CN112458064A - 盖他病毒全长感染性克隆、复制子系统及其制备和应用 - Google Patents
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Abstract
本发明公开了一株GETV毒株(GETV‑GX201808株)，发明人以此构建了以细菌质粒为骨架的病毒感染性克隆pGETV‑GX，并能成功拯救出病毒；然后将报告基因GFP插入GETV感染性克隆病毒基因组中，成功拯救出能表达该报告基因的重组GETV病毒；同时，还构建并表达报告基因GFP的盖他病毒复制子系统。本发明为深入研究GETV的基础和应用研究提供了有效的平台，利于进一步开展病毒分子生物学特性、复制机制、致病机理等研究，为透彻了解GETV从而开发新型的、安全的、高效的GETV诊断试剂和疫苗提供理论依据。
Description
技术领域
本发明涉及生物工程技术领域，尤其涉及盖他病毒全长感染性克隆、复制子系统及其制备和应用。
背景技术
盖他病毒(Getah virus,GETV)是一种单股正链RNA病毒，属于披膜病毒科(Togaviridae)甲病毒属(Alphavirus)成员。甲病毒基因组5ˊ末端的前2/3部分编码非结构蛋白，称为非结构区，该区共编码4种非结构蛋白(nsp1-nsp4)，而靠近3ˊ末端的后1/3称为结构区，编码数种结构蛋白，这些结构蛋白由亚基因组mRNA翻译得到，其包括衣壳蛋白(C)、膜糖蛋白E1、E2、E3以及6K蛋白等。
研究证明，盖他病毒可引起赛马急性发热，后肢伴有麻疹和肿胀。GETV引起猪的疾病时，幼猪症状表现为精神不振、食欲减退或消失、全身震颤、体温升高、腹泻、体表发红、共济失调等，严重者还可出现神经症状，甚至衰竭死亡，但是，耐受强的仔猪则无临床症状。怀孕母猪感染盖他病毒后一般不出现临床症状，但会引起繁殖障碍，导致胚胎死亡并被吸收，从而降低母猪产仔数，妊娠前期感染该病毒可导致胚胎死亡，母猪返情，产死胎等。
1955年首次从马来西亚雪背库蚊(Culex gelidus)中分离到该病毒。血清学检测结果显示，GETV广泛分布于欧洲、亚洲和大洋洲，在猪、马、牛及袋鼠等多种动物体内均检测到GETV抗体。日本自20世纪70年代以来已多次报告马和猪中的GETV感染，1990年印度报告马GETV感染暴发。在中国，自从在海南省首次从蚊虫中鉴定出GETV以来，一些单位开展在媒介昆虫、家畜的病毒分离，以及人和动物GETV血清学调查，结果显示，该病毒迅速传播，在我国东北、西北、西南以及华中广大地区的动物和人群血清中检测到GETV特异性IgM、IgG或中和抗体，并多次在动物中引起疫情，导致家畜感染和致死性疾病。
目前，盖他病毒已经成为危害畜牧业的一个潜在威胁。我国对盖他病毒研究起步晚，对其生物学特殊性、致病机理、诊断试剂等研究的还不够深入。我国尚未报道商品化疫苗的批量生产及使用，国外则有不少报道，如日本用于马的灭活苗、盖他-乙脑二联灭活疫苗，用于猪的弱毒活疫苗，盖他-乙脑-细小三联弱毒活疫苗，上述疫苗主要为弱毒苗且多用于马，但是其保护效率也因为毒株的变异而不一定有很好的保护力。盖他病毒基因工程疫苗尚未见到报道。
目前，已有多种RNA病毒(如脊髓灰质炎病毒、甲病毒、黄病毒、麻疹病毒、流感病毒等)被改造成病毒载体，其中甲病毒表达载体系统是最成功最具前景的RNA病毒载体。甲病毒载体具有许多优势，比如该病毒基因组结构更为简单，载体系统的构建和操作更为容易，病毒本身无广泛的免疫背景，不存在免疫耐受问题，且可感染分裂期细胞和作为抗原递呈细胞的树突状细胞，可望获得更好免疫效果。另外，甲病毒载体瞬时表达和基因不整合的特点使其在肿瘤治疗性疫苗和一些疾病的基因治疗研究中有广阔的前景，所有这些均使其成为新型疫苗研究的有力工具。以辛德毕斯病毒、西门利克森林病毒和委内瑞拉马脑炎病毒为基础的甲病毒表达系统已经成为研究蛋白表达和蛋白修饰的有用工具，已经利用甲病毒载体对大量的来自病毒、细菌的蛋白和肿瘤抗原进行了表达研究。
发明内容
本发明要解决的技术问题是提供盖他病毒全长感染性克隆、复制子系统及其制备和应用，为深入开展GETV的基础和应用研究提供有效的平台。
为解决上述技术问题，本发明采用以下技术方案：
一株盖他病毒，保藏编号为CCTCC NO：V 202069，命名为猪盖他病毒GETV-GX201808。
上述盖他病毒的全长感染性克隆，为该病毒基因组全长感染性克隆pGETV-GX。
pGETV-GX中病毒全长基因为序列表中SEQ.ID.No.27。
上述盖他病毒的复制子系统，为pGETV-rep-GFP。
pGETV-rep-GFP中重组病毒基因为序列表中SEQ.ID.No.29。
上述盖他病毒的全长感染性克隆或复制子系统在制备盖他病毒诊断试剂或盖他病毒基因工程疫苗方面的应用。
盖他病毒全长感染性克隆、复制子系统的制备方法，包括以下步骤：
(1)GETV-GX201808株基因组全序列的扩增；
(2)pGETV-GX重组质粒的构建；
(3)pGEE3GFP重组质粒的构建；
(4)重组质粒pGETV-GX和pGEE3GFP的拯救；
(5)盖他病毒复制子系统(pGETV-rep)的构建。
步骤(1)和(2)中，提取GETV-GX201808株的RNA，经过反转录得到cDNA，以cDNA为模板，分别用S1-S4扩增片段的引物通过PCR扩增得到S1-S4片段；以pBR322为模板，用PBR332PacⅠ381F/PBR332MluⅠ978R扩增得到A片段，将酶切位点PacⅠ、MluⅠ通过A片段插入pBR322载体中，得到pBR322-PM；通过引物CmvF/Cmv-X-M-N R扩增CMV片段，将CMV插入pBR322-PM；将S1通过酶切位点插入pBR322-PM，得到pBR322-PM-S1，将S2通过酶切位点插入pBR322-PM-S1，得到pBR322-PM-S12，将S3通过酶切位点插入pBR322-PM-S12得到pBR322-PM-S123，将S4通过酶切位点插入pBR322-PM-S123得到pBR322-PM-S1234，最后通过引物BGH-MluⅠ-F/BGH-NruⅠ-R扩增BGH，得到片段D，将片段D插入pBR322-PM-S1234得到pGETV-GX；
步骤(3)中，以pGETV-GX为模板，用引物TY G BstbⅠF、ST-44-GFP-1R，GFP3UTRF、TYG MluI R，扩增得到片段B1、B3片段；用ST-44-GFP-2F、GFP3UTRR扩增GFP得到B2片段，将片段B1、B2、B3通过SOE-PCR得到片段B，将片段B插入pGETV-GX得到pGEE3GFP；步骤(4)中，pGETV-GX转染BHK-21细胞后，可拯救亲本病毒rGETV-GX；而pGEE3GFP转染BHK-21细胞后，可拯救重组病毒rGEE3GFP；
步骤(5)中，以pGEE3GFP为模板，用引物TY SwaⅠF，SOE GFP R和SOE GFP F，TY GMluI R扩增出C1与C2片段，将片段C1与C2通过SOE-PCR得到片段C，将片段C与pGEE3GFP同时酶切，将片段C替换插入pGEE3GFP，得到pGETV-rep-GFP，用引物CmvF/SOE G 5utr stru 1R、SOE G 5utr stru 2F/TY SrfI R通过SOE-PCR去除病毒非结构蛋白，构建出辅助质粒pGETV-rep-H。
上述制备方法的引物组，包括序列表中SEQ.ID.No.1至26。
发明人利用一株分离自中国猪血清的GETV毒株(GETV-GX201808株)构建了以细菌质粒为骨架的病毒感染性克隆pGETV-GX，并能成功拯救出病毒；然后将报告基因GFP插入GETV感染性克隆病毒基因组中，成功拯救出能表达该报告基因的重组GETV病毒；同时，还构建并表达报告基因GFP的盖他病毒复制子系统。
结果表明，本发明的rGEE3GFP传到第10代仍然可以观察到强烈的荧光，表明报告基因仍然稳定存在于病毒基因组。而一般病毒传至3-5代后，报告基因出现大量的缺失的现象，观察不到荧光。因此，此种报告病毒可用作工具，在该病毒的基础和应用研究中起重要作用。pGETV-rep-GFP与pGETV-rep-H共转染BHK-21细胞后转染后可以观察到绿色荧光的出现，而没有转染的阴性没有出现荧光，表明所构建的复制子载体系统具有自主复制的能力。总之，本发明为深入研究GETV的基础和应用研究提供了有效的平台，利于进一步开展病毒分子生物学特性、复制机制、致病机理等研究，为透彻了解GETV从而开发新型的、安全的、高效的GETV诊断试剂和疫苗提供理论依据。
附图说明
图1是GETV基因组结构图，图中A：pGETV-GX基因组结构图，B：pGEE3GFP基因组结构图；C：pGETV-rep-GFP基因组结构图；D：pGETV-rep-H基因组结构图。
图2是病毒感染BHK-21细胞图，图中A：rGETV-GX(P3代)感染BHK-21细胞(20X)；B：BHK-21细胞(阴性对照)；C：rGEE3GFP(P3代)感染BHK-21细胞(10X)；D：rGEE3GFP(P3代)感染BHK-21细胞(20X)；E：rGEE3GFP(P10代)感染BHK-21细胞(20X)。
图3是rGEE3GFP感染BHK-21细胞图，图中：1：rGETV-GX；2：rGEE3GFP；3、4、5分别是rGEE3GFP感染BHK-21细胞，获得的P3、P6和P10代病毒液。
图4是病毒感染BHK-21细胞IFA(20X)图，图中A：rGETV-GX感染BHK-21细胞，二抗为绿色荧光；B：rGEE3GFP感染BHK-21细胞，二抗为红色荧光。
图5是rGETV-GX和rGEE3GFP在PK-15细胞上的空斑图，图中A：rGETV-GX B：rGEE3GFP。
图6是GETV-GX201808，rGETV-GX和rGEE3GFP在Marc-145细胞上的生长动力学曲线。
图7是pGETV-rep-GFP与pGETV-rep-H共转染BHK-21细胞图，图中：绿色荧光表达A(20X)B：(40X)。
保藏信息说明
猪盖他病毒GETV-GX201808，保藏编号为CCTCC NO：V 202069，保藏日期：2020年10月13日，保藏地址为：中国.武汉.武汉大学，邮编430072，保藏单位：中国典型培养物保藏中心。
具体实施方式
1材料与方法
1.1病毒、细胞、菌株及参考序列
GETV-GX201808毒株(GenBankaccession numbers：MT269657)于2018年分离自广西某猪场猪血清，由申请人实验室分离保存。BHK-21细胞、PK-15细胞、Marc-145细胞、pBR322由申请人实验室保存，感受态细胞菌DH5α购自康为试剂公司。
1.2主要试剂和仪器
RNA酶抑制剂(RRI,ThermoFisher)、dNTP Mixture(TaKaRa)、ReverseTranscriptase M-MLV(RNase H-，TaKaRa)、Green Taq Mix(Vazyme)、TaKaRa LA
(TaKaRa)、限制性内切酶(NEB)、T4 DNA Ligase(NEB)、Plasmid Mini Kit、Gel ExtractionKit、Viral RNA Kit、Total RNA Kit(OMEGA)、Lipofectamine 2000(Invitrogen)、DNAMarker(TaKaRa)CO2恒温培养箱(Thermo Fisher)、倒置显微镜(Olympus CKX41)、核酸电泳仪(北京君意东方电泳设备)。
1.3引物设计
根据GETV-GX201808毒株全基因组序列设计相关引物(如表1)，由上海杰李生物有限公司合成。
表1盖他病毒全长感染性克隆以及复制子系统所需引物
表1中，F：正向引物，R：反向引物；所述长度(bp)指各基因片段的长度。
1.4 GETV-GX201808株基因组全序列的扩增
首先按Viral RNA Kit(OMEGA)说明书操作，提取GETV-GX201808的RNA，然后用逆转录酶Reverse Transcriptase M-MLV(RNase H-，TaKaRa)和锚定的poly(T)引物Qst进行反转录得到cDNA。以cDNA为模板，分别用引物S1(SEQ.ID.No.5-6)、S2(SEQ.ID.No.7-8)、S3(SEQ.ID.No.9-10)、S4(SEQ.ID.No.11-12)通过PCR扩增，得到S1、S2、S3、S4片段，凝胶纯化回收备用；以pBR322为模板，用引物A(SEQ.ID.No.1-2)，扩增得到A片段，将酶切位点PacI、MluI通过A片段替换插入pBR322载体中，得到pBR322-PM；通过引物CmvF/Cmv-X-M-N R(SEQ.ID.No.3-4)将CMV插入pBR322-PM；将S1、S2、S3、S4片段通过酶切位点依次连入pBR322-PM，得到pBR322-PM-S1234，最后通过引物BGH-MluI-F/BGH-NruI-R(SEQ.ID.No.13-14)扩增BGH得到片段D，将片段D插入pBR322-PM-S1234得到pGETV-GX。将其送到华大基因进行全长测序。
pGETV-GX感染性克隆中病毒全长基因见序列SEQ.ID.No.27。
1.5 pGEE3GFP重组质粒的构建
以pGETV-GX为模板，用引物B1(SEQ.ID.No.15-16)、B3(SEQ.ID.No.19-20)扩增得到B1、B3片段；用引物B2(SEQ.ID.No.17-18)扩增GFP得到B2片段，将片段B1、B2通过SOE-PCR得到片段B1-B2，再将片段B1-B2与B3通过SOE-PCR得到片段B，将片段B插入pGETV-GX得到pGEE3GFP。将其送到华大基因进行全长测序。
pGEE3GFP感染性克隆质粒中重组病毒全长基因见序列SEQ.ID.No.28，基因结构图见图1。
1.6病毒的拯救
按照lipfectamine 2000说明书将构建好的质粒转染到生长良好的BHK-21细胞6孔板上，转染剂量为2μg，6孔板中更换300μL opti-MEM培养液，并将转染的细胞板放置于含有5％CO2的37℃恒温培养箱中培养，6h后更换2mL 2％胎牛血清的MEM维持液，48h收取上清，取收取的上清300μL重新接种到生长良好的6孔板BHK-21细胞上，放置于含有5％CO2的37℃培养箱中培养，48h观察到明显的细胞病变，细胞脱落60％时收取上清，离心后于-80℃保存。将拯救出的病毒名命rGETV-GX，而pGEE3GFP拯救出的病毒命名rGEE3GFP。
1.7拯救病毒的鉴定
1.7.1 rGEE3GFP在BHK-21细胞上遗传稳定性分析
取拯救的rGEE3GFP在BHK-21细胞上连续传代10次，获得P1至P10代病毒液；传代期间观察荧光稳定性，待病毒能够稳定传代后，分别提取P3，P6和P10代病毒液RNA，采用RT-PCR分析病毒液，检测rGEE3GFP在BHK-21细胞上的遗传稳定性。
1.7.2间接免疫荧光检测抗原
取rGEE3GFP与rGETV-GX的P3代病毒液，按MOI＝0.1接种到BHK-21细胞上，同时设置空白对照，接毒后1h换维持液，18h后用PBS洗2遍，加入冰甲醇4℃固定15min，用PBS洗2遍；用1％BSA室温封闭30min，用PBS洗2遍；用抗E2蛋白的兔多克隆抗体按1:1000的比例进行稀释，37℃孵育2h，用PBS洗5遍；在暗室条件下将FITC标记的山羊抗兔IgG(H+L)使用PBS按照1:500的比例进行稀释，37℃孵育1h，用PBS洗5遍，用倒置荧光显微镜观察并拍照。
1.7.3空斑表型
取rGEE3GFP与rGETV-GX P3代病毒液，依次稀释成100TCID50、1000TCID50、10000TCID50等，接种到PK-15细胞上，将接种好病毒液的六孔板置于37℃5％CO2的培养箱中吸附1h，弃掉上清，将2×DMEM(含有4％FBS+2％双抗)和2％Agarose等体积混匀，每孔加入2mL，置于室温凝固，然后倒置于37℃，5％CO2的培养箱中静置培养，72h后用1％福尔马林固定过夜，然后将培养基弃掉，用0.5％的结晶紫染色30min后观察。
1.7.4生长曲线的测定
取GETV-GX201808、rGEE3GFP和rGETV-GX的P3代病毒液，按MOI＝0.1接种到Marc-145细胞上(6孔板)，每个孔设置复孔，1h换维持液，然后分别在接种病毒液毒后6h、12h、24h、36h、48h、60h收毒，测TCID50。
1.8盖他病毒复制子系统(pGETV-rep-GFP)的构建
以pGEE3GFP为模板，用引物TY SwaI F/SOE GFP R(SEQ.ID.No.21-22)和SOE GFPF(SEQ.ID.No.23)，TY G MluI R(SEQ.ID.No.20)扩增出C1与C2片段，将片段C1与C2通过SOE-PCR得到片段C，将片段C与pGEE3GFP同时酶切，将片段C替换插入pGEE3GFP，得到pGETV-rep-GFP；同时，以pGETV-GX为模板，用引物CmvF(SEQ.ID.No.3)/SOE G 5utr stru 1R(SEQ.ID.No.24)、SOE G 5utr stru 2F(SEQ.ID.No.25)/TY SrfI R(SEQ.ID.No.26)扩增H1与H2片段，通过SOE-PCR去除病毒非结构蛋白，构建出辅助质粒pGETV-rep-H。
盖他病毒复制子系统pGETV-rep-GFP中重组病毒基因见序列SEQ.ID.No.29，基因结构图见图1；辅助质粒中病毒基因序列SEQ.ID.No.30，基因结构图见图1。
1.9盖他病毒复制子系统报告基因(pGETV-rep-GFP)的表达
按照1.6中的转染步骤，将pGETV-rep-GFP转染BHK-21细胞，同时设置阴性对照。
2结果
2.1 rGEE3GFP遗传稳定性分析
取拯救的rGEE3GFP在BKH-21细胞上连续传代10次，获得P0至P10代病毒液，传代期间观察荧光稳定性，发现荧光较稳定(图2)；采用RT-PCR分析rGEE3GFP在BHK-21细胞上获得的P3，P6和P10代病毒液，证实rGEE3GFP在BHK-21细胞上能够稳定遗传(图3)。
2.2间接免疫荧光检测抗原
取rGEE3GFP的P3代病毒液，按MOI＝0.1接种到BHK-21细胞上，同时设置空白对照，分别对其进行间接免疫荧光染色，检测病毒蛋白的表达。共聚焦显微镜观察，结果表明，将rGETV-GX与rGGEE3GFP接种在BHK-21细胞上，分别有绿色与红色荧光产生，而作为空白对照的正常BHK-21细胞，无特异荧光产生(图4)。表明rGETV-GX与rGGEE3GFP感染的BHK-21细胞，有GETV蛋白的表达，同时也表明拯救出了具有感染性盖他病毒。
2.3空斑表型分析
为了描述病毒的生长特性和复制能力，分别对rGETV-GX和rGEE3GFP进行了空斑表型分析。空斑实验表明，rGETV-GX和rGEE3GFP均可在PK-15细胞上形成空斑，空斑大小一致(图5)。
2.4拯救病毒的生长曲线
为了进一步了解rGEE3GFP和rGETV-GX的生长特性，将GETV-GX201808与拯救的rGEE3GFP和rGETV-GX的P3代病毒，按照MOI＝0.1接种到Marc-145细胞上，做病毒生长曲线分析(图6)。结果表明，rGEE3GFP和rGETV-GX具有相似的复制能力和增殖特性，均在感染后36h达到最高滴度。
2.5复制子载体系统自主复制能力的定性检测
转染后分别在6h、12h、24h、36h、48h观察荧光，6h便可以观察到绿色荧光的出现(图7)，而没有转染的阴性没有出现荧光。表明所构建的复制子载体系统具有的自主复制能力。
序列表
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<120> 猪盖他病毒全长感染性克隆、复制子系统及其制备和应用
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<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gagagacaaa caacccagat gaattacatc ccaactc 37
<210> 26
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
gagcagatcg tagtagcccg ggcggtccac gttgtcctcc 40
<210> 27
<211> 11691
<212> DNA
<213> getah virus
<400> 27
atggcggacg tgtgacatca ccgttcgctc tttctaggat cctttgctac tccacatagt 60
gagagacaaa caacccagat gaaggtaacc gtggacgttg aggctgacag cccattcctt 120
aaggcccttc agaaggcgtt tcccgccttt gaggttgaat cacagcaggt cacaccgaat 180
gaccatgcta acgctagagc attttcgcat ctggctacta aactgattga gcaagaggtt 240
ccaacaggcg tcaccatcct ggacgtgggt agtgcacccg caaggaggtt gatgtctgac 300
cacacctacc actgcatctg ccccatgaaa agtgcggaag acccagagag gctggcgaat 360
tacgctcgaa agctggcgaa agcatcgggg actgtgctag acaagaatgt gtccggaaag 420
ataacggacc tgcaagacgt catggccact ccagacttgg aatccccgac tttttgcctg 480
cacactgacg agacgtgccg cactagggct gaggtcgccg tgtaccagga cgtatacgct 540
gtgcacgcac cgacgtctct gtaccaccag gccatcaaag gtgtcaggac ggcgtattgg 600
attggatttg acaccactcc attcatgttc gaggcgttag cgggtgcgta tcctgcgtac 660
tcgaccaact gggcagatga gcaagtgctg caggctcgta acatcggcct gtgcgcgaca 720
ggcctctccg aggggcgtcg cggcaaactc tctattatga gaaagaagtg cttgcgaccg 780
agcgacagag taatgttttc ggtcgggtcc accttgtaca ccgagagccg aaagctgctg 840
cgcagctggc acctaccttc cgtgtttcac ctgaagggta agaacagttt tacctgcagg 900
tgcgacacgg tggtgtcatg cgaaggttac gtggtaaaga agatcaccat aagcccaggc 960
atatatggaa aaacagttga ttacgcagtt acccatcacg cagagggttt cctgatgtgt 1020
aagatcactg atacagtcag aggagaaaga gtctctttcc cggtctgtac ctatgtgcct 1080
gcaaccatat gtgaccagat gacgggtata cttgccactg acgtgacacc agaggatgcc 1140
cagaagctcc tggttggatt gaaccaacgc atagtggtga atggtaggac gcaaagaaac 1200
acaaacacaa tgaaaaacta cctactgcca gtggtagcgc aagcattcag taaatgggca 1260
cgagaggcgc gcgcagacat ggaggatgag aaacccctag gcaccagaga acgcacgttg 1320
acgtgttgtt gcctgtgggc gtttaaaagc cacaaaatcc acaccatgta caagcggcct 1380
gaaacgcaaa ctatcgtcaa agtgccttcc acttttgatt cctttgtgat accgagcctg 1440
tggtcatcca gtctttccat gggtatcaga cagagaatca aactgctact cagcgcaaga 1500
atgacccaag gcctaccata ctcaggagac cgcaccgaag ctcgcgcggc agaagaagaa 1560
gagaaggagg tgcaggaggc tgaacttacg agggcagcgc tgccaccgct agtgagcggc 1620
tcttgtgcag acgatatcgc ccaggtggat gtggaggaat taaccttcag agccggagcc 1680
ggggttgtgg aaacacccag gaatgccctg aaggttacac cgcaagcaca cgaccacctc 1740
ataggctcct acttgatcct ttccccccaa acggtgttga aaagcgagaa gctggcaccc 1800
atccaccctc ttgctgagca agtcacggtc atgacccact ccggaagatc cggaagatac 1860
ccagtcgaca agtacgacgg acgggtattg atcccgacag gagcggccat cccagtgagc 1920
gagttccagg cactcagcga gagcgcaacc atggtgtaca atgagaggga atttataaat 1980
cgcaagctgc accacatagc gctatatggg ccagccttga ataccgacga ggaaagctat 2040
gaaaaagtga gagctgagag ggcagagacc gagtatgtgt ttgacgtgga caagaaggca 2100
tgtatcaaga aggaggaggc atcaggcctt gtgttaacag gggacctaat caatccacct 2160
ttccacgaat tcgcatacga aggactcaag atccgcccag cagccccgta ccacacgacg 2220
atcatcggtg tgtttggcgt tccaggttcg ggcaagtcgg ctatcattaa gaacatggtg 2280
acgactcgcg atctggtggc cagtggaaag aaggagaact gccaagagat catgaatgat 2340
gtaaagaggc aacgcgggtt ggacgtgacc gctaggaccg tcgactcaat cctattgaat 2400
gggtgcaaga aaggcgtaga aaacctttac gtcgatgagg cgttcgcgtg tcactcgggt 2460
actttgctag cgctcatcgc gctggtgaga ccgtcaggta aggtagtact gtgcggcgac 2520
cctaagcagt gcggtttctt caatttgatg caactgaagg tgcactataa ccacaacatt 2580
tgtacaaggg tgctccataa gagcatctcc agaagatgca ctctacctgt tacggcgatc 2640
gtgtccacct tgcactacca agggaagatg agaacgacga accgatgcaa cacccccatc 2700
cagattgaca ccaccggttc ctccaaacca gcctcaggag atatcgtgtt aacgtgcttc 2760
cgcggctggg tgaagcaact gcaaatagac tatcgtggac acgaggtgat gactgcagct 2820
gcttcccagg gtttgacaag gaaaggcgtg tacgccgtga gacagaaagt gaacgaaaac 2880
ccactgtact cacctctgtc ggagcacgtc aatgtgttgt tgacccgaac tgaaaaccga 2940
ctggtgtgga agacactgtc gggtgacccg tggataaagg tgttaaccaa tgtcccacgt 3000
ggggatttca gtgcaactct ggaggaatgg caagaagagc atgacggtat catgagagtg 3060
ttgaccgagc gtccagcgga ggttgatcca ttccaaaaca aggctaaagt gtgctgggca 3120
aaatgtctgg tgcaagttct tgagacggcc ggaatacgta tgacggcaga tgaatggaac 3180
accatcttgg ctttcagaga ggacagagcg tactcgccag aagtcgctct caacgagatt 3240
tgcacccgtt actacggcgt tgacctagac agcggcctat tctcagcgca gtcagtttcc 3300
ctcttttatg agaacaacca ctgggacaac aggcctggag gacgcatgta cgggttcaac 3360
catgaagtag ccaggaaata cgcagccaga tttccttttc tacgtggcaa catgaactcg 3420
gggctgcaac taaacgtccc tgagaggaag ctccaacctt tcagcgccga atgcaacata 3480
gtcccatcca atcgtcggtt accgcatgct ctggtcacta gttatcagca gtgccgtgga 3540
gagagggtag agtggttgct gaaaaagatt ccaggtcacc aaatgttact tgtaagtgag 3600
tacaacctgg tgatacctca caaaagagtc ttctggattg cacctccgcg ggtgtcaggc 3660
gcggaccgca cgtacgactt ggacctaggg ttacctatgg atgcaggccg ttacgacctg 3720
gtattcgtca acatacacac tgagtaccgg caacaccact accaacaatg cgtcgaccat 3780
tcaatgcgcc tgcagatgct gggaggggat tcactacacc tgcttagacc aggaggctca 3840
ctgctgatga gagcatatgg ttacgcagac agagtcagcg agatggtggt gacagccctg 3900
gctaggaaat tctcggcgtt ccgtgtcctg agaccggcgt gcgtgacgag caacacagaa 3960
gtgttcctgc tgttttctaa ctttgataac ggcagaagag cggtaacctt gcaccaagct 4020
aaccagaaac ttagctcaat gtatgcctgc aacggattgc acactgctgg ctgtgcaccg 4080
tcatacaggg tccgccgcgc agatatatca ggacacggtg aggaagcggt cgtaaatgct 4140
gccaatgcca aaggtaccgt gagcgatgga gtgtgcaggg cggtcgctaa gaagtggcca 4200
tcatctttca aaggggctgc aactccagtt ggcacagcca aaatgatccg cgcagatggc 4260
atgaccgtaa tccacgcggt gggaccaaat ttctccaccg taacagaagc cgaaggggac 4320
agagagctag cggccgcgta ccgagctgtg gctagcataa ttagcaccaa caacataaag 4380
agcgtcgcag taccgctgct gtccacaggc accttttctg gcggtaagga cagagtgatg 4440
cagtccttga accacttatt cacggcattg gacgccaccg atgcagacgt ggttatctac 4500
tgcagagata aaaactggga aaagaagatt caggaagcca tcgacaggcg gacggcaatc 4560
gagctcgtat ctgaagacgt gaccttggaa accgatctgg ttagggtaca cccggacagt 4620
tgcttagtcg gcagaaatgg ttacagtgca accgacggta aactgtattc ttacctagag 4680
ggtacaaggt tccaccagac ggcggtcgac atggctgaaa tatcaacttt atggccaaga 4740
ctccaagatg ctaacgagca gatctgcctg tacgccctag gggagacgat ggacagtata 4800
cgtactaaat gcccagtaga ggacgccgat tcgtctacgc cgccgaaaac ggtgccgtgt 4860
ctatgtcggt acgcgatgac cgcggagcgg gttgccagac ttaggatgaa taacaccaaa 4920
aacatcatcg tgtgctcctc cttcccattg ccgaagtaca ggatagaagg agtgcagaag 4980
gtgaagtgtg accgagtgct aatttttgac cagaccgtcc cgtcactagt aagtcccaga 5040
aagtacatac aacagccgcc ggaacagctg gataatgtga gcctgacttc cacgacgtcg 5100
acgggatccg catggtcatt tccatcggaa acgacctacg aaactatgga ggtcgtagcc 5160
gaggtacaca ccgaacctcc gatccctccg cctcgccgac gtagagcagc cgtcgcccaa 5220
cttagacaga atctggaagt caccgaggag ctcgagcctt acgtgacaca gcaagcagag 5280
atcatggtca tggagagggt cgtgacgaca gacatacgcg ctatcccagt cccggcacga 5340
cgggccatta caatgccagt cccagccccc agggttcgta aggtcgctac cgatcctcca 5400
tcagaaccgg aagctcctat cccggcacca agaaagcgaa gaaccactag tacctcacct 5460
ccgcataacc cagaggattt cgttcccagg gtaccggttg agttaccgtg ggagccggaa 5520
gacctagaca tccaattcgg tgacttggag ccacgccgtc ggaacaccag ggaccgagat 5580
agcagcacag gaatacagtt cggtgatatc gactttaacc agtcctgact aggcagggct 5640
ggcgcgtata tcttttcgtc tgacactggt ccgggtcacc tgcagcagaa gtccgtaagg 5700
caacatgaac tgccatgcga gactctgtac gcccatgaag acgaacgcat atacccgccg 5760
gcatttgacg gagagaaaga aaaggtactc caggcaaaga tgcagatggc cccgacagaa 5820
gcgaataaga gcaggtacca gtcgaggaaa gtagagaaca tgaaggcact aattgtagaa 5880
agactacgca aaggagcaaa gttgtacctc catgagcaaa ccgacaaagt acccacatac 5940
accagcaagt accctagacc tgtgtactca ccatcggtgg atgacagcct gagcgatccg 6000
gaagtggcag tggccgcctg taactctttc ttagaggaga attatccgac cgtggcgaac 6060
taccagataa ccgatgagta tgacgcttat ctggacttgg tcgacggctc tgaaagctgc 6120
ctcgacagag ctacgttctg cccggccaaa ctaagatgtt accctaagca ccatgcatac 6180
caccaaccac aaatcaggag cgcagtacct tccccttttc aaaacacgtt acaaaacgta 6240
ctagccgcgg ccaccaaaag aaattgtaat gtcacccaaa tgagagaatt accaaccatg 6300
gactctgcgg tgttcaacgt agaaagcttc aaaaaatacg catgtaccgg cgaatattgg 6360
caagaattta aagacaatcc tatacggatc accaccgaaa acataacgac gtacgtggct 6420
aaactcaagg gtcctaaggc tgctgccctt tttgccaaga cgcataacct ggtgccgctt 6480
caggaggtgc caatggaccg cttcgtgatg gatatgaaga gagatgtgaa agttacacca 6540
ggcaccaagc ataccgaaga aaggcctaaa gtgcaagtga ttcaagcggc ggaaccattg 6600
gccacggcat atctatgcgg aatccacaga gagttagtca ggcggctaaa agccgttctg 6660
accccgaaca ttcacactct gtttgacatg tcggcggagg actttgatgc catcatagcg 6720
gcacatttcc aaccgggaga tgctgtactg gagacagata tcgcatcctt cgacaagagc 6780
caggacgact ccttagcgct aacggcgtta atgcttctgg aagacctcgg ggtcgaccaa 6840
gaactgctgg atcttatcga agctgcgttt ggcgagatca cgagtgtgca tctacctacc 6900
ggtacaagat ttaaattcgg tgctatgatg aagtcaggaa tgtttcttac actcttcatc 6960
aacacgctgc tgaacattgt catagcatgc cgcgtcttac gcgacaaatt atcgtcatcg 7020
gcgtgcgccg ccttcatagg cgatgacaac atagtgcacg gcgtgaggtc agacccgcta 7080
atggcagaaa ggtgtgcgag ttgggtcaac atggaagtga agatcatcga tgccacaatg 7140
tgtgagaaac caccatactt ttgtggtgga ttcatcctgt acgacagcgt cgccggtaca 7200
gcgtgtaggg ttgcagatcc actaaagagg ctgttcaaac tcgggaaacc gctcccggcg 7260
gacgacaacc aggacgaaga cagaagaagg gcactaaagg atgaaacagt taagtggtcc 7320
cgcataggat tgagagagga attagacgtg gcactgagct caagatacca agtcagtggc 7380
gtcgggaaca tcactagagc gatgtccacg ctgtctaaga atttgaagtc ttttaggaaa 7440
ataagaggtc ccatcgtaca tctgtacggc ggtcctaaat agatgcagga ttacactaca 7500
tctaaagacc acgtattaca gacatcatga attacatccc aactcaaacc ttttacggac 7560
gccgttggcg accacgcccg gcgtaccgtc catggcgggt gccgatgcag ccggccccac 7620
ccatggtgat tcctgagctg caaactccga tcgtccaggc ccaacagatg cagcagctaa 7680
tcagtgcagt ttctgccctg acgaccaagc aaaatggcaa agcaccgaag aagccaaaga 7740
agaagccaca aaaagcgaag gctaagaaaa acgaacagca aaagaaaaac gagaacaaga 7800
aaccaccacc taagcagaag aatccggcta agaagaagaa accaggaaaa agggaacgca 7860
tgtgcatgaa gatagagaat gattgcatct tcgaggtcaa gcttgacggt aaggtaacgg 7920
gctacgcctg cctagtcggg gataaagtga tgaagccggc acacgttaaa ggtgtgatcg 7980
acaaccccga cctagcgaag cttacctaca agaaatcgag caagtatgac ctagagtgcg 8040
cccagatacc ggtgcacatg aagtcagatg cttcaaagta cacccatgaa aaaccagaag 8100
ggcactacaa ttggcatcac ggtgcagtgc agtacagcgg tggcaggttc acaatcccga 8160
caggcgcagg taaaccagga gacagcggcc ggccgatctt cgacaacaaa ggacgtgtgg 8220
tggccattgt cctgggaggg gccaacgaag gagccaggac tgccctatct gtcgtgacct 8280
ggaccaaaga catggtcaca cggtacaccc cagaaggaac agaagaatgg tccgccgcct 8340
tgatgatgtg cgtcttagcc aacgttacat tcccatgctc agagcccgca tgtgcaccct 8400
gttgctatga aaaacaacca gaacagacac tgaggatgtt ggaggacaac gtggaccgcc 8460
cgggctacta cgatctgctc gaggccacga tgacgtgtaa caatagtgca cgccaccgcc 8520
gcagtgtgac ggaacacttc aatgtctaca aggccacgaa accatatcta gcgtattgcg 8580
cggactgcgg agacgggcag ttctgttaca gcccggtggc tatagaaaaa attagggatg 8640
aggcttccga tggcatgata aagatccaga tcgcagcgca aatcggcatc aacaaaggag 8700
gaacacacga acacaacaaa atcaggtaca tcgccgggca tgacatgaaa gaggcgaacc 8760
gggattcttt acaagtgcat acttccggtg tgtgcgctat ccgaggcacg atgggccact 8820
tcatcgtggc ctactgccct ccaggggacg aactaaaggt ccagttccaa gatgcagaat 8880
cgcacacaca ggcctgcaaa gtgcagtaca aacacgcacc ggccccagta ggcagagaaa 8940
aattcactgt caggccccac ttcggtatcg aagtgccatg cacaacgtac cagctgacta 9000
ccgcaccaac ggaggaagag atcgacatgc ataccccacc ggatatccca gatataacgt 9060
tgctgtcgca acagtcaggc aatgtaaaga tcacagcagg aggaaagacc atcagataca 9120
actgcacgtg cggtagtggc catgtgggca ccaccagtag cgacaagact atcaattcgt 9180
gcaaaatagc gcagtgccat gctgcggtga ctaaccacga caagtggcag tacacctcct 9240
cgtttgtccc tagagccgac cagttgtctc gcaaaggtaa agtgcacgta cccttccctc 9300
tgaccaactc cacatgcagg gtgcccgttg cacgtacacc aggtgtcaca tacggaaaga 9360
gagaactgac agtgaaactg cacccagatc atcccacgct gttgacgtac cgaagtctag 9420
gagcagatcc gcgcccgtat gaggagtgga tagaccgata cgtcgaacgg accataccgg 9480
tgaccgaaga tgggatcgag tacagatggg gaaacaaccc acccgtgcgc ttgtgggccc 9540
agctgacaac tgaaggcaaa ccccatggat ggccgcacga gatcatactc tattactatg 9600
ggctataccc agcagccacc atcgccgccg tctcagccgc gggtctcgca gccatattat 9660
cgctgctggc gtcatgctac atgttcgcca ctgcacgccg caagtgcttg accccatacg 9720
ccctgacccc cggggccgtc attccggtaa cactaggtgt actatgctgc gcgccacgag 9780
cgcatgccgc gtcatttgca gaatctatgg cgtatctatg ggatgagaat caaaccctgt 9840
tttggctgga gcttgcaacg ccgctcgctg ccataatcat acttgtatgc tgcctgaaga 9900
acctgctctg ctgctgcaaa ccgctttctt ttttagtgtt ggtgagcctg ggaactcccg 9960
tcgtaaaatc ttacgaacac accgcaacga tcccgaacgt ggtgggattc ccgtataagg 10020
ctcacattga gaggaacggc ttctccccga tgaccctaca gcttgaggta cttggaacca 10080
gcttggaacc cacgctaaac ttagagtaca taacctgcga atacaagaca gtcgtgccat 10140
caccttatat taagtgctgc gggacatcag aatgcagatc catggagcgc cccgactatc 10200
aatgccaggt ctacacagga gtgtacccat ttatgtgggg cggcgcatac tgcttctgcg 10260
acactgagaa cacccagctg agtgaagcat acgttgatag atcggacgta tgcaagcacg 10320
accatgccgc cgcctacaag gcgcacactg cggcaatgaa agccaccatc cgaataagtt 10380
acgggaacct caatcagaca acaacggcgt tcgtcaacgg ggagcataca gtgaccgtcg 10440
gaggcagcag gtttactttt ggtccaatct ccactgcctg gacgcctttc gacaacaaga 10500
tcgtcgtcta caagaacgac gtctacaatc aggacttccc accctacggg tcaggacaac 10560
cagggaggtt cggagacatc cagagcagga cggtagagag caaggacctg tatgccaaca 10620
ccgccctcaa gttgtcgaga ccttcgtccg gtactgttca cgtgccttac acacagaccc 10680
cttctggctt taagtactgg ataaaagaga gaggcacgtc gctgaatgac aaggctcctt 10740
ttggatgcgt aatcaagacc aacccagtca gagcagaaaa ttgcgccgtt ggcaacatcc 10800
cggtctccat ggacatcccg gacaccgctt ttacgcgcgt gattgatgca cctgccgtca 10860
caaacctgga gtgccaagtg gcggtctgca cgcactcatc ggacttcggc gggatcgcaa 10920
ctctgacttt caaaaccgac aaacccggaa aatgtgctgt ccattctcat tcgaatgtag 10980
ccaccataca ggaggcagct gtggacatca aaacagatgg caagataacc ctgcatttct 11040
ctacagcatc tgcatccccg gcattcaagg tatctgtgtg cagtgccaaa acgacatgca 11100
tggcagcgtg tgagccgccg aaggatcaca tcgtccctta tggggcgagc cacaacaacc 11160
aagtttttcc tgacatgtct ggcacggcaa tgacatgggt gcagcgggta gccggcggac 11220
tcggcgggct aacactcgcc gcagtggcag tacttatact ggtgacgtgt gtgactatgc 11280
gccgctaacc gggaggcttg acataatgta tatatataag catcatagtt tttagtaaag 11340
catataaata atcaagtaga tcaaagggct acctaacccc tgaatagtaa caaaacgcaa 11400
aatacaaaaa cattagttca aagggccagt aacccctgaa tagtaacaaa acataaaaac 11460
caaaaacagt agttcaaagg gctatacaac ccctgaatag taacaaaata cagaaaaacc 11520
ataaaaatta taaaaatcaa ctaatcagat catctaaatt tgaccaattg gaaatagccg 11580
aactctacgg agatgtaggc gtccgaactc cacggagacg taggacaaaa ttctgccgaa 11640
ccccagacca tcggggacgt aggcgtctaa tttgtttttt taatatttta c 11691
<210> 28
<211> 12404
<212> DNA
<213> getah virus
<400> 28
atggcggacg tgtgacatca ccgttcgctc tttctaggat cctttgctac tccacatagt 60
gagagacaaa caacccagat gaaggtaacc gtggacgttg aggctgacag cccattcctt 120
aaggcccttc agaaggcgtt tcccgccttt gaggttgaat cacagcaggt cacaccgaat 180
gaccatgcta acgctagagc attttcgcat ctggctacta aactgattga gcaagaggtt 240
ccaacaggcg tcaccatcct ggacgtgggt agtgcacccg caaggaggtt gatgtctgac 300
cacacctacc actgcatctg ccccatgaaa agtgcggaag acccagagag gctggcgaat 360
tacgctcgaa agctggcgaa agcatcgggg actgtgctag acaagaatgt gtccggaaag 420
ataacggacc tgcaagacgt catggccact ccagacttgg aatccccgac tttttgcctg 480
cacactgacg agacgtgccg cactagggct gaggtcgccg tgtaccagga cgtatacgct 540
gtgcacgcac cgacgtctct gtaccaccag gccatcaaag gtgtcaggac ggcgtattgg 600
attggatttg acaccactcc attcatgttc gaggcgttag cgggtgcgta tcctgcgtac 660
tcgaccaact gggcagatga gcaagtgctg caggctcgta acatcggcct gtgcgcgaca 720
ggcctctccg aggggcgtcg cggcaaactc tctattatga gaaagaagtg cttgcgaccg 780
agcgacagag taatgttttc ggtcgggtcc accttgtaca ccgagagccg aaagctgctg 840
cgcagctggc acctaccttc cgtgtttcac ctgaagggta agaacagttt tacctgcagg 900
tgcgacacgg tggtgtcatg cgaaggttac gtggtaaaga agatcaccat aagcccaggc 960
atatatggaa aaacagttga ttacgcagtt acccatcacg cagagggttt cctgatgtgt 1020
aagatcactg atacagtcag aggagaaaga gtctctttcc cggtctgtac ctatgtgcct 1080
gcaaccatat gtgaccagat gacgggtata cttgccactg acgtgacacc agaggatgcc 1140
cagaagctcc tggttggatt gaaccaacgc atagtggtga atggtaggac gcaaagaaac 1200
acaaacacaa tgaaaaacta cctactgcca gtggtagcgc aagcattcag taaatgggca 1260
cgagaggcgc gcgcagacat ggaggatgag aaacccctag gcaccagaga acgcacgttg 1320
acgtgttgtt gcctgtgggc gtttaaaagc cacaaaatcc acaccatgta caagcggcct 1380
gaaacgcaaa ctatcgtcaa agtgccttcc acttttgatt cctttgtgat accgagcctg 1440
tggtcatcca gtctttccat gggtatcaga cagagaatca aactgctact cagcgcaaga 1500
atgacccaag gcctaccata ctcaggagac cgcaccgaag ctcgcgcggc agaagaagaa 1560
gagaaggagg tgcaggaggc tgaacttacg agggcagcgc tgccaccgct agtgagcggc 1620
tcttgtgcag acgatatcgc ccaggtggat gtggaggaat taaccttcag agccggagcc 1680
ggggttgtgg aaacacccag gaatgccctg aaggttacac cgcaagcaca cgaccacctc 1740
ataggctcct acttgatcct ttccccccaa acggtgttga aaagcgagaa gctggcaccc 1800
atccaccctc ttgctgagca agtcacggtc atgacccact ccggaagatc cggaagatac 1860
ccagtcgaca agtacgacgg acgggtattg atcccgacag gagcggccat cccagtgagc 1920
gagttccagg cactcagcga gagcgcaacc atggtgtaca atgagaggga atttataaat 1980
cgcaagctgc accacatagc gctatatggg ccagccttga ataccgacga ggaaagctat 2040
gaaaaagtga gagctgagag ggcagagacc gagtatgtgt ttgacgtgga caagaaggca 2100
tgtatcaaga aggaggaggc atcaggcctt gtgttaacag gggacctaat caatccacct 2160
ttccacgaat tcgcatacga aggactcaag atccgcccag cagccccgta ccacacgacg 2220
atcatcggtg tgtttggcgt tccaggttcg ggcaagtcgg ctatcattaa gaacatggtg 2280
acgactcgcg atctggtggc cagtggaaag aaggagaact gccaagagat catgaatgat 2340
gtaaagaggc aacgcgggtt ggacgtgacc gctaggaccg tcgactcaat cctattgaat 2400
gggtgcaaga aaggcgtaga aaacctttac gtcgatgagg cgttcgcgtg tcactcgggt 2460
actttgctag cgctcatcgc gctggtgaga ccgtcaggta aggtagtact gtgcggcgac 2520
cctaagcagt gcggtttctt caatttgatg caactgaagg tgcactataa ccacaacatt 2580
tgtacaaggg tgctccataa gagcatctcc agaagatgca ctctacctgt tacggcgatc 2640
gtgtccacct tgcactacca agggaagatg agaacgacga accgatgcaa cacccccatc 2700
cagattgaca ccaccggttc ctccaaacca gcctcaggag atatcgtgtt aacgtgcttc 2760
cgcggctggg tgaagcaact gcaaatagac tatcgtggac acgaggtgat gactgcagct 2820
gcttcccagg gtttgacaag gaaaggcgtg tacgccgtga gacagaaagt gaacgaaaac 2880
ccactgtact cacctctgtc ggagcacgtc aatgtgttgt tgacccgaac tgaaaaccga 2940
ctggtgtgga agacactgtc gggtgacccg tggataaagg tgttaaccaa tgtcccacgt 3000
ggggatttca gtgcaactct ggaggaatgg caagaagagc atgacggtat catgagagtg 3060
ttgaccgagc gtccagcgga ggttgatcca ttccaaaaca aggctaaagt gtgctgggca 3120
aaatgtctgg tgcaagttct tgagacggcc ggaatacgta tgacggcaga tgaatggaac 3180
accatcttgg ctttcagaga ggacagagcg tactcgccag aagtcgctct caacgagatt 3240
tgcacccgtt actacggcgt tgacctagac agcggcctat tctcagcgca gtcagtttcc 3300
ctcttttatg agaacaacca ctgggacaac aggcctggag gacgcatgta cgggttcaac 3360
catgaagtag ccaggaaata cgcagccaga tttccttttc tacgtggcaa catgaactcg 3420
gggctgcaac taaacgtccc tgagaggaag ctccaacctt tcagcgccga atgcaacata 3480
gtcccatcca atcgtcggtt accgcatgct ctggtcacta gttatcagca gtgccgtgga 3540
gagagggtag agtggttgct gaaaaagatt ccaggtcacc aaatgttact tgtaagtgag 3600
tacaacctgg tgatacctca caaaagagtc ttctggattg cacctccgcg ggtgtcaggc 3660
gcggaccgca cgtacgactt ggacctaggg ttacctatgg atgcaggccg ttacgacctg 3720
gtattcgtca acatacacac tgagtaccgg caacaccact accaacaatg cgtcgaccat 3780
tcaatgcgcc tgcagatgct gggaggggat tcactacacc tgcttagacc aggaggctca 3840
ctgctgatga gagcatatgg ttacgcagac agagtcagcg agatggtggt gacagccctg 3900
gctaggaaat tctcggcgtt ccgtgtcctg agaccggcgt gcgtgacgag caacacagaa 3960
gtgttcctgc tgttttctaa ctttgataac ggcagaagag cggtaacctt gcaccaagct 4020
aaccagaaac ttagctcaat gtatgcctgc aacggattgc acactgctgg ctgtgcaccg 4080
tcatacaggg tccgccgcgc agatatatca ggacacggtg aggaagcggt cgtaaatgct 4140
gccaatgcca aaggtaccgt gagcgatgga gtgtgcaggg cggtcgctaa gaagtggcca 4200
tcatctttca aaggggctgc aactccagtt ggcacagcca aaatgatccg cgcagatggc 4260
atgaccgtaa tccacgcggt gggaccaaat ttctccaccg taacagaagc cgaaggggac 4320
agagagctag cggccgcgta ccgagctgtg gctagcataa ttagcaccaa caacataaag 4380
agcgtcgcag taccgctgct gtccacaggc accttttctg gcggtaagga cagagtgatg 4440
cagtccttga accacttatt cacggcattg gacgccaccg atgcagacgt ggttatctac 4500
tgcagagata aaaactggga aaagaagatt caggaagcca tcgacaggcg gacggcaatc 4560
gagctcgtat ctgaagacgt gaccttggaa accgatctgg ttagggtaca cccggacagt 4620
tgcttagtcg gcagaaatgg ttacagtgca accgacggta aactgtattc ttacctagag 4680
ggtacaaggt tccaccagac ggcggtcgac atggctgaaa tatcaacttt atggccaaga 4740
ctccaagatg ctaacgagca gatctgcctg tacgccctag gggagacgat ggacagtata 4800
cgtactaaat gcccagtaga ggacgccgat tcgtctacgc cgccgaaaac ggtgccgtgt 4860
ctatgtcggt acgcgatgac cgcggagcgg gttgccagac ttaggatgaa taacaccaaa 4920
aacatcatcg tgtgctcctc cttcccattg ccgaagtaca ggatagaagg agtgcagaag 4980
gtgaagtgtg accgagtgct aatttttgac cagaccgtcc cgtcactagt aagtcccaga 5040
aagtacatac aacagccgcc ggaacagctg gataatgtga gcctgacttc cacgacgtcg 5100
acgggatccg catggtcatt tccatcggaa acgacctacg aaactatgga ggtcgtagcc 5160
gaggtacaca ccgaacctcc gatccctccg cctcgccgac gtagagcagc cgtcgcccaa 5220
cttagacaga atctggaagt caccgaggag ctcgagcctt acgtgacaca gcaagcagag 5280
atcatggtca tggagagggt cgtgacgaca gacatacgcg ctatcccagt cccggcacga 5340
cgggccatta caatgccagt cccagccccc agggttcgta aggtcgctac cgatcctcca 5400
tcagaaccgg aagctcctat cccggcacca agaaagcgaa gaaccactag tacctcacct 5460
ccgcataacc cagaggattt cgttcccagg gtaccggttg agttaccgtg ggagccggaa 5520
gacctagaca tccaattcgg tgacttggag ccacgccgtc ggaacaccag ggaccgagat 5580
agcagcacag gaatacagtt cggtgatatc gactttaacc agtcctgact aggcagggct 5640
ggcgcgtata tcttttcgtc tgacactggt ccgggtcacc tgcagcagaa gtccgtaagg 5700
caacatgaac tgccatgcga gactctgtac gcccatgaag acgaacgcat atacccgccg 5760
gcatttgacg gagagaaaga aaaggtactc caggcaaaga tgcagatggc cccgacagaa 5820
gcgaataaga gcaggtacca gtcgaggaaa gtagagaaca tgaaggcact aattgtagaa 5880
agactacgca aaggagcaaa gttgtacctc catgagcaaa ccgacaaagt acccacatac 5940
accagcaagt accctagacc tgtgtactca ccatcggtgg atgacagcct gagcgatccg 6000
gaagtggcag tggccgcctg taactctttc ttagaggaga attatccgac cgtggcgaac 6060
taccagataa ccgatgagta tgacgcttat ctggacttgg tcgacggctc tgaaagctgc 6120
ctcgacagag ctacgttctg cccggccaaa ctaagatgtt accctaagca ccatgcatac 6180
caccaaccac aaatcaggag cgcagtacct tccccttttc aaaacacgtt acaaaacgta 6240
ctagccgcgg ccaccaaaag aaattgtaat gtcacccaaa tgagagaatt accaaccatg 6300
gactctgcgg tgttcaacgt agaaagcttc aaaaaatacg catgtaccgg cgaatattgg 6360
caagaattta aagacaatcc tatacggatc accaccgaaa acataacgac gtacgtggct 6420
aaactcaagg gtcctaaggc tgctgccctt tttgccaaga cgcataacct ggtgccgctt 6480
caggaggtgc caatggaccg cttcgtgatg gatatgaaga gagatgtgaa agttacacca 6540
ggcaccaagc ataccgaaga aaggcctaaa gtgcaagtga ttcaagcggc ggaaccattg 6600
gccacggcat atctatgcgg aatccacaga gagttagtca ggcggctaaa agccgttctg 6660
accccgaaca ttcacactct gtttgacatg tcggcggagg actttgatgc catcatagcg 6720
gcacatttcc aaccgggaga tgctgtactg gagacagata tcgcatcctt cgacaagagc 6780
caggacgact ccttagcgct aacggcgtta atgcttctgg aagacctcgg ggtcgaccaa 6840
gaactgctgg atcttatcga agctgcgttt ggcgagatca cgagtgtgca tctacctacc 6900
ggtacaagat ttaaattcgg tgctatgatg aagtcaggaa tgtttcttac actcttcatc 6960
aacacgctgc tgaacattgt catagcatgc cgcgtcttac gcgacaaatt atcgtcatcg 7020
gcgtgcgccg ccttcatagg cgatgacaac atagtgcacg gcgtgaggtc agacccgcta 7080
atggcagaaa ggtgtgcgag ttgggtcaac atggaagtga agatcatcga tgccacaatg 7140
tgtgagaaac caccatactt ttgtggtgga ttcatcctgt acgacagcgt cgccggtaca 7200
gcgtgtaggg ttgcagatcc actaaagagg ctgttcaaac tcgggaaacc gctcccggcg 7260
gacgacaacc aggacgaaga cagaagaagg gcactaaagg atgaaacagt taagtggtcc 7320
cgcataggat tgagagagga attagacgtg gcactgagct caagatacca agtcagtggc 7380
gtcgggaaca tcactagagc gatgtccacg ctgtctaaga atttgaagtc ttttaggaaa 7440
ataagaggtc ccatcgtaca tctgtacggc ggtcctaaat agatgcagga ttacactaca 7500
tctaaagacc acgtattaca gacatcatga attacatccc aactcaaacc ttttacggac 7560
gccgttggcg accacgcccg gcgtaccgtc catggcgggt gccgatgcag ccggccccac 7620
ccatggtgat tcctgagctg caaactccga tcgtccaggc ccaacagatg cagcagctaa 7680
tcagtgcagt ttctgccctg acgaccaagc aaaatggcaa agcaccgaag aagccaaaga 7740
agaagccaca aaaagcgaag gctaagaaaa acgaacagca aaagaaaaac gagaacaaga 7800
aaccaccacc taagcagaag aatccggcta agaagaagaa accaggaaaa agggaacgca 7860
tgtgcatgaa gatagagaat gattgcatct tcgaggtcaa gcttgacggt aaggtaacgg 7920
gctacgcctg cctagtcggg gataaagtga tgaagccggc acacgttaaa ggtgtgatcg 7980
acaaccccga cctagcgaag cttacctaca agaaatcgag caagtatgac ctagagtgcg 8040
cccagatacc ggtgcacatg aagtcagatg cttcaaagta cacccatgaa aaaccagaag 8100
ggcactacaa ttggcatcac ggtgcagtgc agtacagcgg tggcaggttc acaatcccga 8160
caggcgcagg taaaccagga gacagcggcc ggccgatctt cgacaacaaa ggacgtgtgg 8220
tggccattgt cctgggaggg gccaacgaag gagccaggac tgccctatct gtcgtgacct 8280
ggaccaaaga catggtcaca cggtacaccc cagaaggaac agaagaatgg tccgccgcct 8340
tgatgatgtg cgtcttagcc aacgttacat tcccatgctc agagcccgca tgtgcaccct 8400
gttgctatga aaaacaacca gaacagacac tgaggatgtt ggaggacaac gtggaccgcc 8460
cgggctacta cgatctgctc gaggccacga tgacgtgtaa caatagtgca cgccaccgcc 8520
gcagtgtgac ggaacacttc aatgtctaca aggccacgaa accatatcta gcgtattgcg 8580
cggactgcgg agacgggcag ttctgttaca gcccggtggc tatagaaaaa attagggatg 8640
aggcttccga tggcatgata aagatccaga tcgcagcgca aatcggcatc aacaaaggag 8700
gaacacacga acacaacaaa atcaggtaca tcgccgggca tgacatgaaa gaggcgaacc 8760
gggattcttt acaagtgcat acttccggtg tgtgcgctat ccgaggcacg atgggccact 8820
tcatcgtggc ctactgccct ccaggggacg aactaaaggt ccagttccaa gatgcagaat 8880
cgcacacaca ggcctgcaaa gtgcagtaca aacacgcacc ggccccagta ggcagagaaa 8940
aattcactgt caggccccac ttcggtatcg aagtgccatg cacaacgtac cagctgacta 9000
ccgcaccaac ggaggaagag atcgacatgc ataccccacc ggatatccca gatataacgt 9060
tgctgtcgca acagtcaggc aatgtaaaga tcacagcagg aggaaagacc atcagataca 9120
actgcacgtg cggtagtggc catgtgggca ccaccagtag cgacaagact atcaattcgt 9180
gcaaaatagc gcagtgccat gctgcggtga ctaaccacga caagtggcag tacacctcct 9240
cgtttgtccc tagagccgac cagttgtctc gcaaaggtaa agtgcacgta cccttccctc 9300
tgaccaactc cacatgcagg gtgcccgttg cacgtacacc aggtgtcaca tacggaaaga 9360
gagaactgac agtgaaactg cacccagatc atcccacgct gttgacgtac cgaagtctag 9420
gagcagatcc gcgcccgtat gaggagtgga tagaccgata cgtcgaacgg accataccgg 9480
tgaccgaaga tgggatcgag tacagatggg gaaacaaccc acccgtgcgc ttgtgggccc 9540
agctgacaac tgaaggcaaa ccccatggat ggccgcacga gatcatactc tattactatg 9600
ggctataccc agcagccacc atcgccgccg tctcagccgc gggtctcgca gccatattat 9660
cgctgctggc gtcatgctac atgttcgcca ctgcacgccg caagtgcttg accccatacg 9720
ccctgacccc cggggccgtc attccggtaa cactaggtgt actatgctgc gcgccacgag 9780
cgcatgccgc gtcatttgca gaatctatgg cgtatctatg ggatgagaat caaaccctgt 9840
tttggctgga gcttgcaacg ccgctcgctg ccataatcat acttgtatgc tgcctgaaga 9900
acctgctctg ctgctgcaaa ccgctttctt ttttagtgtt ggtgagcctg ggaactcccg 9960
tcgtaaaatc ttacgaacac accgcaacga tcccgaacgt ggtgggattc ccgtataagg 10020
ctcacattga gaggaacggc ttctccccga tgaccctaca gcttgaggta cttggaacca 10080
gcttggaacc cacgctaaac ttagagtaca taacctgcga atacaagaca gtcgtgccat 10140
caccttatat taagtgctgc gggacatcag aatgcagatc catggagcgc cccgactatc 10200
aatgccaggt ctacacagga gtgtacccat ttatgtgggg cggcgcatac tgcttctgcg 10260
acactgagaa cacccagctg agtgaagcat acgttgatag atcggacgta tgcaagcacg 10320
accatgccgc cgcctacaag gcgcacactg cggcaatgaa agccaccatc cgaataagtt 10380
acgggaacct caatcagaca acaacggcgt tcgtcaacgg ggagcataca gtgaccgtcg 10440
gaggcagcag gtttactttt ggtccaatct ccactgcctg gacgcctttc gacaacaaga 10500
tcgtcgtcta caagaacgac gtctacaatc aggacttccc accctacggg tcaggacaac 10560
cagggaggtt cggagacatc cagagcagga cggtagagag caaggacctg tatgccaaca 10620
ccgccctcaa gttgtcgaga ccttcgtccg gtactgttca cgtgccttac acacagaccc 10680
cttctggctt taagtactgg ataaaagaga gaggcacgtc gctgaatgac aaggctcctt 10740
ttggatgcgt aatcaagacc aacccagtca gagcagaaaa ttgcgccgtt ggcaacatcc 10800
cggtctccat ggacatcccg gacaccgctt ttacgcgcgt gattgatgca cctgccgtca 10860
caaacctgga gtgccaagtg gcggtctgca cgcactcatc ggacttcggc gggatcgcaa 10920
ctctgacttt caaaaccgac aaacccggaa aatgtgctgt ccattctcat tcgaatgtag 10980
ccaccataca ggaggcagct gtggacatca aaacagatgg caagataacc ctgcatttct 11040
ctacagcatc tgcatccccg gcattcaagg tatctgtgtg cagtgccaaa acgacatgca 11100
tggcagcgtg tgagccgccg aaggatcaca tcgtccctta tggggcgagc cacaacaacc 11160
aagtttttcc tgacatgtct ggcacggcaa tgacatgggt gcagcgggta gccggcggac 11220
tcggcgggct aacactcgcc gcagtggcag tacttatact ggtgacgtgt gtgactatgc 11280
gccgctaaat gcaggattac actacatcta aagaccacgt attacagaca tcatgcccgc 11340
catgaagatc gagtgccgca tcaccggcac cctgaacggc gtggagttcg agctggtggg 11400
cggcggagag ggcacccccg agcagggccg catgaccaac aagatgaaga gcaccaaagg 11460
cgccctgacc ttcagcccct acctgctgag ccacgtgatg ggctacggct tctaccactt 11520
cggcacctac cccagcggct acgagaaccc cttcctgcac gccatcaaca acggcggcta 11580
caccaacacc cgcatcgaga agtacgagga cggcggcgtg ctgcacgtga gcttcagcta 11640
ccgctacgag gccggccgcg tgatcggcga cttcaaggtg gtgggcaccg gcttccccga 11700
ggacagcgtg atcttcaccg acaagatcat ccgcagcaac gccaccgtgg agcacctgca 11760
ccccatgggc gataacgtgc tggtgggcag cttcgcccgc accttcagcc tgcgcgacgg 11820
cggctactac agcttcgtgg tggacagcca catgcacttc aagagcgcca tccaccccag 11880
catcctgcag aacgggggcc ccatgttcgc cttccgccgc gtggaggagc tgcacagcaa 11940
caccgagctg ggcatcgtgg agtaccagca cgccttcaag accccgatcg cattcgccta 12000
accgggaggc ttgacataat gtatatatat aagcatcata gtttttagta aagcatataa 12060
ataatcaagt agatcaaagg gctacctaac ccctgaatag taacaaaacg caaaatacaa 12120
aaacattagt tcaaagggcc agtaacccct gaatagtaac aaaacataaa aaccaaaaac 12180
agtagttcaa agggctatac aacccctgaa tagtaacaaa atacagaaaa accataaaaa 12240
ttataaaaat caactaatca gatcatctaa atttgaccaa ttggaaatag ccgaactcta 12300
cggagatgta ggcgtccgaa ctccacggag acgtaggaca aaattctgcc gaaccccaga 12360
ccatcgggga cgtaggcgtc taatttgttt ttttaatatt ttac 12404
<210> 29
<211> 8598
<212> DNA
<213> getah virus
<400> 29
atggcggacg tgtgacatca ccgttcgctc tttctaggat cctttgctac tccacatagt 60
gagagacaaa caacccagat gaaggtaacc gtggacgttg aggctgacag cccattcctt 120
aaggcccttc agaaggcgtt tcccgccttt gaggttgaat cacagcaggt cacaccgaat 180
gaccatgcta acgctagagc attttcgcat ctggctacta aactgattga gcaagaggtt 240
ccaacaggcg tcaccatcct ggacgtgggt agtgcacccg caaggaggtt gatgtctgac 300
cacacctacc actgcatctg ccccatgaaa agtgcggaag acccagagag gctggcgaat 360
tacgctcgaa agctggcgaa agcatcgggg actgtgctag acaagaatgt gtccggaaag 420
ataacggacc tgcaagacgt catggccact ccagacttgg aatccccgac tttttgcctg 480
cacactgacg agacgtgccg cactagggct gaggtcgccg tgtaccagga cgtatacgct 540
gtgcacgcac cgacgtctct gtaccaccag gccatcaaag gtgtcaggac ggcgtattgg 600
attggatttg acaccactcc attcatgttc gaggcgttag cgggtgcgta tcctgcgtac 660
tcgaccaact gggcagatga gcaagtgctg caggctcgta acatcggcct gtgcgcgaca 720
ggcctctccg aggggcgtcg cggcaaactc tctattatga gaaagaagtg cttgcgaccg 780
agcgacagag taatgttttc ggtcgggtcc accttgtaca ccgagagccg aaagctgctg 840
cgcagctggc acctaccttc cgtgtttcac ctgaagggta agaacagttt tacctgcagg 900
tgcgacacgg tggtgtcatg cgaaggttac gtggtaaaga agatcaccat aagcccaggc 960
atatatggaa aaacagttga ttacgcagtt acccatcacg cagagggttt cctgatgtgt 1020
aagatcactg atacagtcag aggagaaaga gtctctttcc cggtctgtac ctatgtgcct 1080
gcaaccatat gtgaccagat gacgggtata cttgccactg acgtgacacc agaggatgcc 1140
cagaagctcc tggttggatt gaaccaacgc atagtggtga atggtaggac gcaaagaaac 1200
acaaacacaa tgaaaaacta cctactgcca gtggtagcgc aagcattcag taaatgggca 1260
cgagaggcgc gcgcagacat ggaggatgag aaacccctag gcaccagaga acgcacgttg 1320
acgtgttgtt gcctgtgggc gtttaaaagc cacaaaatcc acaccatgta caagcggcct 1380
gaaacgcaaa ctatcgtcaa agtgccttcc acttttgatt cctttgtgat accgagcctg 1440
tggtcatcca gtctttccat gggtatcaga cagagaatca aactgctact cagcgcaaga 1500
atgacccaag gcctaccata ctcaggagac cgcaccgaag ctcgcgcggc agaagaagaa 1560
gagaaggagg tgcaggaggc tgaacttacg agggcagcgc tgccaccgct agtgagcggc 1620
tcttgtgcag acgatatcgc ccaggtggat gtggaggaat taaccttcag agccggagcc 1680
ggggttgtgg aaacacccag gaatgccctg aaggttacac cgcaagcaca cgaccacctc 1740
ataggctcct acttgatcct ttccccccaa acggtgttga aaagcgagaa gctggcaccc 1800
atccaccctc ttgctgagca agtcacggtc atgacccact ccggaagatc cggaagatac 1860
ccagtcgaca agtacgacgg acgggtattg atcccgacag gagcggccat cccagtgagc 1920
gagttccagg cactcagcga gagcgcaacc atggtgtaca atgagaggga atttataaat 1980
cgcaagctgc accacatagc gctatatggg ccagccttga ataccgacga ggaaagctat 2040
gaaaaagtga gagctgagag ggcagagacc gagtatgtgt ttgacgtgga caagaaggca 2100
tgtatcaaga aggaggaggc atcaggcctt gtgttaacag gggacctaat caatccacct 2160
ttccacgaat tcgcatacga aggactcaag atccgcccag cagccccgta ccacacgacg 2220
atcatcggtg tgtttggcgt tccaggttcg ggcaagtcgg ctatcattaa gaacatggtg 2280
acgactcgcg atctggtggc cagtggaaag aaggagaact gccaagagat catgaatgat 2340
gtaaagaggc aacgcgggtt ggacgtgacc gctaggaccg tcgactcaat cctattgaat 2400
gggtgcaaga aaggcgtaga aaacctttac gtcgatgagg cgttcgcgtg tcactcgggt 2460
actttgctag cgctcatcgc gctggtgaga ccgtcaggta aggtagtact gtgcggcgac 2520
cctaagcagt gcggtttctt caatttgatg caactgaagg tgcactataa ccacaacatt 2580
tgtacaaggg tgctccataa gagcatctcc agaagatgca ctctacctgt tacggcgatc 2640
gtgtccacct tgcactacca agggaagatg agaacgacga accgatgcaa cacccccatc 2700
cagattgaca ccaccggttc ctccaaacca gcctcaggag atatcgtgtt aacgtgcttc 2760
cgcggctggg tgaagcaact gcaaatagac tatcgtggac acgaggtgat gactgcagct 2820
gcttcccagg gtttgacaag gaaaggcgtg tacgccgtga gacagaaagt gaacgaaaac 2880
ccactgtact cacctctgtc ggagcacgtc aatgtgttgt tgacccgaac tgaaaaccga 2940
ctggtgtgga agacactgtc gggtgacccg tggataaagg tgttaaccaa tgtcccacgt 3000
ggggatttca gtgcaactct ggaggaatgg caagaagagc atgacggtat catgagagtg 3060
ttgaccgagc gtccagcgga ggttgatcca ttccaaaaca aggctaaagt gtgctgggca 3120
aaatgtctgg tgcaagttct tgagacggcc ggaatacgta tgacggcaga tgaatggaac 3180
accatcttgg ctttcagaga ggacagagcg tactcgccag aagtcgctct caacgagatt 3240
tgcacccgtt actacggcgt tgacctagac agcggcctat tctcagcgca gtcagtttcc 3300
ctcttttatg agaacaacca ctgggacaac aggcctggag gacgcatgta cgggttcaac 3360
catgaagtag ccaggaaata cgcagccaga tttccttttc tacgtggcaa catgaactcg 3420
gggctgcaac taaacgtccc tgagaggaag ctccaacctt tcagcgccga atgcaacata 3480
gtcccatcca atcgtcggtt accgcatgct ctggtcacta gttatcagca gtgccgtgga 3540
gagagggtag agtggttgct gaaaaagatt ccaggtcacc aaatgttact tgtaagtgag 3600
tacaacctgg tgatacctca caaaagagtc ttctggattg cacctccgcg ggtgtcaggc 3660
gcggaccgca cgtacgactt ggacctaggg ttacctatgg atgcaggccg ttacgacctg 3720
gtattcgtca acatacacac tgagtaccgg caacaccact accaacaatg cgtcgaccat 3780
tcaatgcgcc tgcagatgct gggaggggat tcactacacc tgcttagacc aggaggctca 3840
ctgctgatga gagcatatgg ttacgcagac agagtcagcg agatggtggt gacagccctg 3900
gctaggaaat tctcggcgtt ccgtgtcctg agaccggcgt gcgtgacgag caacacagaa 3960
gtgttcctgc tgttttctaa ctttgataac ggcagaagag cggtaacctt gcaccaagct 4020
aaccagaaac ttagctcaat gtatgcctgc aacggattgc acactgctgg ctgtgcaccg 4080
tcatacaggg tccgccgcgc agatatatca ggacacggtg aggaagcggt cgtaaatgct 4140
gccaatgcca aaggtaccgt gagcgatgga gtgtgcaggg cggtcgctaa gaagtggcca 4200
tcatctttca aaggggctgc aactccagtt ggcacagcca aaatgatccg cgcagatggc 4260
atgaccgtaa tccacgcggt gggaccaaat ttctccaccg taacagaagc cgaaggggac 4320
agagagctag cggccgcgta ccgagctgtg gctagcataa ttagcaccaa caacataaag 4380
agcgtcgcag taccgctgct gtccacaggc accttttctg gcggtaagga cagagtgatg 4440
cagtccttga accacttatt cacggcattg gacgccaccg atgcagacgt ggttatctac 4500
tgcagagata aaaactggga aaagaagatt caggaagcca tcgacaggcg gacggcaatc 4560
gagctcgtat ctgaagacgt gaccttggaa accgatctgg ttagggtaca cccggacagt 4620
tgcttagtcg gcagaaatgg ttacagtgca accgacggta aactgtattc ttacctagag 4680
ggtacaaggt tccaccagac ggcggtcgac atggctgaaa tatcaacttt atggccaaga 4740
ctccaagatg ctaacgagca gatctgcctg tacgccctag gggagacgat ggacagtata 4800
cgtactaaat gcccagtaga ggacgccgat tcgtctacgc cgccgaaaac ggtgccgtgt 4860
ctatgtcggt acgcgatgac cgcggagcgg gttgccagac ttaggatgaa taacaccaaa 4920
aacatcatcg tgtgctcctc cttcccattg ccgaagtaca ggatagaagg agtgcagaag 4980
gtgaagtgtg accgagtgct aatttttgac cagaccgtcc cgtcactagt aagtcccaga 5040
aagtacatac aacagccgcc ggaacagctg gataatgtga gcctgacttc cacgacgtcg 5100
acgggatccg catggtcatt tccatcggaa acgacctacg aaactatgga ggtcgtagcc 5160
gaggtacaca ccgaacctcc gatccctccg cctcgccgac gtagagcagc cgtcgcccaa 5220
cttagacaga atctggaagt caccgaggag ctcgagcctt acgtgacaca gcaagcagag 5280
atcatggtca tggagagggt cgtgacgaca gacatacgcg ctatcccagt cccggcacga 5340
cgggccatta caatgccagt cccagccccc agggttcgta aggtcgctac cgatcctcca 5400
tcagaaccgg aagctcctat cccggcacca agaaagcgaa gaaccactag tacctcacct 5460
ccgcataacc cagaggattt cgttcccagg gtaccggttg agttaccgtg ggagccggaa 5520
gacctagaca tccaattcgg tgacttggag ccacgccgtc ggaacaccag ggaccgagat 5580
agcagcacag gaatacagtt cggtgatatc gactttaacc agtcctgact aggcagggct 5640
ggcgcgtata tcttttcgtc tgacactggt ccgggtcacc tgcagcagaa gtccgtaagg 5700
caacatgaac tgccatgcga gactctgtac gcccatgaag acgaacgcat atacccgccg 5760
gcatttgacg gagagaaaga aaaggtactc caggcaaaga tgcagatggc cccgacagaa 5820
gcgaataaga gcaggtacca gtcgaggaaa gtagagaaca tgaaggcact aattgtagaa 5880
agactacgca aaggagcaaa gttgtacctc catgagcaaa ccgacaaagt acccacatac 5940
accagcaagt accctagacc tgtgtactca ccatcggtgg atgacagcct gagcgatccg 6000
gaagtggcag tggccgcctg taactctttc ttagaggaga attatccgac cgtggcgaac 6060
taccagataa ccgatgagta tgacgcttat ctggacttgg tcgacggctc tgaaagctgc 6120
ctcgacagag ctacgttctg cccggccaaa ctaagatgtt accctaagca ccatgcatac 6180
caccaaccac aaatcaggag cgcagtacct tccccttttc aaaacacgtt acaaaacgta 6240
ctagccgcgg ccaccaaaag aaattgtaat gtcacccaaa tgagagaatt accaaccatg 6300
gactctgcgg tgttcaacgt agaaagcttc aaaaaatacg catgtaccgg cgaatattgg 6360
caagaattta aagacaatcc tatacggatc accaccgaaa acataacgac gtacgtggct 6420
aaactcaagg gtcctaaggc tgctgccctt tttgccaaga cgcataacct ggtgccgctt 6480
caggaggtgc caatggaccg cttcgtgatg gatatgaaga gagatgtgaa agttacacca 6540
ggcaccaagc ataccgaaga aaggcctaaa gtgcaagtga ttcaagcggc ggaaccattg 6600
gccacggcat atctatgcgg aatccacaga gagttagtca ggcggctaaa agccgttctg 6660
accccgaaca ttcacactct gtttgacatg tcggcggagg actttgatgc catcatagcg 6720
gcacatttcc aaccgggaga tgctgtactg gagacagata tcgcatcctt cgacaagagc 6780
caggacgact ccttagcgct aacggcgtta atgcttctgg aagacctcgg ggtcgaccaa 6840
gaactgctgg atcttatcga agctgcgttt ggcgagatca cgagtgtgca tctacctacc 6900
ggtacaagat ttaaattcgg tgctatgatg aagtcaggaa tgtttcttac actcttcatc 6960
aacacgctgc tgaacattgt catagcatgc cgcgtcttac gcgacaaatt atcgtcatcg 7020
gcgtgcgccg ccttcatagg cgatgacaac atagtgcacg gcgtgaggtc agacccgcta 7080
atggcagaaa ggtgtgcgag ttgggtcaac atggaagtga agatcatcga tgccacaatg 7140
tgtgagaaac caccatactt ttgtggtgga ttcatcctgt acgacagcgt cgccggtaca 7200
gcgtgtaggg ttgcagatcc actaaagagg ctgttcaaac tcgggaaacc gctcccggcg 7260
gacgacaacc aggacgaaga cagaagaagg gcactaaagg atgaaacagt taagtggtcc 7320
cgcataggat tgagagagga attagacgtg gcactgagct caagatacca agtcagtggc 7380
gtcgggaaca tcactagagc gatgtccacg ctgtctaaga atttgaagtc ttttaggaaa 7440
ataagaggtc ccatcgtaca tctgtacggc ggtcctaaat agatgcagga ttacactaca 7500
tctaaagacc acgtattaca gacatcatgc ccgccatgaa gatcgagtgc cgcatcaccg 7560
gcaccctgaa cggcgtggag ttcgagctgg tgggcggcgg agagggcacc cccgagcagg 7620
gccgcatgac caacaagatg aagagcacca aaggcgccct gaccttcagc ccctacctgc 7680
tgagccacgt gatgggctac ggcttctacc acttcggcac ctaccccagc ggctacgaga 7740
accccttcct gcacgccatc aacaacggcg gctacaccaa cacccgcatc gagaagtacg 7800
aggacggcgg cgtgctgcac gtgagcttca gctaccgcta cgaggccggc cgcgtgatcg 7860
gcgacttcaa ggtggtgggc accggcttcc ccgaggacag cgtgatcttc accgacaaga 7920
tcatccgcag caacgccacc gtggagcacc tgcaccccat gggcgataac gtgctggtgg 7980
gcagcttcgc ccgcaccttc agcctgcgcg acggcggcta ctacagcttc gtggtggaca 8040
gccacatgca cttcaagagc gccatccacc ccagcatcct gcagaacggg ggccccatgt 8100
tcgccttccg ccgcgtggag gagctgcaca gcaacaccga gctgggcatc gtggagtacc 8160
agcacgcctt caagaccccg atcgcattcg cctaaccggg aggcttgaca taatgtatat 8220
atataagcat catagttttt agtaaagcat ataaataatc aagtagatca aagggctacc 8280
taacccctga atagtaacaa aacgcaaaat acaaaaacat tagttcaaag ggccagtaac 8340
ccctgaatag taacaaaaca taaaaaccaa aaacagtagt tcaaagggct atacaacccc 8400
tgaatagtaa caaaatacag aaaaaccata aaaattataa aaatcaacta atcagatcat 8460
ctaaatttga ccaattggaa atagccgaac tctacggaga tgtaggcgtc cgaactccac 8520
ggagacgtag gacaaaattc tgccgaaccc cagaccatcg gggacgtagg cgtctaattt 8580
gtttttttaa tattttac 8598
<210> 30
<211> 4287
<212> DNA
<213> getah virus
<400> 30
atggcggacg tgtgacatca ccgttcgctc tttctaggat cctttgctac tccacatagt 60
gagagacaaa caacccagat gcaggattac actacatcta aagaccacgt attacagaca 120
tcatgaatta catcccaact caaacctttt acggacgccg ttggcgacca cgcccggcgt 180
accgtccatg gcgggtgccg atgcagccgg ccccacccat ggtgattcct gagctgcaaa 240
ctccgatcgt ccaggcccaa cagatgcagc agctaatcag tgcagtttct gccctgacga 300
ccaagcaaaa tggcaaagca ccgaagaagc caaagaagaa gccacaaaaa gcgaaggcta 360
agaaaaacga acagcaaaag aaaaacgaga acaagaaacc accacctaag cagaagaatc 420
cggctaagaa gaagaaacca ggaaaaaggg aacgcatgtg catgaagata gagaatgatt 480
gcatcttcga ggtcaagctt gacggtaagg taacgggcta cgcctgccta gtcggggata 540
aagtgatgaa gccggcacac gttaaaggtg tgatcgacaa ccccgaccta gcgaagctta 600
cctacaagaa atcgagcaag tatgacctag agtgcgccca gataccggtg cacatgaagt 660
cagatgcttc aaagtacacc catgaaaaac cagaagggca ctacaattgg catcacggtg 720
cagtgcagta cagcggtggc aggttcacaa tcccgacagg cgcaggtaaa ccaggagaca 780
gcggccggcc gatcttcgac aacaaaggac gtgtggtggc cattgtcctg ggaggggcca 840
acgaaggagc caggactgcc ctatctgtcg tgacctggac caaagacatg gtcacacggt 900
acaccccaga aggaacagaa gaatggtccg ccgccttgat gatgtgcgtc ttagccaacg 960
ttacattccc atgctcagag cccgcatgtg caccctgttg ctatgaaaaa caaccagaac 1020
agacactgag gatgttggag gacaacgtgg accgcccggg ctactacgat ctgctcgagg 1080
ccacgatgac gtgtaacaat agtgcacgcc accgccgcag tgtgacggaa cacttcaatg 1140
tctacaaggc cacgaaacca tatctagcgt attgcgcgga ctgcggagac gggcagttct 1200
gttacagccc ggtggctata gaaaaaatta gggatgaggc ttccgatggc atgataaaga 1260
tccagatcgc agcgcaaatc ggcatcaaca aaggaggaac acacgaacac aacaaaatca 1320
ggtacatcgc cgggcatgac atgaaagagg cgaaccggga ttctttacaa gtgcatactt 1380
ccggtgtgtg cgctatccga ggcacgatgg gccacttcat cgtggcctac tgccctccag 1440
gggacgaact aaaggtccag ttccaagatg cagaatcgca cacacaggcc tgcaaagtgc 1500
agtacaaaca cgcaccggcc ccagtaggca gagaaaaatt cactgtcagg ccccacttcg 1560
gtatcgaagt gccatgcaca acgtaccagc tgactaccgc accaacggag gaagagatcg 1620
acatgcatac cccaccggat atcccagata taacgttgct gtcgcaacag tcaggcaatg 1680
taaagatcac agcaggagga aagaccatca gatacaactg cacgtgcggt agtggccatg 1740
tgggcaccac cagtagcgac aagactatca attcgtgcaa aatagcgcag tgccatgctg 1800
cggtgactaa ccacgacaag tggcagtaca cctcctcgtt tgtccctaga gccgaccagt 1860
tgtctcgcaa aggtaaagtg cacgtaccct tccctctgac caactccaca tgcagggtgc 1920
ccgttgcacg tacaccaggt gtcacatacg gaaagagaga actgacagtg aaactgcacc 1980
cagatcatcc cacgctgttg acgtaccgaa gtctaggagc agatccgcgc ccgtatgagg 2040
agtggataga ccgatacgtc gaacggacca taccggtgac cgaagatggg atcgagtaca 2100
gatggggaaa caacccaccc gtgcgcttgt gggcccagct gacaactgaa ggcaaacccc 2160
atggatggcc gcacgagatc atactctatt actatgggct atacccagca gccaccatcg 2220
ccgccgtctc agccgcgggt ctcgcagcca tattatcgct gctggcgtca tgctacatgt 2280
tcgccactgc acgccgcaag tgcttgaccc catacgccct gacccccggg gccgtcattc 2340
cggtaacact aggtgtacta tgctgcgcgc cacgagcgca tgccgcgtca tttgcagaat 2400
ctatggcgta tctatgggat gagaatcaaa ccctgttttg gctggagctt gcaacgccgc 2460
tcgctgccat aatcatactt gtatgctgcc tgaagaacct gctctgctgc tgcaaaccgc 2520
tttctttttt agtgttggtg agcctgggaa ctcccgtcgt aaaatcttac gaacacaccg 2580
caacgatccc gaacgtggtg ggattcccgt ataaggctca cattgagagg aacggcttct 2640
ccccgatgac cctacagctt gaggtacttg gaaccagctt ggaacccacg ctaaacttag 2700
agtacataac ctgcgaatac aagacagtcg tgccatcacc ttatattaag tgctgcggga 2760
catcagaatg cagatccatg gagcgccccg actatcaatg ccaggtctac acaggagtgt 2820
acccatttat gtggggcggc gcatactgct tctgcgacac tgagaacacc cagctgagtg 2880
aagcatacgt tgatagatcg gacgtatgca agcacgacca tgccgccgcc tacaaggcgc 2940
acactgcggc aatgaaagcc accatccgaa taagttacgg gaacctcaat cagacaacaa 3000
cggcgttcgt caacggggag catacagtga ccgtcggagg cagcaggttt acttttggtc 3060
caatctccac tgcctggacg cctttcgaca acaagatcgt cgtctacaag aacgacgtct 3120
acaatcagga cttcccaccc tacgggtcag gacaaccagg gaggttcgga gacatccaga 3180
gcaggacggt agagagcaag gacctgtatg ccaacaccgc cctcaagttg tcgagacctt 3240
cgtccggtac tgttcacgtg ccttacacac agaccccttc tggctttaag tactggataa 3300
aagagagagg cacgtcgctg aatgacaagg ctccttttgg atgcgtaatc aagaccaacc 3360
cagtcagagc agaaaattgc gccgttggca acatcccggt ctccatggac atcccggaca 3420
ccgcttttac gcgcgtgatt gatgcacctg ccgtcacaaa cctggagtgc caagtggcgg 3480
tctgcacgca ctcatcggac ttcggcggga tcgcaactct gactttcaaa accgacaaac 3540
ccggaaaatg tgctgtccat tctcattcga atgtagccac catacaggag gcagctgtgg 3600
acatcaaaac agatggcaag ataaccctgc atttctctac agcatctgca tccccggcat 3660
tcaaggtatc tgtgtgcagt gccaaaacga catgcatggc agcgtgtgag ccgccgaagg 3720
atcacatcgt cccttatggg gcgagccaca acaaccaagt ttttcctgac atgtctggca 3780
cggcaatgac atgggtgcag cgggtagccg gcggactcgg cgggctaaca ctcgccgcag 3840
tggcagtact tatactggtg acgtgtgtga ctatgcgccg ctaaccggga ggcttgacat 3900
aatgtatata tataagcatc atagttttta gtaaagcata taaataatca agtagatcaa 3960
agggctacct aacccctgaa tagtaacaaa acgcaaaata caaaaacatt agttcaaagg 4020
gccagtaacc cctgaatagt aacaaaacat aaaaaccaaa aacagtagtt caaagggcta 4080
tacaacccct gaatagtaac aaaatacaga aaaaccataa aaattataaa aatcaactaa 4140
tcagatcatc taaatttgac caattggaaa tagccgaact ctacggagat gtaggcgtcc 4200
gaactccacg gagacgtagg acaaaattct gccgaacccc agaccatcgg ggacgtaggc 4260
gtctaatttg tttttttaat attttac 4287
Claims (9)
1.一株盖他病毒，保藏编号为CCTCC NO：V 202069，命名为猪盖他病毒GETV-GX201808。
2.权利要求1所述盖他病毒的全长感染性克隆，其特征在于为该病毒基因组全长感染性克隆pGETV-GX。
3.根据权利要求2所述盖他病毒的全长感染性克隆，其特征在于：所述pGETV-GX中病毒全长基因为序列表中SEQ.ID.No.27。
4.权利要求1所述盖他病毒的复制子系统，其特征在于为pGETV-rep-GFP。
5.根据权利要求4所述盖他病毒的复制子系统，其特征在于：所述pGETV-rep-GFP中重组病毒基因为序列表中SEQ.ID.No.29。
6.权利要求2或4所述盖他病毒的全长感染性克隆或复制子系统在制备盖他病毒诊断试剂或盖他病毒基因工程疫苗方面的应用。
7.一种盖他病毒全长感染性克隆、复制子系统的制备方法，其特征在于包括以下步骤：
(1)GETV-GX201808株基因组全序列的扩增；
(2)pGETV-GX重组质粒的构建；
(3)pGEE3GFP重组质粒的构建；
(4)重组质粒pGETV-GX和pGEE3GFP的拯救；
(5)盖他病毒复制子系统(pGETV-rep)的构建。
8.根据权利要求7所述的制备方法，其特征在于：
步骤(1)和(2)中，提取GETV-GX201808株的RNA，经过反转录得到cDNA，以cDNA为模板，分别用S1-S4扩增片段的引物通过PCR扩增得到S1-S4片段；以pBR322为模板，用PBR332PacⅠ 381F/PBR332Mlu Ⅰ 978R扩增得到A片段，将酶切位点Pac Ⅰ、Mlu Ⅰ通过A片段插入pBR322载体中，得到pBR322-PM；通过引物CmvF/Cmv-X-M-N R扩增CMV片段，将CMV插入pBR322-PM；将S1通过酶切位点插入pBR322-PM，得到pBR322-PM-S1，将S2通过酶切位点插入pBR322-PM-S1，得到pBR322-PM-S12，将S3通过酶切位点插入pBR322-PM-S12得到pBR322-PM-S123，将S4通过酶切位点插入pBR322-PM-S123得到pBR322-PM-S1234，最后通过引物BGH-Mlu Ⅰ-F/BGH-Nru Ⅰ-R扩增BGH，得到片段D，将片段D插入pBR322-PM-S1234得到pGETV-GX；
步骤(3)中，以pGETV-GX为模板，用引物TY G Bstb Ⅰ F、ST-44-GFP-1R，GFP3UTRF、TY GMluI R，扩增得到片段B1、B3片段；用ST-44-GFP-2F、GFP3UTRR扩增GFP得到B2片段，将片段B1、B2、B3通过SOE-PCR得到片段B，将片段B插入pGETV-GX得到pGEE3GFP；
步骤(4)中，pGETV-GX转染BHK-21细胞后，可拯救亲本病毒rGETV-GX；而pGEE3GFP转染BHK-21细胞后，可拯救重组病毒rGEE3GFP；
步骤(5)中，以pGEE3GFP为模板，用引物TY Swa Ⅰ F，SOE GFP R和SOE GFP F，TY GMluI R扩增出C1与C2片段，将片段C1与C2通过SOE-PCR得到片段C，将片段C与pGEE3GFP同时酶切，将片段C替换插入pGEE3GFP，得到pGETV-rep-GFP，用引物CmvF/SOE G 5utr stru 1R、SOE G 5utr stru 2F/TY SrfI R通过SOE-PCR去除病毒非结构蛋白，构建出辅助质粒pGETV-rep-H。
9.权利要求8所述制备方法的引物组，其特征在于包括序列表中SEQ.ID.No.1至26。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112029781A (zh) * | 2020-08-14 | 2020-12-04 | 中山大学 | 一种新型冠状病毒SARS-CoV-2的安全型复制子系统及其应用 |
| CN113736750A (zh) * | 2021-09-22 | 2021-12-03 | 中牧实业股份有限公司 | 一株盖他病毒毒株及其应用 |
| CN114940978A (zh) * | 2022-04-08 | 2022-08-26 | 广西大学 | 表达盖他病毒e2蛋白的重组猪繁殖与呼吸综合征病毒的构建方法 |
| CN115433741A (zh) * | 2022-06-23 | 2022-12-06 | 广西大学 | 盖他病毒为载体表达报告蛋白的重组病毒的构建方法 |
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| CN115433741A (zh) * | 2022-06-23 | 2022-12-06 | 广西大学 | 盖他病毒为载体表达报告蛋白的重组病毒的构建方法 |
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