CN107446951A - 一种通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法及其应用 - Google Patents

一种通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法及其应用 Download PDF

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CN107446951A
CN107446951A CN201710470317.5A CN201710470317A CN107446951A CN 107446951 A CN107446951 A CN 107446951A CN 201710470317 A CN201710470317 A CN 201710470317A CN 107446951 A CN107446951 A CN 107446951A
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周庆丰
林丽苗
李群辉
余国莲
李薇
杜云平
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Guangdong Wens Foodstuff Group Co Ltd
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Abstract

本发明涉及生物技术领域，具体是指一种通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法及其应用。所述方法步骤包括：设计靶基因的sgRNA双链寡核苷酸序列；将所述序列与线性化的质粒载体连接，得到sgRNA表达载体；将sgRNA表达载体、表达外源基因的重组鸡痘病毒质粒、鸡痘病毒共转染鸡胚成纤维细胞(CEF)；纯化并验证纯化效果。本发明通过CRISPR/Cas9方法将重组鸡痘病毒筛选代次降低至3‑4代，大大提高了疫苗生产的效率，操作简便，降低了生产成本。

Description

一种通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法及 其应用
技术领域
本发明涉及生物技术领域，具体是指一种通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法及其应用。
背景技术
鸡痘病毒基因组的高保守区的非复制区域可以整合外源DNA，已经成为普遍的表达载体，在病毒分子生物学、载体疫苗生产等领域得到广泛应用。鸡痘病毒载体疫苗不仅能预防鸡痘的发生，而且还能通过插入其他病原的保护性抗原基因，诱导表达插入基因提高相对应病原的免疫抵抗力。由于重组鸡痘病毒所表达的外源基因在机体内随载体病毒的复制而表达，与灭活疫苗相比，免疫剂量小，接种方法简单，免疫一次能获得长期的免疫效果，很大程度上降低了生产成本。目前，国内外已有多种表达外源基因的重组鸡痘疫苗得到生产应用。
CRISPR/Cas9是继第一代人工核酸内切酶—锌指核酸酶(zinc-fingernucleases,ZFN)和第二代人工核酸内切酶—转录激活因子样效应因子核酸酶(transcription activator-like effectornucleases,TALEN)后产生的第三代人工核酸内切酶,可用于各种复杂基因组的定点修饰,修饰类型包括基因的定点突变、基因定点插入、多个位点同时突变和小片段的缺失(Tremblay JP.The CRISPR system can corrector modify the expression of genes responsible for hereditary diseases.Med Sci(Paris)2015；31(11):1014-22.)(Spencer NY,Yan Z,Cong L,Zhang Y,Engelhardt JF,Stanton RC.Definitive localization of intracellular proteins:Novel approachusing CRISPR-Cas9genome editing,with glucose 6-phosphate dehydrogenase as amodel.Anal Biochem 2016；494:55-67.)。目前,CRISPR/Cas9已成功应用于小鼠、斑马鱼和人类细胞甚至细菌的基因组精确编辑(Li JF,Zhang D,Sheen J.Targeted plant genomeediting via the CRISPR/Cas9technology.Methods Mol Biol 2015；1284:239-55.)。由于CRISPR/Cas9技术具有突变效率高、制作简单而且成本低廉等优势,已被公认为是一种具有广阔应用前景的基因组定点改造分子工具。
CRISPR/Cas9系统的工作原理是crRNA(CRISPR-derived RNA)通过碱基配对与tracrRNA(trans-activating RNA)结合形成tracrRNA/crRNA复合物，此复合物引导核酸酶Cas9蛋白在与crRNA配对的序列靶位点剪切双链DNA。而通过人工设计这两种RNA，可以改造形成具有引导作用的sgRNA(short guide RNA)，足以引导Cas9对DNA的定点切割。目前，有报道显示通过CRISPR/Cas9对痘病毒进行切割抑制病毒复制，但在提高重组鸡痘病毒筛选方面还没有报道(王娇娇，张新敏，倪爱民等CRISPR/Cas9切割痘病毒DNA抑制病毒复制中国细胞生物学学报Chinese Journal of Cell Biology 2016,38(4))。在重组鸡痘病毒的构建过程中，目前使用的方法是使用脂质体或电转等方法将含有表达外源基因的质粒与鸡痘病毒共转染，再通过报告基团EGFP、lacz进行空斑筛选，要经过十几代的筛选才能得到纯的重组鸡痘病毒，生产周期长。足见，现有技术有待改善。
发明内容
有鉴于此，有必要针对上述的问题，提供了一种通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法及其应用，通过CRISPR/Cas9系统方法将重组鸡痘病毒筛选代次降低至3-4代，大大提高了疫苗生产的效率。
本发明目的通过以下技术方案实现：
一种通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法，其方法步骤如下所示：
(1)选取靶基因序列：根据重组鸡痘病毒同源重组序列，选取待编辑序列，在待编辑序列上游寻找PAM位点，用Cas9核酸酶识别的PAM位点上的序列为NGG(N:A、T、C、G)，该PAM位点上游的20个碱基序列为靶基因序列；
(2)根据靶基因序列设计sgRNA序列，为两条反向互补的上下游引物，再分别在上下游引物5’端加上CCGG、AAAC即为sgRNA双链寡核苷酸序列；所述sgRNA双链寡核苷酸序列结构为：
Forward oligo：5’CCGG......3’
Reverse oligo：5’AAAC......3’；
其中“......”代表引物序列；
(3)将靶基因的sgRNA双链寡核苷酸序列与线性化的质粒载体(clontech产品Guide-itTMCRISPR/Cas9sgRNA克隆、表达系统中线性化质粒pGuide-it-ZsGreen1 Vector)连接，转化提取得到sgRNA表达载体；
(4)按照脂质体转染法步骤，将上述sgRNA表达载体、表达外源基因的重组鸡痘病毒质粒、鸡痘病毒共转染鸡胚成纤维细胞(CEF)；
(5)将表达外源基因重组鸡痘病毒再经过3-4代纯化从而得到表达外源基因重组鸡痘病毒并验证纯化效果。
进一步的，步骤(3)中具体操作步骤包括：
(3.1)先将上述步骤(2)中的sgRNA双链寡核苷酸序列分别稀释；
(3.2)将(3.1)中稀释的sgRNA双链寡核苷酸序列与Guide-it Oligo AnnealingBuffer(寡核苷酸退火缓冲液)进行PCR反应；
(3.3)将(3.2)中所得的PCR反应产物用Guide-it Oligo Annealing Buffer做100倍稀释；
(3.4)将(3.3)所得稀释产物进行连接反应；
(3.5)将(3.4)的连接产物进行转化放映，即得sgRNA表达载体。
进一步的，所述步骤(3.1)中sgRNA双链寡核苷酸序列的稀释浓度均为100μmol/L。
进一步的，所述步骤(3.2)中PCR反应体系为：sgRNA双链寡核苷酸序列各1ul、Guide-it Oligo Annealing Buffer：8ul。
进一步的，所述步骤(3.2)中PCR反应程序为：95℃，2min；然后10min内缓慢退火从85℃退至30℃；然后25℃时停止。
进一步的，所述步骤(3.4)中连接反应的反应体系为：步骤(3.3)中所得的稀释液：1ul、pGuide-it Vector(Linear)(7.5ng/ul)：2ul、ddH2O：2ul、DNA Ligation Mighty Mix(DNA连接混合液)：5ul。
进一步的，所述步骤(3.4)中连接反应的反应条件为：16℃反应30min。
进一步的，所述步骤(3.5)中转化操作包括：取5ul步骤(3.4)中所得的连接反应产物于50ulDH5a感受态中，冰浴30min，42℃热激90s，冰浴2min，加500ulLB培养基于37℃摇床中，130rpm活化30min。
进一步的，上述通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法在疫苗制备中的应用。
一种利用本发明方法筛选的重组鸡痘病毒制备的载体疫苗。
本发明有益效果：
CRISPR/Cas9在基因编辑上具有效率高、操作简便、成本低等特点，本发明通过CRISPR/Cas9方法将重组鸡痘病毒筛选代次降低至3-4代，大大提高了疫苗生产的效率，降低了生产成本。
附图说明
图1重组病毒rFPV-FADV4 fiber2转移质粒图谱。
图2重组病毒rFPV-FADV4 fiber2感染CEF后蚀斑图。
图3TYB引物鉴定重组病毒rFPV-FADV4 fiber2PCR结果。1-4：单独转移载体rFPV-FADV4 fiber2与FPV共转染CEF细胞后纯化第3、7、9、12代后抽提DNA；5-7：转移载体rFPV-FADV4 fiber2、表达sgRNA的CRISPR/Cas9质粒以1:1比例与FPV共转染CEF细胞纯化第3、4、5抽提的DNA；8：转移载体rFPV-FADV4 fiber2阳性对照；9：FPV DNA阳性对照；10：DL10000。
图4为FADV4 fiber2引物鉴定重组鸡痘病毒rFPV-FADV4 fiber2PCR图。1：质粒pMD22-TYB-lacz-F4与鸡痘病毒共转染CEF细胞纯化第7代抽提的DNA；2：质粒pMD22-TYB-lacz-F4和CRISPR/Cas9表达质粒以1:1比例与鸡痘病毒共转染CEF细胞纯化第4代抽提的DNA；3：阳性4：阴性 5：DL2000。
图5为重组鸡痘病毒rFPV-FADV4 fiber2感染CEF细胞的fiber2基因表达验证试验结果。
图6为正常CEF细胞的fiber2基因表达验证试验结果。
图7为感染了鸡痘病毒的CEF细胞的fiber2基因表达验证试验结果。
具体实施方式
为了更好地说明本发明所解决的问题、所采用的技术方案和所达到的效果，现以CRISPR/Cas9系统快速筛选表达鸡4型腺病毒fiber2基因的重组鸡痘病毒(rFPV-FADV4fiber2)为例进行具体实施例和相关资料的进一步阐述。需要说明的是，本发明实施例仅采用表达鸡4型腺病毒fiber2基因的重组鸡痘病毒(rFPV-FADV4 fiber2)为例，本发明方法不仅局限于用于快速筛选表达鸡4型腺病毒fiber2基因的重组鸡痘病毒(rFPV-FADV4fiber2)，也可用于其他病毒和其他基因，本发明内容包含但不限于以下实施例及其组合实施方式。
实施例1
1.材料
1.1 病毒毒株和细胞
鸡痘病毒鹌鹑化弱毒株(CVCC AV1003)购自本公司大华农生物科技公司，鸡4型腺病毒毒株由鸡场分离并保存，鸡胚成纤维细胞(CEF)用温氏公司的SPF鸡胚制备。
1.2 质粒和菌种
感受态JM109购自宝生物公司，表达FADV4 fiber2重组鸡痘病毒(rFPV-FADV4fiber2)转移质粒pMD22-TYB-lacz-F4为自行构建，构建流程见图1。
1.3 主要试剂
CRISPR/Cas9试剂盒、IPTG、X-gal购自TAKARA公司，DNA回收试剂盒、质粒抽提试剂盒购自OMEGA公司，DNA抽提试剂盒购自Axygen公司，胎牛血清(FCS)和DMEM培养基购自Thermo公司，转染试剂购自life公司。
2.方法步骤：
2.1、选取靶基因序列
重组鸡痘病毒转移质粒pMDTYB22-lacz-F4构建(其构建路线路见图1)，具体步骤如下：
(1)构建含同源重组臂基因的质粒pMD-TYB：以购自本公司大华农生物科技公司的鹌鹑化弱毒株(CVCC AV1003)鸡痘病毒疫苗所抽提的核酸作为模板，使用引物设计软件premier5.0根据GenBank上FPV毒株(GenBank:AF198100.1)的序列设计引物LTYB-F/R、RTYB-F/R(见表1)分别扩增左右同源臂序列，先将扩增的左同源臂序列(SEQ ID NO:3)TA克隆至pMD19T-Simple载体，为质粒pMD-LTYB，再将扩增的右同源臂序列(SEQ ID NO:4)插入到NotI和EcoRI位点间，为含有左右同源重组臂的质粒pMD-TYB；
(2)构建质粒pMD22：合成含鸡痘病毒早晚期启动子LP2EP2、P11启动子的多克隆位点序列(SEQ ID NO:6)，并通过TA克隆至pMD19T-Simple载体中，形成质粒pMD22；
(3)构建质粒pMD22-lacz：以质粒pSV-β-Galactosidase Control Vector为模板，设计引物lacz-F/R(见表1)扩增lacz序列(SEQ ID NO:7)，使用infusion酶将扩增的lacz序列插入质粒pMD22的XhoI位点，形成质粒pMD22-lacz；
(4)构建中间载体pMD22-TYB-lacz：将质粒pMD22-lacz、pMDTYB用NotI酶切连接，形成含有左右同源臂、鸡痘病毒早晚期启动子LP2EP2的多克隆位点、P11启动下lacz基因的中间转移载体pMD22-TYB-lacz(SEQ ID NO:8)；
(5)扩增FADV4 fiber2基因：根据FADV4HB151株fiber2基因序列设计引物F4-fiber2-F/R(见表1)；以本公司某鸡场采集样品分离的病毒，并标号为F4的毒株为模板扩增FADV4 fiber2基因，所得扩增产物序列为SEQ ID NO：9所示；
(6)构建重组鸡痘病毒转移载体pMD22-TYB-lacz-F4：将(5)中所得的FADV4fiber2基因(SEQ ID NO:9)插入到质粒pMD22-TYB-lacz(SEQ ID NO:8)的SmaI位点，即得表达鸡4型腺病毒fiber2基因的重组鸡痘病毒转移载体pMD22-TYB-lacz-F4，序列如SEQ IDNO：1所示；
在构建上述质粒pMD22-TYB-lacz-F4过程中，根据鸡痘病毒(FPV)毒株(GenBank:AF198100.1)序列先选择鸡痘病毒的一段非复制必需区(见序列SEQ ID NO：2)作为同源重组位置TYB，再在这个序列基础上选择左同源臂LTYB(SEQ ID NO：3)和右同源臂RTYB(SEQID NO：4)，同时在左右同源臂间预留一小段序列作为设计sgRNA序列的待编辑序列(SEQ IDNO：5)，在待编辑序列中寻找PAM位点，Cas9核酸酶可识别的待编辑序列中的PAM序列为NGG(N:A、T、C、G)，PAM位点上游的20个碱基序列为靶基因序列。
表1引物序列表
2.2 根据靶基因序列设计合成sgRNA序列
根据靶基因序列运用clontech网上设计软件(http://crispr.mit.edu/)设计sgRNA序列，为两条反向互补引物；再在上游引物5’端加上CCGG四个碱基，在下游引物5’端加上AAAC四个碱基，设计合成sgRNA双链寡核苷酸序列，序列分别为SEQ ID NO：10和SEQ IDNO：11：
SEQ ID NO：10：5’CCGGCCGATAGACTATGGCGATGA3’；
SEQ ID NO：11：5’AAACTCATCGCCATAGTCTATCGG3’。
2.3 将上述合成sgRNA双链寡核苷酸序列连接至线性化表达载体上
将上述SEQ ID NO：10和SEQ ID NO：11按照TAKARA公司CRISPR/Cas9试剂盒方法通过变性、退火之后连接至线性化质粒pGuide-it-ZsGreen1 Vector(Linear)(7.5ng/ul)上，步骤如下所示：
(1)先将SEQ ID NO：10和SEQ ID NO：11分别稀释至浓度为100μmol/L；
(2)在200ul PCR管中配以下体系(总计10ul)：
SEQ ID NO：10(100μmol/L)：1ul、SEQ ID NO：11(100μmol/L)：1ul、Guide-itOligo Annealing Buffer(寡核苷酸退火缓冲液)：8ul；
在PCR仪中按以下程序反应：95℃，2min；然后10min内缓慢退火从85℃退至30℃；然后25℃时停止，双链DNA形成；
(3)将上述反应产物1ul加至99ul Guide-it Oligo Annealing Buffer中，混匀；稀释退火形成的双链DNA，稀释液备用；
(4)连接
反应体系(总计10ul)如下：(3)中所得稀释液1ul、pGuide-it-ZsGreen1 Vector(Linear)(7.5ng/ul)线性化质粒2ul、ddH2O 2ul、DNA Ligation Mighty Mix(DNA连接混合液)5ul；
反应条件：16℃连接反应30min；
(5)转化：取5ul(4)中所得的连接反应产物于50ulDH5a感受态中，冰浴30min，42℃热激90s，冰浴2min，加500ulLB培养基于37℃摇床中，130rpm活化30min；再取100ul菌液涂于含有氨苄青霉素的LB板上，37℃培养16h左右，挑取单个克隆，溶于含有氨苄青霉素的LB液体培养基中，按照OMEGA质粒小量抽提试剂盒说明书抽提质粒。
2.4 阳性质粒判断
将抽提的质粒使用试剂盒中引物dGuide-it Sequencing Primer 1(序列NO：8)送测序公司测序，根据测序所得序列寻找CCGG碱基后面20个碱基是否为序列：AATGGACTATCATATGCTTACCGT，若是则为阳性质粒，即为表达sgRNA的CRISPR/Cas9表达质粒。
2.5 将表达FADV4 fiber2重组鸡痘病毒转移质粒pMD22-TYB-lacz-F4、表达sgRNA的CRISPR/Cas9表达质粒与鸡痘病毒共转染CEF细胞，同时设置单独的转移质粒pMD22-TYB-lacz-F4与鸡痘病毒共转染CEF细胞作为对照，具体操作如下：
先制备好CEF细胞:①将选好的两枚9-10日龄的发育良好的SPF胚用5％的碘棉消毒蛋壳气室部位，再用酒精棉脱碘；②无菌取出鸡胚，放入灭菌的玻璃皿内，用PH7.2的PBS冲洗一次，去头，四肢和内脏；③再用PH7.2的PBS冲洗两次；④用手术剪剪成小块(2-3mm)，用PH7.2的PBS冲洗两次；⑤约加20ml0.25％胰酶溶液，37℃消化15min，期间5min轻摇一次；⑥消化结束，将细胞消化倒入带6-8层纱布的100ml烧杯中过滤；⑦将过滤液2000rpm离心5min，弃去上清液，将细胞用含10％FCS的DMEM培养基溶解；⑧用适当体积接于细胞培养皿中，37℃，5％CO2培养箱培养；
待CEF细胞长满单层后，接种鸡痘病毒液，37℃，5％CO2培养箱孵育2h，去病毒液，洗两遍，按照Lipofectamine 2000试剂使用说明方法将质粒pMD22-TYB-lacz-F4和CRISPR/Cas9表达质粒以1:1比例与鸡痘病毒共转染CEF细胞，同时设单独的质粒pMD22-TYB-lacz-F4与鸡痘病毒共转染CEF细胞，待细胞产生80％病变时收集细胞，反复冻融三次，收集重组病毒液。
2.6 重组鸡痘病毒的纯化
将上述收集的病毒液用X-gal进行蓝白斑筛选纯化，挑取蓝斑，进行数代纯化直至出现的空斑全部为蓝斑，如图2所示；同时抽提纯化的不同代次的病毒液DNA，用鸡痘病毒同源臂上引物TYB-JC-F/R(见表1)及FADV4 fiber2引物F4-fiber2-F/R(见表1)分别对样本进行PCR检测，结果如图3、4所示。
图3为引物TYB-JC-F/R PCR扩增图：编号8为以转移载体pMD22-TYB-lacz-F4为模板的阳性对照，片段大小为5315bp，编号9为以FPV抽提DNA为模板的对照，大小为703bp，若是PCR结果只出现5315bp条带,则说明病毒为rFPV-FADV4 fiber2重组病毒，若是只出现703bp片段，则说明病毒为FPV病毒，若同时出现两条带则说明病毒为rFPV-FADV4 fiber2重组病毒与FPV病毒混合病毒，rFPV-FADV4 fiber2重组病毒不纯，编号1至4为单独的质粒pMD22-TYB-lacz-F4与鸡痘病毒共转染CEF细胞纯化第4、7、9、12代抽提的DNA,可看出重组病毒纯化至12代才纯化好，编号5、6、7为质粒pMD22-TYB-lacz-F4和CRISPR/Cas9表达质粒以1:1比例与鸡痘病毒共转染CEF细胞纯化第3、4、5代后抽提DNA，由此结果可看出使用CRISPR/Cas9表达系统从第3代开始就只有5315bp出有一条条带，及已纯化，可见本发明方法筛选的效率要高很多，节省了时间。
图4为FADV4 fiber2引物F4-fiber2-F/RPCR检测结果图：编号1为单独的质粒pMD22-TYB-lacz-F4与鸡痘病毒共转染CEF细胞纯化第7代抽提的DNA，编号2为质粒pMD22-TYB-lacz-F4和CRISPR/Cas9表达质粒以1:1比例与鸡痘病毒共转染CEF细胞纯化第4代抽提的DNA，从PCR结果可看出FADV4 fiber2基因已重组到FPV病毒中。
实施例3本发明方法筛选重组鸡痘病毒的方法在制备抗鸡4型腺病毒FPV载体疫苗中的应用
按照图1所示构建好rFPV-FADV4 fiber2转移载体，将构建好的载体与上述构建的表达sgRNA的CRISPR/Cas9质粒按1:1比例共转染已感染鸡痘病毒的CEF细胞，培养，通过X-GAL进行蓝白斑筛选，经过四代筛选得到了纯化好的rFPV-FADV4 fiber2病毒，将其在CEF细胞上进行数次传代，收集好病毒液，作为制备抗鸡4型腺病毒FPV载体疫苗提供病毒液，将所收集的病毒液按常规操作制备方法操作，制得抗鸡4型腺病毒FPV载体疫苗。
表达鸡4型腺病毒fiber2基因的重组鸡痘病毒表达效果的验证：
将上述病毒液中的表达鸡4型腺病毒fiber2基因的重组鸡痘病毒(rFPV-FADV4fiber2)进行多次筛选纯化，将纯化得到的rFPV-FADV4 fiber2接种于用24孔板培养的CEF细胞中，置于37℃、5％的二氧化碳培养箱培养，待出现明显病变后，用PBS洗两遍；冷甲醇固定，PBS洗三遍；加1％BSA封闭1h，用PBS洗三遍；加入1:100稀释的鸡抗FADV4的一抗37℃孵育2h，用PBST洗三遍；再加入1∶200稀释的兔抗鸡IgG-FITC荧光二抗，室温孵育1h，PBST洗三遍；置于倒置荧光显微镜下观察结果，同时以正常CEF细胞及感染了鸡痘病毒的CEF细胞经同样的方式处理，作为对照。结果如图5-7所示，可以看出只有感染了rFPV-FADV4 fiber2的CEF细胞上出现了特异性的绿色荧光，表明重组鸡痘病毒rFPV-FADV4 fiber2感染了CEF细胞后有效的表达了FADV4的fiber2抗原。
抗鸡4型腺病毒FPV载体疫苗临床试验验证
本发明还验证了重组鸡痘病毒rFPV-FADV4 fiber2(纯化得到的疫苗)在动物体内的免疫效果，结果显示该重组病毒在动物体内可产生良好的免疫效果。
用重组病毒(rFPV-FADV4 fiber2)(5×105PFU/羽)经翅皮下注射接种于10天龄的SPF鸡上，同时设空白组、免疫某公司灭活疫苗组做对照，分别于免疫前、免后三周采血，通过ELISA方法检测机体抗体水平变化，步骤如下：用包被缓冲液将表达FADV4 fiber2的蛋白稀释至3μg/ml，每孔加100ul，包被于96孔板中37℃2h，1％BSA，37℃封闭1h，加入所采的稀释了200倍的血清，37℃孵育2h，用PBST洗三遍，每次洗3min，再加入羊抗鸡HRP酶标记二抗37℃孵育1h，用PBST洗三遍，每次洗3min，加底物显色，37℃20min，最后加终止液终止，将板置于酶标仪中，在OD450下读数；结果如下：免疫前整体抗体水平OD值为0.31，免疫后三周，空白组平均OD值为0.27，某公司灭活疫苗为0.75，rFPV-FADV4 fiber2重组病毒为0.77，结果显示该重组病毒在动物体内可产生良好免疫应答。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
<110> 广东温氏食品集团股份有限公司
<120>一种通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法及其应用
<160> 11
<210> 1
<211> 10579
<212> DNA
<213> 人工序列
<400> 1
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggt acgcgcggat 420
cttccagaga ttacaacggc atgactaccg agtatagata aagatttaga aatcatttct 480
aagaaattta gatactgatg tatacaagta taactaatag cgagtaattt attttgttct 540
atagcttcat tatatttttt taaagcagat acaagttgtg tatttagagc gtcgttaaag 600
taaacagtat ttccttctac atttaatgaa ctcaataaca tatttcccga aggtaataat 660
cttgaccaag ggaaaataga atattcacgg tataagaaac atacagaata accattttct 720
attaactttt caacagaaat agctcctctc atacccgtac taaaattctc taaaaatctg 780
actggttttt tttctaaaga tactctagtc cctccagatg tgactaaagc tacacgcctg 840
tttttctctt tttgtaattt tacccaatta ttaatgttag tagtcgtgtc cattttttta 900
atataagaat ttatattagg ttaatttata agaaaccaat actttaaatc tctaattcgt 960
tgttctaaac aacagttatg gtttcttaaa ttgttgattc atgataatat tatcgtaata 1020
attctattat tgaaatatct agtctcgttt ttgagataaa tattacgaat aaagcatatt 1080
catatcaaag caacattagc tttacattta agttgtacta cgcatacgca cgaagtacct 1140
attcttatat attcccagga aggcattcca tttttaataa ctatagagtt aacaaaagaa 1200
tgagacgtag aacaataaga ggaccaaaat cgtgtatcta ttcctaaaca gccactaata 1260
gccggatatt ccttacactt ggtttcgaat aggtactgat agtaaacttg tttattatgt 1320
actatttgat ccaatagttc tagtttatta cctctgtgat caaagactgt agttttgtta 1380
gcgacccatg tagaactact ttcacaagat aagtatattc cttcactggt attacctaca 1440
gacaataatt catctatgct tcgtttacca cgatgttcta tattcggagt acgagtacta 1500
aaaacaactt tagatgtatc taatttatcg tttataagat aaggattagt aaattggagt 1560
aacgatccct tgcatactat acctaataca catataaaga ttagccttct aaaattacag 1620
gggtgcgtat ggtatgccat tcttatttat atatgaactt actaattaag taatagaata 1680
tgtctcagta ataattgacg gtacactgta gtatttgatt ccactagtaa acacataaat 1740
tccttaccat tatgtttatt atccactaat agttctctaa taaaaaatgt agagttttgt 1800
aacggaattg ttacaggact tttatgaatt accgattcca tttcgctaat gggtttacca 1860
ccgggaccgg cccaaaacat tctcgctgac gaacctttcc taccacaccc tacacatatt 1920
aagctagtag tatttatatc ttcaggcagt ctagtaatat taacgtaggt acaatcttcg 1980
cgtgttacag gacagcattc tcgcacaccg tcggaatttt ttcttgcgtt ttccggaaga 2040
tatccttcta ggtctagaag cggccgccct gcccggttat tattattttt gacaccagac 2100
caactggtaa tggtagcgac cggcgctcag ctggaattcc gccgatactg acgggctcca 2160
ggagtcgtcg ccaccaatcc ccatatggaa accgtcgata ttcagccatg tgccttcttc 2220
cgcgtgcagc agatggcgat ggctggtttc catcagttgc tgttgactgt agcggctgat 2280
gttgaactgg aagtcgccgc gccactggtg tgggccataa ttcaattcgc gcgtcccgca 2340
gcgcagaccg ttttcgctcg ggaagacgta cggggtatac atgtctgaca atggcagatc 2400
ccagcggtca aaacaggcgg cagtaaggcg gtcgggatag ttttcttgcg gccctaatcc 2460
gagccagttt acccgctctg ctacctgcgc cagctggcag ttcaggccaa tccgcgccgg 2520
atgcggtgta tcgctcgcca cttcaacatc aacggtaatc gccatttgac cactaccatc 2580
aatccggtag gttttccggc tgataaataa ggttttcccc tgatgctgcc acgcgtgagc 2640
ggtcgtaatc agcaccgcat cagcaagtgt atctgccgtg cactgcaaca acgctgcttc 2700
ggcctggtaa tggcccgccg ccttccagcg ttcgacccag gcgttagggt caatgcgggt 2760
cgcttcactt acgccaatgt cgttatccag cggtgcacgg gtgaactgat cgcgcagcgg 2820
cgtcagcagt tgttttttat cgccaatcca catctgtgaa agaaagcctg actggcggtt 2880
aaattgccaa cgcttattac ccagctcgat gcaaaaatcc atttcgctgg tggtcagatg 2940
cgggatggcg tgggacgcgg cggggagcgt cacactgagg ttttccgcca gacgccactg 3000
ctgccaggcg ctgatgtgcc cggcttctga ccatgcggtc gcgttcggtt gcactacgcg 3060
tactgtgagc cagagttgcc cggcgctctc cggctgcggt agttcaggca gttcaatcaa 3120
ctgtttacct tgtggagcga catccagagg cacttcaccg cttgccagcg gcttaccatc 3180
cagcgccacc atccagtgca ggagctcgtt atcgctatga cggaacaggt attcgctggt 3240
cacttcgatg gtttgcccgg ataaacggaa ctggaaaaac tgctgctggt gttttgcttc 3300
cgtcagcgct ggatgcggcg tgcggtcggc aaagaccaga ccgttcatac agaactggcg 3360
atcgttcggc gtatcgccaa aatcaccgcc gtaagccgac cacgggttgc cgttttcatc 3420
atatttaatc agcgactgat ccacccagtc ccagacgaag ccgccctgta aacggggata 3480
ctgacgaaac gcctgccagt atttagcgaa accgccaaga ctgttaccca tcgcgtgggc 3540
gtattcgcaa aggatcagcg ggcgcgtctc tccaggtagc gaaagccatt ttttgatgga 3600
ccatttcggc acagccggga agggctggtc ttcatccacg cgcgcgtaca tcgggcaaat 3660
aatatcggtg gccgtggtgt cggctccgcc gccttcatac tgcaccgggc gggaaggatc 3720
gacagatttg atccagcgat acagcgcgtc gtgattagcg ccgtggcctg attcattccc 3780
cagcgaccag atgatcacac tcgggtgatt acgatcgcgc tgcaccattc gcgttacgcg 3840
ttcgctcatc gccggtagcc agcgcggatc atcggtcaga cgattcattg gcaccatgcc 3900
gtgggtttca atattggctt catccaccac atacaggccg tagcggtcgc acagcgtgta 3960
ccacagcgga tggttcggat aatgcgaaca gcgcacggcg ttaaagttgt tctgcttcat 4020
cagcaggata tcctgcacca tcgtctgctc atccatgacc tgaccatgca gaggatgatg 4080
ctcgtgacgg ttaacgcctc gaatcagcaa cggcttgccg ttcagcagca gcagaccatt 4140
ttcaatccgc acctcgcgga aaccgacatc gcaggcttct gcttcaatca gcgtgccgtc 4200
ggcggtgtgc agttcaacca ccgcacgata gagattcggg atttcggcgc tccacagttt 4260
cgggttttcg acgttcagac gtagtgtgac gcgatcggca taaccaccac gctcatcgat 4320
aatttcaccg ccgaaaggcg cggtgccgct ggcgacctgc gtttcaccct gccataaaga 4380
aactgttacc cgtaggtagt cacgcaactc gccgcacatc tgaacttcag cctccagtac 4440
agcgcggctg aaatcatcat taaagcgagt ggcaacatgg aaatcgctga tttgtgtagt 4500
cggtttatgc agcaacgaga cgtcacggaa aatgccgctc atccgccaca tatcctgatc 4560
ttccagataa ctgccgtcac tccagcgcag caccatcacc gcgaggcggt tttctccggc 4620
gcgtaaaaat gcgctcaggt caaattcaga cggcaaacga ctgtcctggc cgtaaccgac 4680
ccagcgcccg ttgcaccaca gatgaaacgc cgagttaacg ccatcaaaaa taattcgcgt 4740
ctggccttcc tgtagccagc tttcatcaac attaaatgtg agcgagtaac aacccgtcgg 4800
attctccgtg ggaacaaacg gcggattgac cgtaatggga taggtcacgt tggtgtagat 4860
gggcgcatcg taaccgtgca tctgccagtt tgaggggacg acgacagtat cggcctcagg 4920
aagatcgcac tccagccagc tttccggcac cgcttctggt gccggaaacc aggcaaagcg 4980
ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct 5040
attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg 5100
gttttcccag tcacgacgtt gtaaaacgac gggatcgctc gaggaattca tttatagcat 5160
agaaaaaaac aaaatgaaat tctactatat ttttacatac atatattcta aatatgaaag 5220
tggtgattgt gactagcgta gcatcgctct agaataaaaa ttaattaatt atggatttac 5280
gggagggagg ccgctggaca gctgtagagc acgggtccca cgatcatggt tccgctgttg 5340
tttggattaa aaatgctcgc gttcgtgcac tgcagactat agcatagaag ggtgtatcgc 5400
tctccggagg ccgaaaccgg cacggggagg acgcggatcc ctatgtttcc cgggttccag 5460
gcacctgtta ccaccgggct gaacacttgg aattccccga tggatggttc atagtatcca 5520
ttggccgagt acgtccatgg gctggtcacg ctcctattgg ccatgggttc aaagtccgtg 5580
agggtggcgg tggaggggct gaccgttccc gcttgaatcc cggaggggtt gcattgctgg 5640
agataggcgg acacccaaaa ggtgaaccat ttggcattgg cggagttgag gtccccaggg 5700
cgattcccca tggtggcgct gtccaatttc aagtagaggg aggtaacaag gagcccctgt 5760
atgttccact gttgaaggta gtaggcgcaa gagaaggcgt tcgcgctgga attgacggtc 5820
gtggcattgt aggtgttgag gctgggactt cccgagacaa aagtagcgat gggtgtggag 5880
acgctccccc ctccgaccac ggttaaagcg cccgcggtca ccgctagggt attagtgtcg 5940
tacttcagcc ctaggccatt ggcggagacc gtaagcggtc cgctgggatc cggtttcact 6000
tccagagtgt tgttgacaat ctgtaggctt tcatccacgg aaacgcccac tcccgaactg 6060
tcggcttgga tgcctccctg cgcttttagg ttgagttcca gcactccgct tccggtcgag 6120
gtgttgaccg tgaacatgtt aggattgatc tcgaggtcga taccactgct atcggcagtg 6180
atgggtcctt gttggttgag gtgtacgccc agttctccct gatccacgag caaggtgtcg 6240
tccacgctga cccccagtcc acccgcggtg gaatccaatc cgccggacgg gtcgactttt 6300
acggccagtt cccagtcatc gttgaccatt acggtcactc cgtcgacctt gacgtccagt 6360
ccatcggggg tgatgtccag ggccccttcg gggtcaacgg ccaccgccag ttgaccttgg 6420
gcgttgacat cgagactggg gtcgtgggcc aagtccaccg atctgttctt gatgatgatg 6480
ggatcggtga cgttgagcgt aagctgtccg ccctggtcca ctaggggtcc tgagcctccc 6540
aaaaaaggcg ggttgagccc tccgacgggg tcggccacgt aatcgaaagg ataaaccagg 6600
tcaagctggg atgctctcac catgcgtttg gcgcgcttga ttggagccgg ggaaggtccc 6660
gcttcggtct cgggcttccc gttttcggaa tgtcttcttt taggggcccg gagcatgggc 6720
atttattatc gataattaca attcaatttt aggatacaga tctatttata tgccaaaaaa 6780
aaaaaaaaaa aaaagagtcg acctaggcgg ccgctatgaa ttccttctgg acacgatatc 6840
tatcctacta agtatgtatg gtatttattt atcaattaat ctgcgtatgt agtaactact 6900
acagcgtttc taagatcatc atgtcctaca attttatttc tttgacgtcg tgtttatatc 6960
attttctgtt ttgggataat aattttctct aatataaaat tatatattaa ttctttttct 7020
atattgaagt gatttaatta aagaaaatat gtaatcttta tctaattagg tttttcctta 7080
tctaataata gaactgtata cctggtgatc ttcctacttg atttacgtga cctaatataa 7140
ttatttagat atttacctgt ttttcgcata aatataattc ctaaaaatat tattattaag 7200
atattaatat ctattatcca tgataatata tagagaaaca ttatattaat cgccaatcga 7260
atatgaataa catacatagt aataataaag atagcagtta atggcaaact aatattattc 7320
atgataactg ctataaaaga agataatata gcaagatata ttgaagtgtc tatcatatct 7380
tattttatgg ataaaccttt aacggcaact tctaagttac ttattttttg gtttattaaa 7440
ctattggttt tttcgtactt ttcttccaat ttttttgtat ttttctttaa ttttaatatc 7500
tcattatcat gaatgtcgta tagtatttta cttataccct cagagaagaa gccgcttcgt 7560
atctgatctt cattatcaga acctttttta agcctcgtgc aataggagtt agaaagatag 7620
gagttaagta tcttggaaaa attaagtgca atactaggaa aaacccaaca gataatatga 7680
ggcacgagat cgatatgcac atatgttcct acaagttcgt atttataggc actatttgat 7740
gctaatccga tttctaaaac ggctttatta tagataccgt ttttatagtt caatgttttt 7800
atgagttttt tagatgactc tagtctacac cactgcctaa agttcttatt tccaagatca 7860
catattttag tagcatttat atatccgttg tattttaaca tgattacttc tatgttcgca 7920
tagttgataa agcaaaagtt ctcatctata tgtttaacgg tgttaggtac aaactccata 7980
ttgtaatact ttcattcaga atagtattgt ttttacattt tttattataa ggaaaaaact 8040
ggtttattca ttttctttta accatgcata cacaatttac aggaactgat acatgtttag 8100
tcattacagc attattttca ccaagataca ttattttttt aatttctgtg accgtagaac 8160
agtaagattc ccatcttgac tcatcaatgc ccttacaagg agatgtagaa ttagggaatc 8220
ccatgcagct aatcatttga atgtattgtg tgtatccatc tcctttctca gaatatctgc 8280
ccaaaaattc tattttactg acaccagttc cattaacaat cgtcgaacgg caggcgtgca 8340
aacttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 8400
ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 8460
taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 8520
cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 8580
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 8640
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 8700
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 8760
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 8820
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 8880
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 8940
gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 9000
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 9060
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 9120
aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 9180
aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 9240
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 9300
ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 9360
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 9420
atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 9480
tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 9540
gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 9600
tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 9660
gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 9720
cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 9780
gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 9840
atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 9900
aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 9960
atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 10020
aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 10080
aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 10140
gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 10200
gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 10260
gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 10320
ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 10380
ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 10440
atatttgaat gtatttagaa aaataaacaa atgggggttc cgcgcacatt tccccgaaaa 10500
gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 10560
atcacgaggc cctttcgtc 10579
<210> 2
<211> 3121
<212> DNA
<213> 人工序列
<400> 2
gttaatggaa ctggtgtcag taaaatagaa tttttgggca gatattctga gaaaggagat 60
ggatacacac aatacattca aatgattagc tgcatgggat tccctaattc tacatctcct 120
tgtaagggca ttgatgagtc aagatgggaa tcttactgtt ctacggtcac agaaattaaa 180
aaaataatgt atcttggtga aaataatgct gtaatgacta aacatgtatc agttcctgta 240
aattgtgtat gcatggttaa aagaaaatga ataaaccagt tttttcctta taataaaaaa 300
tgtaaaaaca atactattct gaatgaaagt attacaatat ggagtttgta cctaacaccg 360
ttaaacatat agatgagaac ttttgcttta tcaactatgc gaacatagaa gtaatcatgt 420
taaaatacaa cggatatata aatgctacta aaatatgtga tcttggaaat aagaacttta 480
ggcagtggtg tagactagag tcatctaaaa aactcataaa aacattgaac tataaaaacg 540
gtatctataa taaagccgtt ttagaaatcg gattagcatc aaatagtgcc tataaatacg 600
aacttgtagg aacatatgtg catatcgatc tcgtgcctca tattatctgt tgggtttttc 660
ctagtattgc acttaatttt tccaagatac ttaactccta tctttctaac tcctattgca 720
cgaggcttaa aaaaggttct gataatgaag atcagatacg aagcggcttc ttctctgagg 780
gtataagtaa aatactatac gacattcatg ataatgagat attaaaatta aagaaaaata 840
caaaaaaatt ggaagaaaag tacgaaaaaa ccaatagttt aataaaccaa aaaataagta 900
acttagaagt tgccgttaaa ggtttatcca taaaataaga tatgatagac acttcaatat 960
atcttgctat attatcttct tttatagcag ttatcatgaa taatattagt ttgccattaa 1020
ctgctatctt tattattact atgtatgtta ttcatattcg attggcgatt aatataatgt 1080
ttctctatat attatcatgg ataatagata ttaatatctt aataataata tttttaggaa 1140
ttatatttat gcgaaaaaca ggtaaatatc taaataatta tattaggtca cgtaaatcaa 1200
gtaggaagat caccaggtat acagttctat tattagataa ggaaaaacct aattagataa 1260
agattacata ttttctttaa ttaaatcact tcaatataga aaaagaatta atatataatt 1320
ttatattaga gaaaattatt atcccaaaac agaaaatgat ataaacacga cgtcaaagaa 1380
ataaaattgt aggacatgat gatcttagaa acgctgtagt agttactaca tacgcagatt 1440
aattgataaa taaataccat acatacttag taggatagat atcgtgtcca gaagttctag 1500
acctagaagg atatcttccg gaaaacgcaa gaaaaaattc cgacggtgtg cgagaatgct 1560
gtcctgtaac acgcgaagat tgtacctacg ttaatattac tagactgcct gaagatataa 1620
atactactag cttaatatgt gtagggtgtg gtaggaaagg ttcgtcagcg agaatgtttt 1680
gggccggtcc cggtggtaaa cccattagcg aaatggaatc ggtaattcat aaaagtcctg 1740
taacaattcc gttacaaaac tctacatttt ttattagaga actattagtg gataataaac 1800
ataatggtaa ggaatttatg tgtttactag tggaatcaaa tactacagtg taccgtcaat 1860
tattactgag acatattcta ttacttaatt agtaagttca tatataaata agaatggcat 1920
accatacgca cccctgtaat tttagaaggc taatctttat atgtgtatta ggtatagtat 1980
gcaagggatc gttactccaa tttactaatc cttatcttat aaacgataaa ttagatacat 2040
ctaaagttgt ttttagtact cgtactccga atatagaaca tcgtggtaaa cgaagcatag 2100
atgaattatt gtctgtaggt aataccagtg aaggaatata cttatcttgt gaaagtagtt 2160
ctacatgggt cgctaacaaa actacagtct ttgatcacag aggtaataaa ctagaactat 2220
tggatcaaat agtacataat aaacaagttt actatcagta cctattcgaa accaagtgta 2280
aggaatatcc ggctattagt ggctgtttag gaatagatac acgattttgg tcctcttatt 2340
gttctacgtc tcattctttt gttaactcta tagttattaa aaatggaatg ccttcctggg 2400
aatatataag aataggtact tcgtgcgtat gcgtagtaca acttaaatgt aaagctaatg 2460
ttgctttgat atgaatatgc tttattcgta atatttatct caaaaacgag actagatatt 2520
tcaataatag aattattacg ataatattat catgaatcaa caatttaaga aaccataact 2580
gttgtttaga acaacgaatt agagatttaa agtattggtt tcttataaat taacctaata 2640
taaattctta tattaaaaaa atggacacga ctactaacat taataattgg gtaaaattac 2700
aaaaagagaa aaacaggcgt gtagctttag tcacatctgg agggactaga gtatctttag 2760
aaaaaaaacc agtcagattt ttagagaatt ttagtacggg tatgagagga gctatttctg 2820
ttgaaaagtt aatagaaaat ggttattctg tatgtttctt ataccgtgaa tattctattt 2880
tcccttggtc aagattatta ccttcgggaa atatgttatt gagttcatta aatgtagaag 2940
gaaatactgt ttactttaac gacgctctaa atacacaact tgtatctgct ttaaaaaaat 3000
ataatgaagc tatagaacaa aataaattac tcgctattag ttatacttgt atacatcagt 3060
atctaaattt cttagaaatg atttctaaat ctttatctat actcggtagt catgccgttg 3120
t 3121
<210> 3
<211> 1645
<212> DNA
<213> 人工序列
<400> 3
acaacggcat gactaccgag tatagataaa gatttagaaa tcatttctaa gaaatttaga 60
tactgatgta tacaagtata actaatagcg agtaatttat tttgttctat agcttcatta 120
tattttttta aagcagatac aagttgtgta tttagagcgt cgttaaagta aacagtattt 180
ccttctacat ttaatgaact caataacata tttcccgaag gtaataatct tgaccaaggg 240
aaaatagaat attcacggta taagaaacat acagaataac cattttctat taacttttca 300
acagaaatag ctcctctcat acccgtacta aaattctcta aaaatctgac tggttttttt 360
tctaaagata ctctagtccc tccagatgtg actaaagcta cacgcctgtt tttctctttt 420
tgtaatttta cccaattatt aatgttagta gtcgtgtcca tttttttaat ataagaattt 480
atattaggtt aatttataag aaaccaatac tttaaatctc taattcgttg ttctaaacaa 540
cagttatggt ttcttaaatt gttgattcat gataatatta tcgtaataat tctattattg 600
aaatatctag tctcgttttt gagataaata ttacgaataa agcatattca tatcaaagca 660
acattagctt tacatttaag ttgtactacg catacgcacg aagtacctat tcttatatat 720
tcccaggaag gcattccatt tttaataact atagagttaa caaaagaatg agacgtagaa 780
caataagagg accaaaatcg tgtatctatt cctaaacagc cactaatagc cggatattcc 840
ttacacttgg tttcgaatag gtactgatag taaacttgtt tattatgtac tatttgatcc 900
aatagttcta gtttattacc tctgtgatca aagactgtag ttttgttagc gacccatgta 960
gaactacttt cacaagataa gtatattcct tcactggtat tacctacaga caataattca 1020
tctatgcttc gtttaccacg atgttctata ttcggagtac gagtactaaa aacaacttta 1080
gatgtatcta atttatcgtt tataagataa ggattagtaa attggagtaa cgatcccttg 1140
catactatac ctaatacaca tataaagatt agccttctaa aattacaggg gtgcgtatgg 1200
tatgccattc ttatttatat atgaacttac taattaagta atagaatatg tctcagtaat 1260
aattgacggt acactgtagt atttgattcc actagtaaac acataaattc cttaccatta 1320
tgtttattat ccactaatag ttctctaata aaaaatgtag agttttgtaa cggaattgtt 1380
acaggacttt tatgaattac cgattccatt tcgctaatgg gtttaccacc gggaccggcc 1440
caaaacattc tcgctgacga acctttccta ccacacccta cacatattaa gctagtagta 1500
tttatatctt caggcagtct agtaatatta acgtaggtac aatcttcgcg tgttacagga 1560
cagcattctc gcacaccgtc ggaatttttt cttgcgtttt ccggaagata tccttctagg 1620
tctagaagcg gccgctatga attca 1645
<210> 4
<211> 1523
<212> DNA
<213> 人工序列
<400> 4
gatgcggccg ctatgaattc cttctggaca cgatatctat cctactaagt atgtatggta 60
tttatttatc aattaatctg cgtatgtagt aactactaca gcgtttctaa gatcatcatg 120
tcctacaatt ttatttcttt gacgtcgtgt ttatatcatt ttctgttttg ggataataat 180
tttctctaat ataaaattat atattaattc tttttctata ttgaagtgat ttaattaaag 240
aaaatatgta atctttatct aattaggttt ttccttatct aataatagaa ctgtatacct 300
ggtgatcttc ctacttgatt tacgtgacct aatataatta tttagatatt tacctgtttt 360
tcgcataaat ataattccta aaaatattat tattaagata ttaatatcta ttatccatga 420
taatatatag agaaacatta tattaatcgc caatcgaata tgaataacat acatagtaat 480
aataaagata gcagttaatg gcaaactaat attattcatg ataactgcta taaaagaaga 540
taatatagca agatatattg aagtgtctat catatcttat tttatggata aacctttaac 600
ggcaacttct aagttactta ttttttggtt tattaaacta ttggtttttt cgtacttttc 660
ttccaatttt tttgtatttt tctttaattt taatatctca ttatcatgaa tgtcgtatag 720
tattttactt ataccctcag agaagaagcc gcttcgtatc tgatcttcat tatcagaacc 780
ttttttaagc ctcgtgcaat aggagttaga aagataggag ttaagtatct tggaaaaatt 840
aagtgcaata ctaggaaaaa cccaacagat aatatgaggc acgagatcga tatgcacata 900
tgttcctaca agttcgtatt tataggcact atttgatgct aatccgattt ctaaaacggc 960
tttattatag ataccgtttt tatagttcaa tgtttttatg agttttttag atgactctag 1020
tctacaccac tgcctaaagt tcttatttcc aagatcacat attttagtag catttatata 1080
tccgttgtat tttaacatga ttacttctat gttcgcatag ttgataaagc aaaagttctc 1140
atctatatgt ttaacggtgt taggtacaaa ctccatattg taatactttc attcagaata 1200
gtattgtttt tacatttttt attataagga aaaaactggt ttattcattt tcttttaacc 1260
atgcatacac aatttacagg aactgataca tgtttagtca ttacagcatt attttcacca 1320
agatacatta tttttttaat ttctgtgacc gtagaacagt aagattccca tcttgactca 1380
tcaatgccct tacaaggaga tgtagaatta gggaatccca tgcagctaat catttgaatg 1440
tattgtgtgt atccatctcc tttctcagaa tatctgccca aaaattctat tttactgaca 1500
ccagttccat taacgaattc agt 1523
<210> 5
<211> 143
<212> DNA
<213> 人工序列
<400> 5
aaatagtttc gtcactatca tcttcctcat aactaactgt actgtaatct ccttcatcgc 60
catagtctat cggattcaag agtacgggta ttatcaaaca tagaataaaa atagttctat 120
acatcatgtt aatttagata ttt 143
<210> 6
<211> 255
<212> DNA
<213> 人工序列
<400> 6
gcggccgcct aggtcgactc tttttttttt tttttttttt ggcatataaa tagatctgta 60
tcctaaaatt gaattgtaat tatcgataat aaatgcccgg gatccataat taattaattt 120
ttattctaga agcgatgcta cgctagtcac aatcaccact ttcatattta gaatatatgt 180
atgtaaaaat atagtagaat ttcattttgt ttttttctat gctataaatg aattcctcga 240
gctgcaggcg gccgc 255
<210> 7
<211> 3070
<212> DNA
<213> 人工序列
<400> 7
ggatcccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg 60
ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg 120
cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc 180
agaagcggtg ccggaaagct ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt 240
cccctcaaac tggcagatgc acggttacga tgcgcccatc tacaccaacg tgacctatcc 300
cattacggtc aatccgccgt ttgttcccac ggagaatccg acgggttgtt actcgctcac 360
atttaatgtt gatgaaagct ggctacagga aggccagacg cgaattattt ttgatggcgt 420
taactcggcg tttcatctgt ggtgcaacgg gcgctgggtc ggttacggcc aggacagtcg 480
tttgccgtct gaatttgacc tgagcgcatt tttacgcgcc ggagaaaacc gcctcgcggt 540
gatggtgctg cgctggagtg acggcagtta tctggaagat caggatatgt ggcggatgag 600
cggcattttc cgtgacgtct cgttgctgca taaaccgact acacaaatca gcgatttcca 660
tgttgccact cgctttaatg atgatttcag ccgcgctgta ctggaggctg aagttcagat 720
gtgcggcgag ttgcgtgact acctacgggt aacagtttct ttatggcagg gtgaaacgca 780
ggtcgccagc ggcaccgcgc ctttcggcgg tgaaattatc gatgagcgtg gtggttatgc 840
cgatcgcgtc acactacgtc tgaacgtcga aaacccgaaa ctgtggagcg ccgaaatccc 900
gaatctctat cgtgcggtgg ttgaactgca caccgccgac ggcacgctga ttgaagcaga 960
agcctgcgat gtcggtttcc gcgaggtgcg gattgaaaat ggtctgctgc tgctgaacgg 1020
caagccgttg ctgattcgag gcgttaaccg tcacgagcat catcctctgc atggtcaggt 1080
catggatgag cagacgatgg tgcaggatat cctgctgatg aagcagaaca actttaacgc 1140
cgtgcgctgt tcgcattatc cgaaccatcc gctgtggtac acgctgtgcg accgctacgg 1200
cctgtatgtg gtggatgaag ccaatattga aacccacggc atggtgccaa tgaatcgtct 1260
gaccgatgat ccgcgctggc taccggcgat gagcgaacgc gtaacgcgaa tggtgcagcg 1320
cgatcgtaat cacccgagtg tgatcatctg gtcgctgggg aatgaatcag gccacggcgc 1380
taatcacgac gcgctgtatc gctggatcaa atctgtcgat ccttcccgcc cggtgcagta 1440
tgaaggcggc ggagccgaca ccacggccac cgatattatt tgcccgatgt acgcgcgcgt 1500
ggatgaagac cagcccttcc cggctgtgcc gaaatggtcc atcaaaaaat ggctttcgct 1560
acctggagag acgcgcccgc tgatcctttg cgaatacgcc cacgcgatgg gtaacagtct 1620
tggcggtttc gctaaatact ggcaggcgtt tcgtcagtat ccccgtttac agggcggctt 1680
cgtctgggac tgggtggatc agtcgctgat taaatatgat gaaaacggca acccgtggtc 1740
ggcttacggc ggtgattttg gcgatacgcc gaacgatcgc cagttctgta tgaacggtct 1800
ggtctttgcc gaccgcacgc cgcatccagc gctgacggaa gcaaaacacc agcagcagtt 1860
tttccagttc cgtttatccg ggcaaaccat cgaagtgacc agcgaatacc tgttccgtca 1920
tagcgataac gagctcctgc actggatggt ggcgctggat ggtaagccgc tggcaagcgg 1980
tgaagtgcct ctggatgtcg ctccacaagg taaacagttg attgaactgc ctgaactacc 2040
gcagccggag agcgccgggc aactctggct cacagtacgc gtagtgcaac cgaacgcgac 2100
cgcatggtca gaagccgggc acatcagcgc ctggcagcag tggcgtctgg cggaaaacct 2160
cagtgtgacg ctccccgccg cgtcccacgc catcccgcat ctgaccacca gcgaaatgga 2220
tttttgcatc gagctgggta ataagcgttg gcaatttaac cgccagtcag gctttctttc 2280
acagatgtgg attggcgata aaaaacaact gctgacgccg ctgcgcgatc agttcacccg 2340
tgcaccgctg gataacgaca ttggcgtaag tgaagcgacc cgcattgacc ctaacgcctg 2400
ggtcgaacgc tggaaggcgg cgggccatta ccaggccgaa gcagcgttgt tgcagtgcac 2460
ggcagataca cttgctgatg cggtgctgat tacgaccgct cacgcgtggc agcatcaggg 2520
gaaaacctta tttatcagcc ggaaaaccta ccggattgat ggtagtggtc aaatggcgat 2580
taccgttgat gttgaagtgg cgagcgatac accgcatccg gcgcggattg gcctgaactg 2640
ccagctggcg caggtagcag agcgggtaaa ctggctcgga ttagggccgc aagaaaacta 2700
tcccgaccgc cttactgccg cctgttttga ccgctgggat ctgccattgt cagacatgta 2760
taccccgtac gtcttcccga gcgaaaacgg tctgcgctgc gggacgcgcg aattgaatta 2820
tggcccacac cagtggcgcg gcgacttcca gttcaacatc agccgctaca gtcaacagca 2880
actgatggaa accagccatc gccatctgct gcacgcggaa gaaggcacat ggctgaatat 2940
cgacggtttc catatgggga ttggtggcga cgactcctgg agcccgtcag tatcggcgga 3000
attccagctg agcgccggtc gctaccatta ccagttggtc tggtgtcaaa aataataata 3060
accgggcagg 3070
<210> 8
<211> 9139
<212> DNA
<213> 人工序列
<400> 8
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt agaactcggt acgcgcggat 420
cttccagaga ttacaacggc atgactaccg agtatagata aagatttaga aatcatttct 480
aagaaattta gatactgatg tatacaagta taactaatag cgagtaattt attttgttct 540
atagcttcat tatatttttt taaagcagat acaagttgtg tatttagagc gtcgttaaag 600
taaacagtat ttccttctac atttaatgaa ctcaataaca tatttcccga aggtaataat 660
cttgaccaag ggaaaataga atattcacgg tataagaaac atacagaata accattttct 720
attaactttt caacagaaat agctcctctc atacccgtac taaaattctc taaaaatctg 780
actggttttt tttctaaaga tactctagtc cctccagatg tgactaaagc tacacgcctg 840
tttttctctt tttgtaattt tacccaatta ttaatgttag tagtcgtgtc cattttttta 900
atataagaat ttatattagg ttaatttata agaaaccaat actttaaatc tctaattcgt 960
tgttctaaac aacagttatg gtttcttaaa ttgttgattc atgataatat tatcgtaata 1020
attctattat tgaaatatct agtctcgttt ttgagataaa tattacgaat aaagcatatt 1080
catatcaaag caacattagc tttacattta agttgtacta cgcatacgca cgaagtacct 1140
attcttatat attcccagga aggcattcca tttttaataa ctatagagtt aacaaaagaa 1200
tgagacgtag aacaataaga ggaccaaaat cgtgtatcta ttcctaaaca gccactaata 1260
gccggatatt ccttacactt ggtttcgaat aggtactgat agtaaacttg tttattatgt 1320
actatttgat ccaatagttc tagtttatta cctctgtgat caaagactgt agttttgtta 1380
gcgacccatg tagaactact ttcacaagat aagtatattc cttcactggt attacctaca 1440
gacaataatt catctatgct tcgtttacca cgatgttcta tattcggagt acgagtacta 1500
aaaacaactt tagatgtatc taatttatcg tttataagat aaggattagt aaattggagt 1560
aacgatccct tgcatactat acctaataca catataaaga ttagccttct aaaattacag 1620
gggtgcgtat ggtatgccat tcttatttat atatgaactt actaattaag taatagaata 1680
tgtctcagta ataattgacg gtacactgta gtatttgatt ccactagtaa acacataaat 1740
tccttaccat tatgtttatt atccactaat agttctctaa taaaaaatgt agagttttgt 1800
aacggaattg ttacaggact tttatgaatt accgattcca tttcgctaat gggtttacca 1860
ccgggaccgg cccaaaacat tctcgctgac gaacctttcc taccacaccc tacacatatt 1920
aagctagtag tatttatatc ttcaggcagt ctagtaatat taacgtaggt acaatcttcg 1980
cgtgttacag gacagcattc tcgcacaccg tcggaatttt ttcttgcgtt ttccggaaga 2040
tatccttcta ggtctagaag cggccgccct gcccggttat tattattttt gacaccagac 2100
caactggtaa tggtagcgac cggcgctcag ctggaattcc gccgatactg acgggctcca 2160
ggagtcgtcg ccaccaatcc ccatatggaa accgtcgata ttcagccatg tgccttcttc 2220
cgcgtgcagc agatggcgat ggctggtttc catcagttgc tgttgactgt agcggctgat 2280
gttgaactgg aagtcgccgc gccactggtg tgggccataa ttcaattcgc gcgtcccgca 2340
gcgcagaccg ttttcgctcg ggaagacgta cggggtatac atgtctgaca atggcagatc 2400
ccagcggtca aaacaggcgg cagtaaggcg gtcgggatag ttttcttgcg gccctaatcc 2460
gagccagttt acccgctctg ctacctgcgc cagctggcag ttcaggccaa tccgcgccgg 2520
atgcggtgta tcgctcgcca cttcaacatc aacggtaatc gccatttgac cactaccatc 2580
aatccggtag gttttccggc tgataaataa ggttttcccc tgatgctgcc acgcgtgagc 2640
ggtcgtaatc agcaccgcat cagcaagtgt atctgccgtg cactgcaaca acgctgcttc 2700
ggcctggtaa tggcccgccg ccttccagcg ttcgacccag gcgttagggt caatgcgggt 2760
cgcttcactt acgccaatgt cgttatccag cggtgcacgg gtgaactgat cgcgcagcgg 2820
cgtcagcagt tgttttttat cgccaatcca catctgtgaa agaaagcctg actggcggtt 2880
aaattgccaa cgcttattac ccagctcgat gcaaaaatcc atttcgctgg tggtcagatg 2940
cgggatggcg tgggacgcgg cggggagcgt cacactgagg ttttccgcca gacgccactg 3000
ctgccaggcg ctgatgtgcc cggcttctga ccatgcggtc gcgttcggtt gcactacgcg 3060
tactgtgagc cagagttgcc cggcgctctc cggctgcggt agttcaggca gttcaatcaa 3120
ctgtttacct tgtggagcga catccagagg cacttcaccg cttgccagcg gcttaccatc 3180
cagcgccacc atccagtgca ggagctcgtt atcgctatga cggaacaggt attcgctggt 3240
cacttcgatg gtttgcccgg ataaacggaa ctggaaaaac tgctgctggt gttttgcttc 3300
cgtcagcgct ggatgcggcg tgcggtcggc aaagaccaga ccgttcatac agaactggcg 3360
atcgttcggc gtatcgccaa aatcaccgcc gtaagccgac cacgggttgc cgttttcatc 3420
atatttaatc agcgactgat ccacccagtc ccagacgaag ccgccctgta aacggggata 3480
ctgacgaaac gcctgccagt atttagcgaa accgccaaga ctgttaccca tcgcgtgggc 3540
gtattcgcaa aggatcagcg ggcgcgtctc tccaggtagc gaaagccatt ttttgatgga 3600
ccatttcggc acagccggga agggctggtc ttcatccacg cgcgcgtaca tcgggcaaat 3660
aatatcggtg gccgtggtgt cggctccgcc gccttcatac tgcaccgggc gggaaggatc 3720
gacagatttg atccagcgat acagcgcgtc gtgattagcg ccgtggcctg attcattccc 3780
cagcgaccag atgatcacac tcgggtgatt acgatcgcgc tgcaccattc gcgttacgcg 3840
ttcgctcatc gccggtagcc agcgcggatc atcggtcaga cgattcattg gcaccatgcc 3900
gtgggtttca atattggctt catccaccac atacaggccg tagcggtcgc acagcgtgta 3960
ccacagcgga tggttcggat aatgcgaaca gcgcacggcg ttaaagttgt tctgcttcat 4020
cagcaggata tcctgcacca tcgtctgctc atccatgacc tgaccatgca gaggatgatg 4080
ctcgtgacgg ttaacgcctc gaatcagcaa cggcttgccg ttcagcagca gcagaccatt 4140
ttcaatccgc acctcgcgga aaccgacatc gcaggcttct gcttcaatca gcgtgccgtc 4200
ggcggtgtgc agttcaacca ccgcacgata gagattcggg atttcggcgc tccacagttt 4260
cgggttttcg acgttcagac gtagtgtgac gcgatcggca taaccaccac gctcatcgat 4320
aatttcaccg ccgaaaggcg cggtgccgct ggcgacctgc gtttcaccct gccataaaga 4380
aactgttacc cgtaggtagt cacgcaactc gccgcacatc tgaacttcag cctccagtac 4440
agcgcggctg aaatcatcat taaagcgagt ggcaacatgg aaatcgctga tttgtgtagt 4500
cggtttatgc agcaacgaga cgtcacggaa aatgccgctc atccgccaca tatcctgatc 4560
ttccagataa ctgccgtcac tccagcgcag caccatcacc gcgaggcggt tttctccggc 4620
gcgtaaaaat gcgctcaggt caaattcaga cggcaaacga ctgtcctggc cgtaaccgac 4680
ccagcgcccg ttgcaccaca gatgaaacgc cgagttaacg ccatcaaaaa taattcgcgt 4740
ctggccttcc tgtagccagc tttcatcaac attaaatgtg agcgagtaac aacccgtcgg 4800
attctccgtg ggaacaaacg gcggattgac cgtaatggga taggtcacgt tggtgtagat 4860
gggcgcatcg taaccgtgca tctgccagtt tgaggggacg acgacagtat cggcctcagg 4920
aagatcgcac tccagccagc tttccggcac cgcttctggt gccggaaacc aggcaaagcg 4980
ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct 5040
attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg 5100
gttttcccag tcacgacgtt gtaaaacgac gggatcgctc gaggaattca tttatagcat 5160
agaaaaaaac aaaatgaaat tctactatat ttttacatac atatattcta aatatgaaag 5220
tggtgattgt gactagcgta gcatcgctct agaataaaaa ttaattaatt atggatgggc 5280
atttattatc gataattaca attcaatttt aggatacaga tctatttata tgccaaaaaa 5340
aaaaaaaaaa aaaagagtcg acctaggcgg ccgctatgaa ttccttctgg acacgatatc 5400
tatcctacta agtatgtatg gtatttattt atcaattaat ctgcgtatgt agtaactact 5460
acagcgtttc taagatcatc atgtcctaca attttatttc tttgacgtcg tgtttatatc 5520
attttctgtt ttgggataat aattttctct aatataaaat tatatattaa ttctttttct 5580
atattgaagt gatttaatta aagaaaatat gtaatcttta tctaattagg tttttcctta 5640
tctaataata gaactgtata cctggtgatc ttcctacttg atttacgtga cctaatataa 5700
ttatttagat atttacctgt ttttcgcata aatataattc ctaaaaatat tattattaag 5760
atattaatat ctattatcca tgataatata tagagaaaca ttatattaat cgccaatcga 5820
atatgaataa catacatagt aataataaag atagcagtta atggcaaact aatattattc 5880
atgataactg ctataaaaga agataatata gcaagatata ttgaagtgtc tatcatatct 5940
tattttatgg ataaaccttt aacggcaact tctaagttac ttattttttg gtttattaaa 6000
ctattggttt tttcgtactt ttcttccaat ttttttgtat ttttctttaa ttttaatatc 6060
tcattatcat gaatgtcgta tagtatttta cttataccct cagagaagaa gccgcttcgt 6120
atctgatctt cattatcaga acctttttta agcctcgtgc aataggagtt agaaagatag 6180
gagttaagta tcttggaaaa attaagtgca atactaggaa aaacccaaca gataatatga 6240
ggcacgagat cgatatgcac atatgttcct acaagttcgt atttataggc actatttgat 6300
gctaatccga tttctaaaac ggctttatta tagataccgt ttttatagtt caatgttttt 6360
atgagttttt tagatgactc tagtctacac cactgcctaa agttcttatt tccaagatca 6420
catattttag tagcatttat atatccgttg tattttaaca tgattacttc tatgttcgca 6480
tagttgataa agcaaaagtt ctcatctata tgtttaacgg tgttaggtac aaactccata 6540
ttgtaatact ttcattcaga atagtattgt ttttacattt tttattataa ggaaaaaact 6600
ggtttattca ttttctttta accatgcata cacaatttac aggaactgat acatgtttag 6660
tcattacagc attattttca ccaagataca ttattttttt aatttctgtg accgtagaac 6720
agtaagattc ccatcttgac tcatcaatgc ccttacaagg agatgtagaa ttagggaatc 6780
ccatgcagct aatcatttga atgtattgtg tgtatccatc tcctttctca gaatatctgc 6840
ccaaaaattc tattttactg acaccagttc cattaacaat cgtcgaacgg caggcgtgca 6900
aacttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 6960
ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 7020
taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 7080
cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 7140
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 7200
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 7260
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 7320
ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 7380
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 7440
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 7500
gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 7560
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 7620
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 7680
aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 7740
aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 7800
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 7860
ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 7920
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 7980
atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 8040
tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 8100
gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 8160
tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 8220
gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 8280
cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 8340
gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 8400
atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 8460
aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 8520
atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 8580
aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 8640
aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 8700
gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 8760
gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 8820
gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 8880
ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 8940
ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 9000
atatttgaat gtatttagaa aaataaacaa atgggggttc cgcgcacatt tccccgaaaa 9060
gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 9120
atcacgaggc cctttcgtc 9139
<210> 9
<211> 1440
<212> DNA
<213> 人工序列
<400> 9
atgctccggg cccctaaaag aagacattcc gaaaacggga agcccgagac cgaagcggga 60
ccttccccgg ctccaatcaa gcgcgccaaa cgcatggtga gagcatccca gcttgacctg 120
gtttatcctt tcgattacgt ggccgacccc gtcggagggc tcaacccgcc ttttttggga 180
ggctcaggac ccctagtgga ccagggcgga cagcttacgc tcaacgtcac cgatcccatc 240
atcatcaaga acagatcggt ggacttggcc cacgacccca gtctcgatgt caacgcccaa 300
ggtcaactgg cggtggccgt tgaccccgaa ggggccctgg acatcacccc cgatggactg 360
gacgtcaagg tcgacggagt gaccgtaatg gtcaacgatg actgggaact ggccgtaaaa 420
gtcgacccgt ccggcggatt ggattccacc gcgggtggac tgggggtcag cgtggacgac 480
accttgctcg tggatcaggg agaactgggc gtacacctca accaacaagg acccatcact 540
gccgatagca gtggtatcga cctcgagatc aatcctaaca tgttcacggt caacacctcg 600
accggaagcg gagtgctgga actcaaccta aaagcgcagg gaggcatcca agccgacagt 660
tcgggagtgg gcgtttccgt ggatgaaagc ctacagattg tcaacaacac tctggaagtg 720
aaaccggatc ccagcggacc gcttacggtc tccgccaatg gcctagggct gaagtacgac 780
actaataccc tagcggtgac cgcgggcgct ttaaccgtgg tcggaggggg gagcgtctcc 840
acacccatcg ctacttttgt ctcgggaagt cccagcctca acacctacaa tgccacgacc 900
gtcaattcca gcgcgaacgc cttctcttgc gcctactacc ttcaacagtg gaacatacag 960
gggctccttg ttacctccct ctacttgaaa ttggacagcg ccaccatggg gaatcgccct 1020
ggggacctca actccgccaa tgccaaatgg ttcacctttt gggtgtccgc ctatctccag 1080
caatgcaacc cctccgggat tcaagcggga acggtcagcc cctccaccgc caccctcacg 1140
gactttgaac ccatggccaa taggagcgtg accagcccat ggacgtactc ggccaatgga 1200
tactatgaac catccatcgg ggaattccaa gtgttcagcc cggtggtaac aggtgcctgg 1260
aacccgggaa acatagggat ccgcgtcctc cccgtgccgg tttcggcctc cggagagcga 1320
tacacccttc tatgctatag tctgcagtgc acgaacgcga gcatttttaa tccaaacaac 1380
agcggaacca tgatcgtggg acccgtgctc tacagctgtc cagcggcctc cctcccgtaa 1440
<210> 10
<211> 24
<212> DNA
<213> 人工序列
<400> 10
ccggccgata gactatggcg atga 24
<210> 11
<211> 24
<212> DNA
<213> 人工序列
<400> 11
aaactcatcg ccatagtcta tcgg 24

Claims (10)

1.一种通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法，其特征在于，所述方法步骤如下所示：
(1)选取靶基因序列；
(2)根(1)中据靶基因序列设计两条反向互补的上下游引物；分别在上下游引物5’端加上CCGG和AAAC，即为sgRNA双链寡核苷酸序列；所述sgRNA双链寡核苷酸序列结构为：
Forward oligo：5’CCGG......3’
Reverse oligo：5’AAAC......3’；
其中“......”代表引物序列；
(3)将(2)中靶基因的sgRNA双链寡核苷酸序列与线性化的质粒载体连接，转化提取得到sgRNA表达载体；
(4)将(3)中sgRNA表达载体、表达外源基因的重组鸡痘病毒质粒、鸡痘病毒共转染鸡胚成纤维细胞；
(5)将(4)中细胞经过3-4代纯化从而得到表达外源基因重组鸡痘病毒；并验证纯化效果。
2.根据权利要求1所述的方法，其特征在于，所述步骤(3)中具体操作步骤包括：
(3.1)先将权利要求1中步骤(2)中的sgRNA双链寡核苷酸序列分别稀释；
(3.2)将(3.1)中稀释的sgRNA双链寡核苷酸序列与Guide-it Oligo AnnealingBuffer(寡核苷酸退火缓冲液)进行PCR反应；
(3.3)将(3.2)中所得的PCR反应产物用Guide-it Oligo Annealing Buffer做100倍稀释；
(3.4)将(3.3)所得稀释产物进行连接反应；
(3.5)将(3.4)的连接产物进行转化放映，即得sgRNA表达载体。
3.根据权利要求2所述的方法，其特征在于，所述步骤(3.1)中sgRNA双链寡核苷酸序列的稀释浓度均为100μmol/L。
4.根据权利要求2所述的方法，其特征在于，所述步骤(3.2)中PCR反应体系为：sgRNA双链寡核苷酸序列各1ul、Guide-it Oligo Annealing Buffer：8ul。
5.根据权利要求2所述的方法，其特征在于，，所述步骤(3.2)中PCR反应程序为：95℃，2min；然后10min内缓慢退火从85℃退至30℃；然后25℃时停止。
6.根据权利要求2所述的方法，其特征在于，所述步骤(3.4)中连接转化的反应体系为：步骤(3.3)中所得的稀释液：1ul、pGuide-it Vector(Linear)(7.5ng/ul)：2ul、ddH2O：2ul、DNA Ligation Mighty Mix：5ul。
7.根据权利要求2所述的方法，其特征在于，所述步骤(3.4)中连接反应的反应条件为：16℃反应30min。
8.根据权利要求2所述的方法，其特征在于，所述步骤(3.5)中转化操作包括：取5ul步骤(3.4)中所得的连接反应产物于50ulDH5a感受态中，冰浴30min，42℃热激90s，冰浴2min，加500ulLB培养基于37℃摇床中，130rpm活化30min。
9.根据权利要求1-8任意一项所述的通过CRISPR/Cas9系统快速筛选重组鸡痘病毒的方法在疫苗制备中的应用。
10.一种载体疫苗，其特征在于，所述疫苗利用1-8任意一项所述的通过CRISPR/Cas9系统快速筛选的重组鸡痘病毒制备。
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