980
cttggtgcgg gtttgatcgc taaatacaaa ccactaccag aatataagtt caatgggagc 2040
ttcaatattt agttcccttt aaactgccaa ggcaaaataa tcaagcaaag agccaagcaa 2100
aatgctactg cctccatgca aaaaacccgc aactaagcgg gtttttactt attaacaact 2160
ttaactactc aggatattac cttcatcgct atcgtaaagt tgctaataga cttaatcagt 2220
taaattaaga taatacgata acatttaccg cagatgggcc tttagcgcca ttctcaatag 2280
agaatgagac ttcctgacct tcttccaatg ttttgaaatt attactctga attgcagaat 2340
aatgagggcc c 2351
<210> 3
<211> 550
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Leu Ala Ala Gly Leu Thr Thr Ala Gly Leu Leu Ala Gly Gly Ser Ala
1 5 10 15
Pro Ser Pro Ser Met Ile Leu Ala Ala Ile Val His Pro Ala Val Ser
20 25 30
Thr Thr Leu Gly Ser Val Ala Ala Ser Ala Ser Leu His Gly Gly Gly
35 40 45
Ile Met Thr Ile Leu Val Gly Ile Ala Gly Pro Gly Ala Ile Gly Ala
50 55 60
Pro Val Pro Ala Ala Ala Gly Gly Ala Ala Ala Ile Gly Val Val Gly
65 70 75 80
Ile Ala Ala Leu Ile Ala Val Ala Thr Met Ala Thr Met Leu Leu Thr
85 90 95
Ala Ser Thr His Gly Ala Pro Ala Gly Thr Val Gly Val Gly Gly Gly
100 105 110
Ala Leu Ile Val Ala Gly Leu Thr Val Ala Val Thr Ala Gly Ala Ala
115 120 125
Pro Gly Ala Leu Leu Thr Ala Gly Ile Gly Val Ala Val Val Ala Gly
130 135 140
Ala Thr Gly Leu Pro Leu Thr Ala Gly Thr Ala Ala Leu His Ile Thr
145 150 155 160
Ala Gly Ala Leu Leu Val Val Leu Thr Gly Pro Ser Leu Ala Ala Thr
165 170 175
Pro Met Pro Val Ala Gly Val Ala Pro Ala Thr Thr Ala Gly Gly Ala
180 185 190
Ile Val Ser Ala Ala Ser Cys Thr Thr Ala Cys Leu Ala Pro Ile Ala
195 200 205
Leu Val Leu Ala Ala Leu Pro Gly Ile Gly Ser Gly Leu Met Thr Thr
210 215 220
Val His Ala Thr Thr Ala Thr Gly Leu Thr Val Ala Gly Pro Ser Ala
225 230 235 240
Leu Ala Thr Ala Gly Gly Ala Gly Ala Ser Gly Ala Ile Ile Pro Ser
245 250 255
Ser Thr Gly Ala Ala Leu Ala Val Gly Val Val Leu Pro Gly Val Ala
260 265 270
Gly Leu Leu Thr Gly Met Ala Pro Ala Val Pro Thr Ala Ala Val Ser
275 280 285
Val Val Ala Leu Thr Val Ala Leu Val Ala Gly Ala Ser Thr Gly Ala
290 295 300
Ile Cys Leu Ala Met Leu Gly Ala Ser Gly Gly Gly Leu Leu Gly Val
305 310 315 320
Leu Gly Thr Thr Gly Ala Ala Val Val Ser Gly Ala Pro Ile Gly Gly
325 330 335
Val Cys Thr Ser Val Pro Ala Ala Leu Ala Gly Ile Ala Leu Thr Ala
340 345 350
Leu Pro Val Leu Val Val Ser Thr Thr Ala Ala Gly Ile Gly Thr Ser
355 360 365
Ala Leu Val Leu Ala Leu Ile Ala His Ile Ser Leu Ala Thr Leu Ala
370 375 380
Ala Ala Ala Leu Gly Ala Thr Leu Ala Ala Ala Ala Leu Ile Ala Thr
385 390 395 400
Leu Ala Ala Ala Ala Leu Gly Gly His Pro Gly Val Leu Ala Thr Gly
405 410 415
Pro Leu Thr Gly Met Thr His Ala Thr His Gly Leu Ile Gly Ala Leu
420 425 430
Pro Ser Leu Leu Gly Val Pro Ala Ser Pro Thr Gly Ala Gly Ile Thr
435 440 445
Thr Leu Gly Val Ala Gly Ala Pro Gly Val Leu Ile Gly Cys Thr His
450 455 460
Ala Ala Thr Leu Gly Leu Pro Cys Gly Leu Gly Ser Gly Ile Ser His
465 470 475 480
Ala Ala Ala Leu Pro Gly Gly His Thr Pro Ala His Val Gly Gly Ala
485 490 495
Leu Ala Gly Gly Ala Ile Leu Thr Ser Gly Ala Pro Leu Gly Gly Val
500 505 510
Ala Val Ala Gly Leu Gly Ala Thr Leu Gly Ala Pro Ile Ala Ala Ile
515 520 525
Gly Gly Ala Leu Thr Pro Ala Thr Thr Gly Gly Pro Leu Ala Pro Pro
530 535 540
Pro Ser Leu Val Ala Val
545 550

Claims (9)

1.一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗，其特征在于：所述二联疫苗为工程菌株DBFF01-T3SS-GAPDH，其拉丁文名称为Aeromonas salmonicida DBFF01-T3SS-GAPDH，该菌株已保藏于中国典型培养物保藏中心，保藏地址：中国，武汉，武汉大学，保藏编号：CCTCC NO：M 2021557，保藏日期：2021年05月19日。
2.如权利要求1所述一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗，其特征在于：所述DBFF01-T3SS-GAPDH菌株的T3SS编码基因来源于P. luminescens TT01，其GAPDH编码基因来源于迟缓爱德华氏菌。
3.如权利要求2所述一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗，其特征在于：所述T3SS的核苷酸序列如SEQ ID NO.1所示。
4.如权利要求2所述一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗，其特征在于：所述P. luminescens TT01菌株中的III型效应因子LopT的N端信号肽编码基因与迟缓爱德华氏菌的GAPDH编码基因融合，并构建了该融合基因与LopT蛋白的伴侣蛋白SlcT编码基因的共表达载体LopT-GAPDH-3FALG-SlcT。
5.如权利要求4所述一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗，其特征在于：所述LopT-GAPDH-3FALG-SlcT的核苷酸序列如SEQ ID NO.2所示，其编码的蛋白质的氨基酸序列如SEQ ID NO.3所示。
6.如权利要求1所述一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗，其特征在于：所述二联疫苗的接种方式为浸泡免疫。
7.一种如权利要求1-6任一项所述具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗的制备方法，其特征在于：包括以下步骤：
1）通过三亲本结合转移将P. luminescens TT01菌株的T3SS插入杀鲑气单胞菌DBFF01的基因组上，获得重组菌株DBFF01-T3SS；
2）构建pBBR1-Cm-GAPDH原核表达质粒；
3）将pBBR1-Cm-GAPDH原核表达质粒电击转化至E. coli MFDpir中，获得接合转移供体菌株MFDpir/pBBR1-Cm-GAPDH；
4）通过双亲本接合转移将pBBR1-Cm-GAPDH原核表达质粒转入重组菌株DBFF01-T3SS中，获得菌株DBFF01-T3SS-GAPDH。
8.如权利要求7所述一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗的制备方法，其特征在于：所述步骤2）的具体操作步骤为：采用PCR扩增方法扩增LopT-GAPDH-3FLAG-SlcT的基因片段，并克隆到表达质粒pBBR1MCS2-Tac-mCherry-Cm，获得重组质粒pBBR1-Cm-GAPDH。
9.一种如权利要求1-6任一项所述具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗的应用，其特征在于：所述二联疫苗用于制备抗杀鲑气单胞菌和迟缓爱德华氏菌感染的药物。
CN202111351867.8A 2021-06-21 2021-11-16 一种具有防治鱼类杀鲑气和迟缓爱德华氏菌感染的二联疫苗及其制备方法与应用 Active CN114164159B (zh)

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