表达狗干扰素(α、β或γ)的重组新城疫弱毒疫苗株的制作方法

专利名称：表达狗干扰素(α、β或γ)的重组新城疫弱毒疫苗株的制作方法
技术领域：
本发明涉及重组病毒疫苗领域，具体地，本发明涉及表达狗干扰素(α、β或 Y-干扰素)的重组新城疫弱毒疫苗株，更具体地，涉及表达狗干扰素(α、β或Υ-干扰 素)的重组新城疫LaSota弱毒疫苗株。本发明还公开了制备所述重组新城疫弱毒疫苗株 的方法和该重组新城疫弱毒疫苗株在制备狗α、β或γ-干扰素中的应用。
背景技术：
干扰素是一组多细胞来源的复杂糖蛋白，它是有核细胞受病毒感染后产生的。另 夕卜，寡聚核苷酸、细菌、内毒素等也能刺激免疫细胞产生干扰素，两类干扰素作用相似，但理 化性质和抗原性存在差异，前者称为I型，后者称为II型[1’2]。白细胞是干扰素的重要来源[3]，人用干扰素大多从血液中提取，产量有限，且价格 昂贵，极大地限制了干扰素的广泛应用。因此，世界各国都在探索大量生产干扰素的途径， 如从脐带血，或高等植物中提取；探询稳定高产细胞株；或改变生产方式。近年来，随着对 细胞因子研究的不断深入和分子生物学技术的发展，大量的细胞因子得以克隆，许多细胞 因子基因工程药物面市，在治疗畜禽疾病提供了新手段的同时创造了巨大的经济和社会效 益Μ。分子生物学的迅速发展，使干扰素的研究日益深入，特别是PCR技术的广泛应用，对 构建动物干扰素基因重组疫苗，提高其免疫效力、降低病毒载体毒性等方面有着重要的意 义。大肠杆菌表达系统已广泛用于制备人和动物干扰素，所表达的重组蛋白在体内外 具有明显的抗病毒活性[5 7]。但大肠杆菌表达的蛋白经尿素溶解、变性后复性率低。而且 成本高，不便于大规模生产。毕赤酵母和杆状病毒表达系统是近年来发展起来的真核表达 系统，表达的外源蛋白可以获得正确折叠和糖基化[8’9]，但两种表达系统同样存在成本高、 纯化困难的缺点。新近发展起来的新城疫活病毒载体系统，使表达的外源蛋白接近天然状 态，无需纯化，成本低廉，利于大规模生产有生物活性的外源蛋白。基因工程载体技术的研究和应用也促进了干扰素的研究和开发。我国学者陈炬 等(陈炬等，中国科学B辑，1990，3:253-260.)首次把人干扰素2Α基因在烟草植株中表达 成功，随后，人干扰素2Β基因在烟草中也表达成功(Edelbaum, 0.，et al.，Expression of active human interferon-beta intransgenic plants. J Interferon Res,1992. 12(6) p. 449-53.)。负链RNA病毒的反向遗传操作(Reverse genetic)是通过操作病毒基因组cDNA 制造新病毒的过程，其基本过程是①组装完整的病毒基因组(或重组型基因组)cDNA克 隆，5’末端精确地缀于T7启动子后，3’末端精确缀于自我剪切的核酸酶序列和T7转录终 止信号之前，构成基因组cDNA转录模板；②以基因组cDNA转录模板与启动病毒复制必须的 转录相关功能结构蛋白如核蛋白(NP)、磷酸蛋白(P)和聚合酶蛋白(L)的表达质粒(T7启 动子)一起，共转染整合表达T7聚合酶的病毒复制许可细胞；③24-72小时后收获培养上 清，过滤后继续敏感细胞传代或接种鸡胚尿囊腔救获(rescue)病毒。对基因组cDNA进行突变、缺失或外源基因插入修饰后，通过反向遗传操作系统(reverse genetic system,RGS 系统)可获得相应的突变或重组的负链RNA病毒。申请人:之前获得授权的中国专利“新城疫LaSota疫苗株反向遗传操作系统及其 应用”(申请号200510097997. 8，申请日2005年9月2日)已经建立了一种新城疫LaSota 弱毒疫苗株的反向遗传操作系统，并且成功救获了野生型病毒株，并且为进一步开展新城 疫病毒活载体疫苗研制及重组新城疫病毒(Newcastle disease virus，NDV)相关基础奠定 了坚实的基础
发明内容
基于重组干扰素在治疗狗病毒性疾病和寄生虫病方面广阔的应用前景，本文建立 了以新城疫病毒疫苗株，如新城疫病毒LaSota疫苗株为载体的新型表达系统，构建了表达 狗α、β、Y干扰素基因的全基因组克隆，实现了狗α、β、γ干扰素在SPF鸡胚内高效、 稳定表达，并对其表达产物进行了抗病毒活性研究。因此，本发明的一个目的是提供以下各项1. 一种表达编码狗干扰素的基因的重组新城疫弱毒疫苗株。2.根据以上1的重组新城疫弱毒疫苗株，其为重组新城疫LaSota弱毒疫苗株。3.根据以上1或2的重组新城疫弱毒疫苗株，其中所述狗干扰素是狗α、β或 Y-干扰素，优选所述狗α、β和Y-干扰素的氨基酸序列分别如SEQID No. USEQ ID No. 2 和SEQ ID No. 3所示，更优选编码所述狗α、β和γ -干扰素的基因分别如SEQ ID No. 4、 SEQ ID No. 5 和 SEQ ID No. 6 所示。4.根据以上1或2的重组新城疫弱毒疫苗株，其为rL-Caninea、rL-Caninei3、或 rL-Canineγ。5.根据以上1-4中任何一项的重组新城疫弱毒疫苗株在制备狗干扰素中的应用。6. 一种生产以上1-4中任何一项的重组新城疫弱毒疫苗株的方法，该方法包括(1)构建转录质粒，该转录质粒包括其中插入编码狗干扰素的基因的所述新城疫 弱毒疫苗株的基因组cDNA序列；(2)构建一个或多个转录辅助质粒，该转录辅助质粒包括编码所述新城疫弱毒疫 苗株的核蛋白(NP)的cDNA序列、编码所述新城疫弱毒疫苗株的磷酸蛋白(P)的cDNA序列、 和编码所述新城疫弱毒疫苗株的大聚合酶蛋白(L)的cDNA序列；(3)将所述转录质粒和转录辅助质粒共转染所述新城疫弱毒疫苗株复制许可的宿 主细胞，培养转染后的宿主细胞；(4)收获上清液，过滤后继续敏感细胞传代或接种鸡胚尿囊腔救获重组病毒株。7.根据以上6的方法，其中所述重组新城疫弱毒疫苗株是重组新城疫LaSota弱毒 疫苗株。8.根据以上6或7的方法，其中所述转录质粒是pBRN-FL-CalFNa、 pBRN-FL-CalFN β、或 pBRN-FL-CalFN γ (基因序列分别见 SEQ ID No. 7、SEQ ID No. 8、SEQ ID No. 9，质粒图谱分别见图4-6)。9.根据以上6或7的方法，其中所述转录辅助质粒是质粒pBSNP、pBSP或pBSL。10.根据以上6-9中任何一项的方法，其中所述宿主细胞是BHK-21。
11.根据以上6-10中任何一项的方法制备的重组新城疫弱毒疫苗株，优选为 rL-Canine α、rL-Canine β、或 rL-Canine γ。12. 一种生产狗干扰素的方法，该方法包括1)将根据以上1-4中任何一项的重组新城疫弱毒疫苗株接种敏感细胞或接种鸡 胚尿囊腔；2)收获细胞上清液或鸡胚尿囊液。13.重组新城疫LaSota弱毒疫苗株rL-Canine α的SPF鸡胚尿囊液，其保藏号 为 CGMCC No. 2622。
12.重组新城疫LaSota弱毒疫苗株rL-Canine β的SPF鸡胚尿囊液，其保藏号为 CGMCC No. 2623。13.重组新城疫LaSota弱毒疫苗株rL-Canine γ的SPF鸡胚尿囊液，其保藏号为 CGMCC No. 2624。本发明利用新城疫病毒LaSota株作为表达载体，分别构建表达狗α、β、γ-干 扰素(Canine interferon-α , Canine interferon-β , Canine interferon- γ )完整开放 阅读框的全基因组质粒 pBRN-FL-CalFN-α、pBRN-FL-CalFN-β、pBRN-FL-CalFN-γ，转染 ΒΗΚ-21细胞获得表达CaIFN-α、β、γ重组新城疫病毒。采用RT-PCR方法，证实收获的鸡胚 尿囊液内的重组病毒含有相应外源基因。重组新城疫(Newcastle disease virus)LaSota 弱毒疫苗株rL-Canine、rL-Canine β和rL-Canine y接种的SPF鸡胚尿囊液于2008年 7月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心，其保藏号分别为CGMCC No. 2622,CGMCC No. 2623和CGMCC No. 2624。将重组病毒尿囊液经紫外线照射灭活新城疫病 毒，利用重组病毒VSV-EGFP在MDCK细胞系上测定rL-Canine α ,rL-Canine β ,rL-Canine Y抗病毒活性，表达CaIFN的重组新城疫病毒均能有效抑制水疱性口炎病毒(VSV)在狗 肾细胞(MDCK)上的复制，rL-Canine α、rL-Canine β、rL-Canine γ 接种 SPF 鸡胚所收 尿囊液抗病毒活性分别为2X106IU/mL、2X106IU/mL、2X106IU/mL。结果表明=CaIFN-α， CaIFN- β，CaIFN- γ 在重组新城疫病毒 rL-Canine α，rL-Canine β，rL-Canine γ 接种的 SPF鸡胚尿囊液中获得良好表达，并具有高效而稳定的抗病毒活性。


图1.狗α、β、Y干扰素基因的全基因组克隆的酶切与PCR鉴定结果(重组克 隆质粒进行 PmeI 酶切鉴定，产物分别为l，pBRN-FL-CaIFNa，610bp ； 2, pBRN-FL-CalFN β， 640bp ；3,pBRN-FL-CaIFN γ , 554bp ；获得质粒 PCR 鉴定产物片分别为4，pBRN_FL_CaIFN α， 1490bp ；5，pBRN-FL-CalFN β，1520bp ；6，pBRN-FL-CalFN γ，1430bp ；Ml, DNA 分子量标准 DL15000 ；M2, DNA 分子量标准 DL2000)；图2利用VSV-EGFP-MDCK系统测定rL-CalFN α/β/γ抗病毒活性(Α C 对接 种rL-CalFN α的SPF鸡胚尿囊液进行稀释(分别地，2 X IO5, 2 X IO6, 2 X IO7) ；D F 对接 种rL-CalFN β的SPF鸡胚尿囊液进行稀释(分别地，2 X IO5, 2 X IO6, 2 X IO7) ；G I 对接 种r L-CaIFN γ的SPF鸡胚尿囊液进行稀释(分别地，2 X 104，2 X 105，2 X IO6) ;J，K:对接种 rLaSota的SPF鸡胚尿囊液进行稀释(分别地，2 X 101，2 X IO2) ；L 阴性对照)；图 3. pBRN-FL-Pmel 的质粒图谱；
图 4. pBRN-FL-CaIFN a 质粒图谱；图 5. pBRN-FL-CaIFN ^ 质粒图谱；图 6. pBRN-FL-CaIFN y 质粒图谱；图7.质粒pBSNP的序列，带下划线的斜体部分是NP基因的编码序列；图8.质粒pBSP的序列，带下划线的斜体部分是P基因的编码序列；图9.质粒pBSL的序列，带下划线的斜体部分是L基因的编码序列。序列表说明SEQIDNo.1狗a干扰素氨基酉堯序列；
SEQIDNo.2狗0干扰素氨基酉堯序列；
SEQIDNo.3狗Y干扰素氨基酉堯序列；
SEQIDNo.4狗a干扰素基因
SEQIDNo.5狗0干扰素基因
SEQIDNo.6狗Y干扰素基因
SEQIDNo.7插入狗a干扰素的LaSota基因组序列的质粒:pBRN-FL-CaIFNa
SEQIDNo.8插入狗0干扰素的LaSota基因组序列的质粒:pBRN-FL-CaIFN旦
SEQIDNo.9插入狗、干扰素的LaSota基因组序列的质粒:pBRN-FL-CaIFNy
SEQIDNo.10pBRN-FL-PmeI的核苷酸序列。
具体实施例方式下文将参考实施例详细描述本发明，所述实施例仅是意图举例说明本发明，而不 是意图限制本发明的范围。本发明的范围由后附的权利要求具体限定。实施例1狗a、0、Y干扰素在新城疫病毒LaSota疫苗株中的高效稳定表达及抗 病毒活性检测材料和方法1 材料表达绿色荧光蛋白报告基因的重组水疱性口炎病毒VSV-GFP商购自中国农业科学 院哈尔滨兽医研究所_。BHK-21细胞(ATCC CCL-10)、MDCK细胞(ATCC No. CCL-22)由本实 验室传代培养，培养基为含10%胎牛血清的DMEM(Dulbecco' s Modified Eagle Media)； SPF鸡胚商购自中国农业科学院哈尔滨兽医研究所；表达T7聚合酶的痘病毒vTF7-3 (ATCC， VR-2153)购自 ATCC;新城疫 LaSota 克隆株 rLaSota (按照 ZL200510097997. 8 制备)由本 实验室保存；rLaSota反向遗传操作系统基因组转录模板质粒pBRN-FL-Pmel (参见葛金 英，温志远，王永，等。表达绿色荧光蛋白重组新城疫病毒LaSota疫苗株的构建[J].微生 物学报，2006，46 (4) 547 551)(病毒基因组转录质粒pBRN-FL-Pmel，含有新城疫病毒的 基因组全长cDNA，pBRN-FL-Pmel的序列信息见SEQ ID No. 10，质粒图谱见图3)。如ZL200510097997. 8或参考文献[11]所述，将表达新城疫病毒(基因组序列 GenBank登录号AY845400)的核蛋白(NP)、磷酸蛋白(P)及大聚合酶蛋白(L)基因的开放阅 读框架(ORF)cDNA分别紧接着克隆在pBlueScript载体(Clontech)质粒T7启动子下游， 分别构建转录辅助质粒pBSNP，pBSP和pBSL(序列分别如图7-9所示)。2表达狗a、3、Y干扰素基因的全基因组克隆的构建
由 Genebank 登录号 AB102731、NM 001135787、EF095772 委托上海捷瑞生物工程 有限公司合成狗干扰素a、0、Y，合成的片段经EcoRV酶切位点连入同样经此酶处理的 pBlueScript 载体(Clontech)上，质粒命名为 pBS-CalFNa、pBS_CaIFN3 , pBS-CalFN y。以质粒 pBS-CalFNa、pBS-CalFN^、pBS-CalFN y 为模板，通过 PCR 引物(表 1) 在干扰素基因0RF的5'端引入Pmel限制酶识别序列和NDV自身聚合酶L识别的转录终 止序列GE(TTAAGAAAAAA)、转录起始序列GS(ACGGGTAGAA)及增强基因表达的Kozak序列 (GCCACC)，在干扰素基因0RF的3'端引入Pmel限制酶识别序列(所用引物见表1)；为 了确保重组基因组cDNA全长总碱基数仍保持6的倍数，在引物GS和Kozak序列之间加 入适当数量碱基进行调整。PCR末端经Pmel酶切插入同样经Pmel处理的pBRN-FL-Pmel 载体内，表达狗a、0、Y干扰素的重组基因组全长cDNA克隆命名为pBRN-FL-CalFNa、 pBRN-FL-CaIFN 旦、pBRN-FL-CaIFN y (序列如 SEQ ID No. 7，SEQ ID No. 8，SEQ ID No. 9 所 示，质粒图谱见图4，5，6)。利用引物冊乂_3# 5' -gtccgtgcattgatcatg-3'和各基因PCR 所用下游引物鉴定方向。3重组病毒的拯救BHK-21细胞(ATCC CCL-10)接种于六孔板内生长达50 80%单层时，转录质粒 pBRN-FL-CaIFN a、pBRN-FL-CalFN 0、pBRN-FL-CaIFN y 分别与转录辅助质粒 pBSNP、pBSP 和pBSL共转染预先感染vTF7-3的BHK-21细胞。转染上清经0. 22um孔径滤器过滤后接种 9 11天的SPF鸡胚尿囊腔；接种后的SPF鸡胚继续培养3-5天，取鸡胚尿囊液50 ill按 常规进行新城疫病毒的血凝(HA)和血凝抑制(HI)试验，详细操作方法参见文献[11]。收获 HA及HI试验结果阳性尿囊液，-70°C冻存，并按常规方法分别于9-10日龄鸡胚尿囊腔接种 进行传代、EID50滴定[11]。重组新城疫LaSota弱毒疫苗株rL-Canine a、rL-Canine 3和 rL-Canine Y接种的SPF鸡胚尿囊液于2008年7月31日保藏于中国微生物菌株保藏管理委 员会普通微生物中心，其保藏号分别为CGMCC No. 2622、CGMCC No. 2623和CGMCC No. 2624。4 rL-CalFN a、rL-CaIFN 3 和 rL-CalFN y 抗病毒活性检测分别取rL-CaIFN a、rL-CalFN 3、rL-CalFN Y 以及 rLaSota 接种的 SPF 鸡胚尿囊 液2ml放入平皿中，置于紫外灯下照射2h，每间隔20分钟摇动一次，以灭活NDV病毒。经紫 外线灭活的尿囊液接种SPF鸡胚以检测病毒灭活是否彻底，每胚100 y L，共接3枚，接种后 的鸡胚置37°C孵化器内孵化，3天后检测鸡胚尿囊液是否HA阳性。HA阴性样品分装，-70°C 保存备用。用VSV-GFP-MDCK 细胞系统测定 rL-CalFN a、rL_CaIFN 3、rL_CaIFN y 的抗病毒活 性，具体为取灭活后重组病毒rL-CaIFNa、rL-CaIFN^、rL-CaIFN y鸡胚尿囊液，每个样 品100 u L，用含5%胎牛血清的DMEM进行10倍系列稀释，在生长于96孔细胞培养板上的 MDCK细胞上加入2X 101 2X 108稀释的尿囊液，同时取相同稀释倍数的灭活rLaSota鸡胚 尿囊液作为对照组，于37°C作用24h。吸出上清后，接种VSV-EGFP (表达增强型绿色荧光蛋 白的重组水泡性口炎病毒，商购自中国农业科学院哈尔滨兽医研究所)，剂量为200PFU (噬 斑形成单位)/孔感染细胞，感作lh后换完全培养液继续培养，同时设空白对照组，24h后荧 光显微镜观察结果。以空白对照组为对照，将减少50% VSV-EGFP感染率的样品的最高稀释 度定为1个病毒抑制单位(IU)。5重组病毒尿囊液抗病毒稳定性检测
取灭活后重组病毒rL-CaIFN α、rL-CaIFN β、rL-CaIFN γ鸡胚尿囊液分装， 100 μ L/管，一部分放4°C冰箱长期保存，每间隔1周取出一份进行抗病毒活性检测；另一部 分放_70°C冰箱，取冻融1次、2次、3次、4次的样品检测抗病毒活性单位的变化，以检测反 复冻融对抗病毒活性的影响。结果1表达狗α、β、γ干扰素基因的全基因组克隆的构建及鉴定通过PCR引物在狗干扰素基因CaIFN α、CaIFN β、CaIFN γ的ORF的5'端和3' 端引入PmeI限制酶识别序列(所用引物见表1)；由于插入的外源基因为PmeI单切连入 pBRN-FL-Pmel载体，因此克隆质粒经PmeI酶切的产物应有两条，一条为载体，约为18kb，另 一条为外源基因及其所携带的GE/GS等长约500 700bp (图1)；而经鉴定引物进行PCR扩 增，应得到比相应外源基因大200bp左右的条带，产物约700 900bp，电泳结果显示与预期 相符(图1)。证明表达狗α、β、γ干扰素的重组基因组全长cDNA克隆构建成功。表1构建表达狗α、β、Y干扰素基因的全基因组克隆所用引物 注下划线部分为干扰素基因特异序列，酶切位点用边框表示，Kozak序列用斜体 表示，基因起始信号和基因结束信号用小写字符表示，用黑体字符调整碱基数以适应6的 倍数规则。2重组病毒的拯救及鉴定全基因组转录质粒pBRN-FL-CaIFN α、pBRN-FL-CalFN β、pBRN-FL-CalFN γ 分别 与转录辅助质粒pBSNP、pBSP和pBSL共转染预先感染vTF7_3的BHK-21细胞。转染上清 接种后SPF胚继续培养3-5天，取鸡胚尿囊液50 μ L按常规进行新城疫病毒的血凝(HA)和 血凝抑止(HI)试验。收获HA及HI试验结果阳性尿囊液，-70°C冻存。同时进行RT-PCR 鉴定，PCR产物测序结果(数据未显示)证明重组病毒基因组内含有相应的外源基因片段， 至此成功拯救出表达狗干扰素α、β、Y的重组新城疫病毒，并分别命名为rL-CalFNa、 rL-CaIFN β 禾口 rL_CaIFN γ。
3 rL-CaIFN α、rL-CaIFN β 和 rL-CalFN y 抗病毒活性检测收获的重组病毒尿囊液经紫外线灭活，进行10倍系列稀释，稀释后的样品处理细胞24h，然后按每孔200PFU的接种量感染VSV-EGFP，24h后经荧光显微镜观察结果。2 X 105 倍稀释 rL-CaIFN a 和 rL-CalFN 3 和 2 X 104 倍稀释 rL-CalFN y 可以抑制 90 % VSV-EGFP 的复制，仅有少量荧光出现(图2A，D，G)，2X106稀释的rL-CaIFNa和rL_CaIFN3以及 2X105倍稀释rL-CaIFN y可部分抑制VSV-EGFP的复制，荧光亮度约为对照组的一半(图 2B，E，H)，2X 102倍稀释的rLaSota病毒接种SPF鸡胚尿囊液组也出现大量荧光(图2K)， 不能抑制VSV-EGFP的复制，但是10倍稀释时可以非特异性的部分抑制VSV-EGFP在MDCK细 胞中的复制(图2J)。未加干扰素样品的对照孔出现大量荧光(图2L)，VSV-EGFP在MDCK 细胞中大量复制。测得rL-CaIFNa、rL_CaIFN0的抗病毒活性为2X 107IU/mL，rL-CalFN y 抗病毒活性为2X106IU/mL。4重组病毒尿囊液抗病毒稳定性检测取灭活后重组病毒rL-CaIFN a、rL-CaIFN ^和L_CaIFN y鸡胚尿囊液分装，分别 放4°C冰箱长期保存，每间隔5天取出一份进行抗病毒活性检测。重组病毒尿囊液在4°C存 放10天时其抗病毒活性未表现明显下降；另一部分放_70°C冰箱，取冻融1次、2次、3次样 品检测抗病毒活性单位的变化，结果样品经3次反复冻融抗病毒活性下降小于10倍。干扰素(IFN)是机体在接触病毒或其他诱生物后分泌的一类蛋白，具有广谱抗病 毒、抑制细胞增殖和免疫调节等多种生物学活性[1]。Camilo等[12]在牛疱疹病毒中表达了 BoIFN-y并进行了体内抗病毒保护实验，取得了良好效果。国内外研究表明，BoIFN在抗水 泡性口炎病毒、口蹄疫病毒、牛病毒性腹泻病毒、牛呼吸道合胞体病毒、牛白血病病毒以及 副流感-3型的试验中均取得了良好效果[5_9]。IFN 2C具有抗弓形虫作用[4]。重组BoIFN-Y 作为免疫佐剂，曾用于奶牛乳房炎、细菌性疾病的治疗[13’14]。干扰素在抗病毒以及免疫调 节方面的杰出表现使之成为研究焦点。随着重组DNA技术的发展，人们尝试利用多种表达 系统探索干扰素大规模生产的捷径。最初研究人员利用大肠杆菌表达干扰素，纯化的蛋白 具有广谱抗病毒生物学活性，但纯化困难，且容易复性不完全[5 8]；之后出现杆状病毒表达 系统[15 17]、酵母表达系统[14]、疱疹病毒表达系统[12]等真核表达系统，表达的干扰素近乎 天然，但成本高，而且纯化困难。重组新城疫病毒表达系统表达的牛干扰素蛋白糖基化等蛋白翻译后加工和折叠 与哺乳动物相似，表达完整的干扰素蛋白更有利于保持蛋白天然构型与生物学活性，因此 新城疫病毒具有绝对的优势成为分泌型外源蛋白表达载体系统。亲本株NDV rLaSota具 有良好的鸡胚生长特性，低毒力，高生长滴度，表达的干扰素无需纯化，便于大量生产。NDV rLaSota株作为疫苗载体和外源蛋白功能研究载体具有广阔的应用前景。本文构建了表达狗a、0、y干扰素的重组新城疫病毒rL-CaIFNa、rL-CaIFN^ 和rL-CalFNy，插入基因为带有信号肽的干扰素基因阅读框，干扰素蛋白能够以分泌形式 释放到培养上清中。收获的SPF鸡胚尿囊液经紫外线灭活NDV病毒后，具有106IU/mL的高 滴度抗病毒活性，与杆状病毒表达系统、酵母表达系统具有相近，甚至更高的抗病毒滴度， 但成本相对更低，具有产业化应用潜力。本研究克隆的狗IFN-a、-0、-Y基因，为进一步 利用基因工程技术生产重组狗IFN及其生物学功能的研究奠定了基础，期望通过进一步开 展深入研究，使重组IFN成为具有抗病毒、抗肿瘤和免疫调节作用的主要药物。参考文献[1]侯云德，分子病毒学[M].北京学苑出版社，1990.
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[2]蔡友忠。干扰素及其在畜牧业中的应用研究进展[J]。福建畜牧兽医，2006， 28(4) 3 5。[3]张继瑜，赵荣材，阎继业.细胞因子基因工程药物研究应用进展[J]。中国兽 医科技，2001，31 (3) 15 17。[4]张君，于三科.IFN 2C介导的抗弓形虫作用及其作用机理[J]。动物医学进展， 2000,21(1) 25 27。[5]汪明，史喜菊，夏春.荷斯坦奶牛IFN-a基因的克隆表达和抗病毒活性分析 [J].中国兽医杂志，2004，40(1) :13 17.[6]许金俊，秦爱建，刘岳农，等.奶牛干扰素基因的克隆及其在大肠埃希氏菌 中表达[J].扬州大学学报(农业与生命科学版)，2003，24(1) :5 9.[7]史喜菊，夏春，汪明.晋南黄牛IFN-a基因的克隆、表达及抗病毒活性研究 [J].中国预防兽医学报，2004，26 (2) :100 114.[8]史喜菊，张灿，韩春来，等。牛干扰素在大肠杆菌与毕赤酵母中的表达及抗 病毒活性比较[J]。农业生物技术学报，2005，13(4) 461 467[9]万建青，吴文学，夏春，等.毕赤酵母表达猪干扰素基因及其抑制蓝耳病病毒 效果[J].生物工程学报，2002，18 (6) :683 686.[10]温志远，葛金英，胡森，等。表达增强绿色荧光蛋白重组水泡性口炎病毒印地 安那株的构建[J]。预防兽医学报，2007，29 (12)，905 910。[11]葛金英，温志远，王永，等。表达绿色荧光蛋白重组新城疫病毒LaSota疫苗株 的构建[J].微生物学报，2006，46 (4) :547 551.[12]Camilo R, Monique H, Lome A and Griebel P. The invivo effects ofrecombinant bovine herpesvirus-1expressing bovine interferon-y [J]. Journal of General Virology,2000,81 2665 2673[13]Gina M P and Lorraine M S. Specific immune responses of dairy cattleafter primary inoculation with recombinant bovine interferon-y as adjuvantvaccinating against mastitis[J].American Journal of Veterinary Research,1996,57(6) :819 824[14]Fernando Diaz, Felipe M, Araceli P, Verala E, Suarez-Guemes F andMontano L F. Secretion of IFN-y by bovine peripheral blood mononuclearcells stimulated with mycobacterium bovi s protein fractions obtained byisoelectric-focusing[J]. Veterinary Immunology and Immunopathology,1999,67 203 212[15]陈伟业，肇慧君，王喜军，等.重组杆状病毒表达牛0干扰素及其生物学活性 研究[J].微生物学报，2007，47 (4)[16]秦立廷，王喜军，胡森，李志中，陈伟业，等.重组杆状病毒表达牛干扰素 及其抗病毒活性研究[J].微生物学报，2007，47 (3)[17]秦立廷，王喜军，胡森，李志中，陈伟业，等.重组杆状病毒表达猪干扰素 及其抗病毒活性的研究[J].生物工程学报，2007，(3)序列表
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100105110
12
〈213〉狗<400>6 atgaattata caagctatat cttagcattt cagctttgcg tgattttgtg ttcttctggc60tgtaactgtc aggccatgtt ttttaaagaa atagaaaacc taaaggaata ttttaatgca120agtaatccag atgtatcgga cggtgggtct cttttcgtag atattttgaa gaaatggaga180gaggagagtg acaaaacaat cattcagagc caaattgtct ctttctactt gaaactgttt240gacaacttta aagataacca gatcattcaa aggagcatgg ataccatcaa ggaagacatg300cttggcaagt tcttaaatag cagcaccagt aagagggagg acttccttaa gctgattcaa360attcctgtga acgatctgca ggtccagcgc aaggcgataa atgaactcat caaagtgatg420aatgatctct caccaagatc caacctaagg aagcggaaaa ggagtcagaa tctgtttcga480ggccgcagag catcgaaata a501<210>7<211>20020<212>DNA〈213〉人工<400>7ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa60ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg120caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt180gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata240tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg300ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac360cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac420aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca480cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg540actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct600caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaccgat660gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt720cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct780ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct840tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa900acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt960cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc1020cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca1080tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc1140gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat1200tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg1260ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga1320attggagcca atcaattctt gcggagaact gtgaatgcgc ttggcagaac1380
ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatcactcatggtta3780tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttttctgtgactg3840
gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagttgctcttgcc3900cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtgctcatcattg3960gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgagatccagttcga4020tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcaccagcgtttctg4080ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcgacacggaaat4140gttgaatact catactcttc ctttttcaat attattgaag catttatcagggttattgtc4200tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggggttccgcgca4260catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatgacattaacct4320ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attctaggcggctatatgcg4380ttggatgcaa tttctatgcg cacccgttct cggagcactg tccgaccgctttggccgccg4440cccagtcctg ctcgcttcgc tacttggagc cactatcgag ctacgcgatcatggcgacca4500cacccgtcct gtggatcccc cgaagtctag aactagtgga tccccctgatacgactcact4560ataggaccaa acagagaatc cgtgagttac gataaaaggc gaaggagcaattgaagtcgc4620acgggtagaa ggtgtgactc tcgagtgcga gcccgaagca caaactcgagaaagccttct4680gccaacatgt cttccgtatt tgatgagtac gaacagctcc tcgcggctcagactcgcccc4740aatggagctc atggaggggg agaaaaaggg agtaccttaa aagtagacgtcccggtattc4800actcttaaca gtgatgaccc agaagataga tggagctttg tggtattctgcctccggatt4860gctgttagcg aagatgccaa caaaccactc aggcaaggtg ctctcatatctcttttatgc4920tcccactcac aggtaatgag gaaccatgtt gcccttgcag ggaaacagaatgaagccaca4980ttggccgtgc ttgagattga tggctttgcc aacggcacgc cccagttcaacaataggagt5040ggagtgtctg aagagagagc acagagattt gcgatgatag caggatctctccctcgggca5100tgcagcaacg gaaccccgtt cgtcacagcc ggggccgaag atgatgcaccagaagacatc5160accgataccc tggagaggat cctctctatc caggctcaag tatgggtcacagtagcaaaa5220gccatgactg cgtatgagac tgcagatgag tcggaaacaa ggcgaatcaataagtatatg5280cagcaaggca gggtccaaaa gaaatacatc ctctaccccg tatgcaggagcacaatccaa5340ctcacgatca gacagtctct tgcagtccgc atctttttgg ttagegagetcaagagaggc5400cgcaacacgg caggtggtac ctctacttat tataacctgg taggggacgtagactcatac5460atcaggaata ccgggcttac tgcattcttc ttgacactca agtacggaatcaacaccaag5520acatcagccc ttgcacttag tagcctctca ggcgacatcc agaagatgaagcagctcatg5580cgtttgtatc ggatgaaagg agataatgcg ccgtacatga cattacttggtgatagtgac5640cagatgagct ttgcgcctgc cgagtatgca caactttact cctttgccatgggtatggca5700tcagtcctag ataaaggtac tgggaaatac caatttgcca gggactttatgagcacatca5760ttctggagac ttggagtaga gtacgctcag gctcagggaa gtagcattaacgaggatatg5820gctgccgagc taaagctaac cccagcagca aggaggggcc tggcagctgctgcccaacgg5880gtctccgagg agaccagcag catagacatg cctactcaac aagtcggagtcctcactggg5940cttagcgagg gggggtccca agctctacaa ggcggatcga atagatcgcaagggcaacca6000gaagccgggg atggggagac ccaattcctg gatctgatga gagcggtagcaaatagcatg6060
atactctaga ccagatgaga gccactacaa aaatgtgaac acagatgagg aacgaaggtt 10800tccctaatag taatttgtgt gaaagttctg gtagtctgtc agttcagagg gttaagaaaa10860aactaccggt tgtagatgac caaaggacga tatacgggta gaacggtaag agaggccgcc10920cctcaattgc gagccaggct tcacaacctc cgttctaccg cttcaccgacaacggtcctc10980aatcatggac cgcgccgtta gccaagttgc gttagagaat gatgaaagag aggcaaaaaa11040tacatggcgc ttgatattcc ggattgcaat cttattctta acagtagtga ccttggctat11100atctgtagcc tcccttttat atagcatggg ggctagcaca cctagcgatcttgtaggcat11160accgactagg aattccaggg cagaagaaaa gattacatct acacttggttccaatcaaga11220tgtagtagat aggatatata agcaagtggc ccttgagtct ccgttggcattgttaaaaac11280tgagaccaca attatgaacg caataacatc tctctcttat cagattaatg gagctgcaaa11340caacagtggg tggggggcac ttatccatga cccagattat atagggggga taggcaaaga11400actcattgta gatgatgcta gtgatgtcac atcattctat ccctctgcatttcaagaaca11460tctgaatttt atcccggcgc ctactacagg atcaggttgc actcgaataccctcatttga11520catgagtgct acccattact gctacaccca taatgtaata ttgtctggatgcagagatca11580ctcacattca tatcagtatt tagcacttgg tgtgctccgg acatctgcaa cagggagggt11640attcttttct actctgcgtt ccatcaacct ggacgacacc caaaatcgga agtcttgcag11700tgtgagtgca actcccctgg gttgtgatat gctgtgctcg aaagtcacgg agacagagga11760agaagattat aactcagctg tccctacgcg gatggtacat gggaggttag ggttcgacgg11820ccagtaccac gaaaaggacc tagatgtcac aacattattc ggggactggg tggccaacta11880cccaggagta gggggtggat cttttattga cagccgcgta tggttctcag tctacggagg11940gttaaaaccc aattcaccca gtgacactgt acaggaaggg aaatatgtga tatacaagcg12000atacaatgac acatgcccag atgagcaaga ctaccagatt cgaatggcca ggtcttcgta12060taagcctgga cggtttggtg ggaaacgcat acagcaggct atcttatcta tcaaggtgtc12120aacatcctta ggcgaagacc cggtactgac tgtaccgccc aacacagtca cactcatggg12180ggccgaaggc agaattctca cagtagggac atctcatttc ttgtatcaacgagggtcatc12240atacttctct cccgcgttat tatatcctat gacagtcagc aacaaaacag ccactcttca12300tagtccttat acattcaatg ccttcactcg gccaggtagt atcccttgccaggcttcagc12360aagatgcccc aacccgtgtg ttactggagt ctatacagat ccacatcccctaatcttcta12420tagaaaccac accttgcgag gggtattcgg gacaatgctt gatggtgtacaagcaagact12480taaccctgcg tctgcagtat tcgatagcac atcccgcagt cgcattactcgagtgagttc12540aagcagtacc aaagcagcat acacaacatc aacttgtttt aaagtggtca agactaataa12600gacctattgt ctcagcattg ctgaaatatc taatactctc ttcggagaattcagaatcgt12660cccgttacta gttgagatcc tcaaagatga cggggttaga gaagccaggtctggctagtt12720gagtcaatta taaaggagtt ggaaagatgg cattgtatca cctatcttctgcgacatcaa12780gaatcaaacc gaatgccggc gcgtgctcga attccatgtt gccagttgaccacaatcagc12840cagtgctcat gcgatcagat taagccttgt caatagtctc ttgattaaga aaaaatgtaa12900gtggcaatga gatacaaggc aaaacagctc atggtaaata atacgggtag aacatggcga12960gctccggtcc tgaaagggca gagcatcaga ttatcctacc agagtcacacctgtcttcac13020cattggtcaa gcacaaacta ctctattact ggaaattaac tgggctaccg cttcctgatg13080
tcccgaaaataagaggcttaactacaaaag
tgtcggatgctgtgaaaccattacttagcc
acataattacattcccagctaatccgtact
gggaaagggaggacagggatactatcctgg
agttcccttctgtgcaagatattggtgctc
cggcatttttgcaagagttagatttgagtg
gtcagattcatcctgaactcacatctccaa
tgttcagagggatagggactgcatcttcct
tacccgaggtaagatgtgcaagacacggga
ccatcatgagtcttctcgaactgcatgtac
tttcaaatgagatgaaccccccgcaatgac
attcggttgtttataggaatctacaggcgg
agttccgtccattatggagagaaaatacag
tggggtatattacatctgcagtgccctaca
aaactcctccagggtccaatcaaagcttac
ttgccatgcattctgtaagggagggcgggg
gatactactttcatctactcatgaacttgt
tctctaatggttatgcatgtcgaggagata
acctgggcgggcctacatttgtacatgagg
ggcacggtacgctcttgtctaaatcagatg
agcggcagcgtgtgacagacatcctatcca
ggaagaatattgacactgcgctgattgaag
cggagagtctggtgagcacgctagcgaaca
acattgacacagttatccggtctgtgatat
cagtatttctatttaccccttacaatctct
aacagtgcacgagacagatcctagaggtta
ataaaataggcgatataatcagtctagagc
tcccactaaggacatacttgaagcatagta
gtattaccaaactcaaagaaatgtttacag
ctacatgaaaactataggca
actcttaacgaaaatcacatattaataggc
tttaatcatattatgttagaaaaaagttga
t C 13-3-3-1gtcttaaaaaaaggttgcgc
caccaaatctttgtttggtgggtcggcatg
gcatccgaaggaggacgtcgtccactcgga
taacaaagcccgaaaggaagctgagttggc
accccttggggcctctaaacgggtcttgag
cggatcgagacctcgatactagtgcggtgg
<210>8
agaaatgttcaatacccactgagtatttac17820ccgatcaagtgagctctatcatgtctccta17880acatgtctcggaagagcctcaatttgatca17940tgttgttgttcccccaagagccattattag18000gagtgaaagatccattcacccgacaacctg18060ctccagcaaggtatgacgcattcacactta18120atccggaggaagactacttagtacgatact18180cttggtataaggcatctcatctcctttctg18240actccttatacttagctgaagggagcggag18300cacatgaaactatctattacaatacgctct18360atttcgggccgaccccaactcagtttttga18420aggtaacatgcaaagatggatttgtccaag18480aggaaagtgacctgacctcagataaagcag18540gatctgtatcattgctgcattgtgacattg18600tagatcaactagctatcaatttatctctga18660tagtaatcatcaaagtgttgtatgcaatgg18720ttgctccgtgggatatattc18780tggagtgttacctggtatttgtcatgggtt18840tggtgaggatggcaaaaactctggtgcagc18900agatcacactgaccaggttattcacctcac18960gtcctttaccaagattaataaagtacttga19020ccgggggacagcccgtccgtccattctgtg19080taactcagataacccagattatcgctagtc19140atatggaagctgagggtgatctcgctgaca19200ctactgacgggaaaaagaggacatcactta19260caatactaggtcttagagtcgaaaatctca19320ttaaaggcatgatctccatggaggacctta19380cctgccctaaatatttgaaggctgtcctag19440acacttctgtactgtacttgactcgtgctc19500atgcagtcaacggatattacagtaactgtg19560tccttttttggccaattgtattcttgttga19620accctgactccttaggactcgaattcgaac19680acaattattcttgagtgtagtctcgtcatt19740gcatctccacctcctcgcggtccgacctgg19800tggctaagggaggggcccccgcggggctgc19860tgctgccaccgctgagcaataactagcata19920gggttttttgctgaaaggaggaactatatc19980agctgaattc20020
<211>20495<212>DNA〈213〉人工
<400>8ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa60ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg120caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt180gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata240tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg300ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac360cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac420aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca480cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg540actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct600caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaccgat660gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt720cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct780ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct840tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa900acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt960cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc1020cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca1080tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc1140gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat1200tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg1260ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga1320attggagcca atcaattctt gcggagaact gtgaatgcgc ttggcagaac1380atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg1440tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg1500ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc1560tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg1620taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca1680ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga1740ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa1800cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt1860ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc1920aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt1980ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac2040gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac2100
gaccggagca agcaactctc tgctgttgat gcttgacaag ctcagcaataaatcgtccaa 6840tgctaaaaag ggcccatggt cgagccccca agaggggaat caccaacgtccgactcaaca6900gcaggggagt caacccagtc gcggaaacag tcaggaaaga ccgcagaaccaagtcaaggc6960cgcccctgga aaccagggca cagacgtgaa cacagcatat catggacaatgggaggagtc7020acaactatca gctggtgcaa cccctcatgc tctccgatca aggcagagccaagacaatac7080ccttgtatct gcggatcatg tccagccacc tgtagacttt gtgcaagcgatgatgtctat7140gatggaggcg atatcacaga gagtaagtaa ggttgactat cagctagatcttgtcttgaa7200acagacatcc tccatcccta tgatgcggtc cgaaatccaa cagctgaaaacatctgttgc7260agtcatggaa gccaacttgg gaatgatgaa gattctggat cccggttgtgccaacatttc7320atctctgagt gatctacggg cagttgcccg atctcacccg gttttagtttcaggccctgg7380agacccctct ccctatgtga cacaaggagg cgaaatggca cttaataaactttcgcaacc7440agtgccacat ccatctgaat tgattaaacc cgccactgca tgcgggcctgatataggagt7500ggaaaaggac actgtccgtg cattgatcat gtcacgccca atgcacccgagttcttcagc7560caagctccta agcaagttag atgcagccgg gtcgatcgag gaaatcaggaaaatcaagcg7620ccttgctcta aatggctaat tactactgcc acacgcagcg ggtccctgtccactcggcat7680cacacggaat ctgcaccgag ttcccccccg cagacccaag gtccaactctgtttaaactt7740agaaaaaata cgggtagaag tgccgccacc atgaggagac tctccgccagctcttgtgac7800cactacacca ctgactttgc cttccccaag gaactgtttg atggccagcggctccaggag7860gcgcaagccc tctctgtggt ccacgtgatg acccagaagg tcttccacctcttctgcacg7920aacatgtcct ctgctccttg gaacatgacc ctcctggagg aattgtgctcggggctctct7980gagcagctgg atgacctgga tgcctgtccc ctgcaggagg cagggctggccgagaccccc8040ctcatgcatg aagactccac cctgaggacc tacttccaaa ggatctccctctacctgcaa8100gacaggaacc acagcccgtg tgcctgggag atggtccgag cagaaatcgggagatccttc8160ttctccttga ccatcttgca agaaagagta aggaggagga aatgagtttaaacccaagcg8220gcaatcctct ctcgcttcct cagccccact gaatggacgc gtaaccgtaattaatctagc8280tacatttaag attaagaaaa aatacgggta gaattggagt gccccaattgtgccaagatg8340gactcatcta ggacaattgg gctgtacttt gattctgccc attcttctagcaacctgtta8400gcatttccga tcgtcctaca aggcacagga gatgggaaga agcaaatcgccccgcaatat8460aggatccagc gccttgactt gtggactgat agtaaggagg actcagtattcatcaccacc8520tatggattca tctttcaagt tgggaatgaa gaagccactg tcggcatgatcgatgataaa8580cccaagcgcg agttactttc cgctgcgatg ctctgcctag gaagcgtcccaaataccgga8640gaccttattg agctggcaag ggcctgtctc actatgatag tcacatgcaagaagagtgca8700actaatactg agagaatggt tttctcagta gtgcaggcac cccaagtgctgcaaagctgt8760agggttgtgg caaacaaata ctcatcagtg aatgcagtca agcacgtgaaagcgccagag8820aagattcccg ggagtggaac cctagaatat aaggtgaact ttgtctccttgactgtggta8880ccgaagaagg atgtctacaa gatcccagct gcagtattga aggtttctggctcgagtctg8940tacaatcttg cgctcaatgt cactattaat gtggaggttg acccgaggagtcctttggtt9000aaatctctgt ctaagtctga cagcggatac tatgctaacc tcttcttgcatattggactt9060atgaccaccg tagataggaa ggggaagaaa gtgacatttg acaagctggaaaagaaaata9120
aggagccttgatctatctgtcgggctcagtgatgtgctcgggccttccgtgttggtaaaa9180
gcaagaggtgcacggactaagcttttggcacctttcttctctagcagtgggacagcctgc9240
tatcccatagcaaatgcttctcctcaggtggccaagatactctggagtcaaaccgcgtgc9300
ctgcggagcgttaaaatcattatccaagcaggtacccaacgcgctgtcgcagtgaccgcc9360
gaccacgaggttacctctactaagctggagaaggggcacacccttgccaaatacaatcct9420
tttaagaaataagctgcgtctctgagattgcgctccgcccactcacccagatcatcatga9480
C 3- C 3.3.3.3.3.3. Ctaatctgtcttgattatttacagttagtttacctgcctatcaagttagaa9540
aaaacacgggtagaagattctggatcccggttggcgccctccaggtgcaagatgggctcc9600
agaccttctaccaagaacccagcacctatgatgctgactatccgggttgcgctggtactg9660
agttgcatctgtccggcaaactccattgatggcaggcctcttgcagctgcaggaattgtg9720
gttacaggagacaaagccgtcaacatatacacctcatcccagacaggatcaatcatagtt9780
aagctcctcccgaatctgcccaaggataaggaggcatgtgcgaaagcccccttggatgca9840
tacaacaggacattgaccactttgctcaccccccttggtgactctatccgtaggatacaa9900
gagtctgtgactacatctggaggggggagacaggggcgccttataggcgccattattggc9960
ggtgtggctcttggggttgcaactgccgcacaaataacagcggccgcagctctgatacaa10020
gccaaacaaaatgctgccaacatcctccgacttaaagagagcattgccgcaaccaatgag10080
gctgtgcatgaggtcactgacggattatcgcaactagcagtggcagttgggaagatgcag10140
cagtttgttaatgaccaacttaataaaacagctcaggaattagactgcatcaaaattgca10200
cagcaagttggtgtagagctcaacctgtacctaaccgaattgactacagtattcggacca10260
caaatcacttcacctgctttaaacaagctgactattcaggcactttacaatctagctggt10320
ggaaatatggattacttattgactaagttaggtgtagggacagctcatta10380
atcggtagcggcttaatcaccggtaaccctattctatacgactcacagactcaactcttg10440
ggtatacgggtaactctaccttcagtcgggaacctaaataatatgcgtgccacctacttg10500
gaaaccttatccgtaagcacaaccaggggatttgcctcggcacttgtccccaaagtggtg10560
acacaggtcggttctgtgatagaagaacttgacacctcatactgtatagaaactgactta10620
gatttatattgtacaagaatagtaacgttccctatgtcccctggtatttattcctgcttg10680
agcggcaatacgtcggcctgtatgtactcaaagaccgaaggcgcacttactacaccatac10740
atgactatcaaaggttcagtcatcgccaactgcaagatgacaacatgtagatgtgtaaac10800
cccccgggtatcatatcgcaaaactatggagaagccgtgtctctaatagataaacaat ca10860
tgcaatgttttatccttaggcgggataactttaaggctcagtggggaattcgatgtaact10920
tatcagaagaatatctcaatacaagattctcaagtaataataacaggcaatcttgatatc10980
tcaactgagcttgggaatgtcaacaactcgatcagtaatgctttgaataagttagaggaa11040
agcaacagaaaactagacaaagtcaatgtcaaactgactagcacatctgctctcattacc11100
tatatcgttttgactatcatatctcttgtttttggtatacttagcctgattctagcatgc11160
tacctaatgtacaagcaaaaggcgcaacaaaagaccttattatggcttgggaataatact11220
ctagaccagatgagagccactacaaaaatgtgaacacagatgaggaacgaaggtttccct11280
aatagtaatttgtgtgaaagttctggtagtctgtcagttcagagggttaagaaaaaacta11340
ccggttgtagatgaccaaaggacgatatacgggtagaacggtaagagaggccgcccctca11400
attgcgagccaggcttcacaacctccgttctaccgcttcaccgacaacggtcctcaatca11460
tggaccgcgccgttagccaagttgcgttagagaatgatgaaagagaggca13-C at11520
ggcgcttgatattccggattgcaatcttattcttaacagtagtgaccttggctatatctg11580
tagcctcccttttatatagcatgggggctagcacacctagcgatcttgtaggcataccga11640
ctaggaattccagggcagaagaaaagattacatctacacttggttccaatcaagatgtag11700
tagataggatatataagcaagtggcccttgagtctccgttggcattgttaaaaactgaga11760
ccacaattatgaacgcaataacatctctctcttatcagattaatggagctgcaaacaaca11820
gtgggtggggggcacttatccatgacccagattatataggggggataggcaaagaactca11880
ttgtagatgatgctagtgatgtcacatcattctatccctctgcatttcaagaacatctga11940
attttatcccggcgcctactacaggatcaggttgcactcgaataccctcatttgacatga12000
gtgctacccattactgctacacccataatgtaatattgtctggatgcagagatcactcac12060
attcatatcagtatttagcacttggtgtgctccggacatctgcaacagggagggtattct12120
tttctactctgcgttccatcaacctggacg3-C 3-C C C 3.3.3.3.tcggaagtcttgcagtgtga12180
gtgcaactcccctgggttgtgatatgctgtgctcgaaagtcacggagacagaggaagaag12240
attataactcagctgtccctacgcggatggtacatgggaggttagggttcgacggccagt12300
accacgaaaaggacctagatgtcacaacattattcggggactgggtggccaactacccag12360
gagtagggggtggatcttttattgacagccgcgtatggttctcagtctacggagggttaa12420
aacccaattcacccagtgacactgtacaggaagggaaatatgtgatatacaagcgataca12480
atgacacatgcccagatgagcaagactaccagattcgaatggccaggtcttcgtataagc12540
ctggacggtttggtgggaaacgcatacagcaggctatcttatctatcaaggtgtcaacat12600
ccttaggcgaagacccggtactgactgtaccgcccaacacagtcacactcatgggggccg12660
aaggcagaattctcacagtagggacatctcatttcttgtatcaacgagggtcatcatact12720
tctctcccgcgttattatatcctatgacagtcagcaacaaaacagccactcttcatagtc12780
cttatacattcaatgccttcactcggccaggtagtatcccttgccaggcttcagcaagat12840
gccccaacccgtgtgttactggagtctatacagatccacatcccctaatcttctatagaa12900
accacaccttgcgaggggtattcgggacaatgcttgatggtgtacaagcaagacttaacc12960
ctgcgtctgcagtattcgatagcacatcccgcagtcgcattactcgagtgagttcaagca13020
gtaccaaagcagcatacacaacatcaacttgttttaaagtggtcaagactaataagacct13080
attgtctcagcattgctgaaatatctaatactctcttcggagaattcagaatcgtcccgt13140
tactagttgagatcctcaaagatgacggggttagagaagccaggtctggctagttgagtc13200
aattataaaggagttggaaagatggcattgtatcacctatcttctgcgacatcaagaatc13260
aaaccgaatgccggcgcgtgctcgaattccatgttgccagttgaccacaatcagccagtg13320
ctcatgcgatcagattaagccttgtcaatagtctcttgattaagaaaaaatgtaagtggc13380
aatgagatacaaggcaaaacagctcatggtaaataatacgggtagaacatggcgagctcc13440
ggtcctgaaagggcagagcatcagattatcctaccagagtcacacctgtcttcaccattg13500
gtcaagcacaaactactctattactggaaattaactgggctaccgcttcctgatgaatgt13560
gacttcgaccacctcattctcagccgacaatggaaaaaaatacttgaatcggcctctcct13620
gatactgagagaatgataaaactcggaagggcagtacaccaaactcttaaccacaattcc13680
agaataaccggagtgctccaccccaggtgtttagaagaactggctaatattgaggtccca13740
gattcaaccaacaaatttcggaagattgagaagaagatcc11C 3-C 3-3-cacgagatat13800
28
ggagaactgt tcacaaggct gtgtacgcat atagagaaga aactgctggg gtcatcttgg13860tctaacaatg tcccccggtc agaggagttc agcagcatcc gtacggacccggcattctgg13920
tttcactcaa aatggtccac agccaagttt gcatggctcc atataaaaca gatccagagg13980catctgatgg tggcagctag gacaaggtct gcggccaaca aattggtgatgctaacccat14040aaggtaggcc aagtctttgt cactcctgaa cttgtcgttg tgacgcatacgaatgagaac14100aagttcacat gtcttaccca ggaacttgta ttgatgtatg cagatatgatggagggcaga14160gatatggtca acataatatc aaccacggcg gtgcatctca gaagcttatcagagaaaatt14220gatgacattt tgcggttaat agacgctctg gcaaaagact tgggtaatca agtctacgat14280gttgtatcac taatggaggg atttgcatac ggagctgtcc agctactcga gccgtcaggt14340acatttgcag gagatttctt cgcattcaac ctgcaggagc ttaaagacattctaattggc14400ctcctcccca atgatatagc agaatccgtg actcatgcaa tcgctactgtattctctggt14460ttagaacaga atcaagcagc tgagatgttg tgtctgttgc gtctgtgggg tcacccactg14520cttgagtccc gtattgccgc aaaggcagtc aggagccaaa tgtgcgcaccgaaaatggta14580gactttgata tgatccttca ggtactgtct ttcttcaagg gaacaatcatcaacgggtac14640agaaagaaga atgcaggtgt gtggccgcga gtcaaagtgg atacaatata tgggaaggtc14700attgggcaac tacatgcaga ttcagcagag atttcacacg atatcatgttgagagagtat14760aagagtttat ctgcacttga atttgagcca tgtatagaat atgaccctgtcaccaacctg14820agcatgttcc taaaagacaa ggcaatcgca caccccaacg ataattggcttgcctcgttt14880aggcggaacc ttctctccga agaccagaag aaacatgtaa aagaagcaacttcgactaat14940cgcctcttga tagagttttt agagtcaaat gattttgatc catataaaga gatggaatat15000ctgacgaccc ttgagtacct tagagatgac aatgtggcag tatcatactcgctcaaggag15060aaggaagtga aagttaatgg acggatcttc gctaagctga caaagaagttaaggaactgt15120caggtgatgg cggaagggat cctagccgat cagattgcac ctttctttca gggaaatgga15180gtcattcagg atagcatatc cttgaccaag agtatgctag cgatgagtca actgtctttt15240aacagcaata agaaacgtat cactgactgt aaagaaagag tatcttcaaa ccgcaatcat15300gatccgaaaa gcaagaaccg tcggagagtt gcaaccttca taacaactga cctgcaaaag15360tactgtctta attggagata tcagacaatc aaattgttcg ctcatgccatcaatcagttg15420atgggcctac ctcacttctt cgaatggatt cacctaagac tgatggacactacgatgttc15480gtaggagacc ctttcaatcc tccaagtgac cctactgact gtgacctctcaagagtccct15540aatgatgaca tatatattgt cagtgccaga gggggtatcg aaggattatg ccagaagcta15600tggacaatga tctcaattgc tgcaatccaa cttgctgcag ctagatcgca ttgtcgtgtt15660gcctgtatgg tacagggtga taatcaagta atagcagtaa cgagagaggtaagatcagac15720gactctccgg agatggtgtt gacacagttg catcaagcca gtgataatttcttcaaggaa15780ttaattcatg tcaatcattt gattggccat aatttgaagg atcgtgaaaccatcaggtca15840gacacattct tcatatacag caaacgaatc ttcaaagatg gagcaatcctcagtcaagtc15900ctcaaaaatt catctaaatt agtgctagtg tcaggtgatc tcagtgaaaa caccgtaatg15960tcctgtgcca acattgcctc tactgtagca cggctatgcg agaacgggcttcccaaagac16020ttctgttact atttaaacta tataatgagt tgtgtgcaga catactttga ctctgagttc16080tccatcacca acaattcgca ccccgatctt aatcagtcgt ggattgagga catctctttt16140
ccttctgtgcaagatattggtgctcgagtg
tttttgcaagagttagatttgagtgctcca
attcatcctgaactcacatctccaaatccg
agagggatagggactgcatcttcctcttgg
gaggtaagatgtgcaagacacgggaactcc
atgagtcttctcgaactgcatgtaccacat
aatgagatgaaccccccgcaatgacatttc
gttgtttataggaatctacaggcggaggta
cgtccattatggagagaaaatacagaggaa
tatattacatctgcagtgccctacagatct
cctccagggtccaatcaaagcttactagat
atgcattctgtaagggagggcggggtagta
tactttcatctactcatgaacttgtttgct
aatggttatgcatgtcgaggagatatggag
ggcgggcctacatttgtacatgaggtggtg
ggtacgctcttgtctaaatcagatgagatc
cagcgtgtgacagacatcctatccagtcct
aatattgacactgcgctgattgaagccggg
agtctggtgagcacgctagcgaacataact
gacacagttatccggtctgtgatatatatg
tttctatttaccccttacaatctctctact
tgcacgagacagatcctagaggttacaata
ataggcgatataatcagtctagagcttaaa
ctaaggacatacttgaagcatagtacctgc
3-CC3.3-3.CtC3.aagaaatgtttacagacact
aaattctacatgaaaactataggcaatgca
taacgaaaatcacatattaataggctcctt
tcatattatgttagaaaaaagttgaaccct
taaatgtcttaaaaaaaggttgcgcacaat
aatctttgtttggtgggtcggcatggcatc
cgaaggaggacgtcgtccactcggatggct
aagcccgaaaggaagctgagttggctgctg
ttggggcctctaaacgggtcttgaggggtt
cgagacctcgatactagtgcggtggagctg
<210>9
<211>20651
<212>DNA
〈213〉人工
<400>9
aaagatccattcacccgacaacctgcggca18540gcaaggtatgacgcattcacacttagtcag18600gaggaagactacttagtacgatacttgttc18660tataaggcatctcatctcctttctgtaccc18720ttatacttagctgaagggagcggagccatc18780gaaactatctattacaatacgctcttttca18840gggccgaccccaactcagtttttgaattcg18900acatgcaaagatggatttgtccaagagttc18960agtgacctgacctcagataaagcagtgggg19020gtatcattgctgcattgtgacattgaaact19080caactagctatcaatttatctctgattgcc19140atcatcaaagtgttgtatgcaatgggatac19200ccgtgttccacaaaaggatatattctctct19260tgttacctggtatttgtcatgggttacctg19320aggatggcaaaaactctggtgcagcggcac19380acactgaccaggttattcacctcacagcgg19440ttaccaagattaataaagtacttgaggaag19500ggacagcccgtccgtccattctgtgcggag19560cagataacccagattatcgctagtcacatt19620gaagctgagggtgatctcgctgacacagta19680gacgggaaaaagaggacatcacttaaacag19740ctaggtcttagagtcgaaaatctcaataaa19800ggcatgatctccatggaggaccttatccca19860cctaaatatttgaaggctgtcctaggtatt19920tctgtactgtacttgactcgtgctcaacaa19980gtcaacggatattacagtaactgtgactct20040ttttggccaattgtattcttgttgatttaa20100gactccttaggactcgaattcgaactcaaa20160tattcttgagtgtagtctcgtcattcacca20220tccacctcctcgcggtccgacctgggcatc20280aagggaggggcccccgcggggctgctaaca20340ccaccgctgagcaataactagcataacccc20400ttttgctgaaaggaggaactatatccggat20460aattc20495
ttctcatgtttgacagcttatcatcgataagctttaatgcggtagtttatcacagttaaa60
ttgctaacgcagtcaggcaccgtgtatgaaatctaacaatgcgctcatcgtcatcctcgg120
caccgtcaccctggatgctgtaggcataggcttggttatgccggtactgccgggcctctt180
gcgggatatcgtccattccgacagcatcgccagtcactatggcgtgctgctagcgctata240
tgcgttgatgcaatttctatgcgcacccgttctcggagcactgtccgaccgctttggccg300
ccgcccagtcctgctcgcttcgctacttggagccactatcgactacgcgatcatggcgac360
cacacccgtcctgtggatcctctacgccggacgcatcgtggccggcatcaccggcgccac420
aggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgcca480
cttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccggggg540
actgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcct600
caacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaccgat660
gcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgt720
cgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgct780
ctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgct840
tgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaa900
acgtttcggcgagaagcaggccattatcgccggcatggcggccgacgcgctgggctacgt960
cttgctggcgttcgcgacgcgaggctggatggccttccccattatgattcttctcgcttc1020
cggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgacca1080
tcagggacagcttcaaggatcgctcgcggctcttaccagcctaacttcgatcactggacc1140
gctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggttggcatggat1200
tgtaggcgccgccctataccttgtctgcctccccgcgttgcgtcgcggtgcatggagccg1260
ggccacctcgacctgaatggaagccggcggcacctcgctaacggattcaccactccaaga1320
attggagccaatcaattcttgcggagaactgtgaatgcgc3.3.3.CC3.3.CCCttggcagaac1380
atatccatcgcgtccgccatctccagcagccgcacgcggcgcatctcgggcagcgttggg1440
tcctggccacgggtgcgcatgatcgtgctcctgtcgttgaggacccggctaggctggcgg1500
ggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgc1560
tgctgcaaaacgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcg1620
taaagtctggaaacgcggaagtcagcgccctgcaccattatgttccggatctgcatcgca1680
ggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattga1740
ccctgagtgatttttctctggtcccgccgcatccataccgccagttgtttaccctcacaa1800
cgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatcctctctcgt1860
ttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgacc1920
aaacaggaaaaaaccgcccttaacatggcccgctttatcagaagccagacattaacgctt1980
ctggagaaactcaacgagctggacgcggatgaacaggcagacatctgtgaatcgcttcac2040
gaccacgctgatgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaac2100
ctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagc2160
agacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacc2220
cagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattg2280
tactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaatacc2340
gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtcgttcggctgc 2400ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata2460acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgtaaaaaggccg2520cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct2580caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgtttccccctggaa2640gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc2700tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctcagttcggtgt2760aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagcccgaccgctgcg2820ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg2880cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgctacagagttct2940tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatctgcgctctgc3000tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg3060ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc3120aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt3180aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatccttttaaattaaa3240aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgacagttaccaat3300gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatccatagttgcct3360gactccccgt cgtgtagata actacgatac gggagggctt accatctggccccagtgctg3420caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag3480ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatccagtctatta3540attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgcaacgttgttg3600ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg3660gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct3720ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta3780tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttttctgtgactg3840gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagttgctcttgcc3900cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg3960gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga4020tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcaccagcgtttctg4080ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat4140gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc4200tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca4260catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct4320ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attctaggcg gctatatgcg4380ttggatgcaa tttctatgcg cacccgttct cggagcactg tccgaccgctttggccgccg4440cccagtcctg ctcgcttcgc tacttggagc cactatcgag ctacgcgatcatggcgacca4500cacccgtcct gtggatcccc cgaagtctag aactagtgga tccccctgatacgactcact4560ataggaccaa acagagaatc cgtgagttac gataaaaggc gaaggagcaa ttgaagtcgc4620acgggtagaa ggtgtgactc tcgagtgcga gcccgaagca caaactcgag aaagccttct4680
gccaacatgt cttccgtatt tgatgagtac gaacagctcc tcgcggctca gactcgcccc 4740aatggagctc atggaggggg agaaaaaggg agtaccttaa aagtagacgt cccggtattc4800actcttaaca gtgatgaccc agaagataga tggagctttg tggtattctg cctccggatt4860gctgttagcg aagatgccaa caaaccactc aggcaaggtg ctctcatatc tcttttatgc4920tcccactcac aggtaatgag gaaccatgtt gcccttgcag ggaaacagaa tgaagccaca 4980ttggccgtgc ttgagattga tggctttgcc aacggcacgc cccagttcaa caataggagt5040ggagtgtctg aagagagagc acagagattt gcgatgatag caggatctct ccctcgggca 5100tgcagcaacg gaaccccgtt cgtcacagcc ggggccgaag atgatgcacc agaagacatc5160accgataccc tggagaggat cctctctatc caggctcaag tatgggtcac agtagcaaaa 5220gccatgactg cgtatgagac tgcagatgag tcggaaacaa ggcgaatcaa taagtatatg 5280cagcaaggca gggtccaaaa gaaatacatc ctctaccccg tatgcaggag cacaatccaa 5340ctcacgatca gacagtctct tgcagtccgc atctttttgg ttagegaget caagagaggc5400cgcaacacgg caggtggtac ctctacttat tataacctgg taggggacgt agactcatac5460atcaggaata ccgggcttac tgcattcttc ttgacactca agtacggaat caacaccaag 5520acatcagccc ttgcacttag tagcctctca ggcgacatcc agaagatgaa gcagctcatg 5580cgtttgtatc ggatgaaagg agataatgcg ccgtacatga cattacttgg tgatagtgac5640cagatgagct ttgcgcctgc cgagtatgca caactttact cctttgccat gggtatggca 5700tcagtcctag ataaaggtac tgggaaatac caatttgcca gggactttat gagcacatca 5760ttctggagac ttggagtaga gtacgctcag gctcagggaa gtagcattaa cgaggatatg 5820gctgccgagc taaagctaac cccagcagca aggaggggcc tggcagctgc tgcccaacgg 5880gtctccgagg agaccagcag catagacatg cctactcaac aagtcggagt cctcactggg 5940cttagcgagg gggggtccca agctctacaa ggcggatcga atagatcgca agggcaacca 6000gaagccgggg atggggagac ccaattcctg gatctgatga gagcggtagc aaatagcatg 6060agggaggcgc caaactctgc acagggcact ccccaatcgg ggcctccccc aactcctggg 6120ccatcccaag ataacgacac cgactggggg tattgataaa caaaacccag cctgcttcca 6180caaaaacatc ccaatgccct cacccgtagt cgacccctcg atttgcggct ctatatgacc6240acaccctcaa acaaacatcc ccctctttcc tccctccccc tgctgtacaa ctccgcacgc6300cctagatacc acaggcacaa tgcggctcac taacaatcaa aacagagccg agggaattag 6360aaaaaagtac gggtagaaga gggatattca gagatcaggg caagtctccc gagtctctgc6420tctctcctct acctgataga ccaggacaaa catggccacc tttacggatg cagagatcga 6480cgagctattt gagacaagtg gaactgtcat tgacaacata attacagccc agggtaaacc6540agcagagact gttggaagga gtgcaatccc acaaggcaag accaaggtgc tgagcgcagc6600atgggagaag catgggagca tccagccacc ggccagtcaa gacaaccccg atcgacagga 6660cagatctgac aaacaaccat ccacacccga gcaaacgacc ccgcatgaca gcccgccggc6720cacatccgcc gaccagcccc ccacccaggc cacagacgaa gccgtcgaca cacagctcag 6780gaccggagca agcaactctc tgctgttgat gcttgacaag ctcagcaata aatcgtccaa 6840tgctaaaaag ggcccatggt cgagccccca agaggggaat caccaacgtc cgactcaaca 6900gcaggggagt caacccagtc gcggaaacag tcaggaaaga ccgcagaacc aagtcaaggc6960cgcccctgga aaccagggca cagacgtgaa cacagcatat catggacaat gggaggagtc7020[1038]acaactatca gctggtgcaa cccctcatgc tctccgatca aggcagagcc aagacaatac7080 ccttgtatct gcggatcatg tccagccacc tgtagacttt gtgcaagcga tgatgtctat7140gatggaggcg atatcacaga gagtaagtaa ggttgactat cagctagatc ttgtcttgaa 7200acagacatcc tccatcccta tgatgcggtc cgaaatccaa cagctgaaaa catctgttgc7260agtcatggaa gccaacttgg gaatgatgaa gattctggat cccggttgtg ccaacatttc7320atctctgagt gatctacggg cagttgcccg atctcacccg gttttagttt caggccctgg 7380agacccctct ccctatgtga cacaaggagg cgaaatggca cttaataaac tttcgcaacc7440agtgccacat ccatctgaat tgattaaacc cgccactgca tgcgggcctg atataggagt7500ggaaaaggac actgtccgtg cattgatcat gtcacgccca atgcacccga gttcttcagc7560caagctccta agcaagttag atgcagccgg gtcgatcgag gaaatcagga aaatcaagcg 7620ccttgctcta aatggctaat tactactgcc acacgcagcg ggtccctgtc cactcggcat7680cacacggaat ctgcaccgag ttcccccccg cagacccaag gtccaactct gtttaaactt7740agaaaaaata cgggtagaag cgccaccatg gtaataggtg agactcttcc cattttcatc7800atgaccagta gatgcatcct ccaaacaact ctcctgttgt atttctccac catggctctt7860gccatgagca acgacttgct tcgatcccag ctaagcagca gcagtttgga gtgtcaggag 7920ctcctattac agttgaatgg aaccactgaa tattgcctca aggacaggat aaacttcgag 7980atccctgagg aaatcgagaa atcacgccag ttccagaagg aggacatcat attgatcacc8040catgagatgt tccagaagat ctttgatatt ttcaggagaa atatctctag aacaggatgg 8100aatgagacca ctgtcgagaa ccttcttgtg aagctccact ggcagaagga acatctggag 8160ataatcctgg aggacgtcaa agagaaggaa aacttcacct gggacaacag gactcttctg 8220cacctgaaga aatattactt aaggattgtg cagtacctga aggccaagga gtacagcatc8280tgtgcctgga caatagtcca agcagaaatc tgcaggaact ttttcttcct taatatactt8340acagattatc tccagaactg agtttaaacc caagcggcaa tcctctctcg cttcctcagc8400cccactgaat ggacgcgtaa ccgtaattaa tctagctaca tttaagatta agaaaaaata 8460cgggtagaat tggagtgccc caattgtgcc aagatggact catctaggac aattgggctg 8520tactttgatt ctgcccattc ttctagcaac ctgttagcat ttccgatcgt cctacaaggc8580acaggagatg ggaagaagca aatcgccccg caatatagga tccagcgcct tgacttgtgg 8640actgatagta aggaggactc agtattcatc accacctatg gattcatctt tcaagttggg 8700aatgaagaag ccactgtcgg catgatcgat gataaaccca agcgcgagtt actttccgct8760gcgatgctct gcctaggaag cgtcccaaat accggagacc ttattgagct ggcaagggcc8820tgtctcacta tgatagtcac atgcaagaag agtgcaacta atactgagag aatggttttc8880tcagtagtgc aggcacccca agtgctgcaa agctgtaggg ttgtggcaaa caaatactca 8940tcagtgaatg cagtcaagca cgtgaaagcg ccagagaaga ttcccgggag tggaacccta 9000gaatataagg tgaactttgt ctccttgact gtggtaccga agaaggatgt ctacaagatc9060ccagctgcag tattgaaggt ttctggctcg agtctgtaca atcttgcgct caatgtcact9120attaatgtgg aggttgaccc gaggagtcct ttggttaaat ctctgtctaa gtctgacagc9180ggatactatg ctaacctctt cttgcatatt ggacttatga ccaccgtaga taggaagggg 9240aagaaagtga catttgacaa gctggaaaag aaaataagga gccttgatct atctgtcggg 9300ctcagtgatg tgctcgggcc ttccgtgttg gtaaaagcaa gaggtgcacg gactaagctt9360[1077]ttggcacctt tcttctctag cagtgggaca gcctgctatc ccatagcaaa tgcttctcct9420caggtggcca agatactctg gagtcaaacc gcgtgcctgc ggagcgttaa aatcattatc9480 caagcaggta cccaacgcgc tgtcgcagtg accgccgacc acgaggttac ctctactaag9540ctggagaagg ggcacaccct tgccaaatac aatcctttta agaaataagc tgcgtctctg9600agattgcgct ccgcccactc acccagatca tcatgacaca aaaaactaat ctgtcttgat9660tatttacagt tagtttacct gcctatcaag ttagaaaaaa cacgggtaga agattctgga9720tcccggttgg cgccctccag gtgcaagatg ggctccagac cttctaccaa gaacccagca9780cctatgatgc tgactatccg ggttgcgctg gtactgagtt gcatctgtcc ggcaaactcc9840attgatggca ggcctcttgc agctgcagga attgtggtta caggagacaa agccgtcaac9900atatacacct catcccagac aggatcaatc atagttaagc tcctcccgaa tctgcccaag9960gataaggagg catgtgcgaa agcccccttg gatgcataca acaggacatt gaccactttg 10020ctcacccccc ttggtgactc tatccgtagg atacaagagt ctgtgactac atctggaggg 10080gggagacagg ggcgccttat aggcgccatt attggcggtg tggctcttgg ggttgcaact10140gccgcacaaa taacagcggc cgcagctctg atacaagcca aacaaaatgc tgccaacatc10200ctccgactta aagagagcat tgccgcaacc aatgaggctg tgcatgaggt cactgacgga 10260ttatcgcaac tagcagtggc agttgggaag atgcagcagt ttgttaatga ccaacttaat10320aaaacagctc aggaattaga ctgcatcaaa attgcacagc aagttggtgt agagctcaac10380ctgtacctaa ccgaattgac tacagtattc ggaccacaaa tcacttcacc tgctttaaac10440aagctgacta ttcaggcact ttacaatcta gctggtggaa atatggatta cttattgact10500aagttaggtg tagggaacaa tcaactcagc tcattaatcg gtagcggctt aatcaccggt10560aaccctattc tatacgactc acagactcaa ctcttgggta tacgggtaac tctaccttca 10620gtcgggaacc taaataatat gcgtgccacc tacttggaaa ccttatccgt aagcacaacc10680aggggatttg cctcggcact tgtccccaaa gtggtgacac aggtcggttc tgtgatagaa 10740gaacttgaca cctcatactg tatagaaact gacttagatt tatattgtac aagaatagta 10800acgttcccta tgtcccctgg tatttattcc tgcttgagcg gcaatacgtc ggcctgtatg 10860tactcaaaga ccgaaggcgc acttactaca ccatacatga ctatcaaagg ttcagtcatc10920gccaactgca agatgacaac atgtagatgt gtaaaccccc cgggtatcat atcgcaaaac10980tatggagaag ccgtgtctct aatagataaa caatcatgca atgttttatc cttaggcggg 11040ataactttaa ggctcagtgg ggaattcgat gtaacttatc agaagaatat ctcaatacaa 11100gattctcaag taataataac aggcaatctt gatatctcaa ctgagcttgg gaatgtcaac11160aactcgatca gtaatgcttt gaataagtta gaggaaagca acagaaaact agacaaagtc11220aatgtcaaac tgactagcac atctgctctc attacctata tcgttttgac tatcatatct11280cttgtttttg gtatacttag cctgattcta gcatgctacc taatgtacaa gcaaaaggcg 11340caacaaaaga ccttattatg gcttgggaat aatactctag accagatgag agccactaca 11400aaaatgtgaa cacagatgag gaacgaaggt ttccctaata gtaatttgtg tgaaagttct11460ggtagtctgt cagttcagag ggttaagaaa aaactaccgg ttgtagatga ccaaaggacg 11520atatacgggt agaacggtaa gagaggccgc ccctcaattg cgagccaggc ttcacaacct11580ccgttctacc gcttcaccga caacggtcct caatcatgga ccgcgccgtt agccaagttg 11640cgttagagaa tgatgaaaga gaggcaaaaa atacatggcg cttgatattc cggattgcaa 11700[1116]tcttattctt aacagtagtg accttggcta tatctgtagc ctccctttta tatagcatgg 11760gggctagcac acctagcgat cttgtaggca taccgactag gaattccagg gcagaagaaa 11820agattacatc tacacttggt tccaatcaag atgtagtaga taggatatat aagcaagtgg 11880cccttgagtc tccgttggca ttgttaaaaa ctgagaccac aattatgaac gcaataacat 11940ctctctctta tcagattaat ggagctgcaa acaacagtgg gtggggggca cttatccatg 12000acccagatta tatagggggg ataggcaaag aactcattgt agatgatgct agtgatgtca 12060catcattcta tccctctgca tttcaagaac atctgaattt tatcccggcg cctactacag 12120gatcaggttg cactcgaata ccctcatttg acatgagtgc tacccattac tgctacaccc12180ataatgtaat attgtctgga tgcagagatc actcacattc atatcagtat ttagcacttg 12240gtgtgctccg gacatctgca acagggaggg tattcttttc tactctgcgt tccatcaacc12300tggacgacac ccaaaatcgg aagtcttgca gtgtgagtgc aactcccctg ggttgtgata 12360tgctgtgctc gaaagtcacg gagacagagg aagaagatta taactcagct gtccctacgc12420ggatggtaca tgggaggtta gggttcgacg gccagtacca cgaaaaggac ctagatgtca 12480caacattatt cggggactgg gtggccaact acccaggagt agggggtgga tcttttattg 12540acagccgcgt atggttctca gtctacggag ggttaaaacc caattcaccc agtgacactg 12600tacaggaagg gaaatatgtg atatacaagc gatacaatga cacatgccca gatgagcaag 12660actaccagat tcgaatggcc aggtcttcgt ataagcctgg acggtttggt gggaaacgca 12720tacagcaggc tatcttatct atcaaggtgt caacatcctt aggcgaagac ccggtactga 12780ctgtaccgcc caacacagtc acactcatgg gggccgaagg cagaattctc acagtaggga 12840catctcattt cttgtatcaa cgagggtcat catacttctc tcccgcgtta ttatatccta 12900tgacagtcaggccactcttc atagtcctta tacattcaat gccttcactc12960ggccaggtag tatcccttgc caggcttcag caagatgccc caacccgtgt gttactggag 13020tctatacaga tccacatccc ctaatcttct atagaaacca caccttgcga ggggtattcg 13080ggacaatgct tgatggtgta caagcaagac ttaaccctgc gtctgcagta ttcgatagca 13140catcccgcag tcgcattact cgagtgagtt caagcagtac caaagcagca tacacaacat13200caacttgttt taaagtggtc aagactaata agacctattg tctcagcatt gctgaaatat13260ctaatactct cttcggagaa ttcagaatcg tcccgttact agttgagatc ctcaaagatg 13320acggggttag agaagccagg tctggctagt tgagtcaatt ataaaggagt tggaaagatg 13380gcattgtatc acctatcttc tgcgacatca agaatcaaac cgaatgccgg cgcgtgctcg 13440aattccatgt tgccagttga ccacaatcag ccagtgctca tgcgatcaga ttaagccttg 13500tcaatagtct cttgattaag aaaaaatgta agtggcaatg agatacaagg caaaacagct13560catggtaaat aatacgggta gaacatggcg agctccggtc ctgaaagggc agagcatcag 13620attatcctac cagagtcaca cctgtcttca ccattggtca agcacaaact actctattac13680tggaaattaa ctgggctacc gcttcctgat gaatgtgact tcgaccacct cattctcagc13740cgacaatgga aaaaaatact tgaatcggcc tctcctgata ctgagagaat gataaaactc13800ggaagggcag tacaccaaac tcttaaccac aattccagaa taaccggagt gctccacccc13860aggtgtttag aagaactggc taatattgag gtcccagatt
atttcggaag 13920attgagaaga agatccaaat tcacaacacg agatatggag aactgttcac aaggctgtgt13980acgcatatag agaagaaact gctggggtca tcttggtcta acaatgtccc ccggtcagag 14040[1155]gagttcagca gcatccgtac ggacccggca ttctggtttc actcaaaatg gtccacagcc14100aagtttgcat ggctccatat aaaacagatc cagaggcatc tgatggtggc agctaggaca 14160 [1157]aggtctgcgg ccaacaaatt ggtgatgcta acccataagg taggccaagt ctttgtcact14220cctgaacttg tcgttgtgac gcatacgaat gagaacaagt tcacatgtct tacccaggaa 14280cttgtattga tgtatgcaga tatgatggag ggcagagata tggtcaacat aatatcaacc14340acggcggtgc atctcagaag cttatcagag aaaattgatg acattttgcg gttaatagac14400gctctggcaa aagacttggg taatcaagtc tacgatgttg tatcactaat ggagggattt14460gcatacggag ctgtccagct actcgagccg tcaggtacat ttgcaggaga tttcttcgca 14520ttcaacctgc aggagcttaa agacattcta attggcctcc tccccaatga tatagcagaa 14580tccgtgactc atgcaatcgc tactgtattc tctggtttag aacagaatca agcagctgag 14640atgttgtgtc tgttgcgtct gtggggtcac ccactgcttg agtcccgtat tgccgcaaag 14700gcagtcagga gccaaatgtg cgcaccgaaa atggtagact ttgatatgat ccttcaggta 14760ctgtctttct tcaagggaac aatcatcaac gggtacagaa agaagaatgc aggtgtgtgg 14820ccgcgagtca aagtggatac aatatatggg aaggtcattg ggcaactaca tgcagattca 14880gcagagattt cacacgatat catgttgaga gagtataaga gtttatctgc acttgaattt14940gagccatgta tagaatatga ccctgtcacc aacctgagca tgttcctaaa agacaaggca 15000atcgcacacc ccaacgataa ttggcttgcc tcgtttaggc ggaaccttct ctccgaagac15060cagaagaaac atgtaaaaga agcaacttcg actaatcgcc tcttgataga gtttttagag 15120tcaaatgatt ttgatccata taaagagatg gaatatctga cgacccttga gtaccttaga 15180gatgacaatg tggcagtatc atactcgctc aaggagaagg aagtgaaagt taatggacgg 15240atcttcgcta agctgacaaa gaagttaagg aactgtcagg tgatggcgga agggatccta 15300gccgatcaga ttgcaccttt ctttcaggga aatggagtca ttcaggatag catatccttg 15360accaagagta tgctagcgat gagtcaactg tcttttaaca gcaataagaa acgtatcact15420gactgtaaag aaagagtatc ttcaaaccgc aatcatgatc cgaaaagcaa gaaccgtcgg 15480agagttgcaa ccttcataac aactgacctg caaaagtact gtcttaattg gagatatcag 15540acaatcaaat tgttcgctca tgccatcaat cagttgatgg gcctacctca cttcttcgaa 15600tggattcacc taagactgat ggacactacg atgttcgtag gagacccttt caatcctcca 15660agtgacccta ctgactgtga cctctcaaga gtccctaatg atgacatata tattgtcagt15720gccagagggg gtatcgaagg attatgccag aagctatgga caatgatctc aattgctgca 15780atccaacttg ctgcagctag atcgcattgt cgtgttgcct gtatggtaca gggtgataat15840caagtaatag cagtaacgag agaggtaaga tcagacgact ctccggagat ggtgttgaca 15900cagttgcatc aagccagtga taatttcttc aaggaattaa ttcatgtcaa tcatttgatt15960ggccataatt tgaaggatcg tgaaaccatc aggtcagaca cattcttcat atacagcaaa 16020cgaatcttca aagatggagc aatcctcagt caagtcctca aaaattcatc taaattagtg 16080ctagtgtcag gtgatctcag tgaaaacacc gtaatgtcct gtgccaacat tgcctctact16140gtagcacggc tatgcgagaa cgggcttccc aaagacttct gttactattt aaactatata 16200atgagttgtg tgcagacata ctttgactct gagttctcca tcaccaacaa ttcgcacccc16260gatcttaatc agtcgtggat tgaggacatc tcttttgtgc actcatatgt tctgactcct16320gcccaattag ggggactgag taaccttcaa tactcaaggc tctacactag aaatatcggt16380[1194]gacccgggga ctactgcttt tgcagagatc cagcgactag aagcagtgggattactgagt16440cctaacatta tgactaatat cttaactagg ccgcctggga atggagattgggccagtctg16500tgcaacgacc catactcttt caattttgag actgttgcaa gcccaaatattgttcttaag16560aaacatacgc gaagagtcct atttgaaact tgttcaaatc ccttattgtctggagtgcac16620acagaggata atgaggcaga agagaaggca ttggctgaat tcttgcttaatcaagaggtg16680attcatcccc gcgttgcgca tgccatcatg gaggcaagct ctgtaggtaggagaaagcaa16740attcaagggc ttgttgacac 3.3. C 3.3.3. C 3. C C gtaattaaga ttgcgcttactaggaggcca16800ttaggcataa agaggctgat gcggatagtc aattattcta gcatgcatgcaatgctgttt16860agagacgatg ttttttcctc cagtagatcc aaccacccct tagtctcttctaatatgtgt16920tctctgacac tggcagacta tgcacggaat agaagctggt cacctttgacgggaggcagg16980aaaatactgg gtgtatctaa tcctgatacg atagaactcg tagagggtgagattcttagt17040gtaagcggag ggtgtacaag atgtgacagc ggagatgaac aatttacttggttccatctt17100ccaagcaata tagaattgac cgatgacacc agcaagaatc ctccgatgagggtaccatat17160ctcgggtcga agacacagga gaggagagct gcctcacttg caaaaatagctcatatgtcg17220ccacatgtaa aggctgccct aagggcatca tccgtgttga tctgggcttatggggataat17280gacgtaaatt ggactgctgc tcttacgatt gcaaaatctc ggtgtaatgtaaacttagag17340tatcttcggt tactgtcccc tttacccacg gctgggaatc ttcaacatagactagatgat17400ggtataactc agatgacatt cacccctgca tctctctaca ggtgtcaccttacattcaca17460tatccaatga ttctcaaagg ctgttcactg aagaaggagt caaagaggggaatgtggttt17520accaacagag tcatgctctt gggtttatct ctaatcgaat cgatatttccaatgacagca17580accaggacat atgatgagat cacactgcac ctacatagta aatttagttgctgtatcaga17640gaagcacctg ttgcggttcc tttcgagcta cttggggtgg taccggaactgaggacagtg17700acctcaaata agtttatgta tgatcctagc cctggatcgg agggagactttgcgagactt17760gacttagcta tcttcaagag ttatgagctt aatctggagt catatcccacgatagagcta17820atgaacattc tttcaatatc cagcgggaag ttgattggcc agtctgtggtttcttatgat17880gaagatacct ccataaagaa tgacgccata atagtgtacg acaatacccgaaattggatc17940agtgaagctc agaattcaga tgtggtccgc ctatttgaat atgcagcacttgaagtgctc18000 [1221]ctcaactgtt cttaccaact ctattacctg agagtaagag gcctagacaatattgtctta18060tatatgggtg atttatacaa gaatatgcca ggaattctac tttccaacattgcagctaca18120atatctcatc ccgtcattca ttcaaggtta catgcagtgg gcctggtcaaccatgacgga18180tcacaccaac ttgcagatac ggattttatc gaaatgtctg caaaactattagtatcttgc18240acccgacgtg tgatctccgg cttatattca ggaaataagt atgatctgctgttcccatct18300gtcttagatg ataacctgaa tgagaagatg cttcagctga tatcccggttatgctgtctg18360tacacggtac tctttgctac aacaagagaa atcccgaaaa taagaggcttaactacaaaa18420gagaaatgtt caatacccac tgagtattta ctgtcggatg ctgtgaaaccattacttagc18480cccgatcaag tgagctctat catgtctcct aacataatta cattcccagctaatccgtac18540tacatgtctc ggaagagcct caatttgatc agggaaaggg aggacagggatactatcctg18600gtgttgttgt tcccccaaga gccattatta gagttccctt ctgtgcaagatattggtgct18660cgagtgaaag atccattcac ccgacaacct gcggcatttt tgcaagagttagatttgagt18720[1233]gctccagcaa ggtatgacgc attcacactt agtcagattcatcctgaactcacatctcca 18780aatccggagg aagactactt agtacgatac ttgttcagagggatagggactgcatcttcc18840tcttggtata aggcatctca tctcctttct gtacccgaggtaagatgtgcaagacacggg18900aactccttat acttagctga agggagcgga gccatcatgagtcttctcgaactgcatgta18960ccacatgaaa ctatctatta caatacgctc ttttcaaatgagatgaaccccccgcaatga19020catttcgggc cgaccccaac tcagtttttg aattcggttgtttataggaatctacaggcg19080gaggtaacat gcaaagatgg atttgtccaa gagttccgtccattatggagagaaaataca19140gaggaaagtg acctgacctc agataaagca gtggggtatattacatctgcagtgccctac19200agatctgtat cattgctgca ttgtgacatt gaaactcctccagggtccaatcaaagctta19260ctagatcaac tagctatcaa tttatctctg attgccatgcattctgtaagggagggcggg19320gtagtaatca tcaaagtgtt gtatgcaatg ggatactactttcatctactcatgaacttg19380tttgctccgt gttccacaaa aggatatatt ctctctaatggttatgcatgtcgaggagat19440atggagtgtt acctggtatt tgtcatgggt tacctgggcgggcctacatttgtacatgag19500gtggtgagga tggcaaaaac tctggtgcag cggcacggtacgctcttgtctaaatcagat19560gagatcacac tgaccaggtt attcacctca cagcggcagcgtgtgacagacatcctatcc19620agtcctttac caagattaat aaagtacttg aggaagaatattgacactgcgctgattgaa19680gccgggggac agcccgtccg tccattctgt gcggagagtctggtgagcacgctagcgaac19740ataactcaga taacccagat tatcgctagt cacattgacacagttatccggtctgtgata19800tatatggaag ctgagggtga tctcgctgac acagtatttctatttaccccttacaatctc19860tctactgacg ggaaaaagag gacatcactt aaacagtgcacgagacagatcctagaggtt19920acaatactag gtcttagagt cgaaaatctc aataaaataggcgatataatcagtctagag19980cttaaaggca tgatctccat ggaggacctt atcccactaaggacatacttgaagcatagt20040acctgcccta aatatttgaa ggctgtccta ggtattaccaaactcaaagaaatgtttaca20100gacacttctg tactgtactt gactcgtgct caacaaaaattctacatgaaaactataggc20160aatgcagtca acggatatta cagtaactgt gactcttaacgaaaatcacatattaatagg20220ctcctttttt ggccaattgt attcttgttg atttaatcatattatgttagaaaaaagttg20280aaccctgact ccttaggact cgaattcgaa ctcaaataaatgtcttaaaaaaaggttgcg20340cacaattatt cttgagtgta gtctcgtcat tcaccaaatctttgtttggtgggtcggcat20400ggcatctcca cctcctcgcg gtccgacctg ggcatccgaaggaggacgtcgtccactcgg20460atggctaagg gaggggcccc cgcggggctg ctaacaaagcccgaaaggaagctgagttgg20520ctgctgccac cgctgagcaa taactagcat aaccccttggggcctctaaacgggtcttga20580ggggtttttt gctgaaagga ggaactatat ccggatcgagacctcgatactagtgcggtg20640gagctgaatt c20651<210>10<211>20558<212>DNA〈213〉人工<400>10ttctcatgtt tgacagctta tcatcgataa gctttaatgcggtagtttatcacagttaaa 60[1272]ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg 120caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt180gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata 240 [1275]tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg 300ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac360cacacccgtc ctgtggatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac420aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca 480cttcgggctc atgagcgctt gtttcggcgt gggtatggtg gcaggccccg tggccggggg 540actgttgggc gccatctcct tgcatgcacc attccttgcg gcggcggtgc tcaacggcct600caacctacta ctgggctgct tcctaatgca ggagtcgcat aagggagagc gtcgaccgat660gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt720cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct780ctgggtcatt ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct840tgcggtattc ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa 900acgtttcggc gagaagcagg ccattatcgc cggcatggcg gccgacgcgc tgggctacgt960cttgctggcg ttcgcgacgc gaggctggat ggccttcccc attatgattc ttctcgcttc1020cggcggcatc gggatgcccg cgttgcaggc catgctgtcc aggcaggtag atgacgacca 1080tcagggacag cttcaaggat cgctcgcggc tcttaccagc ctaacttcga tcactggacc1140gctgatcgtc acggcgattt atgccgcctc ggcgagcaca tggaacgggt tggcatggat1200tgtaggcgcc gccctatacc ttgtctgcct ccccgcgttg cgtcgcggtg catggagccg 1260ggccacctcg acctgaatgg aagccggcgg cacctcgcta acggattcac cactccaaga 1320attggagcca atcaattctt gcggagaact gtgaatgcgc ttggcagaac1380atatccatcg cgtccgccat ctccagcagc cgcacgcggc gcatctcggg cagcgttggg 1440tcctggccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg 1500ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc1560tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg 1620taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca 1680ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga 1740ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa 1800cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt1860ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc1920aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt1980ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac2040gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac2100ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc2160agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc2220cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg 2280tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc2340gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc2400[1311]ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata 2460acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg 2520cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct2580 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 2640gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc2700tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt2760aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 2820ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 2880cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct2940tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc3000tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg 3060ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc3120aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt3180aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa 3240aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat3300gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct3360gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg 3420caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag 3480ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta 3540attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg 3600ccattgctgc aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg 3660gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct3720ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta 3780tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg 3840gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc3900cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg 3960gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga 4020tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg 4080ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat4140gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc4200tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca 4260catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct4320ataaaaatag gcgtatcacg aggccctttc gtcttcaaga attctaggcg gctatatgcg 4380ttggatgcaa tttctatgcg cacccgttct cggagcactg tccgaccgct ttggccgccg 4440cccagtcctg ctcgcttcgc tacttggagc cactatcgag ctacgcgatc atggcgacca 4500cacccgtcct gtggatcccc cgaagtctag aactagtgga tccccctgat acgactcact4560ataggaccaa acagagaatc cgtgagttac gataaaaggc gaaggagcaa ttgaagtcgc4620acgggtagaa ggtgtgactc tcgagtgcga gcccgaagca caaactcgag aaagccttct4680gccaacatgt cttccgtatt tgatgagtac gaacagctcc tcgcggctca gactcgcccc4740[1350]aatggagctc atggaggggg agaaaaaggg agtaccttaa aagtagacgt cccggtattc4800actcttaaca gtgatgaccc agaagataga tggagctttg tggtattctg cctccggatt4860gctgttagcg aagatgccaa caaaccactc aggcaaggtg ctctcatatc tcttttatgc4920 [1353]tcccactcac aggtaatgag gaaccatgtt gcccttgcag ggaaacagaa tgaagccaca 4980ttggccgtgc ttgagattga tggctttgcc aacggcacgc cccagttcaa caataggagt5040ggagtgtctg aagagagagc acagagattt gcgatgatag caggatctct ccctcgggca 5100tgcagcaacg gaaccccgtt cgtcacagcc ggggccgaag atgatgcacc agaagacatc5160accgataccc tggagaggat cctctctatc caggctcaag tatgggtcac agtagcaaaa 5220gccatgactg cgtatgagac tgcagatgag tcggaaacaa ggcgaatcaa taagtatatg 5280cagcaaggca gggtccaaaa gaaatacatc ctctaccccg tatgcaggag cacaatccaa 5340ctcacgatca gacagtctct tgcagtccgc atctttttgg ttagegaget caagagaggc5400cgcaacacgg caggtggtac ctctacttat tataacctgg taggggacgt agactcatac5460atcaggaata ccgggcttac tgcattcttc ttgacactca agtacggaat caacaccaag 5520acatcagccc ttgcacttag tagcctctca ggcgacatcc agaagatgaa gcagctcatg 5580cgtttgtatc ggatgaaagg agataatgcg ccgtacatga cattacttgg tgatagtgac5640cagatgagct ttgcgcctgc cgagtatgca caactttact cctttgccat gggtatggca 5700tcagtcctag ataaaggtac tgggaaatac caatttgcca gggactttat gagcacatca 5760ttctggagac ttggagtaga gtacgctcag gctcagggaa gtagcattaa cgaggatatg 5820gctgccgagc taaagctaac cccagcagca aggaggggcc tggcagctgc tgcccaacgg 5880gtctccgagg agaccagcag catagacatg cctactcaac aagtcggagt cctcactggg 5940cttagcgagg gggggtccca agctctacaa ggcggatcga atagatcgca agggcaacca 6000gaagccgggg atggggagac ccaattcctg gatctgatga gagcggtagc aaatagcatg 6060agggaggcgc caaactctgc acagggcact ccccaatcgg ggcctccccc aactcctggg 6120ccatcccaag ataacgacac cgactggggg tattgataaa caaaacccag cctgcttcca 6180caaaaacatc ccaatgccct cacccgtagt cgacccctcg atttgcggct ctatatgacc6240acaccctcaa acaaacatcc ccctctttcc tccctccccc tgctgtacaa ctccgcacgc6300cctagatacc acaggcacaa tgcggctcac taacaatcaa aacagagccg agggaattag 6360aaaaaagtac gggtagaaga gggatattca gagatcaggg caagtctccc gagtctctgc6420tctctcctct acctgataga ccaggacaaa catggccacc tttacggatg cagagatcga 6480cgagctattt gagacaagtg gaactgtcat tgacaacata attacagccc agggtaaacc6540agcagagact gttggaagga gtgcaatccc acaaggcaag accaaggtgc tgagcgcagc6600atgggagaag catgggagca tccagccacc ggccagtcaa gacaaccccg atcgacagga 6660cagatctgac aaacaaccat ccacacccga gcaaacgacc ccgcatgaca gcccgccggc6720cacatccgcc gaccagcccc ccacccaggc cacagacgaa gccgtcgaca cacagctcag 6780gaccggagca agcaactctc tgctgttgat gcttgacaag ctcagcaata aatcgtccaa 6840tgctaaaaag ggcccatggt cgagccccca agaggggaat caccaacgtc cgactcaaca 6900gcaggggagt caacccagtc gcggaaacag tcaggaaaga ccgcagaacc aagtcaaggc6960cgcccctgga aaccagggca cagacgtgaa cacagcatat catggacaat gggaggagtc7020acaactatca gctggtgcaa cccctcatgc tctccgatca aggcagagcc aagacaatac7080[1389]ccttgtatct gcggatcatg tccagccacc tgtagacttt gtgcaagcga tgatgtctat7140 [1390]gatggaggcg atatcacaga gagtaagtaa ggttgactat cagctagatc ttgtcttgaa 7200acagacatcc tccatcccta tgatgcggtc cgaaatccaa cagctgaaaa catctgttgc7260agtcatggaa gccaacttgg gaatgatgaa gattctggat cccggttgtg ccaacatttc7320atctctgagt gatctacggg cagttgcccg atctcacccg gttttagttt caggccctgg 7380agacccctct ccctatgtga cacaaggagg cgaaatggca cttaataaac tttcgcaacc7440agtgccacat ccatctgaat tgattaaacc cgccactgca tgcgggcctg atataggagt7500ggaaaaggac actgtccgtg cattgatcat gtcacgccca atgcacccga gttcttcagc7560caagctccta agcaagttag atgcagccgg gtcgatcgag gaaatcagga aaatcaagcg 7620ccttgctcta aatggctaat tactactgcc acacgcagcg ggtccctgtc cactcggcat7680cacacggaat ctgcaccgag ttcccccccg cagacccaag gtccaactct gtttaaactt7740agaaaaaata cgggtagaag tgccaccatg aattatacaa gctatatctt agcatttcag 7800ctttgcgtga ttttgtgttc ttctggctgt aactgtcagg ccatgttttt taaagaaata 7860gaaaacctaa aggaatattt taatgcaagt aatccagatg tatcggacgg tgggtctctt7920ttcgtagata ttttgaagaa atggagagag gagagtgaca aaacaatcat tcagagccaa 7980attgtctctt tctacttgaa actgtttgac aactttaaag ataaccagat cattcaaagg 8040agcatggata ccatcaagga agacatgctt ggcaagttct taaatagcag caccagtaag 8100agggaggact tccttaagct gattcaaatt cctgtgaacg atctgcaggt ccagcgcaag 8160gcgataaatg aactcatcaa agtgatgaat gatctctcac caagatccaa cctaaggaag 8220cggaaaagga gtcagaatct gtttcgaggc cgcagagcat cgaaataagt ttaaacccaa 8280gcggcaatcc tctctcgctt cctcagcccc actgaatgga cgcgtaaccg taattaatct8340agctacattt aagattaaga aaaaatacgg gtagaattgg agtgccccaa ttgtgccaag 8400atggactcat ctaggacaat tgggctgtac tttgattctg cccattcttc tagcaacctg 8460ttagcatttc cgatcgtcct acaaggcaca ggagatggga agaagcaaat cgccccgcaa 8520tataggatcc agcgccttga cttgtggact gatagtaagg aggactcagt attcatcacc8580acctatggat tcatctttca agttgggaat gaagaagcca ctgtcggcat gatcgatgat8640aaacccaagc gcgagttact ttccgctgcg atgctctgcc taggaagcgt cccaaatacc8700ggagacctta ttgagctggc aagggcctgt ctcactatga tagtcacatg caagaagagt8760gcaactaata ctgagagaat ggttttctca gtagtgcagg caccccaagt gctgcaaagc8820tgtagggttg tggcaaacaa atactcatca gtgaatgcag tcaagcacgt gaaagcgcca 8880gagaagattc ccgggagtgg aaccctagaa tataaggtga actttgtctc cttgactgtg 8940gtaccgaaga aggatgtcta caagatccca gctgcagtat tgaaggtttc tggctcgagt9000ctgtacaatc ttgcgctcaa tgtcactatt aatgtggagg ttgacccgag gagtcctttg 9060gttaaatctc tgtctaagtc tgacagcgga tactatgcta acctcttctt gcatattgga 9120cttatgacca ccgtagatag gaaggggaag aaagtgacat ttgacaagct ggaaaagaaa 9180ataaggagcc ttgatctatc tgtcgggctc agtgatgtgc tcgggccttc cgtgttggta9240aaagcaagag gtgcacggac taagcttttg gcacctttct tctctagcag tgggacagcc9300tgctatccca tagcaaatgc ttctcctcag gtggccaaga tactctggag tcaaaccgcg9360tgcctgcgga gcgttaaaat cattatccaa gcaggtaccc aacgcgctgt cgcagtgacc9420[1428]gccgaccacg aggttacctc tactaagctg gagaaggggc acacccttgc caaatacaat9480ccttttaaga aataagctgc gtctctgaga ttgcgctccg cccactcacc cagatcatca9540tgacacaaaa aactaatctg tcttgattat ttacagttag tttacctgcc tatcaagtta9600gaaaaaacac gggtagaaga ttctggatcc cggttggcgc cctccaggtg caagatgggc9660tccagacctt ctaccaagaa cccagcacct atgatgctga ctatccgggt tgcgctggta9720ctgagttgca tctgtccggc aaactccatt gatggcaggc ctcttgcagc tgcaggaatt9780 [1434]gtggttacag gagacaaagc cgtcaacata tacacctcat cccagacagg atcaatcata9840gttaagctcc tcccgaatct gcccaaggat aaggaggcat gtgcgaaagc ccccttggat9900gcatacaaca ggacattgac cactttgctc accccccttg gtgactctat ccgtaggata9960caagagtctg tgactacatc tggagggggg agacaggggc gccttatagg cgccattatt10020ggcggtgtgg ctcttggggt tgcaactgcc gcacaaataa cagcggccgc agctctgata 10080caagccaaac aaaatgctgc caacatcctc cgacttaaag agagcattgc cgcaaccaat10140gaggctgtgc atgaggtcac tgacggatta tcgcaactag cagtggcagt tgggaagatg 10200cagcagtttg ttaatgacca acttaataaa acagctcagg aattagactg catcaaaatt10260gcacagcaag ttggtgtaga gctcaacctg tacctaaccg aattgactac agtattcgga 10320ccacaaatca cttcacctgc tttaaacaag ctgactattc aggcacttta caatctagct10380ggtggaaata tggattactt attgactaag ttaggtgtag ggaacaatca actcagctca 10440ttaatcggta gcggcttaat caccggtaac cctattctat acgactcaca gactcaactc10500ttgggtatac gggtaactct accttcagtc gggaacctaa ataatatgcg tgccacctac10560ttggaaacct tatccgtaag cacaaccagg ggatttgcct cggcacttgt ccccaaagtg 10620gtgacacagg tcggttctgt gatagaagaa cttgacacct catactgtat agaaactgac10680ttagatttat attgtacaag aatagtaacg ttccctatgt cccctggtat ttattcctgc10740ttgagcggca atacgtcggc ctgtatgtac tcaaagaccg aaggcgcact tactacacca 10800tacatgacta tcaaaggttc agtcatcgcc aactgcaaga tgacaacatg tagatgtgta 10860aaccccccgg gtatcatatc gcaaaactat ggagaagccg tgtctctaat agataaacaa 10920tcatgcaatg ttttatcctt aggcgggata actttaaggc tcagtgggga attcgatgta 10980acttatcaga agaatatctc aatacaagat tctcaagtaa taataacagg caatcttgat11040atctcaactg agcttgggaa tgtcaacaac tcgatcagta atgctttgaa taagttagag 11100gaaagcaaca gaaaactaga caaagtcaat gtcaaactga ctagcacatc tgctctcatt11160acctatatcg ttttgactat catatctctt gtttttggta tacttagcct gattctagca 11220tgctacctaa tgtacaagca aaaggcgcaa caaaagacct tattatggct tgggaataat11280actctagacc agatgagagc cactacaaaa atgtgaacac agatgaggaa cgaaggtttc11340cctaatagta atttgtgtga aagttctggt agtctgtcag ttcagagggt taagaaaaaa 11400ctaccggttg tagatgacca aaggacgata tacgggtaga acggtaagag aggccgcccc11460tcaattgcga gccaggcttc acaacctccg ttctaccgct tcaccgacaa cggtcctcaa 11520tcatggaccg cgccgttagc caagttgcgt tagagaatga tgaaagagag gcaaaaaata 11580catggcgctt gatattccgg attgcaatct tattcttaac agtagtgacc ttggctatat11640ctgtagcctc ccttttatat agcatggggg ctagcacacc tagcgatctt gtaggcatac11700cgactaggaa ttccagggca gaagaaaaga ttacatctac acttggttcc aatcaagatg 11760[1467]tagtagatag gatatataag caagtggccc ttgagtctcc gttggcattg ttaaaaactg11820agaccacaat tatgaacgca ataacatctc tctcttatca gattaatgga gctgcaaaca11880acagtgggtg gggggcactt atccatgacc cagattatat aggggggata ggcaaagaac11940tcattgtaga tgatgctagt gatgtcacat cattctatcc ctctgcattt caagaacatc12000tgaattttat cccggcgcct actacaggat caggttgcac tcgaataccc tcatttgaca12060tgagtgctac ccattactgc tacacccata atgtaatatt gtctggatgc agagatcact12120cacattcata tcagtattta gcacttggtg tgctccggac atctgcaaca gggagggtat12180tcttttctac tctgcgttcc atcaacctgg acgacaccca aaatcggaag tcttgcagtg12240tgagtgcaac tcccctgggt tgtgatatgc tgtgctcgaa agtcacggag acagaggaag12300aagattataa ctcagctgtc cctacgcgga tggtacatgg gaggttaggg ttcgacggcc12360agtaccacga aaaggaccta gatgtcacaa cattattcgg ggactgggtg gccaactacc12420caggagtagg gggtggatct tttattgaca gccgcgtatg gttctcagtc tacggagggt12480taaaacccaa ttcacccagt gacactgtac aggaagggaa atatgtgata tacaagcgat12540acaatgacac atgcccagat gagcaagact accagattcg aatggccagg tcttcgtata12600agcctggacg gtttggtggg aaacgcatac agcaggctat cttatctatc aaggtgtcaa12660catccttagg cgaagacccg gtactgactg taccgcccaa cacagtcaca ctcatggggg12720ccgaaggcag aattctcaca gtagggacat ctcatttctt gtatcaacga gggtcatcat12780acttctctcc cgcgttatta tatcctatga cagtcagcaa caaaacagcc actcttcata12840gtccttatac attcaatgcc ttcactcggc caggtagtat cccttgccag gcttcagcaa12900gatgccccaa cccgtgtgtt actggagtct atacagatcc acatccccta atcttctata12960gaaaccacac cttgcgaggg gtattcggga caatgcttga tggtgtacaa gcaagactta13020accctgcgtc tgcagtattc gatagcacat cccgcagtcg cattactcga gtgagttcaa13080gcagtaccaa agcagcatac acaacatcaa cttgttttaa agtggtcaag actaataaga13140cctattgtct cagcattgct gaaatatcta atactctctt cggagaattc agaatcgtcc13200cgttactagt tgagatcctc aaagatgacg gggttagaga agccaggtct ggctagttga13260gtcaattata aaggagttgg aaagatggca ttgtatcacc tatcttctgc gacatcaaga13320atcaaaccga atgccggcgc gtgctcgaat tccatgttgc cagttgacca caatcagcca 13380gtgctcatgc gatcagatta agccttgtca atagtctctt gattaagaaa aaatgtaagt13440ggcaatgaga tacaaggcaa aacagctcat ggtaaataat acgggtagaa catggcgagc13500tccggtcctg aaagggcaga gcatcagatt atcctaccag agtcacacct gtcttcacca13560ttggtcaagc acaaactact ctattactgg aaattaactg ggctaccgct tcctgatgaa13620tgtgacttcg accacctcat tctcagccga caatggaaaa aaatacttga atcggcctct13680cctgatactg agagaatgat aaaactcgga agggcagtac accaaactct taaccacaat13740tccagaataa ccggagtgct ccaccccagg tgtttagaag aactggctaa tattgaggtc13800ccagattcaa ccaacaaatt tcggaagatt gagaagaaga tccaaattca caacacgaga13860tatggagaac tgttcacaag gctgtgtacg catatagaga agaaactgct ggggtcatct13920tggtctaaca atgtcccccg gtcagaggag ttcagcagca tccgtacgga cccggcattc13980tggtttcact caaaatggtc cacagccaag tttgcatggc tccatataaa acagatccag14040aggcatctga tggtggcagc taggacaagg tctgcggcca acaaattggt gatgctaacc14100[1506]cataaggtag gccaagtctt tgtcactcct gaacttgtcg ttgtgacgca tacgaatgag 14160aacaagttca catgtcttac ccaggaactt gtattgatgt atgcagatat gatggagggc14220agagatatgg tcaacataat atcaaccacg gcggtgcatc tcagaagctt atcagagaaa 14280 [1509]attgatgaca ttttgcggtt aatagacgct ctggcaaaag acttgggtaa tcaagtctac14340gatgttgtat cactaatgga gggatttgca tacggagctg tccagctact cgagccgtca 14400ggtacatttg caggagattt cttcgcattc aacctgcagg agcttaaaga cattctaatt14460ggcctcctcc ccaatgatat agcagaatcc gtgactcatg caatcgctac tgtattctct14520ggtttagaac agaatcaagc agctgagatg ttgtgtctgt tgcgtctgtg gggtcaccca 14580ctgcttgagt cccgtattgc cgcaaaggca gtcaggagcc aaatgtgcgc accgaaaatg 14640gtagactttg atatgatcct tcaggtactg tctttcttca agggaacaat catcaacggg 14700tacagaaaga agaatgcagg tgtgtggccg cgagtcaaag tggatacaat atatgggaag 14760gtcattgggc aactacatgc agattcagca gagatttcac acgatatcat gttgagagag 14820tataagagtt tatctgcact tgaatttgag ccatgtatag aatatgaccc tgtcaccaac14880ctgagcatgt tcctaaaaga caaggcaatc gcacacccca acgataattg gcttgcctcg 14940tttaggcgga accttctctc cgaagaccag aagaaacatg taaaagaagc aacttcgact15000aatcgcctct tgatagagtt tttagagtca aatgattttg atccatataa agagatggaa 15060tatctgacga cccttgagta ccttagagat gacaatgtgg cagtatcata ctcgctcaag 15120gagaaggaag tgaaagttaa tggacggatc ttcgctaagc tgacaaagaa gttaaggaac15180tgtcaggtga tggcggaagg gatcctagcc gatcagattg cacctttctt tcagggaaat15240ggagtcattc aggatagcat atccttgacc aagagtatgc tagcgatgag tcaactgtct15300tttaacagca ataagaaacg tatcactgac tgtaaagaaa gagtatcttc aaaccgcaat15360catgatccga aaagcaagaa ccgtcggaga gttgcaacct tcataacaac tgacctgcaa 15420aagtactgtc ttaattggag atatcagaca atcaaattgt tcgctcatgc catcaatcag 15480ttgatgggcc tacctcactt cttcgaatgg attcacctaa gactgatgga cactacgatg 15540ttcgtaggag accctttcaa tcctccaagt gaccctactg actgtgacct ctcaagagtc15600cctaatgatg acatatatat tgtcagtgcc agagggggta tcgaaggatt atgccagaag 15660ctatggacaa tgatctcaat tgctgcaatc caacttgctg cagctagatc gcattgtcgt15720gttgcctgta tggtacaggg tgataatcaa gtaatagcag taacgagaga ggtaagatca 15780gacgactctc cggagatggt gttgacacag ttgcatcaag ccagtgataa tttcttcaag 15840gaattaattc atgtcaatca tttgattggc cataatttga aggatcgtga aaccatcagg 15900tcagacacat tcttcatata cagcaaacga atcttcaaag atggagcaat cctcagtcaa 15960gtcctcaaaa attcatctaa attagtgcta gtgtcaggtg atctcagtga aaacaccgta 16020atgtcctgtg ccaacattgc ctctactgta gcacggctat gcgagaacgg gcttcccaaa 16080gacttctgtt actatttaaa ctatataatg agttgtgtgc agacatactt tgactctgag 16140ttctccatca ccaacaattc gcaccccgat cttaatcagt cgtggattga ggacatctct16200tttgtgcact catatgttct gactcctgcc caattagggg gactgagtaa ccttcaatac16260tcaaggctct acactagaaa tatcggtgac ccggggacta ctgcttttgc agagatccag 16320cgactagaag cagtgggatt actgagtcct aacattatga ctaatatctt aactaggccg 16380cctgggaatg gagattgggc cagtctgtgc aacgacccat actctttcaa ttttgagact16440[1545]gttgcaagcc caaatattgt tcttaagaaa catacgcgaa gagtcctatt tgaaacttgt16500tcaaatccct tattgtctgg agtgcacaca gaggataatg aggcagaaga gaaggcattg16560gctgaattct tgcttaatca agaggtgatt catccccgcg ttgcgcatgc catcatggag16620gcaagctctg taggtaggag aaagcaaatt caagggcttg ttgacacaac aaacaccgta16680attaagattg cgcttactag gaggccatta ggcataaaga ggctgatgcg gatagtcaat16740 [1550]tattctagca tgcatgcaat gctgtttaga gacgatgttt tttcctccag tagatccaac16800caccccttag tctcttctaa tatgtgttct ctgacactgg cagactatgc acggaataga16860agctggtcac ctttgacggg aggcaggaaa atactgggtg tatctaatcc tgatacgata16920gaactcgtag agggtgagat tcttagtgta agcggagggt gtacaagatg tgacagcgga16980gatgaacaat ttacttggtt ccatcttcca agcaatatag aattgaccga tgacaccagc17040aagaatcctc cgatgagggt accatatctc gggtcgaaga cacaggagag gagagctgcc17100tcacttgcaa aaatagctca tatgtcgcca catgtaaagg ctgccctaag ggcatcatcc17160gtgttgatct gggcttatgg ggataatgac gtaaattgga ctgctgctct tacgattgca17220aaatctcggt gtaatgtaaa cttagagtat cttcggttac tgtccccttt acccacggct17280gggaatcttc aacatagact agatgatggt ataactcaga tgacattcac ccctgcatct17340ctctacaggt gtcaccttac attcacatat ccaatgattc tcaaaggctg ttcactgaag17400aaggagtcaa agaggggaat gtggtttacc aacagagtca tgctcttggg tttatctcta17460atcgaatcga tatttccaat gacagcaacc aggacatatg atgagatcac actgcaccta17520catagtaaat ttagttgctg tatcagagaa gcacctgttg cggttccttt cgagctactt17580ggggtggtac cggaactgag gacagtgacc tcaaataagt ttatgtatga tcctagccct17640ggatcggagg gagactttgc gagacttgac ttagctatct tcaagagtta tgagcttaat17700ctggagtcat atcccacgat agagctaatg aacattcttt caatatccag cgggaagttg17760attggccagt ctgtggtttc ttatgatgaa gatacctcca taaagaatga cgccataata17820gtgtacgaca atacccgaaa ttggatcagt gaagctcaga attcagatgt ggtccgccta17880tttgaatatg cagcacttga agtgctcctc aactgttctt accaactcta ttacctgaga17940gtaagaggcc tagacaatat tgtcttatat atgggtgatt tatacaagaa tatgccagga18000attctacttt ccaacattgc agctacaata tctcatcccg tcattcattc aaggttacat18060gcagtgggcc tggtcaacca tgacggatca caccaacttg cagatacgga ttttatcgaa18120atgtctgcaa aactattagt atcttgcacc cgacgtgtga tctccggctt atattcagga18180aataagtatg atctgctgtt cccatctgtc ttagatgata acctgaatga gaagatgctt18240cagctgatat cccggttatg ctgtctgtac acggtactct ttgctacaac aagagaaatc18300ccgaaaataa gaggcttaac tacaaaagag aaatgttcaa tacccactga gtatttactg18360tcggatgctg tgaaaccatt acttagcccc gatcaagtga gctctatcat gtctcctaac18420ataattacat tcccagctaa tccgtactac atgtctcgga agagcctcaa tttgatcagg18480gaaagggagg acagggatac tatcctggtg ttgttgttcc cccaagagcc attattagag18540ttcccttctg tgcaagatat tggtgctcga gtgaaagatc cattcacccg acaacctgcg18600gcatttttgc aagagttaga tttgagtgct ccagcaaggt atgacgcatt cacacttagt18660cagattcatc ctgaactcac atctccaaat ccggaggaag actacttagt acgatacttg18720ttcagaggga tagggactgc atcttcctct tggtataagg catctcatct cctttctgta18780[1584]cccgaggtaa gatgtgcaag acacgggaac tccttatact tagctgaagg gagcggagcc18840atcatgagtc ttctcgaact gcatgtacca catgaaacta tctattacaa tacgctcttt18900tcaaatgaga tgaacccccc gcaatgacat ttcgggccga ccccaactca gtttttgaat18960tcggttgttt ataggaatct acaggcggag gtaacatgca aagatggatt tgtccaagag19020 [1588]ttccgtccat tatggagaga aaatacagag gaaagtgacc tgacctcaga taaagcagtg19080gggtatatta catctgcagt gccctacaga tctgtatcat tgctgcattg tgacattgaa19140actcctccag ggtccaatca aagcttacta gatcaactag ctatcaattt atctctgatt19200gccatgcatt ctgtaaggga gggcggggta gtaatcatca aagtgttgta tgcaatggga19260tactactttc atctactcat gaacttgttt gctccgtgtt ccacaaaagg atatattctc19320tctaatggtt atgcatgtcg aggagatatg gagtgttacc tggtatttgt catgggttac19380ctgggcgggc ctacatttgt acatgaggtg gtgaggatgg caaaaactct ggtgcagcgg19440cacggtacgc tcttgtctaa atcagatgag atcacactga ccaggttatt cacctcacag19500cggcagcgtg tgacagacat cctatccagt cctttaccaa gattaataaa gtacttgagg19560aagaatattg acactgcgct gattgaagcc gggggacagc ccgtccgtcc attctgtgcg19620gagagtctgg tgagcacgct agcgaacata actcagataa cccagattat cgctagtcac19680attgacacag ttatccggtc tgtgatatat atggaagctg agggtgatct cgctgacaca19740gtatttctat ttacccctta caatctctct actgacggga aaaagaggac atcacttaaa19800cagtgcacga gacagatcct agaggttaca atactaggtc ttagagtcga aaatctcaat19860aaaataggcg atataatcag tctagagctt aaaggcatga tctccatgga ggaccttatc19920ccactaagga catacttgaa gcatagtacc tgccctaaat atttgaaggc tgtcctaggt19980attaccaaac tcaaagaaat gtttacagac acttctgtac tgtacttgac tcgtgctcaa20040caaaaattct acatgaaaac tataggcaat gcagtcaacg gatattacag taactgtgac20100tcttaacgaa aatcacatat taataggctc cttttttggc caattgtatt cttgttgatt20160taatcatatt atgttagaaa aaagttgaac cctgactcct taggactcga attcgaactc20220aaataaatgt cttaaaaaaa ggttgcgcac aattattctt gagtgtagtc tcgtcattca20280ccaaatcttt gtttggtggg tcggcatggc atctccacct cctcgcggtc cgacctgggc20340atccgaagga ggacgtcgtc cactcggatg gctaagggag gggcccccgc ggggctgcta20400acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac20460cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg20520gatcgagacc tcgatactag tgcggtggag ctgaattc20558
权利要求
一种表达编码狗干扰素的基因的重组新城疫弱毒疫苗株。
2.根据权利要求1的重组新城疫弱毒疫苗株，其为重组新城疫LaSota弱毒疫苗株。
3.根据权利要求1或2的重组新城疫弱毒疫苗株，其中所述狗干扰素是狗α、β或 Y-干扰素，优选所述狗α、β和Y-干扰素的氨基酸序列分别如SEQ ID No. 1、SEQ ID No. 2和SEQ ID No. 3所示，更优选编码所述狗α、β和γ -干扰素的基因分别如SEQ ID No. 4、SEQ ID No. 5 和 SEQ ID No. 6 所示。
4.根据权利要求1或2的重组新城疫弱毒疫苗株，其为rL-Canineα ,rL-Canine β、或 rL-Canineγ。
5.根据权利要求1-4中任何一项的重组新城疫弱毒疫苗株在制备狗干扰素中的应用。
6.一种生产权利要求1-4中任何一项的重组新城疫弱毒疫苗株的方法，该方法包括(1)构建转录质粒，该转录质粒包括其中插入编码狗干扰素的基因的所述新城疫弱毒 疫苗株的基因组cDNA序列；(2)构建一个或多个转录辅助质粒，该转录辅助质粒包括编码所述新城疫弱毒疫苗株 的核蛋白(NP)的cDNA序列、编码所述新城疫弱毒疫苗株的磷酸蛋白(P)的cDNA序列、和 编码所述新城疫弱毒疫苗株的大聚合酶蛋白(L)的cDNA序列；(3)将所述转录质粒和转录辅助质粒共转染所述新城疫弱毒疫苗株复制许可的宿主细 胞，培养转染后的宿主细胞；(4)收获上清液，过滤后继续敏感细胞传代或接种鸡胚尿囊腔救获重组病毒株。
7.根据权利要求6的方法，其中所述重组新城疫弱毒疫苗株是重组新城疫LaSota弱毒 疫苗株。
8.根据权利要求6或7的方法，其中所述转录质粒是pBRN-FL-CalFNa、 pBRN-FL-CalFN β、或 pBRN-FL-CalFN γ。
9.根据权利要求6或7的方法，其中所述转录辅助质粒是质粒pBSNP、pBSP或pBSL。
10.根据权利要求6-9中任何一项的方法，其中所述宿主细胞是BHK-21。
11.根据权利要求6-10中任何一项的方法制备的重组新城疫弱毒疫苗株，优选为 rL-Canine α、rL-Canine β、或 rL-Canine γ。
12.—种生产狗干扰素的方法，该方法包括1)将根据权利要求1-4中任何一项的重组新城疫弱毒疫苗株接种敏感细胞或接种鸡 胚尿囊腔；2)收获细胞上清液或鸡胚尿囊液。
13.重组新城疫LaSota弱毒疫苗株rL-Canineα的SPF鸡胚尿囊液，其保藏号为CGMCC No.2622。
14.重组新城疫LaSota弱毒疫苗株rL-Canineβ的SPF鸡胚尿囊液，其保藏号为CGMCC No.2623。
15.重组新城疫LaSota弱毒疫苗株rL-Canineγ的SPF鸡胚尿囊液，其保藏号为CGMCC No.2624。
全文摘要
本发明涉及表达狗干扰素(α、β或γ-干扰素)的重组新城疫弱毒疫苗株，具体地，涉及表达狗干扰素(α、β或γ-干扰素)的重组新城疫LaSota弱毒疫苗株。本发明还公开了制备所述重组新城疫弱毒疫苗株的方法和该重组新城疫弱毒疫苗株在制备狗α、β或γ-干扰素中的应用。
文档编号C12N15/85GK101870963SQ20091008845
公开日2010年10月27日 申请日期2009年7月1日 优先权日2009年7月1日
发明者步志高, 葛金英 申请人:中国农业科学院哈尔滨兽医研究所