CN107201346B - 3b蛋白优势表位缺失的口蹄疫标记疫苗株及其构建方法和应用 - Google Patents

3b蛋白优势表位缺失的口蹄疫标记疫苗株及其构建方法和应用 Download PDF

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CN107201346B
CN107201346B CN201710175378.9A CN201710175378A CN107201346B CN 107201346 B CN107201346 B CN 107201346B CN 201710175378 A CN201710175378 A CN 201710175378A CN 107201346 B CN107201346 B CN 107201346B
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李平花
刘在新
卢曾军
寻广谨
孙普
白兴文
包慧芳
曹轶梅
付元芳
陈应理
李冬
马雪青
张婧
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Lanzhou Veterinary Research Institute of CAAS
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Abstract

本发明提供一种3B蛋白优势表位缺失的口蹄疫标记疫苗株及其构建方法和应用，该标记疫苗株3B1和3B2非结构蛋白编码的氨基酸序列如序列表SEQ ID No.8所示。本发明构建的疫苗株具有3B1和3B2非结构蛋白氨基酸的突变修饰（4AGP6‑4TAA6），其中3B1和3B2蛋白氨基酸的突变导致重组病毒完全丧失了与识别3B1和3B2优势表位PYxGP单克隆抗体3B4B1的结合能力，并且经标记的病毒具有与亲本病毒相似的复制能力。因而本发明构建的口蹄疫标记疫苗株可以用来发展具有区分自然感染与疫苗免疫的口蹄疫标记疫苗，为我国口蹄疫的控制、净化和无疫区的建设提供一种可靠的技术支撑。

Description

3B蛋白优势表位缺失的口蹄疫标记疫苗株及其构建方法和 应用
技术领域
本发明属于基因工程技术领域，具体涉及3B蛋白优势表位缺失的口蹄疫标记疫苗株及其构建方法和应用。
背景技术
口蹄疫(Foot-and-Mouth Disease,FMD)是猪、牛、羊等主要家畜和野生偶蹄动物感染的一种急性、热性、高度接触性传染病。该病传播迅速、发病率高，危害巨大，因此国际兽疫局(OIE)将其列为必报疫病之首，我国规定为一类动物传染病。该病的暴发和流行使家畜的生产能力下降，活畜及畜产品贸易停滞，经济损失巨大；同时给公共卫生和国家声誉造成严重的负面影响，因此世界各国都十分重视对该病的防控。
我国是口蹄疫流行较为严重的国家，口蹄疫的暴发常年不断，给我国畜牧业发展造成了巨大的危害。传统灭活疫苗的免疫接种是我国预防和控制口蹄疫的最有效手段，但常规灭活疫苗不能够区分疫苗免疫和野毒感染动物，致使我国口蹄疫不能有效的控制和净化。近年来，随着RNA病毒反向遗传操作和基因重组等技术的不断成熟以及伪狂犬病毒、猪瘟病毒、猪繁殖与呼吸障碍病毒和鸡马立克病病毒等优势表位缺失标记病毒的成功研制为口蹄疫标记疫苗的发展提供了新的思路。近年来，研究者通过缺失口蹄疫病毒结构蛋白VP1G-H环，缺失前导蛋白L和3B1或L和3D蛋白的修饰，缺失3A优势表位等构建口蹄疫标记病毒，发展能够鉴别诊断的口蹄疫疫苗。研究表明这些疫苗可以满足口蹄疫的免疫预防和鉴别诊断的目的。但是，口蹄疫病毒结构蛋白VP1的G-H环是诱导机体产生中和抗体的主要免疫基因，缺失会影响疫苗的免疫原性；弱毒疫苗存在毒力返祖的风险，非结构蛋白基因的缺失会影响病毒的复制能力，因此本领域仍然需要创新发展更好的口蹄疫标记病毒疫苗候选株，为口蹄疫的有效预防、控制和净化以及我国无疫区的建设提供有效的技术支撑。
发明内容
为了解决现有技术中存在的缺陷，本发明提供了3B蛋白优势表位缺失的口蹄疫标记疫苗株及其构建方法和应用。
本申请人丙氨酸扫描的研究表明，实验室筛选的口蹄疫病毒抗3B非结构蛋白(口蹄疫病毒非结构蛋白3B由3B1、3B2和3B3三个蛋白组成)的单克隆抗体3B4B1识别位于3B1和3B2上的优势表位PYxGP。鉴于此，本申请人以O型口蹄疫疫苗毒株O/ZK/93-08全长感染性克隆为骨架，利用反向遗传操作技术，突变修饰口蹄疫非结构蛋白3B1和3B2优势表位的关键氨基酸，构建了一株3B1和3B2第4～6位氨基酸突变(4AGP6-4TAA6)的重组病毒，该毒株用免疫印迹(Western Blot，WB)和免疫荧光(Indirect Immunofluorescence，IIF)分析，均不与抗口蹄疫病毒3B单抗3B4B1发生反应，而且重组病毒与亲本病毒具有相似的复制能力。因此本发明构建的口蹄疫标记病毒可以用来发展具有鉴别诊断的口蹄疫标记疫苗，用于口蹄疫的预防和控制。
口蹄疫病毒存在7个血清型：O、A、Asia1、C、STAⅠ、SATⅡ和SATIII型，这7个血清型的3B蛋白编码的氨基酸非常保守，因此，本发明的构建方法不仅适用于O型口蹄疫病毒的改造，还适用于其他血清型口蹄疫病毒的改造，任何血清型口蹄疫病毒在同等位置的替换均包含在本发明的保护范围之内。
本发明提供3B蛋白优势表位缺失的口蹄疫标记疫苗株，其3B1和3B2非结构蛋白编码的氨基酸序列如序列表SEQ ID No.8所示。
作为优选，所述口蹄疫标记疫苗株3B1和3B2非结构蛋白的核苷酸序列如序列表SEQ ID No.7所示。
作为优选，所述口蹄疫标记疫苗株编码蛋白的氨基酸序列如序列表SEQ ID No.6所示。
作为优选，所述口蹄疫标记疫苗株的基因组RNA对应的cDNA核苷酸序列如序列表SEQ ID No.5所示。
本发明还提供3B蛋白优势表位缺失的口蹄疫标记疫苗株的构建方法，步骤如下：
合成含口蹄疫疫苗毒株非结构蛋白3B1和3B2第4～6位氨基酸修饰(4AGP6-4TAA6)的Z4片段，将该片段克隆在载体中，得到重组质粒，将重组质粒用内切酶消化，回收修饰的Z4片段，将其插入至用同样内切酶消化的口蹄疫病毒毒株的全长感染性克隆中，得到重组全长质粒；用内切酶将重组全长质粒线化后，转染细胞，拯救获得的病毒，即为3B蛋白优势表位缺失的口蹄疫标记疫苗株。
作为优选：所述含口蹄疫疫苗毒株非结构蛋白3B1和3B2蛋白第4～6位氨基酸修饰的氨基酸序列如SEQ ID No.8所示。
作为优选，对应的核苷酸序列如SEQ ID No.7所示。
作为优选：所述口蹄疫病毒毒株为O型口蹄疫病毒毒株。
作为优选，所述口蹄疫病毒毒株的基因组RNA对应的cDNA的核苷酸序列如SEQ IDNo.5所示。
本发明构建的口蹄疫标记疫苗株不与识别口蹄疫病毒3B1和3B2优势表位PYxGP的抗口蹄疫的3B单抗3B4B1反应。
本发明还提供上述口蹄疫标记疫苗株在制备口蹄疫标记疫苗中的应用。
作为优选，所述口蹄疫标记疫苗为O型口蹄疫标记疫苗。
本发明还提供一种疫苗，其活性成分为权利要求1-4任一所述的口蹄疫标记疫苗株。在制备疫苗时，以现有技术中的方法进行制备即可。
本发明构建的口蹄疫病毒具有3B1和3B2非结构蛋白第4～6位氨基酸的突变修饰(4AGP6-4TAA6)，经标记的病毒具有与亲本病毒相似的复制能力。其中3B1和3B2蛋白的突变导致重组病毒完全丧失了与识别3B1和3B2优势表位PYxGP的单克隆抗体3B4B1的结合能力。因而本发明构建的标记病毒可以用来发展鉴别诊断的口蹄疫标记疫苗，为我国口蹄疫的控制、净化和无疫区的建设提供一种可靠的技术支撑。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。在附图中：
图1为口蹄疫病毒3B1和3B2蛋白氨基酸突变的示意图。
图2为重组质粒pOFS/3BM的酶切鉴定图；其中，左侧泳道为pOFS/3BM质粒Bgl II/Not I酶切结果；右侧泳道为DL12000DNA marker。
图3为重组质粒pOFS/3BM 3B蛋白的测序峰图；其中，A:pOFS/3BM重组质粒3B1的部分测序峰图；B：pOFS/3BM重组质粒3B2的部分测序峰图；C:pOFS质粒3B1的部分测序峰图；D:pOFS质粒3B2的部分测序峰图。
图4为重组质粒pOFS/3BM转染BSR/T7细胞60h后引起的CPE(A：正常的BSR/T7细胞，B：出现CPE的BSR/T7细胞)。
图5为重组病毒O/rV-1/3BM与O/ZK/93-08 3B蛋白氨基酸的比对图。
图6为间接免疫荧光检测重组病毒在BHK-21上的复制。
图7为用Western blot分析重组病毒和亲本病毒的结果。
图8为P10、P15和P20代重组病毒O/rV-1/3BM与O/ZK/93-08 3B蛋白氨基酸的比对图。
图9为重组病毒O/rV-1/3BM与O/ZK/93-08病毒在不同时间的复制滴度比对图。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为市售。
实施例1
1、3B1和3B2蛋白氨基酸修饰的口蹄疫病毒全长克隆的构建
以本申请人已经构建的O型口蹄疫疫苗毒株O/ZK/93-08(口蹄疫疫苗毒株O/ZK/93-08的基因组RNA对应的cDNA核苷酸序列如SEQ ID No.1，口蹄疫疫苗毒株O/ZK/93-08蛋白编码的氨基酸序列如序列表SEQ ID No.2，其中1057～8022的核苷酸编码的病毒蛋白序列如序列表SEQ ID No.2，口蹄疫疫苗毒株O/ZK/93-08 3B1和3B2的核苷酸序列见SEQ IDNo.3，口蹄疫疫苗毒株O/ZK/93-08 3B1和3B2蛋白编码的氨基酸序列见SEQ ID No.4)的全长感染性克隆pOZKF-Z1234(见公开专利：用反向遗传操作拓展口蹄疫疫苗株抗原谱及疫苗制备方法，专利公布号CN101948811A)为骨架，构建非结构蛋白3B1和3B2第4～6位氨基酸突变的全长克隆。口蹄疫病毒3B1和3B2蛋白氨基酸突变的示意图见图1。金唯志生物科技有限公司全基因合成含口蹄疫疫苗毒株O/ZK/93-08非结构蛋白3B1和3B2第4～6位氨基酸修饰(4AGP6-4TAA6)的Z4片段(该片段被克隆在pUC57载体中，重组质粒命名为pUC-Z4/Δ3B)，将公司合成构建的pUC-Z4/Δ3B重组质粒用Bgl II和Not I内切酶消化，回收修饰的Z4片段，将其插入用同样内切酶消化的O/ZK/93-08疫苗株的全长感染性克隆pOZKF-Z1234(又命名为pOFS)中，得到重组全长质粒pOFS/3BM。重组全长质粒用Bgl II和Not I酶切鉴定，结果切出与预期相符的目的条带，见图2。全长质粒测序结果也表明，构建的重组质粒含预期的突变修饰，见图3。
2、重组病毒的拯救
BHK-21细胞购自中国兽医监察所，产品目录号为BHK-21F5620071213。
BSR/T7细胞(表达T7RNA聚合酶的BHK-21稳定细胞系)在文献《中国预防兽医学报》2016年第2期，袁红，李平花等“O型与A型口蹄疫重组标记疫苗病毒株的制备及鉴定”中公开过，公众可从中国农业科学院兰州兽医研究所获得。
用QIAGEN Plasmid Midi Kits(购自QIAGEN公司)制备质粒pOFS/3BM，用NotⅠ内切酶线化后用DNA片段回收试剂盒纯化回收作为转染模板。常规培养的单层BSR/T7细胞生长至70％～80％时用于转染。取2个灭菌的1.5ml离心管，加入250μl的Opti-MEMI无血清培养液(购自Gibco公司)，然后在其中一管中加入2.5μg的线化质粒，另一管中加入20μl的LipofectamineTM2000(购自Gibco公司)吹打混匀；室温放置5min后将两管溶液混合，室温再放置20min。与此同时，将6孔板中的BSR/T7细胞用Opti-MEMI温和冲洗两遍，20min后，将DNA-脂质体混合液缓慢地加入到6孔细胞培养板中，轻轻混匀，置含5％CO2的37℃培养箱中培养。5h后，加入1ml含10％胎牛血清的GMEM(购自Gibco公司)培养基，置5％CO2的37℃培养箱中继续培养，观察细胞出现致细胞病变(cytopathogenic effct,CPE)的情况。转染60h后转染细胞出现典型的CPE，(见图4)。转染72h后收获细胞，反复冻融2～3次后，收集病毒液，即为目标病毒，命名为O/rV-1/3BM。收集的重组病毒在BHK21细胞上连续传代增殖20代，-70℃以下保存各代病毒备用。
3、重组病毒的鉴定
3.1、RT-PCR鉴定
取转染的上清用RNAasy Mini Kit(购自QIAGEN公司)提取O/rV-1/3BM细胞毒总RNA，RT-PCR扩增重组病毒的囊括全基因的5个片段，并进行序列的测定，验证重组病毒的正确性。测序结果表明：重组病毒O/rV-1/3BM为正确的构建，含3B1和3B2预期氨基酸的突变(4AGP6-4TAA6)。重组病毒基因组RNA对应的cDNA核苷酸序列如序列表SEQ ID No.5，其中1057～8022的核苷酸编码病毒蛋白的氨基酸序列如序列表SEQ ID No.6，重组病毒3B1和3B2蛋白的核苷酸序列如序列表SEQ ID No.7，重组病毒3B1和3B2蛋白编码的氨基酸序列如序列表SEQ ID No.8)。O/ZK/93-08疫苗毒株与重组病毒3B蛋白氨基酸序列比对结果见图5所示，测序及氨基酸序列比对分析结果表明本发明成功构建了含预期3B蛋白突变的重组病毒。
3.2、间接免疫荧光((Indirect immunofluorescence,IFA))鉴定病毒蛋白
BHK-21单层细胞(六孔培养板)生长至70％时，分别接种转染的上清(即重组病毒O/rV-1/3BM)和O/ZK/93-08疫苗毒，37℃孵育6h后弃培养液，PBS洗涤2次，4％冷多聚甲醛固定20min，PBS洗三遍，0.2％Triton X-100通透10min，PBS洗三遍，1:200稀释的口蹄疫病毒抗3A(3A24)和抗3B(3B4B1)的单克隆抗体37℃孵育1小时，PBS洗三遍。1:100稀释的FITC标记的山羊抗鼠的抗体37℃孵育1小时，PBS洗三遍。最后加入0.5ug/ml DAPI(PBS配制)染色10分钟，PBS洗三遍，去除多余的DAPI，置共聚焦荧光显微镜下拍照。同时设正常细胞对照。结果表明接种转染上清的BHK细胞用3A单抗3A24作用，能看到可见的绿色荧光，而用3B单抗3B4B1作用，则看不到任何可见荧光；而接种O/ZK/93-08病毒的BHK细胞用3A和3B单抗作用，均能看到可见的绿色荧光，对照细胞用3A和3B单抗作用均看不到可见荧光。说明本发明拯救的重组病毒为正确的构建，3B1和3B2氨基酸的突变(4AGP6-4TAA6)取消了重组口蹄疫病毒O/rV-1/3BM与3B单抗3B4B1的反应能力，见图6。
文中提到的3A单抗3A24在文献《口蹄疫病毒非结构蛋白3A单克隆抗体的制备和鉴定》，中国兽医科学2010,40(04):331-336中公开过，3B单抗3B4B1在文献《口蹄疫病毒非结构蛋白3B单克隆抗体的制备与鉴定》，江苏农业学报,2009,25(2):296～300中公开过，公众均可从中国农业科学院兰州兽医研究所获得。
3.3Western blot分析结果
BHK-21单层细胞生长至90％满时，分别接种转染上清和O/ZK/93-08病毒，感染10h后弃培养液，用PBS(0.01mol/L，pH值7.2)漂洗2次后收集细胞，用PBS重悬，反复冻融裂解3次后离心，上清经SDS-PAGE电泳后将分离的蛋白转印到NC膜，然后用口蹄疫病毒非结构蛋白抗3A单抗3A24和抗3B单抗3B4B1进行免疫印记。结果表明：转染上清感染的BHK-21细胞能与3A单抗反应，而与3B单抗不反应，而O/ZK/93-08病毒感染的BHK-21细胞均能与抗3A和3B的单抗反应。说明拯救的病毒为正确的构建，3B1和3B2氨基酸的修饰完全取消了重组口蹄疫病毒与3B单抗3B4B1的反应能力，适合做标记疫苗候选毒株，结果见图7。
3.4、拯救病毒的遗传稳定性分析
将重组病毒O/rV-1/3BM按10％接种量接种BHK-21细胞，连续传代，观察每代病毒出现95％CPE的时间。结果P4代以后拯救病毒出现95％CPE时间稳定在8-10h左右，与亲本病毒基本一致。取重组病毒的P10，P15，P20细胞毒，提取总RNA后，用引物OZ5269(+)/OZ6811(-)(OZ5269(+):caagaagtgattgagcgggt；OZ6811(-):tttgtcctcttcagacatct)进行RT-PCR扩增，测序验证3B修饰蛋白的基因稳定性。结果表明，3B蛋白氨基酸的突变修饰在连续传代过程中未发生任何变化，适合做遗传标记。P10、P15和P20代重组病毒与亲本病毒3B蛋白氨基酸的比对分析见图8。
3.5、标记病毒的一步生长曲线
将第六代重组病毒O/rV-1/3BM和亲本病毒O/ZK/93-08以1个MOI的病毒感染量接种长满的单层BHK-21细胞(25mL培养瓶)，吸附1h后弃接种的病毒液，用MEM洗2次后，加5mlMEM培养基置于37℃CO2培养箱继续培养。病毒接种细胞后4h、8h、12h、16h、20h收取样品，在BHK-21单层细胞上(96孔板)滴定病毒效价(实验进行三次重复)，并按照Reed-Muench法计算TCID50，绘制一步生长曲线。结果表明：用反向遗传操作技术构建的口蹄疫重组病毒具有与O/ZK/93-08亲本毒株相似的生长特性，3B蛋白氨基酸的修饰几乎没有影响重组病毒在BHK-21细胞上的复制能力，见图9。
综上，本发明构建的重组病毒用IFA和WB检测，结果该病毒均不与本申请人筛选的口蹄疫病毒抗3B特异的单抗3B4B1反应，说明通过对口蹄疫病毒3B1和3B2蛋白氨基酸的突变修饰(4AGP6-4TAA6)，成功取消了重组病毒与3B单抗3B4B1的结合能力。因此用本发明得到的重组病毒可以用来发展具有鉴别诊断的口蹄疫标记疫苗，用于我国口蹄疫的有效预防、控制和净化。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 中国农业科学院兰州兽医研究所
<120> 3B蛋白优势表位缺失的口蹄疫标记疫苗株及其构建方法和应用
<210> 1
<211> 8137
<212> DNA
<213> 口蹄疫疫苗毒株O/ZK/93-08的核苷酸序列
<400> 1
ttgaaagggg gcgctagggt ctcacccctg gcatgccaac gacagctcct gcgtcgcacc 60
ccgcacttac gtctctgcaa gcgcaggaac cgatggacta tcgttcaccc acctacagct 120
ggactcacga caccgcgtgg ccactttagc tggattgtgc ggacgaacat cgcttgcgca 180
tttcgcgtga ccggttagta ctcttaccac tctccgccta cttggtcgtc agcgctgtct 240
tgggcactcc tgttgggggc tgtccgacgc tccacggtct cctccgtttt aggaccacgg 300
tgttggggcc gccacgtgcg agccgctcgc ctggtgtgct tcggctgtca cccgaagccc 360
gcctttcacc cccccccccc ccccaaccag taccgtcgtt cccgacgttt aaagggaggt 420
aaccacaagc ttgcactacc actcccggtg tcaacgggat gtgaccgcaa gatgagcctt 480
cacccggaag taaaacggca acttcacaca gttttgcccg ttttcatgag aaacgggacg 540
tccgcgcacg aaacgcgccg tcgcttgagg aagacttgta caaacacggt ctaagcaggt 600
ttccacaact gacaaaaaac ccgtgcagct tgaaaccccg cctggtcttt ccaggtctag 660
aggggtgaca ctttgtactg tgctcgactc cacgctcggt ccactggcgg gtgttagtag 720
cagcactgtt gcttcgtagc ggagcatggt ggccgtggga actcctcctt ggtaacaagg 780
acccacgggg ccaaaagcca cgtccagacg gacccaccat gtgtgcaacc ccagcacggc 840
aactttactg tgaacaccac cttaaggtga cactgatact ggtactcggt cactggtgac 900
aggctaagga tgcccttcag gtaccccgag gtaacacgag acactcggga tctgagaagg 960
ggattgggac ttctttaaag tgcccagttt aaaaagcttc tatgcctgaa taggcgaccg 1020
gaggccggcg cctttctatt gaccaccacc aaatccatga acacgactga ctgttttatc 1080
gctctgttac acgttctcag ggagattaaa gcactgtttc tgtcacgaac acaagggaaa 1140
atggaattca cacttcacaa cggtgaaaag aaggtcttct acgccagacc caacaaccac 1200
gacaattgct ggttgaacgc catcctccaa ctgttcaggt acgtcgacga acccttcttc 1260
gactgggtct acgactcacc tgagaacctt actcttgagg cgatcaggcg actcgaagaa 1320
attactggtc ttgagctaca cgagggtgga ccacccgccc ttgtcgtctg gaacattaag 1380
cacttgctct gcaccggaat cggcaccgct tcgcggccta gcgaggtgtg tatggtggac 1440
ggtacagaca tgtgcttggc cgacttccac gctggtatct ttctgaaggg acaagaccac 1500
gccgtattcg cctgtgtcac ctccgacggg tggtacgcga ttgacgacga ggatttttac 1560
ccgtggacac cagacccggc tgacgttttg gtttttgttc cgtacgatca agaaccactt 1620
aatggagaat ggaaagcaaa ggtccagaag cggcttaagg gcgccgggca atccagcccg 1680
acgaccgggt cacagaacca atcaggcaac actggaagca tcattaacaa ctactacatg 1740
cagcaatacc agaactccat ggacacacag cttggtgaca acgccattag cggaggctcc 1800
aacgagggtt ctacggatac cacctccacc cacacgaaca acacccagaa caacgactgg 1860
ttttcaaaac tggccaactc cgctctcagc ggtctcttcg gtgctcttct cgccgacaaa 1920
aagacagagg aaactaccct cctcgaggac cgcattctca ccacccgcaa cggacacacg 1980
acctcgacaa cccagtcgag cgtcggggtg acgtacgggt atgcaacagc tgaggacttc 2040
gtgagcgggc ccaacacctc tggtcttgag accagggttg tccaggccga acggttcttc 2100
aaaacccact tgttcgactg ggtcaccagt gacccgtttg gacggtgcca catgttggag 2160
ctcccgactg accacaaagg cgtctacggc agcctaaccg actcgtacgc gtatatgagg 2220
aacggttggg acgttgaagt caccgcggtg ggaaaccagt tcaacggagg ctgcttgttg 2280
gtggcaatgg taccagagct ttgttccatc aacaagagag agctgtacca gctcacactt 2340
ttcccccacc agttcattaa cccacggacg aacatgacgg cacacatcac tgtgccctac 2400
gttggcgtca acaggtacga ccaatacaag gtgcataaac cctggaccct tgttgtcatg 2460
gtcgtggccc ccttgacggt caacaatgag ggtgctccgc aaatcaaggt gtatgccaac 2520
atcgccccca ccaacgttta cgttgcgggt gaattccctt ccaaggaggg gatcttcccc 2580
gtggcatgca gcgacggtta cggcggtttg gtgaccacgg acccaaagac ggcggacccc 2640
gtgtacggga aagtgttcaa ccccccccgt aacttgttgc cagggcggtt tacaaacctc 2700
cttgatgtgg ccgaggcgtg tcccacgttc ctacacttcg aaggtgacgt accgtacgtg 2760
accacgaaga cggactcaga cagggtgttg gcccaattcg acctgtctct ggcagcaaag 2820
cacatgtcga acactttcct cgcgggtctt gcccagtatt acacacagta cagcggcacc 2880
atcaacctac acttcatgtt cacagggccc accgatgcga aggcgcgcta catgattgcg 2940
tatgcccctc ctggcatgga accgccgaaa acgcctgagg ccgccgcaca ctgcattcac 3000
gctgagtggg acacagggct gaattcaaag ttcacatttt caattcccta cctttcggcc 3060
gctgactacg cgtacaccgc gtccgacgtc gccgaaacca caaacgtgca gggatgggtc 3120
tgcttgttcc agataacaca cgggaaagcc gacggcgatg ctctgattgt gctagctagt 3180
gctggcaaag actttgacct acgcctaccg gttgacgccc gcacgcagac cacctctgcg 3240
ggcgagtccg cggaccccgt taccgccacc gttgagaatt acggtggtga gacacaggtc 3300
cagagacgcc agcacacgga tatctcgttt atactagaca gatttgtgaa agtcacacca 3360
aaagaccaaa tcaatgtgct ggacctgatg cagatccctg cccacacttt agtaggggcc 3420
ctcctgcgga cggccaccta ctacttctcc gacttggagt tggctgtcaa acacaagggt 3480
gatctcacct gggttccgaa cggggcccct gagacagctt tggacaacac caccaaccca 3540
acagcttacc acaaagcacc actcacgcga ctggccttgc cttacacggc cccacaccgc 3600
gtcttagcga ccgtctacaa cggaagttgt aagtacagtg gcgcccgcgt gagcaacgtg 3660
aggggtgacc ttcaagtgtt ggctcagaag gcagaaagag ctctgcccac ctcctttaac 3720
tatggtgcca ttaaggcaac ccgggtgact gagttactct accgaatgaa gagagccgag 3780
acatactgcc ccaggcccct tcttgccatt caaccgagtg acgctagaca caagcagaag 3840
atcgtggcac ccgcaaaaca gcttctgaac ttcgacctcc tcaagctggc gggagacgtc 3900
gagtccaacc ctggaccctt cttcttctcc gacgtcaggt cgaacttcgc aaaactggtg 3960
gacaccatca accagatgca ggaggatatg tcaacaaagc acggacccga ctttaatcga 4020
ctggtgtccg cgtttgagga actggccact ggagtgaagg ccatcaggac tggtctcgac 4080
gaggccaagc cctggtacaa gctcatcaaa ctcctaagcc gcttgtcgtg catggccgct 4140
gtagcagcac ggtctaagga cccagtcctt gtggccatca tgctagctga caccggtctt 4200
gagattctgg acagcacctt tgtagtgaag aagatatctg actcgctctc cagtctcttc 4260
cacgtgccgg cccccgcctt cagtttcgga gccccgatcc tattggctgg gttggtcaaa 4320
gtcgcctcga gtttcttccg gtctacgccc gaagaccttg agagagcaga aaagcagctc 4380
aaagcacgtg acattaacga tatatttgcc attctcaaga atggtgagtg gttggtcaag 4440
ctgatcctcg ccatccgcga ctggattaag gcgtggatcg cctctgaaga aaagtttgtc 4500
actatgacag acttggtacc tggtatcctt gaaaaacagc gggatctcaa cgaccccggc 4560
aagtacaagg aggccaagga gtggcttgac aacgcgcgtc aagcgtgttt gaagagcggg 4620
aacgttcaca ttgccaacct gtgcaaagtg atcgctccag cgcccagcaa gtcgagacct 4680
gaaccagtgg tcgtttgcct ccgcggcaaa tctggccagg ggaaaagttt ccttgcgaac 4740
gtgctcgcgc aagcaatttc ctcacacttc actggcagga ccgactcggt ctggtactgc 4800
ccgcccgatc ctgaccactt cgacggttac aatcagcaga ccgttgttgt gatggacgac 4860
ttgggccaaa accctgacgg caaggatttc aagtactttg cccaaatggt ttcaaccacg 4920
gggttcatcc cgcccatggc ctcgcttgag gacaaaggca aacctttcaa cagcaaagtc 4980
atcatagcca ctaccaactt gtactcgggt ttcaccccga gaaccatggt gtgtcccgac 5040
gcactgaacc ggaggttcca ctttgacatc gatgtgagcg ccaaggacgg atacaagatc 5100
gacaacaaac tggacatagt caaagccctc gaggacaccc acactaaccc agtggcgatg 5160
ttccaatacg attgcgccct tctcaacggc atggctgttg aaatgaagag aatgcaacaa 5220
gacatgttca agcctcagcc acctcttcag aacgtctacc aacttgttca agaagtgatt 5280
gagcgggtgg aactccacga aaaggtgtcg agtcacccga tttttaaaca gatctcaatc 5340
ccttcccaaa agtctgtgct gtactttctc attgagaaag ggcagcacga agcagcaatc 5400
gagttctttg aggggatggt tcacgattct atcaaggagg agctccgacc cctcattcaa 5460
cagacctcat ttgtgaagcg cgccttcaag cgcctgaagg agaattttga gattgtagcc 5520
ctgtgtttaa ccctcttggc aaacatagtg atcatgctac gcgaagcgcg caagaggcgc 5580
cagtcagtgg atgactcact ggatgacgac gcggctcttg acgatgcgga aaagaaccct 5640
ctagaggcga gtggcgccag cgccgttggt ttcagagaga gatcccccac cgagcaaaag 5700
acgtgcgacg acgtgaacac tgagcccgtt gtgcccggga gggaacaacc gcgagctgaa 5760
ggaccctacg ccgggccact cgaacgtcag aaacctctta aagtgaaagc caggttgcca 5820
caacaagagg gaccttacgc cggtccgatg gagcggcaga aaccgctgaa agtgaaagca 5880
aaagcccccg tcgtgaagga aggaccctac gaggggccgg tgaaaaagcc tgtcgctttg 5940
aaagtgagag caaagaactt gatcgtcact gagagtggag caccaccgac cgacttgcaa 6000
aagatggtca tgggcaacac taaacccgtc gagctcatcc tcgatggcaa gacggtggct 6060
atctgctgtg ctactggagt gtttggcact gcctacctcg tgcctcgtca tctcttcgca 6120
gagaggtatg acaagatcat gttggacggc agagccttga cagacagtga ctacagagtg 6180
tttgagtttg agattaaagt aaaaggacag gacatgctct cagacgctgc tctcatggtg 6240
ttacaccgtg ggaaccgcgt gcgagatatc acgaagcatt ttcgcgatgt agcgagaatg 6300
aagaagggaa cccccgtcgt cggcgtgatc aacaacgctg atgtcgggag actcatattc 6360
tctggtgaag ccctcactta caaggacatt gtcgtgtgca tggacggaga caccatgcct 6420
gggctctttg cctacagagc ttccaccaag gcgggctact gtggaggagc cgtcctggca 6480
aaagacgggg ccgagacgtt catcgtcggc acccactctg caggtggcaa cggtgtggga 6540
tattgttcat gcgtttcccg ctcaatgctt ctgaaaatga aggcacacat cgatcccgaa 6600
ccacaccacg aggggttgat tgtcgacacc agagacgtgg aagagcgcgt gcacgtgatg 6660
cgcaaaacca agctcgcgcc caccgtagcg cacggtgtgt tcaaccccga attcgggcct 6720
gccgctctgt ccaacaagga cccacgcctg aatgaggggg ttgtcctcga cgatgtcatt 6780
ttctccaaac acaaaggaga cacaaagatg tctgaagagg acaaagtgct gttccggcgc 6840
tgtgctgctg actacgcgtc acgcttacac agcgtgttgg ggacggcaaa tgccccactg 6900
agcatttacg aggctatcaa aggcgtcgac ggactcgacg ccatggaacc ggataccgcg 6960
cccggtctcc cctgggctct ccaggggaaa cgccgcggtg ccctgatcga ctttgaaaac 7020
ggcaccgtcg ggcccgaggt cgaggcagcc ctcaagctca tggagagacg tgagtacaag 7080
ttcgtctgcc agaccttcct gaaggacgag attcgcccgc tggagaaggt gcgcgctggc 7140
aagacacgca ttgtcgacgt cctgcctgtt gaacacatcc tctacaccag gatgatgatt 7200
ggtagattct gcgcccaaat gcactcaaac aacggaccgc aaattggctc ggcggtcggt 7260
tgcaaccctg acgttgattg gcaaagattt ggcacacatt tcgcccagta caaaaacgtg 7320
tgggatgtgg actattcggc ctttgatgct aaccactgca gtgatgcgat gaacatcatg 7380
ttcgaggagg tgttccgcac ggagtttggc ttccacccga acgccgagtg gattctgaag 7440
actctagtga acacggagca cgcctatgag aacaagcgta tcaccgtcga gggtggaatg 7500
ccatctggtt gttccgcaac aagcattatc aacacaattt tgaacaacat ctacgtgctc 7560
tacgccctgc gcagacacta tgagggagtc gagctggaca cttacaccat gatctcctac 7620
ggagacgaca tcgtggtggc gagtgattac gacctggact ttgaggccct taagcctcac 7680
ttcaagtccc ttggtcaaac cattactcca gccgacaaaa gcgacaaagg ttttgttctt 7740
ggtcactcca ttaccgatgt cactttcctc aaaagacact tccacatgga ttacggaact 7800
gggttttaca aacctgtgat ggcctcgaag accctcgagg ccatcctctc ctttgcacgc 7860
cgtgggacca tacaggagaa gttgatctcc gtggcaggac tcgccgtcca ttctggaccc 7920
gacgagtacc ggcgtctctt tgagcccttc caaggcctct ttgagattcc aagctacaga 7980
tcactttacc tgcgttgggt gaacgccgtg tgcggtgacg cataatccct cagatgtcac 8040
tactggcaaa aagaccctga ggcgcgcgac gccgtaggag tgaaaaaccg caaaggtttt 8100
tcccacttcc tatttcaaaa aaaaaaaaaa aaaaaaa 8137
<210> 2
<211> 2322
<212> PRT
<213> 口蹄疫疫苗毒株O/ZK/93-08编码蛋白的氨基酸序列
<400> 2
Met Asn Thr Thr Asp Cys Phe Ile Ala Leu Leu His Val Leu Arg Glu
1 5 10 15
Ile Lys Ala Leu Phe Leu Ser Arg Thr Gln Gly Lys Met Glu Phe Thr
20 25 30
Leu His Asn Gly Glu Lys Lys Val Phe Tyr Ala Arg Pro Asn Asn His
35 40 45
Asp Asn Cys Trp Leu Asn Ala Ile Leu Gln Leu Phe Arg Tyr Val Asp
50 55 60
Glu Pro Phe Phe Asp Trp Val Tyr Asp Ser Pro Glu Asn Leu Thr Leu
65 70 75 80
Glu Ala Ile Arg Arg Leu Glu Glu Ile Thr Gly Leu Glu Leu His Glu
85 90 95
Gly Gly Pro Pro Ala Leu Val Val Trp Asn Ile Lys His Leu Leu Cys
100 105 110
Thr Gly Ile Gly Thr Ala Ser Arg Pro Ser Glu Val Cys Met Val Asp
115 120 125
Gly Thr Asp Met Cys Leu Ala Asp Phe His Ala Gly Ile Phe Leu Lys
130 135 140
Gly Gln Asp His Ala Val Phe Ala Cys Val Thr Ser Asp Gly Trp Tyr
145 150 155 160
Ala Ile Asp Asp Glu Asp Phe Tyr Pro Trp Thr Pro Asp Pro Ala Asp
165 170 175
Val Leu Val Phe Val Pro Tyr Asp Gln Glu Pro Leu Asn Gly Glu Trp
180 185 190
Lys Ala Lys Val Gln Lys Arg Leu Lys Gly Ala Gly Gln Ser Ser Pro
195 200 205
Thr Thr Gly Ser Gln Asn Gln Ser Gly Asn Thr Gly Ser Ile Ile Asn
210 215 220
Asn Tyr Tyr Met Gln Gln Tyr Gln Asn Ser Met Asp Thr Gln Leu Gly
225 230 235 240
Asp Asn Ala Ile Ser Gly Gly Ser Asn Glu Gly Ser Thr Asp Thr Thr
245 250 255
Ser Thr His Thr Asn Asn Thr Gln Asn Asn Asp Trp Phe Ser Lys Leu
260 265 270
Ala Asn Ser Ala Leu Ser Gly Leu Phe Gly Ala Leu Leu Ala Asp Lys
275 280 285
Lys Thr Glu Glu Thr Thr Leu Leu Glu Asp Arg Ile Leu Thr Thr Arg
290 295 300
Asn Gly His Thr Thr Ser Thr Thr Gln Ser Ser Val Gly Val Thr Tyr
305 310 315 320
Gly Tyr Ala Thr Ala Glu Asp Phe Val Ser Gly Pro Asn Thr Ser Gly
325 330 335
Leu Glu Thr Arg Val Val Gln Ala Glu Arg Phe Phe Lys Thr His Leu
340 345 350
Phe Asp Trp Val Thr Ser Asp Pro Phe Gly Arg Cys His Met Leu Glu
355 360 365
Leu Pro Thr Asp His Lys Gly Val Tyr Gly Ser Leu Thr Asp Ser Tyr
370 375 380
Ala Tyr Met Arg Asn Gly Trp Asp Val Glu Val Thr Ala Val Gly Asn
385 390 395 400
Gln Phe Asn Gly Gly Cys Leu Leu Val Ala Met Val Pro Glu Leu Cys
405 410 415
Ser Ile Asn Lys Arg Glu Leu Tyr Gln Leu Thr Leu Phe Pro His Gln
420 425 430
Phe Ile Asn Pro Arg Thr Asn Met Thr Ala His Ile Thr Val Pro Tyr
435 440 445
Val Gly Val Asn Arg Tyr Asp Gln Tyr Lys Val His Lys Pro Trp Thr
450 455 460
Leu Val Val Met Val Val Ala Pro Leu Thr Val Asn Asn Glu Gly Ala
465 470 475 480
Pro Gln Ile Lys Val Tyr Ala Asn Ile Ala Pro Thr Asn Val Tyr Val
485 490 495
Ala Gly Glu Phe Pro Ser Lys Glu Gly Ile Phe Pro Val Ala Cys Ser
500 505 510
Asp Gly Tyr Gly Gly Leu Val Thr Thr Asp Pro Lys Thr Ala Asp Pro
515 520 525
Val Tyr Gly Lys Val Phe Asn Pro Pro Arg Asn Leu Leu Pro Gly Arg
530 535 540
Phe Thr Asn Leu Leu Asp Val Ala Glu Ala Cys Pro Thr Phe Leu His
545 550 555 560
Phe Glu Gly Asp Val Pro Tyr Val Thr Thr Lys Thr Asp Ser Asp Arg
565 570 575
Val Leu Ala Gln Phe Asp Leu Ser Leu Ala Ala Lys His Met Ser Asn
580 585 590
Thr Phe Leu Ala Gly Leu Ala Gln Tyr Tyr Thr Gln Tyr Ser Gly Thr
595 600 605
Ile Asn Leu His Phe Met Phe Thr Gly Pro Thr Asp Ala Lys Ala Arg
610 615 620
Tyr Met Ile Ala Tyr Ala Pro Pro Gly Met Glu Pro Pro Lys Thr Pro
625 630 635 640
Glu Ala Ala Ala His Cys Ile His Ala Glu Trp Asp Thr Gly Leu Asn
645 650 655
Ser Lys Phe Thr Phe Ser Ile Pro Tyr Leu Ser Ala Ala Asp Tyr Ala
660 665 670
Tyr Thr Ala Ser Asp Val Ala Glu Thr Thr Asn Val Gln Gly Trp Val
675 680 685
Cys Leu Phe Gln Ile Thr His Gly Lys Ala Asp Gly Asp Ala Leu Ile
690 695 700
Val Leu Ala Ser Ala Gly Lys Asp Phe Asp Leu Arg Leu Pro Val Asp
705 710 715 720
Ala Arg Thr Gln Thr Thr Ser Ala Gly Glu Ser Ala Asp Pro Val Thr
725 730 735
Ala Thr Val Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg Gln
740 745 750
His Thr Asp Ile Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro
755 760 765
Lys Asp Gln Ile Asn Val Leu Asp Leu Met Gln Ile Pro Ala His Thr
770 775 780
Leu Val Gly Ala Leu Leu Arg Thr Ala Thr Tyr Tyr Phe Ser Asp Leu
785 790 795 800
Glu Leu Ala Val Lys His Lys Gly Asp Leu Thr Trp Val Pro Asn Gly
805 810 815
Ala Pro Glu Thr Ala Leu Asp Asn Thr Thr Asn Pro Thr Ala Tyr His
820 825 830
Lys Ala Pro Leu Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg
835 840 845
Val Leu Ala Thr Val Tyr Asn Gly Ser Cys Lys Tyr Ser Gly Ala Arg
850 855 860
Val Ser Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Glu
865 870 875 880
Arg Ala Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys Ala Thr Arg
885 890 895
Val Thr Glu Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro
900 905 910
Arg Pro Leu Leu Ala Ile Gln Pro Ser Asp Ala Arg His Lys Gln Lys
915 920 925
Ile Val Ala Pro Ala Lys Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu
930 935 940
Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Phe Phe Phe Ser Asp Val
945 950 955 960
Arg Ser Asn Phe Ala Lys Leu Val Asp Thr Ile Asn Gln Met Gln Glu
965 970 975
Asp Met Ser Thr Lys His Gly Pro Asp Phe Asn Arg Leu Val Ser Ala
980 985 990
Phe Glu Glu Leu Ala Thr Gly Val Lys Ala Ile Arg Thr Gly Leu Asp
995 1000 1005
Glu Ala Lys Pro Trp Tyr Lys Leu Ile Lys Leu Leu Ser Arg Leu
1010 1015 1020
Ser Cys Met Ala Ala Val Ala Ala Arg Ser Lys Asp Pro Val Leu
1025 1030 1035
Val Ala Ile Met Leu Ala Asp Thr Gly Leu Glu Ile Leu Asp Ser
1040 1045 1050
Thr Phe Val Val Lys Lys Ile Ser Asp Ser Leu Ser Ser Leu Phe
1055 1060 1065
His Val Pro Ala Pro Ala Phe Ser Phe Gly Ala Pro Ile Leu Leu
1070 1075 1080
Ala Gly Leu Val Lys Val Ala Ser Ser Phe Phe Arg Ser Thr Pro
1085 1090 1095
Glu Asp Leu Glu Arg Ala Glu Lys Gln Leu Lys Ala Arg Asp Ile
1100 1105 1110
Asn Asp Ile Phe Ala Ile Leu Lys Asn Gly Glu Trp Leu Val Lys
1115 1120 1125
Leu Ile Leu Ala Ile Arg Asp Trp Ile Lys Ala Trp Ile Ala Ser
1130 1135 1140
Glu Glu Lys Phe Val Thr Met Thr Asp Leu Val Pro Gly Ile Leu
1145 1150 1155
Glu Lys Gln Arg Asp Leu Asn Asp Pro Gly Lys Tyr Lys Glu Ala
1160 1165 1170
Lys Glu Trp Leu Asp Asn Ala Arg Gln Ala Cys Leu Lys Ser Gly
1175 1180 1185
Asn Val His Ile Ala Asn Leu Cys Lys Val Ile Ala Pro Ala Pro
1190 1195 1200
Ser Lys Ser Arg Pro Glu Pro Val Val Val Cys Leu Arg Gly Lys
1205 1210 1215
Ser Gly Gln Gly Lys Ser Phe Leu Ala Asn Val Leu Ala Gln Ala
1220 1225 1230
Ile Ser Ser His Phe Thr Gly Arg Thr Asp Ser Val Trp Tyr Cys
1235 1240 1245
Pro Pro Asp Pro Asp His Phe Asp Gly Tyr Asn Gln Gln Thr Val
1250 1255 1260
Val Val Met Asp Asp Leu Gly Gln Asn Pro Asp Gly Lys Asp Phe
1265 1270 1275
Lys Tyr Phe Ala Gln Met Val Ser Thr Thr Gly Phe Ile Pro Pro
1280 1285 1290
Met Ala Ser Leu Glu Asp Lys Gly Lys Pro Phe Asn Ser Lys Val
1295 1300 1305
Ile Ile Ala Thr Thr Asn Leu Tyr Ser Gly Phe Thr Pro Arg Thr
1310 1315 1320
Met Val Cys Pro Asp Ala Leu Asn Arg Arg Phe His Phe Asp Ile
1325 1330 1335
Asp Val Ser Ala Lys Asp Gly Tyr Lys Ile Asp Asn Lys Leu Asp
1340 1345 1350
Ile Val Lys Ala Leu Glu Asp Thr His Thr Asn Pro Val Ala Met
1355 1360 1365
Phe Gln Tyr Asp Cys Ala Leu Leu Asn Gly Met Ala Val Glu Met
1370 1375 1380
Lys Arg Met Gln Gln Asp Met Phe Lys Pro Gln Pro Pro Leu Gln
1385 1390 1395
Asn Val Tyr Gln Leu Val Gln Glu Val Ile Glu Arg Val Glu Leu
1400 1405 1410
His Glu Lys Val Ser Ser His Pro Ile Phe Lys Gln Ile Ser Ile
1415 1420 1425
Pro Ser Gln Lys Ser Val Leu Tyr Phe Leu Ile Glu Lys Gly Gln
1430 1435 1440
His Glu Ala Ala Ile Glu Phe Phe Glu Gly Met Val His Asp Ser
1445 1450 1455
Ile Lys Glu Glu Leu Arg Pro Leu Ile Gln Gln Thr Ser Phe Val
1460 1465 1470
Lys Arg Ala Phe Lys Arg Leu Lys Glu Asn Phe Glu Ile Val Ala
1475 1480 1485
Leu Cys Leu Thr Leu Leu Ala Asn Ile Val Ile Met Leu Arg Glu
1490 1495 1500
Ala Arg Lys Arg Arg Gln Ser Val Asp Asp Ser Leu Asp Asp Asp
1505 1510 1515
Ala Ala Leu Asp Asp Ala Glu Lys Asn Pro Leu Glu Ala Ser Gly
1520 1525 1530
Ala Ser Ala Val Gly Phe Arg Glu Arg Ser Pro Thr Glu Gln Lys
1535 1540 1545
Thr Cys Asp Asp Val Asn Thr Glu Pro Val Val Pro Gly Arg Glu
1550 1555 1560
Gln Pro Arg Ala Glu Gly Pro Tyr Ala Gly Pro Leu Glu Arg Gln
1565 1570 1575
Lys Pro Leu Lys Val Lys Ala Arg Leu Pro Gln Gln Glu Gly Pro
1580 1585 1590
Tyr Ala Gly Pro Met Glu Arg Gln Lys Pro Leu Lys Val Lys Ala
1595 1600 1605
Lys Ala Pro Val Val Lys Glu Gly Pro Tyr Glu Gly Pro Val Lys
1610 1615 1620
Lys Pro Val Ala Leu Lys Val Arg Ala Lys Asn Leu Ile Val Thr
1625 1630 1635
Glu Ser Gly Ala Pro Pro Thr Asp Leu Gln Lys Met Val Met Gly
1640 1645 1650
Asn Thr Lys Pro Val Glu Leu Ile Leu Asp Gly Lys Thr Val Ala
1655 1660 1665
Ile Cys Cys Ala Thr Gly Val Phe Gly Thr Ala Tyr Leu Val Pro
1670 1675 1680
Arg His Leu Phe Ala Glu Arg Tyr Asp Lys Ile Met Leu Asp Gly
1685 1690 1695
Arg Ala Leu Thr Asp Ser Asp Tyr Arg Val Phe Glu Phe Glu Ile
1700 1705 1710
Lys Val Lys Gly Gln Asp Met Leu Ser Asp Ala Ala Leu Met Val
1715 1720 1725
Leu His Arg Gly Asn Arg Val Arg Asp Ile Thr Lys His Phe Arg
1730 1735 1740
Asp Val Ala Arg Met Lys Lys Gly Thr Pro Val Val Gly Val Ile
1745 1750 1755
Asn Asn Ala Asp Val Gly Arg Leu Ile Phe Ser Gly Glu Ala Leu
1760 1765 1770
Thr Tyr Lys Asp Ile Val Val Cys Met Asp Gly Asp Thr Met Pro
1775 1780 1785
Gly Leu Phe Ala Tyr Arg Ala Ser Thr Lys Ala Gly Tyr Cys Gly
1790 1795 1800
Gly Ala Val Leu Ala Lys Asp Gly Ala Glu Thr Phe Ile Val Gly
1805 1810 1815
Thr His Ser Ala Gly Gly Asn Gly Val Gly Tyr Cys Ser Cys Val
1820 1825 1830
Ser Arg Ser Met Leu Leu Lys Met Lys Ala His Ile Asp Pro Glu
1835 1840 1845
Pro His His Glu Gly Leu Ile Val Asp Thr Arg Asp Val Glu Glu
1850 1855 1860
Arg Val His Val Met Arg Lys Thr Lys Leu Ala Pro Thr Val Ala
1865 1870 1875
His Gly Val Phe Asn Pro Glu Phe Gly Pro Ala Ala Leu Ser Asn
1880 1885 1890
Lys Asp Pro Arg Leu Asn Glu Gly Val Val Leu Asp Asp Val Ile
1895 1900 1905
Phe Ser Lys His Lys Gly Asp Thr Lys Met Ser Glu Glu Asp Lys
1910 1915 1920
Val Leu Phe Arg Arg Cys Ala Ala Asp Tyr Ala Ser Arg Leu His
1925 1930 1935
Ser Val Leu Gly Thr Ala Asn Ala Pro Leu Ser Ile Tyr Glu Ala
1940 1945 1950
Ile Lys Gly Val Asp Gly Leu Asp Ala Met Glu Pro Asp Thr Ala
1955 1960 1965
Pro Gly Leu Pro Trp Ala Leu Gln Gly Lys Arg Arg Gly Ala Leu
1970 1975 1980
Ile Asp Phe Glu Asn Gly Thr Val Gly Pro Glu Val Glu Ala Ala
1985 1990 1995
Leu Lys Leu Met Glu Arg Arg Glu Tyr Lys Phe Val Cys Gln Thr
2000 2005 2010
Phe Leu Lys Asp Glu Ile Arg Pro Leu Glu Lys Val Arg Ala Gly
2015 2020 2025
Lys Thr Arg Ile Val Asp Val Leu Pro Val Glu His Ile Leu Tyr
2030 2035 2040
Thr Arg Met Met Ile Gly Arg Phe Cys Ala Gln Met His Ser Asn
2045 2050 2055
Asn Gly Pro Gln Ile Gly Ser Ala Val Gly Cys Asn Pro Asp Val
2060 2065 2070
Asp Trp Gln Arg Phe Gly Thr His Phe Ala Gln Tyr Lys Asn Val
2075 2080 2085
Trp Asp Val Asp Tyr Ser Ala Phe Asp Ala Asn His Cys Ser Asp
2090 2095 2100
Ala Met Asn Ile Met Phe Glu Glu Val Phe Arg Thr Glu Phe Gly
2105 2110 2115
Phe His Pro Asn Ala Glu Trp Ile Leu Lys Thr Leu Val Asn Thr
2120 2125 2130
Glu His Ala Tyr Glu Asn Lys Arg Ile Thr Val Glu Gly Gly Met
2135 2140 2145
Pro Ser Gly Cys Ser Ala Thr Ser Ile Ile Asn Thr Ile Leu Asn
2150 2155 2160
Asn Ile Tyr Val Leu Tyr Ala Leu Arg Arg His Tyr Glu Gly Val
2165 2170 2175
Glu Leu Asp Thr Tyr Thr Met Ile Ser Tyr Gly Asp Asp Ile Val
2180 2185 2190
Val Ala Ser Asp Tyr Asp Leu Asp Phe Glu Ala Leu Lys Pro His
2195 2200 2205
Phe Lys Ser Leu Gly Gln Thr Ile Thr Pro Ala Asp Lys Ser Asp
2210 2215 2220
Lys Gly Phe Val Leu Gly His Ser Ile Thr Asp Val Thr Phe Leu
2225 2230 2235
Lys Arg His Phe His Met Asp Tyr Gly Thr Gly Phe Tyr Lys Pro
2240 2245 2250
Val Met Ala Ser Lys Thr Leu Glu Ala Ile Leu Ser Phe Ala Arg
2255 2260 2265
Arg Gly Thr Ile Gln Glu Lys Leu Ile Ser Val Ala Gly Leu Ala
2270 2275 2280
Val His Ser Gly Pro Asp Glu Tyr Arg Arg Leu Phe Glu Pro Phe
2285 2290 2295
Gln Gly Leu Phe Glu Ile Pro Ser Tyr Arg Ser Leu Tyr Leu Arg
2300 2305 2310
Trp Val Asn Ala Val Cys Gly Asp Ala
2315 2320
<210> 3
<211> 141
<212> DNA
<213> 口蹄疫疫苗毒株O/ZK/93-08 3B1和3B2蛋白的核苷酸序列
<400> 3
ggaccctacg ccgggccact cgaacgtcag aaacctctta aagtgaaagc caggttgcca 60
caacaagagg gaccttacgc cggtccgatg gagcggcaga aaccgctgaa agtgaaagca 120
aaagcccccg tcgtgaagga a 141
<210> 4
<211> 47
<212> PRT
<213> 口蹄疫疫苗毒株O/ZK/93-08 3B1和3B2蛋白编码的氨基酸序列
<400> 4
Gly Pro Tyr Ala Gly Pro Leu Glu Arg Gln Lys Pro Leu Lys Val Lys
1 5 10 15
Ala Arg Leu Pro Gln Gln Glu Gly Pro Tyr Ala Gly Pro Met Glu Arg
20 25 30
Gln Lys Pro Leu Lys Val Lys Ala Lys Ala Pro Val Val Lys Glu
35 40 45
<210> 5
<211> 8137
<212> DNA
<213> 重组病毒O/rV-1/3BM核苷酸序列
<400> 5
ttgaaagggg gcgctagggt ctcacccctg gcatgccaac gacagctcct gcgtcgcacc 60
ccgcacttac gtctctgcaa gcgcaggaac cgatggacta tcgttcaccc acctacagct 120
ggactcacga caccgcgtgg ccactttagc tggattgtgc ggacgaacat cgcttgcgca 180
tttcgcgtga ccggttagta ctcttaccac tctccgccta cttggtcgtc agcgctgtct 240
tgggcactcc tgttgggggc tgtccgacgc tccacggtct cctccgtttt aggaccacgg 300
tgttggggcc gccacgtgcg agccgctcgc ctggtgtgct tcggctgtca cccgaagccc 360
gcctttcacc cccccccccc ccccaaccag taccgtcgtt cccgacgttt aaagggaggt 420
aaccacaagc ttgcactacc actcccggtg tcaacgggat gtgaccgcaa gatgagcctt 480
cacccggaag taaaacggca acttcacaca gttttgcccg ttttcatgag aaacgggacg 540
tccgcgcacg aaacgcgccg tcgcttgagg aagacttgta caaacacggt ctaagcaggt 600
ttccacaact gacaaaaaac ccgtgcagct tgaaaccccg cctggtcttt ccaggtctag 660
aggggtgaca ctttgtactg tgctcgactc cacgctcggt ccactggcgg gtgttagtag 720
cagcactgtt gcttcgtagc ggagcatggt ggccgtggga actcctcctt ggtaacaagg 780
acccacgggg ccaaaagcca cgtccagacg gacccaccat gtgtgcaacc ccagcacggc 840
aactttactg tgaacaccac cttaaggtga cactgatact ggtactcggt cactggtgac 900
aggctaagga tgcccttcag gtaccccgag gtaacacgag acactcggga tctgagaagg 960
ggattgggac ttctttaaag tgcccagttt aaaaagcttc tatgcctgaa taggcgaccg 1020
gaggccggcg cctttctatt gaccaccacc aaatccatga acacgactga ctgttttatc 1080
gctctgttac acgttctcag ggagattaaa gcactgtttc tgtcacgaac acaagggaaa 1140
atggaattca cacttcacaa cggtgaaaag aaggtcttct acgccagacc caacaaccac 1200
gacaattgct ggttgaacgc catcctccaa ctgttcaggt acgtcgacga acccttcttc 1260
gactgggtct acgactcacc tgagaacctt actcttgagg cgatcaggcg actcgaagaa 1320
attactggtc ttgagctaca cgagggtgga ccacccgccc ttgtcgtctg gaacattaag 1380
cacttgctct gcaccggaat cggcaccgct tcgcggccta gcgaggtgtg tatggtggac 1440
ggtacagaca tgtgcttggc cgacttccac gctggtatct ttctgaaggg acaagaccac 1500
gccgtattcg cctgtgtcac ctccgacggg tggtacgcga ttgacgacga ggatttttac 1560
ccgtggacac cagacccggc tgacgttttg gtttttgttc cgtacgatca agaaccactt 1620
aatggagaat ggaaagcaaa ggtccagaag cggcttaagg gcgccgggca atccagcccg 1680
acgaccgggt cacagaacca atcaggcaac actggaagca tcattaacaa ctactacatg 1740
cagcaatacc agaactccat ggacacacag cttggtgaca acgccattag cggaggctcc 1800
aacgagggtt ctacggatac cacctccacc cacacgaaca acacccagaa caacgactgg 1860
ttttcaaaac tggccaactc cgctctcagc ggtctcttcg gtgctcttct cgccgacaaa 1920
aagacagagg aaactaccct cctcgaggac cgcattctca ccacccgcaa cggacacacg 1980
acctcgacaa cccagtcgag cgtcggggtg acgtacgggt atgcaacagc tgaggacttc 2040
gtgagcgggc ccaacacctc tggtcttgag accagggttg tccaggccga acggttcttc 2100
aaaacccact tgttcgactg ggtcaccagt gacccgtttg gacggtgcca catgttggag 2160
ctcccgactg accacaaagg cgtctacggc agcctaaccg actcgtacgc gtatatgagg 2220
aacggttggg acgttgaagt caccgcggtg ggaaaccagt tcaacggagg ctgcttgttg 2280
gtggcaatgg taccagagct ttgttccatc aacaagagag agctgtacca gctcacactt 2340
ttcccccacc agttcattaa cccacggacg aacatgacgg cacacatcac tgtgccctac 2400
gttggcgtca acaggtacga ccaatacaag gtgcataaac cctggaccct tgttgtcatg 2460
gtcgtggccc ccttgacggt caacaatgag ggtgctccgc aaatcaaggt gtatgccaac 2520
atcgccccca ccaacgttta cgttgcgggt gaattccctt ccaaggaggg gatcttcccc 2580
gtggcatgca gcgacggtta cggcggtttg gtgaccacgg acccaaagac ggcggacccc 2640
gtgtacggga aagtgttcaa ccccccccgt aacttgttgc cagggcggtt tacaaacctc 2700
cttgatgtgg ccgaggcgtg tcccacgttc ctacacttcg aaggtgacgt accgtacgtg 2760
accacgaaga cggactcaga cagggtgttg gcccaattcg acctgtctct ggcagcaaag 2820
cacatgtcga acactttcct cgcgggtctt gcccagtatt acacacagta cagcggcacc 2880
atcaacctac acttcatgtt cacagggccc accgatgcga aggcgcgcta catgattgcg 2940
tatgcccctc ctggcatgga accgccgaaa acgcctgagg ccgccgcaca ctgcattcac 3000
gctgagtggg acacagggct gaattcaaag ttcacatttt caattcccta cctttcggcc 3060
gctgactacg cgtacaccgc gtccgacgtc gccgaaacca caaacgtgca gggatgggtc 3120
tgcttgttcc agataacaca cgggaaagcc gacggcgatg ctctgattgt gctagctagt 3180
gctggcaaag actttgacct acgcctaccg gttgacgccc gcacgcagac cacctctgcg 3240
ggcgagtccg cggaccccgt taccgccacc gttgagaatt acggtggtga gacacaggtc 3300
cagagacgcc agcacacgga tatctcgttt atactagaca gatttgtgaa agtcacacca 3360
aaagaccaaa tcaatgtgct ggacctgatg cagatccctg cccacacttt agtaggggcc 3420
ctcctgcgga cggccaccta ctacttctcc gacttggagt tggctgtcaa acacaagggt 3480
gatctcacct gggttccgaa cggggcccct gagacagctt tggacaacac caccaaccca 3540
acagcttacc acaaagcacc actcacgcga ctggccttgc cttacacggc cccacaccgc 3600
gtcttagcga ccgtctacaa cggaagttgt aagtacagtg gcgcccgcgt gagcaacgtg 3660
aggggtgacc ttcaagtgtt ggctcagaag gcagaaagag ctctgcccac ctcctttaac 3720
tatggtgcca ttaaggcaac ccgggtgact gagttactct accgaatgaa gagagccgag 3780
acatactgcc ccaggcccct tcttgccatt caaccgagtg acgctagaca caagcagaag 3840
atcgtggcac ccgcaaaaca gcttctgaac ttcgacctcc tcaagctggc gggagacgtc 3900
gagtccaacc ctggaccctt cttcttctcc gacgtcaggt cgaacttcgc aaaactggtg 3960
gacaccatca accagatgca ggaggatatg tcaacaaagc acggacccga ctttaatcga 4020
ctggtgtccg cgtttgagga actggccact ggagtgaagg ccatcaggac tggtctcgac 4080
gaggccaagc cctggtacaa gctcatcaaa ctcctaagcc gcttgtcgtg catggccgct 4140
gtagcagcac ggtctaagga cccagtcctt gtggccatca tgctagctga caccggtctt 4200
gagattctgg acagcacctt tgtagtgaag aagatatctg actcgctctc cagtctcttc 4260
cacgtgccgg cccccgcctt cagtttcgga gccccgatcc tattggctgg gttggtcaaa 4320
gtcgcctcga gtttcttccg gtctacgccc gaagaccttg agagagcaga aaagcagctc 4380
aaagcacgtg acattaacga tatatttgcc attctcaaga atggtgagtg gttggtcaag 4440
ctgatcctcg ccatccgcga ctggattaag gcgtggatcg cctctgaaga aaagtttgtc 4500
actatgacag acttggtacc tggtatcctt gaaaaacagc gggatctcaa cgaccccggc 4560
aagtacaagg aggccaagga gtggcttgac aacgcgcgtc aagcgtgttt gaagagcggg 4620
aacgttcaca ttgccaacct gtgcaaagtg atcgctccag cgcccagcaa gtcgagacct 4680
gaaccagtgg tcgtttgcct ccgcggcaaa tctggccagg ggaaaagttt ccttgcgaac 4740
gtgctcgcgc aagcaatttc ctcacacttc actggcagga ccgactcggt ctggtactgc 4800
ccgcccgatc ctgaccactt cgacggttac aatcagcaga ccgttgttgt gatggacgac 4860
ttgggccaaa accctgacgg caaggatttc aagtactttg cccaaatggt ttcaaccacg 4920
gggttcatcc cgcccatggc ctcgcttgag gacaaaggca aacctttcaa cagcaaagtc 4980
atcatagcca ctaccaactt gtactcgggt ttcaccccga gaaccatggt gtgtcccgac 5040
gcactgaacc ggaggttcca ctttgacatc gatgtgagcg ccaaggacgg atacaagatc 5100
gacaacaaac tggacatagt caaagccctc gaggacaccc acactaaccc agtggcgatg 5160
ttccaatacg attgcgccct tctcaacggc atggctgttg aaatgaagag aatgcaacaa 5220
gacatgttca agcctcagcc acctcttcag aacgtctacc aacttgttca agaagtgatt 5280
gagcgggtgg aactccacga aaaggtgtcg agtcacccga tttttaaaca gatctcaatc 5340
ccttcccaaa agtctgtgct gtactttctc attgagaaag ggcagcacga agcagcaatc 5400
gagttctttg aggggatggt tcacgattct atcaaggagg agctccgacc cctcattcaa 5460
cagacctcat ttgtgaagcg cgccttcaag cgcctgaagg agaattttga gattgtagcc 5520
ctgtgtttaa ccctcttggc aaacatagtg atcatgctac gcgaagcgcg caagaggcgc 5580
cagtcagtgg atgactcact ggatgacgac gcggctcttg acgatgcgga aaagaaccct 5640
ctagaggcga gtggcgccag cgccgttggt ttcagagaga gatcccccac cgagcaaaag 5700
acgtgcgacg acgtgaacac tgagcccgtt gtgcccggga gggaacaacc gcgagctgaa 5760
ggaccctaca ccgcggcact cgaacgtcag aaacctctta aagtgaaagc caggttgcca 5820
caacaagagg gaccttacac cgctgcgatg gagcggcaga aaccgctgaa agtgaaagca 5880
aaagcccccg tcgtgaagga aggaccctac gaggggccgg tgaaaaagcc tgtcgctttg 5940
aaagtgagag caaagaactt gatcgtcact gagagtggag caccaccgac cgacttgcaa 6000
aagatggtca tgggcaacac taaacccgtc gagctcatcc tcgatggcaa gacggtggct 6060
atctgctgtg ctactggagt gtttggcact gcctacctcg tgcctcgtca tctcttcgca 6120
gagaggtatg acaagatcat gttggacggc agagccttga cagacagtga ctacagagtg 6180
tttgagtttg agattaaagt aaaaggacag gacatgctct cagacgctgc tctcatggtg 6240
ttacaccgtg ggaaccgcgt gcgagatatc acgaagcatt ttcgcgatgt agcgagaatg 6300
aagaagggaa cccccgtcgt cggcgtgatc aacaacgctg atgtcgggag actcatattc 6360
tctggtgaag ccctcactta caaggacatt gtcgtgtgca tggacggaga caccatgcct 6420
gggctctttg cctacagagc ttccaccaag gcgggctact gtggaggagc cgtcctggca 6480
aaagacgggg ccgagacgtt catcgtcggc acccactctg caggtggcaa cggtgtggga 6540
tattgttcat gcgtttcccg ctcaatgctt ctgaaaatga aggcacacat cgatcccgaa 6600
ccacaccacg aggggttgat tgtcgacacc agagacgtgg aagagcgcgt gcacgtgatg 6660
cgcaaaacca agctcgcgcc caccgtagcg cacggtgtgt tcaaccccga attcgggcct 6720
gccgctctgt ccaacaagga cccacgcctg aatgaggggg ttgtcctcga cgatgtcatt 6780
ttctccaaac acaaaggaga cacaaagatg tctgaagagg acaaagtgct gttccggcgc 6840
tgtgctgctg actacgcgtc acgcttacac agcgtgttgg ggacggcaaa tgccccactg 6900
agcatttacg aggctatcaa aggcgtcgac ggactcgacg ccatggaacc ggataccgcg 6960
cccggtctcc cctgggctct ccaggggaaa cgccgcggtg ccctgatcga ctttgaaaac 7020
ggcaccgtcg ggcccgaggt cgaggcagcc ctcaagctca tggagagacg tgagtacaag 7080
ttcgtctgcc agaccttcct gaaggacgag attcgcccgc tggagaaggt gcgcgctggc 7140
aagacacgca ttgtcgacgt cctgcctgtt gaacacatcc tctacaccag gatgatgatt 7200
ggtagattct gcgcccaaat gcactcaaac aacggaccgc aaattggctc ggcggtcggt 7260
tgcaaccctg acgttgattg gcaaagattt ggcacacatt tcgcccagta caaaaacgtg 7320
tgggatgtgg actattcggc ctttgatgct aaccactgca gtgatgcgat gaacatcatg 7380
ttcgaggagg tgttccgcac ggagtttggc ttccacccga acgccgagtg gattctgaag 7440
actctagtga acacggagca cgcctatgag aacaagcgta tcaccgtcga gggtggaatg 7500
ccatctggtt gttccgcaac aagcattatc aacacaattt tgaacaacat ctacgtgctc 7560
tacgccctgc gcagacacta tgagggagtc gagctggaca cttacaccat gatctcctac 7620
ggagacgaca tcgtggtggc gagtgattac gacctggact ttgaggccct taagcctcac 7680
ttcaagtccc ttggtcaaac cattactcca gccgacaaaa gcgacaaagg ttttgttctt 7740
ggtcactcca ttaccgatgt cactttcctc aaaagacact tccacatgga ttacggaact 7800
gggttttaca aacctgtgat ggcctcgaag accctcgagg ccatcctctc ctttgcacgc 7860
cgtgggacca tacaggagaa gttgatctcc gtggcaggac tcgccgtcca ttctggaccc 7920
gacgagtacc ggcgtctctt tgagcccttc caaggcctct ttgagattcc aagctacaga 7980
tcactttacc tgcgttgggt gaacgccgtg tgcggtgacg cataatccct cagatgtcac 8040
tactggcaaa aagaccctga ggcgcgcgac gccgtaggag tgaaaaaccg caaaggtttt 8100
tcccacttcc tatttcaaaa aaaaaaaaaa aaaaaaa 8137
<211> 2322
<212> PRT
<213> 重组病毒O/rV-1/3BM编码蛋白的氨基酸序列
<400> 6
Met Asn Thr Thr Asp Cys Phe Ile Ala Leu Leu His Val Leu Arg Glu
1 5 10 15
Ile Lys Ala Leu Phe Leu Ser Arg Thr Gln Gly Lys Met Glu Phe Thr
20 25 30
Leu His Asn Gly Glu Lys Lys Val Phe Tyr Ala Arg Pro Asn Asn His
35 40 45
Asp Asn Cys Trp Leu Asn Ala Ile Leu Gln Leu Phe Arg Tyr Val Asp
50 55 60
Glu Pro Phe Phe Asp Trp Val Tyr Asp Ser Pro Glu Asn Leu Thr Leu
65 70 75 80
Glu Ala Ile Arg Arg Leu Glu Glu Ile Thr Gly Leu Glu Leu His Glu
85 90 95
Gly Gly Pro Pro Ala Leu Val Val Trp Asn Ile Lys His Leu Leu Cys
100 105 110
Thr Gly Ile Gly Thr Ala Ser Arg Pro Ser Glu Val Cys Met Val Asp
115 120 125
Gly Thr Asp Met Cys Leu Ala Asp Phe His Ala Gly Ile Phe Leu Lys
130 135 140
Gly Gln Asp His Ala Val Phe Ala Cys Val Thr Ser Asp Gly Trp Tyr
145 150 155 160
Ala Ile Asp Asp Glu Asp Phe Tyr Pro Trp Thr Pro Asp Pro Ala Asp
165 170 175
Val Leu Val Phe Val Pro Tyr Asp Gln Glu Pro Leu Asn Gly Glu Trp
180 185 190
Lys Ala Lys Val Gln Lys Arg Leu Lys Gly Ala Gly Gln Ser Ser Pro
195 200 205
Thr Thr Gly Ser Gln Asn Gln Ser Gly Asn Thr Gly Ser Ile Ile Asn
210 215 220
Asn Tyr Tyr Met Gln Gln Tyr Gln Asn Ser Met Asp Thr Gln Leu Gly
225 230 235 240
Asp Asn Ala Ile Ser Gly Gly Ser Asn Glu Gly Ser Thr Asp Thr Thr
245 250 255
Ser Thr His Thr Asn Asn Thr Gln Asn Asn Asp Trp Phe Ser Lys Leu
260 265 270
Ala Asn Ser Ala Leu Ser Gly Leu Phe Gly Ala Leu Leu Ala Asp Lys
275 280 285
Lys Thr Glu Glu Thr Thr Leu Leu Glu Asp Arg Ile Leu Thr Thr Arg
290 295 300
Asn Gly His Thr Thr Ser Thr Thr Gln Ser Ser Val Gly Val Thr Tyr
305 310 315 320
Gly Tyr Ala Thr Ala Glu Asp Phe Val Ser Gly Pro Asn Thr Ser Gly
325 330 335
Leu Glu Thr Arg Val Val Gln Ala Glu Arg Phe Phe Lys Thr His Leu
340 345 350
Phe Asp Trp Val Thr Ser Asp Pro Phe Gly Arg Cys His Met Leu Glu
355 360 365
Leu Pro Thr Asp His Lys Gly Val Tyr Gly Ser Leu Thr Asp Ser Tyr
370 375 380
Ala Tyr Met Arg Asn Gly Trp Asp Val Glu Val Thr Ala Val Gly Asn
385 390 395 400
Gln Phe Asn Gly Gly Cys Leu Leu Val Ala Met Val Pro Glu Leu Cys
405 410 415
Ser Ile Asn Lys Arg Glu Leu Tyr Gln Leu Thr Leu Phe Pro His Gln
420 425 430
Phe Ile Asn Pro Arg Thr Asn Met Thr Ala His Ile Thr Val Pro Tyr
435 440 445
Val Gly Val Asn Arg Tyr Asp Gln Tyr Lys Val His Lys Pro Trp Thr
450 455 460
Leu Val Val Met Val Val Ala Pro Leu Thr Val Asn Asn Glu Gly Ala
465 470 475 480
Pro Gln Ile Lys Val Tyr Ala Asn Ile Ala Pro Thr Asn Val Tyr Val
485 490 495
Ala Gly Glu Phe Pro Ser Lys Glu Gly Ile Phe Pro Val Ala Cys Ser
500 505 510
Asp Gly Tyr Gly Gly Leu Val Thr Thr Asp Pro Lys Thr Ala Asp Pro
515 520 525
Val Tyr Gly Lys Val Phe Asn Pro Pro Arg Asn Leu Leu Pro Gly Arg
530 535 540
Phe Thr Asn Leu Leu Asp Val Ala Glu Ala Cys Pro Thr Phe Leu His
545 550 555 560
Phe Glu Gly Asp Val Pro Tyr Val Thr Thr Lys Thr Asp Ser Asp Arg
565 570 575
Val Leu Ala Gln Phe Asp Leu Ser Leu Ala Ala Lys His Met Ser Asn
580 585 590
Thr Phe Leu Ala Gly Leu Ala Gln Tyr Tyr Thr Gln Tyr Ser Gly Thr
595 600 605
Ile Asn Leu His Phe Met Phe Thr Gly Pro Thr Asp Ala Lys Ala Arg
610 615 620
Tyr Met Ile Ala Tyr Ala Pro Pro Gly Met Glu Pro Pro Lys Thr Pro
625 630 635 640
Glu Ala Ala Ala His Cys Ile His Ala Glu Trp Asp Thr Gly Leu Asn
645 650 655
Ser Lys Phe Thr Phe Ser Ile Pro Tyr Leu Ser Ala Ala Asp Tyr Ala
660 665 670
Tyr Thr Ala Ser Asp Val Ala Glu Thr Thr Asn Val Gln Gly Trp Val
675 680 685
Cys Leu Phe Gln Ile Thr His Gly Lys Ala Asp Gly Asp Ala Leu Ile
690 695 700
Val Leu Ala Ser Ala Gly Lys Asp Phe Asp Leu Arg Leu Pro Val Asp
705 710 715 720
Ala Arg Thr Gln Thr Thr Ser Ala Gly Glu Ser Ala Asp Pro Val Thr
725 730 735
Ala Thr Val Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg Gln
740 745 750
His Thr Asp Ile Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro
755 760 765
Lys Asp Gln Ile Asn Val Leu Asp Leu Met Gln Ile Pro Ala His Thr
770 775 780
Leu Val Gly Ala Leu Leu Arg Thr Ala Thr Tyr Tyr Phe Ser Asp Leu
785 790 795 800
Glu Leu Ala Val Lys His Lys Gly Asp Leu Thr Trp Val Pro Asn Gly
805 810 815
Ala Pro Glu Thr Ala Leu Asp Asn Thr Thr Asn Pro Thr Ala Tyr His
820 825 830
Lys Ala Pro Leu Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg
835 840 845
Val Leu Ala Thr Val Tyr Asn Gly Ser Cys Lys Tyr Ser Gly Ala Arg
850 855 860
Val Ser Asn Val Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Glu
865 870 875 880
Arg Ala Leu Pro Thr Ser Phe Asn Tyr Gly Ala Ile Lys Ala Thr Arg
885 890 895
Val Thr Glu Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro
900 905 910
Arg Pro Leu Leu Ala Ile Gln Pro Ser Asp Ala Arg His Lys Gln Lys
915 920 925
Ile Val Ala Pro Ala Lys Gln Leu Leu Asn Phe Asp Leu Leu Lys Leu
930 935 940
Ala Gly Asp Val Glu Ser Asn Pro Gly Pro Phe Phe Phe Ser Asp Val
945 950 955 960
Arg Ser Asn Phe Ala Lys Leu Val Asp Thr Ile Asn Gln Met Gln Glu
965 970 975
Asp Met Ser Thr Lys His Gly Pro Asp Phe Asn Arg Leu Val Ser Ala
980 985 990
Phe Glu Glu Leu Ala Thr Gly Val Lys Ala Ile Arg Thr Gly Leu Asp
995 1000 1005
Glu Ala Lys Pro Trp Tyr Lys Leu Ile Lys Leu Leu Ser Arg Leu
1010 1015 1020
Ser Cys Met Ala Ala Val Ala Ala Arg Ser Lys Asp Pro Val Leu
1025 1030 1035
Val Ala Ile Met Leu Ala Asp Thr Gly Leu Glu Ile Leu Asp Ser
1040 1045 1050
Thr Phe Val Val Lys Lys Ile Ser Asp Ser Leu Ser Ser Leu Phe
1055 1060 1065
His Val Pro Ala Pro Ala Phe Ser Phe Gly Ala Pro Ile Leu Leu
1070 1075 1080
Ala Gly Leu Val Lys Val Ala Ser Ser Phe Phe Arg Ser Thr Pro
1085 1090 1095
Glu Asp Leu Glu Arg Ala Glu Lys Gln Leu Lys Ala Arg Asp Ile
1100 1105 1110
Asn Asp Ile Phe Ala Ile Leu Lys Asn Gly Glu Trp Leu Val Lys
1115 1120 1125
Leu Ile Leu Ala Ile Arg Asp Trp Ile Lys Ala Trp Ile Ala Ser
1130 1135 1140
Glu Glu Lys Phe Val Thr Met Thr Asp Leu Val Pro Gly Ile Leu
1145 1150 1155
Glu Lys Gln Arg Asp Leu Asn Asp Pro Gly Lys Tyr Lys Glu Ala
1160 1165 1170
Lys Glu Trp Leu Asp Asn Ala Arg Gln Ala Cys Leu Lys Ser Gly
1175 1180 1185
Asn Val His Ile Ala Asn Leu Cys Lys Val Ile Ala Pro Ala Pro
1190 1195 1200
Ser Lys Ser Arg Pro Glu Pro Val Val Val Cys Leu Arg Gly Lys
1205 1210 1215
Ser Gly Gln Gly Lys Ser Phe Leu Ala Asn Val Leu Ala Gln Ala
1220 1225 1230
Ile Ser Ser His Phe Thr Gly Arg Thr Asp Ser Val Trp Tyr Cys
1235 1240 1245
Pro Pro Asp Pro Asp His Phe Asp Gly Tyr Asn Gln Gln Thr Val
1250 1255 1260
Val Val Met Asp Asp Leu Gly Gln Asn Pro Asp Gly Lys Asp Phe
1265 1270 1275
Lys Tyr Phe Ala Gln Met Val Ser Thr Thr Gly Phe Ile Pro Pro
1280 1285 1290
Met Ala Ser Leu Glu Asp Lys Gly Lys Pro Phe Asn Ser Lys Val
1295 1300 1305
Ile Ile Ala Thr Thr Asn Leu Tyr Ser Gly Phe Thr Pro Arg Thr
1310 1315 1320
Met Val Cys Pro Asp Ala Leu Asn Arg Arg Phe His Phe Asp Ile
1325 1330 1335
Asp Val Ser Ala Lys Asp Gly Tyr Lys Ile Asp Asn Lys Leu Asp
1340 1345 1350
Ile Val Lys Ala Leu Glu Asp Thr His Thr Asn Pro Val Ala Met
1355 1360 1365
Phe Gln Tyr Asp Cys Ala Leu Leu Asn Gly Met Ala Val Glu Met
1370 1375 1380
Lys Arg Met Gln Gln Asp Met Phe Lys Pro Gln Pro Pro Leu Gln
1385 1390 1395
Asn Val Tyr Gln Leu Val Gln Glu Val Ile Glu Arg Val Glu Leu
1400 1405 1410
His Glu Lys Val Ser Ser His Pro Ile Phe Lys Gln Ile Ser Ile
1415 1420 1425
Pro Ser Gln Lys Ser Val Leu Tyr Phe Leu Ile Glu Lys Gly Gln
1430 1435 1440
His Glu Ala Ala Ile Glu Phe Phe Glu Gly Met Val His Asp Ser
1445 1450 1455
Ile Lys Glu Glu Leu Arg Pro Leu Ile Gln Gln Thr Ser Phe Val
1460 1465 1470
Lys Arg Ala Phe Lys Arg Leu Lys Glu Asn Phe Glu Ile Val Ala
1475 1480 1485
Leu Cys Leu Thr Leu Leu Ala Asn Ile Val Ile Met Leu Arg Glu
1490 1495 1500
Ala Arg Lys Arg Arg Gln Ser Val Asp Asp Ser Leu Asp Asp Asp
1505 1510 1515
Ala Ala Leu Asp Asp Ala Glu Lys Asn Pro Leu Glu Ala Ser Gly
1520 1525 1530
Ala Ser Ala Val Gly Phe Arg Glu Arg Ser Pro Thr Glu Gln Lys
1535 1540 1545
Thr Cys Asp Asp Val Asn Thr Glu Pro Val Val Pro Gly Arg Glu
1550 1555 1560
Gln Pro Arg Ala Glu Gly Pro Tyr Thr Ala Ala Leu Glu Arg Gln
1565 1570 1575
Lys Pro Leu Lys Val Lys Ala Arg Leu Pro Gln Gln Glu Gly Pro
1580 1585 1590
Tyr Thr Ala Ala Met Glu Arg Gln Lys Pro Leu Lys Val Lys Ala
1595 1600 1605
Lys Ala Pro Val Val Lys Glu Gly Pro Tyr Glu Gly Pro Val Lys
1610 1615 1620
Lys Pro Val Ala Leu Lys Val Arg Ala Lys Asn Leu Ile Val Thr
1625 1630 1635
Glu Ser Gly Ala Pro Pro Thr Asp Leu Gln Lys Met Val Met Gly
1640 1645 1650
Asn Thr Lys Pro Val Glu Leu Ile Leu Asp Gly Lys Thr Val Ala
1655 1660 1665
Ile Cys Cys Ala Thr Gly Val Phe Gly Thr Ala Tyr Leu Val Pro
1670 1675 1680
Arg His Leu Phe Ala Glu Arg Tyr Asp Lys Ile Met Leu Asp Gly
1685 1690 1695
Arg Ala Leu Thr Asp Ser Asp Tyr Arg Val Phe Glu Phe Glu Ile
1700 1705 1710
Lys Val Lys Gly Gln Asp Met Leu Ser Asp Ala Ala Leu Met Val
1715 1720 1725
Leu His Arg Gly Asn Arg Val Arg Asp Ile Thr Lys His Phe Arg
1730 1735 1740
Asp Val Ala Arg Met Lys Lys Gly Thr Pro Val Val Gly Val Ile
1745 1750 1755
Asn Asn Ala Asp Val Gly Arg Leu Ile Phe Ser Gly Glu Ala Leu
1760 1765 1770
Thr Tyr Lys Asp Ile Val Val Cys Met Asp Gly Asp Thr Met Pro
1775 1780 1785
Gly Leu Phe Ala Tyr Arg Ala Ser Thr Lys Ala Gly Tyr Cys Gly
1790 1795 1800
Gly Ala Val Leu Ala Lys Asp Gly Ala Glu Thr Phe Ile Val Gly
1805 1810 1815
Thr His Ser Ala Gly Gly Asn Gly Val Gly Tyr Cys Ser Cys Val
1820 1825 1830
Ser Arg Ser Met Leu Leu Lys Met Lys Ala His Ile Asp Pro Glu
1835 1840 1845
Pro His His Glu Gly Leu Ile Val Asp Thr Arg Asp Val Glu Glu
1850 1855 1860
Arg Val His Val Met Arg Lys Thr Lys Leu Ala Pro Thr Val Ala
1865 1870 1875
His Gly Val Phe Asn Pro Glu Phe Gly Pro Ala Ala Leu Ser Asn
1880 1885 1890
Lys Asp Pro Arg Leu Asn Glu Gly Val Val Leu Asp Asp Val Ile
1895 1900 1905
Phe Ser Lys His Lys Gly Asp Thr Lys Met Ser Glu Glu Asp Lys
1910 1915 1920
Val Leu Phe Arg Arg Cys Ala Ala Asp Tyr Ala Ser Arg Leu His
1925 1930 1935
Ser Val Leu Gly Thr Ala Asn Ala Pro Leu Ser Ile Tyr Glu Ala
1940 1945 1950
Ile Lys Gly Val Asp Gly Leu Asp Ala Met Glu Pro Asp Thr Ala
1955 1960 1965
Pro Gly Leu Pro Trp Ala Leu Gln Gly Lys Arg Arg Gly Ala Leu
1970 1975 1980
Ile Asp Phe Glu Asn Gly Thr Val Gly Pro Glu Val Glu Ala Ala
1985 1990 1995
Leu Lys Leu Met Glu Arg Arg Glu Tyr Lys Phe Val Cys Gln Thr
2000 2005 2010
Phe Leu Lys Asp Glu Ile Arg Pro Leu Glu Lys Val Arg Ala Gly
2015 2020 2025
Lys Thr Arg Ile Val Asp Val Leu Pro Val Glu His Ile Leu Tyr
2030 2035 2040
Thr Arg Met Met Ile Gly Arg Phe Cys Ala Gln Met His Ser Asn
2045 2050 2055
Asn Gly Pro Gln Ile Gly Ser Ala Val Gly Cys Asn Pro Asp Val
2060 2065 2070
Asp Trp Gln Arg Phe Gly Thr His Phe Ala Gln Tyr Lys Asn Val
2075 2080 2085
Trp Asp Val Asp Tyr Ser Ala Phe Asp Ala Asn His Cys Ser Asp
2090 2095 2100
Ala Met Asn Ile Met Phe Glu Glu Val Phe Arg Thr Glu Phe Gly
2105 2110 2115
Phe His Pro Asn Ala Glu Trp Ile Leu Lys Thr Leu Val Asn Thr
2120 2125 2130
Glu His Ala Tyr Glu Asn Lys Arg Ile Thr Val Glu Gly Gly Met
2135 2140 2145
Pro Ser Gly Cys Ser Ala Thr Ser Ile Ile Asn Thr Ile Leu Asn
2150 2155 2160
Asn Ile Tyr Val Leu Tyr Ala Leu Arg Arg His Tyr Glu Gly Val
2165 2170 2175
Glu Leu Asp Thr Tyr Thr Met Ile Ser Tyr Gly Asp Asp Ile Val
2180 2185 2190
Val Ala Ser Asp Tyr Asp Leu Asp Phe Glu Ala Leu Lys Pro His
2195 2200 2205
Phe Lys Ser Leu Gly Gln Thr Ile Thr Pro Ala Asp Lys Ser Asp
2210 2215 2220
Lys Gly Phe Val Leu Gly His Ser Ile Thr Asp Val Thr Phe Leu
2225 2230 2235
Lys Arg His Phe His Met Asp Tyr Gly Thr Gly Phe Tyr Lys Pro
2240 2245 2250
Val Met Ala Ser Lys Thr Leu Glu Ala Ile Leu Ser Phe Ala Arg
2255 2260 2265
Arg Gly Thr Ile Gln Glu Lys Leu Ile Ser Val Ala Gly Leu Ala
2270 2275 2280
Val His Ser Gly Pro Asp Glu Tyr Arg Arg Leu Phe Glu Pro Phe
2285 2290 2295
Gln Gly Leu Phe Glu Ile Pro Ser Tyr Arg Ser Leu Tyr Leu Arg
2300 2305 2310
Trp Val Asn Ala Val Cys Gly Asp Ala
2315 2320
<210> 7
<211> 141
<212> DNA
<213> 重组病毒O/ rV-1/3BM 3B1和3B2蛋白的核苷酸序列
<400> 7
ggaccctaca ccgcggcact cgaacgtcag aaacctctta aagtgaaagc caggttgcca 60
caacaagagg gaccttacac cgctgcgatg gagcggcaga aaccgctgaa agtgaaagca 120
aaagcccccg tcgtgaagga a 141
<210> 8
<211> 47
<212> PRT
<213> 重组病毒O/ rV-1/3BM 3B1和3B2蛋白编码的氨基酸序列
<400> 8
Gly Pro Tyr Thr Ala Ala Leu Glu Arg Gln Lys Pro Leu Lys Val Lys
1 5 10 15
Ala Arg Leu Pro Gln Gln Glu Gly Pro Tyr Thr Ala Ala Met Glu Arg
20 25 30
Gln Lys Pro Leu Lys Val Lys Ala Lys Ala Pro Val Val Lys Glu
35 40 45

Claims (8)

1.3B蛋白优势表位缺失的口蹄疫标记疫苗株，其特征在于：所述口蹄疫标记疫苗株编码蛋白的氨基酸序列如SEQ ID No.6所示。
2.根据权利要求1所述的口蹄疫标记疫苗株，其特征在于：所述口蹄疫标记疫苗株的基因组RNA对应的cDNA的核苷酸序列如SEQ ID No.5所示。
3.3B蛋白优势表位缺失的口蹄疫标记疫苗株的构建方法，其特征在于：步骤如下：合成含口蹄疫疫苗毒株非结构蛋白3B1和3B2 第4～6位氨基酸修饰的片段，将该片段克隆在载体中，得到重组质粒，将重组质粒用内切酶消化，回收修饰的片段，将其插入至用同样内切酶消化的口蹄疫病毒毒株的全长感染性克隆中，得到重组全长质粒；用内切酶将重组全长质粒线化后，转染细胞，拯救获得的病毒，即为3B蛋白优势表位缺失的口蹄疫标记疫苗株，其中，所述口蹄疫标记疫苗株第4～6位氨基酸修饰的非结构蛋白3B1和3B2 蛋白氨基酸序列如SEQ ID No.8所示，所述口蹄疫标记疫苗株的基因组RNA对应的cDNA的核苷酸序列如SEQ ID No.5所示。
4.根据权利要求3所述的构建方法，其特征在于：所述口蹄疫标记疫苗株第4～6位氨基酸修饰的非结构蛋白3B1和3B2 蛋白氨基酸序列对应的核苷酸序列如SEQ ID No.7所示。
5.根据权利要求3所述的构建方法，其特征在于：所述口蹄疫标记疫苗株为O型口蹄疫苗株。
6.根据权利要求1或2所述口蹄疫标记疫苗株在制备口蹄疫标记疫苗中的应用。
7.根据权利要求6所述的应用，其特征在于，所述口蹄疫标记疫苗为O型口蹄疫标记疫苗。
8.一种疫苗，其特征在于，其活性成分为权利要求1-2任一所述的口蹄疫标记疫苗株。
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