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与流程

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-mouthdisease,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。

技术实现要素：

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(westernblot，wb)和免疫荧光(indirectimmunofluorescence，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seqidno.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seqidno.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seqidno.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seqidno.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seqidno.8所示。

作为优选，对应的核苷酸序列如seqidno.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seqidno.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bglii/noti酶切结果；右侧泳道为dl12000dnamarker。

图3为重组质粒pofs/3bm3b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/93-083b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westernblot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/93-083b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seqidno.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seqidno.2，其中1057～8022的核苷酸编码的病毒蛋白序列如序列表seqidno.2，口蹄疫疫苗毒株o/zk/93-083b1和3b2的核苷酸序列见seqidno.3，口蹄疫疫苗毒株o/zk/93-083b1和3b2蛋白编码的氨基酸序列见seqidno.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bglii和noti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bglii和noti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qiagenplasmidmidikits(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lipofectamine^tm2000(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(cytopathogeniceffct,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rnaasyminikit(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seqidno.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seqidno.6，重组病毒3b1和3b2蛋白的核苷酸序列如序列表seqidno.7，重组病毒3b1和3b2蛋白编码的氨基酸序列如序列表seqidno.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((indirectimmunofluorescence,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％tritonx-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/mldapi(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。

3.3westernblot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

ttgaaagggggcgctagggtctcacccctggcatgccaacgacagctcctgcgtcgcacc60

ccgcacttacgtctctgcaagcgcaggaaccgatggactatcgttcacccacctacagct120

ggactcacgacaccgcgtggccactttagctggattgtgcggacgaacatcgcttgcgca180

tttcgcgtgaccggttagtactcttaccactctccgcctacttggtcgtcagcgctgtct240

tgggcactcctgttgggggctgtccgacgctccacggtctcctccgttttaggaccacgg300

tgttggggccgccacgtgcgagccgctcgcctggtgtgcttcggctgtcacccgaagccc360

gcctttcaccccccccccccccccaaccagtaccgtcgttcccgacgtttaaagggaggt420

aaccacaagcttgcactaccactcccggtgtcaacgggatgtgaccgcaagatgagcctt480

cacccggaagtaaaacggcaacttcacacagttttgcccgttttcatgagaaacgggacg540

tccgcgcacgaaacgcgccgtcgcttgaggaagacttgtacaaacacggtctaagcaggt600

ttccacaactgacaaaaaacccgtgcagcttgaaaccccgcctggtctttccaggtctag660

aggggtgacactttgtactgtgctcgactccacgctcggtccactggcgggtgttagtag720

cagcactgttgcttcgtagcggagcatggtggccgtgggaactcctccttggtaacaagg780

acccacggggccaaaagccacgtccagacggacccaccatgtgtgcaaccccagcacggc840

aactttactgtgaacaccaccttaaggtgacactgatactggtactcggtcactggtgac900

aggctaaggatgcccttcaggtaccccgaggtaacacgagacactcgggatctgagaagg960

ggattgggacttctttaaagtgcccagtttaaaaagcttctatgcctgaataggcgaccg1020

gaggccggcgcctttctattgaccaccaccaaatccatgaacacgactgactgttttatc1080

gctctgttacacgttctcagggagattaaagcactgtttctgtcacgaacacaagggaaa1140

atggaattcacacttcacaacggtgaaaagaaggtcttctacgccagacccaacaaccac1200

gacaattgctggttgaacgccatcctccaactgttcaggtacgtcgacgaacccttcttc1260

gactgggtctacgactcacctgagaaccttactcttgaggcgatcaggcgactcgaagaa1320

attactggtcttgagctacacgagggtggaccacccgcccttgtcgtctggaacattaag1380

cacttgctctgcaccggaatcggcaccgcttcgcggcctagcgaggtgtgtatggtggac1440

ggtacagacatgtgcttggccgacttccacgctggtatctttctgaagggacaagaccac1500

gccgtattcgcctgtgtcacctccgacgggtggtacgcgattgacgacgaggatttttac1560

ccgtggacaccagacccggctgacgttttggtttttgttccgtacgatcaagaaccactt1620

aatggagaatggaaagcaaaggtccagaagcggcttaagggcgccgggcaatccagcccg1680

acgaccgggtcacagaaccaatcaggcaacactggaagcatcattaacaactactacatg1740

cagcaataccagaactccatggacacacagcttggtgacaacgccattagcggaggctcc1800

aacgagggttctacggataccacctccacccacacgaacaacacccagaacaacgactgg1860

ttttcaaaactggccaactccgctctcagcggtctcttcggtgctcttctcgccgacaaa1920

aagacagaggaaactaccctcctcgaggaccgcattctcaccacccgcaacggacacacg1980

acctcgacaacccagtcgagcgtcggggtgacgtacgggtatgcaacagctgaggacttc2040

gtgagcgggcccaacacctctggtcttgagaccagggttgtccaggccgaacggttcttc2100

aaaacccacttgttcgactgggtcaccagtgacccgtttggacggtgccacatgttggag2160

ctcccgactgaccacaaaggcgtctacggcagcctaaccgactcgtacgcgtatatgagg2220

aacggttgggacgttgaagtcaccgcggtgggaaaccagttcaacggaggctgcttgttg2280

gtggcaatggtaccagagctttgttccatcaacaagagagagctgtaccagctcacactt2340

ttcccccaccagttcattaacccacggacgaacatgacggcacacatcactgtgccctac2400

gttggcgtcaacaggtacgaccaatacaaggtgcataaaccctggacccttgttgtcatg2460

gtcgtggcccccttgacggtcaacaatgagggtgctccgcaaatcaaggtgtatgccaac2520

atcgcccccaccaacgtttacgttgcgggtgaattcccttccaaggaggggatcttcccc2580

gtggcatgcagcgacggttacggcggtttggtgaccacggacccaaagacggcggacccc2640

gtgtacgggaaagtgttcaaccccccccgtaacttgttgccagggcggtttacaaacctc2700

cttgatgtggccgaggcgtgtcccacgttcctacacttcgaaggtgacgtaccgtacgtg2760

accacgaagacggactcagacagggtgttggcccaattcgacctgtctctggcagcaaag2820

cacatgtcgaacactttcctcgcgggtcttgcccagtattacacacagtacagcggcacc2880

atcaacctacacttcatgttcacagggcccaccgatgcgaaggcgcgctacatgattgcg2940

tatgcccctcctggcatggaaccgccgaaaacgcctgaggccgccgcacactgcattcac3000

gctgagtgggacacagggctgaattcaaagttcacattttcaattccctacctttcggcc3060

gctgactacgcgtacaccgcgtccgacgtcgccgaaaccacaaacgtgcagggatgggtc3120

tgcttgttccagataacacacgggaaagccgacggcgatgctctgattgtgctagctagt3180

gctggcaaagactttgacctacgcctaccggttgacgcccgcacgcagaccacctctgcg3240

ggcgagtccgcggaccccgttaccgccaccgttgagaattacggtggtgagacacaggtc3300

cagagacgccagcacacggatatctcgtttatactagacagatttgtgaaagtcacacca3360

aaagaccaaatcaatgtgctggacctgatgcagatccctgcccacactttagtaggggcc3420

ctcctgcggacggccacctactacttctccgacttggagttggctgtcaaacacaagggt3480

gatctcacctgggttccgaacggggcccctgagacagctttggacaacaccaccaaccca3540

acagcttaccacaaagcaccactcacgcgactggccttgccttacacggccccacaccgc3600

gtcttagcgaccgtctacaacggaagttgtaagtacagtggcgcccgcgtgagcaacgtg3660

aggggtgaccttcaagtgttggctcagaaggcagaaagagctctgcccacctcctttaac3720

tatggtgccattaaggcaacccgggtgactgagttactctaccgaatgaagagagccgag3780

acatactgccccaggccccttcttgccattcaaccgagtgacgctagacacaagcagaag3840

atcgtggcacccgcaaaacagcttctgaacttcgacctcctcaagctggcgggagacgtc3900

gagtccaaccctggacccttcttcttctccgacgtcaggtcgaacttcgcaaaactggtg3960

gacaccatcaaccagatgcaggaggatatgtcaacaaagcacggacccgactttaatcga4020

ctggtgtccgcgtttgaggaactggccactggagtgaaggccatcaggactggtctcgac4080

gaggccaagccctggtacaagctcatcaaactcctaagccgcttgtcgtgcatggccgct4140

gtagcagcacggtctaaggacccagtccttgtggccatcatgctagctgacaccggtctt4200

gagattctggacagcacctttgtagtgaagaagatatctgactcgctctccagtctcttc4260

cacgtgccggcccccgccttcagtttcggagccccgatcctattggctgggttggtcaaa4320

gtcgcctcgagtttcttccggtctacgcccgaagaccttgagagagcagaaaagcagctc4380

aaagcacgtgacattaacgatatatttgccattctcaagaatggtgagtggttggtcaag4440

ctgatcctcgccatccgcgactggattaaggcgtggatcgcctctgaagaaaagtttgtc4500

actatgacagacttggtacctggtatccttgaaaaacagcgggatctcaacgaccccggc4560

aagtacaaggaggccaaggagtggcttgacaacgcgcgtcaagcgtgtttgaagagcggg4620

aacgttcacattgccaacctgtgcaaagtgatcgctccagcgcccagcaagtcgagacct4680

gaaccagtggtcgtttgcctccgcggcaaatctggccaggggaaaagtttccttgcgaac4740

gtgctcgcgcaagcaatttcctcacacttcactggcaggaccgactcggtctggtactgc4800

ccgcccgatcctgaccacttcgacggttacaatcagcagaccgttgttgtgatggacgac4860

ttgggccaaaaccctgacggcaaggatttcaagtactttgcccaaatggtttcaaccacg4920

gggttcatcccgcccatggcctcgcttgaggacaaaggcaaacctttcaacagcaaagtc4980

atcatagccactaccaacttgtactcgggtttcaccccgagaaccatggtgtgtcccgac5040

gcactgaaccggaggttccactttgacatcgatgtgagcgccaaggacggatacaagatc5100

gacaacaaactggacatagtcaaagccctcgaggacacccacactaacccagtggcgatg5160

ttccaatacgattgcgcccttctcaacggcatggctgttgaaatgaagagaatgcaacaa5220

gacatgttcaagcctcagccacctcttcagaacgtctaccaacttgttcaagaagtgatt5280

gagcgggtggaactccacgaaaaggtgtcgagtcacccgatttttaaacagatctcaatc5340

ccttcccaaaagtctgtgctgtactttctcattgagaaagggcagcacgaagcagcaatc5400

gagttctttgaggggatggttcacgattctatcaaggaggagctccgacccctcattcaa5460

cagacctcatttgtgaagcgcgccttcaagcgcctgaaggagaattttgagattgtagcc5520

ctgtgtttaaccctcttggcaaacatagtgatcatgctacgcgaagcgcgcaagaggcgc5580

cagtcagtggatgactcactggatgacgacgcggctcttgacgatgcggaaaagaaccct5640

ctagaggcgagtggcgccagcgccgttggtttcagagagagatcccccaccgagcaaaag5700

acgtgcgacgacgtgaacactgagcccgttgtgcccgggagggaacaaccgcgagctgaa5760

ggaccctacgccgggccactcgaacgtcagaaacctcttaaagtgaaagccaggttgcca5820

caacaagagggaccttacgccggtccgatggagcggcagaaaccgctgaaagtgaaagca5880

aaagcccccgtcgtgaaggaaggaccctacgaggggccggtgaaaaagcctgtcgctttg5940

aaagtgagagcaaagaacttgatcgtcactgagagtggagcaccaccgaccgacttgcaa6000

aagatggtcatgggcaacactaaacccgtcgagctcatcctcgatggcaagacggtggct6060

atctgctgtgctactggagtgtttggcactgcctacctcgtgcctcgtcatctcttcgca6120

gagaggtatgacaagatcatgttggacggcagagccttgacagacagtgactacagagtg6180

tttgagtttgagattaaagtaaaaggacaggacatgctctcagacgctgctctcatggtg6240

ttacaccgtgggaaccgcgtgcgagatatcacgaagcattttcgcgatgtagcgagaatg6300

aagaagggaacccccgtcgtcggcgtgatcaacaacgctgatgtcgggagactcatattc6360

tctggtgaagccctcacttacaaggacattgtcgtgtgcatggacggagacaccatgcct6420

gggctctttgcctacagagcttccaccaaggcgggctactgtggaggagccgtcctggca6480

aaagacggggccgagacgttcatcgtcggcacccactctgcaggtggcaacggtgtggga6540

tattgttcatgcgtttcccgctcaatgcttctgaaaatgaaggcacacatcgatcccgaa6600

ccacaccacgaggggttgattgtcgacaccagagacgtggaagagcgcgtgcacgtgatg6660

cgcaaaaccaagctcgcgcccaccgtagcgcacggtgtgttcaaccccgaattcgggcct6720

gccgctctgtccaacaaggacccacgcctgaatgagggggttgtcctcgacgatgtcatt6780

ttctccaaacacaaaggagacacaaagatgtctgaagaggacaaagtgctgttccggcgc6840

tgtgctgctgactacgcgtcacgcttacacagcgtgttggggacggcaaatgccccactg6900

agcatttacgaggctatcaaaggcgtcgacggactcgacgccatggaaccggataccgcg6960

cccggtctcccctgggctctccaggggaaacgccgcggtgccctgatcgactttgaaaac7020

ggcaccgtcgggcccgaggtcgaggcagccctcaagctcatggagagacgtgagtacaag7080

ttcgtctgccagaccttcctgaaggacgagattcgcccgctggagaaggtgcgcgctggc7140

aagacacgcattgtcgacgtcctgcctgttgaacacatcctctacaccaggatgatgatt7200

ggtagattctgcgcccaaatgcactcaaacaacggaccgcaaattggctcggcggtcggt7260

tgcaaccctgacgttgattggcaaagatttggcacacatttcgcccagtacaaaaacgtg7320

tgggatgtggactattcggcctttgatgctaaccactgcagtgatgcgatgaacatcatg7380

ttcgaggaggtgttccgcacggagtttggcttccacccgaacgccgagtggattctgaag7440

actctagtgaacacggagcacgcctatgagaacaagcgtatcaccgtcgagggtggaatg7500

ccatctggttgttccgcaacaagcattatcaacacaattttgaacaacatctacgtgctc7560

tacgccctgcgcagacactatgagggagtcgagctggacacttacaccatgatctcctac7620

ggagacgacatcgtggtggcgagtgattacgacctggactttgaggcccttaagcctcac7680

ttcaagtcccttggtcaaaccattactccagccgacaaaagcgacaaaggttttgttctt7740

ggtcactccattaccgatgtcactttcctcaaaagacacttccacatggattacggaact7800

gggttttacaaacctgtgatggcctcgaagaccctcgaggccatcctctcctttgcacgc7860

cgtgggaccatacaggagaagttgatctccgtggcaggactcgccgtccattctggaccc7920

gacgagtaccggcgtctctttgagcccttccaaggcctctttgagattccaagctacaga7980

tcactttacctgcgttgggtgaacgccgtgtgcggtgacgcataatccctcagatgtcac8040

tactggcaaaaagaccctgaggcgcgcgacgccgtaggagtgaaaaaccgcaaaggtttt8100

tcccacttcctatttcaaaaaaaaaaaaaaaaaaaaa8137

<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

metasnthrthraspcyspheilealaleuleuhisvalleuargglu

151015

ilelysalaleupheleuserargthrglnglylysmetgluphethr

202530

leuhisasnglyglulyslysvalphetyralaargproasnasnhis

354045

aspasncystrpleuasnalaileleuglnleupheargtyrvalasp

505560

gluprophepheasptrpvaltyraspserprogluasnleuthrleu

65707580

glualaileargargleuglugluilethrglyleugluleuhisglu

859095

glyglyproproalaleuvalvaltrpasnilelyshisleuleucys

100105110

thrglyileglythralaserargprosergluvalcysmetvalasp

115120125

glythraspmetcysleualaaspphehisalaglyilepheleulys

130135140

glyglnasphisalavalphealacysvalthrseraspglytrptyr

145150155160

alaileaspaspgluaspphetyrprotrpthrproaspproalaasp

165170175

valleuvalphevalprotyraspglngluproleuasnglyglutrp

180185190

lysalalysvalglnlysargleulysglyalaglyglnserserpro

195200205

thrthrglyserglnasnglnserglyasnthrglyserileileasn

210215220

asntyrtyrmetglnglntyrglnasnsermetaspthrglnleugly

225230235240

aspasnalaileserglyglyserasngluglyserthraspthrthr

245250255

serthrhisthrasnasnthrglnasnasnasptrppheserlysleu

260265270

alaasnseralaleuserglyleupheglyalaleuleualaasplys

275280285

lysthrglugluthrthrleuleugluaspargileleuthrthrarg

290295300

asnglyhisthrthrserthrthrglnserservalglyvalthrtyr

305310315320

glytyralathralagluaspphevalserglyproasnthrsergly

325330335

leugluthrargvalvalglnalagluargphephelysthrhisleu

340345350

pheasptrpvalthrserasppropheglyargcyshismetleuglu

355360365

leuprothrasphislysglyvaltyrglyserleuthraspsertyr

370375380

alatyrmetargasnglytrpaspvalgluvalthralavalglyasn

385390395400

glnpheasnglyglycysleuleuvalalametvalprogluleucys

405410415

serileasnlysarggluleutyrglnleuthrleupheprohisgln

420425430

pheileasnproargthrasnmetthralahisilethrvalprotyr

435440445

valglyvalasnargtyraspglntyrlysvalhislysprotrpthr

450455460

leuvalvalmetvalvalalaproleuthrvalasnasngluglyala

465470475480

proglnilelysvaltyralaasnilealaprothrasnvaltyrval

485490495

alaglyglupheproserlysgluglyilepheprovalalacysser

500505510

aspglytyrglyglyleuvalthrthraspprolysthralaasppro

515520525

valtyrglylysvalpheasnproproargasnleuleuproglyarg

530535540

phethrasnleuleuaspvalalaglualacysprothrpheleuhis

545550555560

phegluglyaspvalprotyrvalthrthrlysthraspserasparg

565570575

valleualaglnpheaspleuserleualaalalyshismetserasn

580585590

thrpheleualaglyleualaglntyrtyrthrglntyrserglythr

595600605

ileasnleuhisphemetphethrglyprothraspalalysalaarg

610615620

tyrmetilealatyralaproproglymetgluproprolysthrpro

625630635640

glualaalaalahiscysilehisalaglutrpaspthrglyleuasn

645650655

serlysphethrpheserileprotyrleuseralaalaasptyrala

660665670

tyrthralaseraspvalalagluthrthrasnvalglnglytrpval

675680685

cysleupheglnilethrhisglylysalaaspglyaspalaleuile

690695700

valleualaseralaglylysasppheaspleuargleuprovalasp

705710715720

alaargthrglnthrthrseralaglygluseralaaspprovalthr

725730735

alathrvalgluasntyrglyglygluthrglnvalglnargarggln

740745750

histhraspileserpheileleuaspargphevallysvalthrpro

755760765

lysaspglnileasnvalleuaspleumetglnileproalahisthr

770775780

leuvalglyalaleuleuargthralathrtyrtyrpheseraspleu

785790795800

gluleualavallyshislysglyaspleuthrtrpvalproasngly

805810815

alaprogluthralaleuaspasnthrthrasnprothralatyrhis

820825830

lysalaproleuthrargleualaleuprotyrthralaprohisarg

835840845

valleualathrvaltyrasnglysercyslystyrserglyalaarg

850855860

valserasnvalargglyaspleuglnvalleualaglnlysalaglu

865870875880

argalaleuprothrserpheasntyrglyalailelysalathrarg

885890895

valthrgluleuleutyrargmetlysargalagluthrtyrcyspro

900905910

argproleuleualaileglnproseraspalaarghislysglnlys

915920925

ilevalalaproalalysglnleuleuasnpheaspleuleulysleu

930935940

alaglyaspvalgluserasnproglyprophephepheseraspval

945950955960

argserasnphealalysleuvalaspthrileasnglnmetglnglu

965970975

aspmetserthrlyshisglyproasppheasnargleuvalserala

980985990

pheglugluleualathrglyvallysalaileargthrglyleuasp

99510001005

glualalysprotrptyrlysleuilelysleuleuserargleu

101010151020

sercysmetalaalavalalaalaargserlysaspprovalleu

102510301035

valalailemetleualaaspthrglyleugluileleuaspser

104010451050

thrphevalvallyslysileseraspserleuserserleuphe

105510601065

hisvalproalaproalapheserpheglyalaproileleuleu

107010751080

alaglyleuvallysvalalaserserphepheargserthrpro

108510901095

gluaspleugluargalaglulysglnleulysalaargaspile

110011051110

asnaspilephealaileleulysasnglyglutrpleuvallys

111511201125

leuileleualaileargasptrpilelysalatrpilealaser

113011351140

gluglulysphevalthrmetthraspleuvalproglyileleu

114511501155

glulysglnargaspleuasnaspproglylystyrlysgluala

116011651170

lysglutrpleuaspasnalaargglnalacysleulyssergly

117511801185

asnvalhisilealaasnleucyslysvalilealaproalapro

119011951200

serlysserargprogluprovalvalvalcysleuargglylys

120512101215

serglyglnglylysserpheleualaasnvalleualaglnala

122012251230

ileserserhisphethrglyargthraspservaltrptyrcys

123512401245

proproaspproasphispheaspglytyrasnglnglnthrval

125012551260

valvalmetaspaspleuglyglnasnproaspglylysaspphe

126512701275

lystyrphealaglnmetvalserthrthrglypheilepropro

128012851290

metalaserleugluasplysglylyspropheasnserlysval

129513001305

ileilealathrthrasnleutyrserglyphethrproargthr

131013151320

metvalcysproaspalaleuasnargargphehispheaspile

132513301335

aspvalseralalysaspglytyrlysileaspasnlysleuasp

134013451350

ilevallysalaleugluaspthrhisthrasnprovalalamet

135513601365

pheglntyraspcysalaleuleuasnglymetalavalglumet

137013751380

lysargmetglnglnaspmetphelysproglnproproleugln

138513901395

asnvaltyrglnleuvalglngluvalilegluargvalgluleu

140014051410

hisglulysvalserserhisproilephelysglnileserile

141514201425

proserglnlysservalleutyrpheleuileglulysglygln

143014351440

hisglualaalailegluphephegluglymetvalhisaspser

144514501455

ilelysglugluleuargproleuileglnglnthrserpheval

146014651470

lysargalaphelysargleulysgluasnphegluilevalala

147514801485

leucysleuthrleuleualaasnilevalilemetleuargglu

149014951500

alaarglysargargglnservalaspaspserleuaspaspasp

150515101515

alaalaleuaspaspalaglulysasnproleuglualasergly

152015251530

alaseralavalglyphearggluargserprothrgluglnlys

153515401545

thrcysaspaspvalasnthrgluprovalvalproglyargglu

155015551560

glnproargalagluglyprotyralaglyproleugluarggln

156515701575

lysproleulysvallysalaargleuproglnglngluglypro

158015851590

tyralaglyprometgluargglnlysproleulysvallysala

159516001605

lysalaprovalvallysgluglyprotyrgluglyprovallys

161016151620

lysprovalalaleulysvalargalalysasnleuilevalthr

162516301635

gluserglyalaproprothraspleuglnlysmetvalmetgly

164016451650

asnthrlysprovalgluleuileleuaspglylysthrvalala

165516601665

ilecyscysalathrglyvalpheglythralatyrleuvalpro

167016751680

arghisleuphealagluargtyrasplysilemetleuaspgly

168516901695

argalaleuthraspserasptyrargvalphegluphegluile

170017051710

lysvallysglyglnaspmetleuseraspalaalaleumetval

171517201725

leuhisargglyasnargvalargaspilethrlyshisphearg

173017351740

aspvalalaargmetlyslysglythrprovalvalglyvalile

174517501755

asnasnalaaspvalglyargleuilepheserglyglualaleu

176017651770

thrtyrlysaspilevalvalcysmetaspglyaspthrmetpro

177517801785

glyleuphealatyrargalaserthrlysalaglytyrcysgly

179017951800

glyalavalleualalysaspglyalagluthrpheilevalgly

180518101815

thrhisseralaglyglyasnglyvalglytyrcyssercysval

182018251830

serargsermetleuleulysmetlysalahisileaspproglu

183518401845

prohishisgluglyleuilevalaspthrargaspvalgluglu

185018551860

argvalhisvalmetarglysthrlysleualaprothrvalala

186518701875

hisglyvalpheasnproglupheglyproalaalaleuserasn

188018851890

lysaspproargleuasngluglyvalvalleuaspaspvalile

189519001905

pheserlyshislysglyaspthrlysmetserglugluasplys

191019151920

valleupheargargcysalaalaasptyralaserargleuhis

192519301935

servalleuglythralaasnalaproleuseriletyrgluala

194019451950

ilelysglyvalaspglyleuaspalametgluproaspthrala

195519601965

proglyleuprotrpalaleuglnglylysargargglyalaleu

197019751980

ileaspphegluasnglythrvalglyprogluvalglualaala

198519901995

leulysleumetgluargargglutyrlysphevalcysglnthr

200020052010

pheleulysaspgluileargproleuglulysvalargalagly

201520202025

lysthrargilevalaspvalleuprovalgluhisileleutyr

203020352040

thrargmetmetileglyargphecysalaglnmethisserasn

204520502055

asnglyproglnileglyseralavalglycysasnproaspval

206020652070

asptrpglnargpheglythrhisphealaglntyrlysasnval

207520802085

trpaspvalasptyrseralapheaspalaasnhiscysserasp

209020952100

alametasnilemetpheglugluvalpheargthrgluphegly

210521102115

phehisproasnalaglutrpileleulysthrleuvalasnthr

212021252130

gluhisalatyrgluasnlysargilethrvalgluglyglymet

213521402145

proserglycysseralathrserileileasnthrileleuasn

215021552160

asniletyrvalleutyralaleuargarghistyrgluglyval

216521702175

gluleuaspthrtyrthrmetilesertyrglyaspaspileval

218021852190

valalaserasptyraspleuasppheglualaleulysprohis

219522002205

phelysserleuglyglnthrilethrproalaasplysserasp

221022152220

lysglyphevalleuglyhisserilethraspvalthrpheleu

222522302235

lysarghisphehismetasptyrglythrglyphetyrlyspro

224022452250

valmetalaserlysthrleuglualaileleuserphealaarg

225522602265

argglythrileglnglulysleuileservalalaglyleuala

227022752280

valhisserglyproaspglutyrargargleuphegluprophe

228522902295

glnglyleuphegluileprosertyrargserleutyrleuarg

230023052310

trpvalasnalavalcysglyaspala

23152320

<210>3

<211>141

<212>dna

<213>口蹄疫疫苗毒株o/zk/93-083b1和3b2蛋白的核苷酸序列

<400>3

ggaccctacgccgggccactcgaacgtcagaaacctcttaaagtgaaagccaggttgcca60

caacaagagggaccttacgccggtccgatggagcggcagaaaccgctgaaagtgaaagca120

aaagcccccgtcgtgaaggaa141

<210>4

<211>47

<212>prt

<213>口蹄疫疫苗毒株o/zk/93-083b1和3b2蛋白编码的氨基酸序列

<400>4

glyprotyralaglyproleugluargglnlysproleulysvallys

151015

alaargleuproglnglngluglyprotyralaglyprometgluarg

202530

glnlysproleulysvallysalalysalaprovalvallysglu

354045

<210>5

<211>8137

<212>dna

<213>重组病毒o/rv-1/3bm核苷酸序列

<400>5

ttgaaagggggcgctagggtctcacccctggcatgccaacgacagctcctgcgtcgcacc60

ccgcacttacgtctctgcaagcgcaggaaccgatggactatcgttcacccacctacagct120

ggactcacgacaccgcgtggccactttagctggattgtgcggacgaacatcgcttgcgca180

tttcgcgtgaccggttagtactcttaccactctccgcctacttggtcgtcagcgctgtct240

tgggcactcctgttgggggctgtccgacgctccacggtctcctccgttttaggaccacgg300

tgttggggccgccacgtgcgagccgctcgcctggtgtgcttcggctgtcacccgaagccc360

gcctttcaccccccccccccccccaaccagtaccgtcgttcccgacgtttaaagggaggt420

aaccacaagcttgcactaccactcccggtgtcaacgggatgtgaccgcaagatgagcctt480

cacccggaagtaaaacggcaacttcacacagttttgcccgttttcatgagaaacgggacg540

tccgcgcacgaaacgcgccgtcgcttgaggaagacttgtacaaacacggtctaagcaggt600

ttccacaactgacaaaaaacccgtgcagcttgaaaccccgcctggtctttccaggtctag660

aggggtgacactttgtactgtgctcgactccacgctcggtccactggcgggtgttagtag720

cagcactgttgcttcgtagcggagcatggtggccgtgggaactcctccttggtaacaagg780

acccacggggccaaaagccacgtccagacggacccaccatgtgtgcaaccccagcacggc840

aactttactgtgaacaccaccttaaggtgacactgatactggtactcggtcactggtgac900

aggctaaggatgcccttcaggtaccccgaggtaacacgagacactcgggatctgagaagg960

ggattgggacttctttaaagtgcccagtttaaaaagcttctatgcctgaataggcgaccg1020

gaggccggcgcctttctattgaccaccaccaaatccatgaacacgactgactgttttatc1080

gctctgttacacgttctcagggagattaaagcactgtttctgtcacgaacacaagggaaa1140

atggaattcacacttcacaacggtgaaaagaaggtcttctacgccagacccaacaaccac1200

gacaattgctggttgaacgccatcctccaactgttcaggtacgtcgacgaacccttcttc1260

gactgggtctacgactcacctgagaaccttactcttgaggcgatcaggcgactcgaagaa1320

attactggtcttgagctacacgagggtggaccacccgcccttgtcgtctggaacattaag1380

cacttgctctgcaccggaatcggcaccgcttcgcggcctagcgaggtgtgtatggtggac1440

ggtacagacatgtgcttggccgacttccacgctggtatctttctgaagggacaagaccac1500

gccgtattcgcctgtgtcacctccgacgggtggtacgcgattgacgacgaggatttttac1560

ccgtggacaccagacccggctgacgttttggtttttgttccgtacgatcaagaaccactt1620

aatggagaatggaaagcaaaggtccagaagcggcttaagggcgccgggcaatccagcccg1680

acgaccgggtcacagaaccaatcaggcaacactggaagcatcattaacaactactacatg1740

cagcaataccagaactccatggacacacagcttggtgacaacgccattagcggaggctcc1800

aacgagggttctacggataccacctccacccacacgaacaacacccagaacaacgactgg1860

ttttcaaaactggccaactccgctctcagcggtctcttcggtgctcttctcgccgacaaa1920

aagacagaggaaactaccctcctcgaggaccgcattctcaccacccgcaacggacacacg1980

acctcgacaacccagtcgagcgtcggggtgacgtacgggtatgcaacagctgaggacttc2040

gtgagcgggcccaacacctctggtcttgagaccagggttgtccaggccgaacggttcttc2100

aaaacccacttgttcgactgggtcaccagtgacccgtttggacggtgccacatgttggag2160

ctcccgactgaccacaaaggcgtctacggcagcctaaccgactcgtacgcgtatatgagg2220

aacggttgggacgttgaagtcaccgcggtgggaaaccagttcaacggaggctgcttgttg2280

gtggcaatggtaccagagctttgttccatcaacaagagagagctgtaccagctcacactt2340

ttcccccaccagttcattaacccacggacgaacatgacggcacacatcactgtgccctac2400

gttggcgtcaacaggtacgaccaatacaaggtgcataaaccctggacccttgttgtcatg2460

gtcgtggcccccttgacggtcaacaatgagggtgctccgcaaatcaaggtgtatgccaac2520

atcgcccccaccaacgtttacgttgcgggtgaattcccttccaaggaggggatcttcccc2580

gtggcatgcagcgacggttacggcggtttggtgaccacggacccaaagacggcggacccc2640

gtgtacgggaaagtgttcaaccccccccgtaacttgttgccagggcggtttacaaacctc2700

cttgatgtggccgaggcgtgtcccacgttcctacacttcgaaggtgacgtaccgtacgtg2760

accacgaagacggactcagacagggtgttggcccaattcgacctgtctctggcagcaaag2820

cacatgtcgaacactttcctcgcgggtcttgcccagtattacacacagtacagcggcacc2880

atcaacctacacttcatgttcacagggcccaccgatgcgaaggcgcgctacatgattgcg2940

tatgcccctcctggcatggaaccgccgaaaacgcctgaggccgccgcacactgcattcac3000

gctgagtgggacacagggctgaattcaaagttcacattttcaattccctacctttcggcc3060

gctgactacgcgtacaccgcgtccgacgtcgccgaaaccacaaacgtgcagggatgggtc3120

tgcttgttccagataacacacgggaaagccgacggcgatgctctgattgtgctagctagt3180

gctggcaaagactttgacctacgcctaccggttgacgcccgcacgcagaccacctctgcg3240

ggcgagtccgcggaccccgttaccgccaccgttgagaattacggtggtgagacacaggtc3300

cagagacgccagcacacggatatctcgtttatactagacagatttgtgaaagtcacacca3360

aaagaccaaatcaatgtgctggacctgatgcagatccctgcccacactttagtaggggcc3420

ctcctgcggacggccacctactacttctccgacttggagttggctgtcaaacacaagggt3480

gatctcacctgggttccgaacggggcccctgagacagctttggacaacaccaccaaccca3540

acagcttaccacaaagcaccactcacgcgactggccttgccttacacggccccacaccgc3600

gtcttagcgaccgtctacaacggaagttgtaagtacagtggcgcccgcgtgagcaacgtg3660

aggggtgaccttcaagtgttggctcagaaggcagaaagagctctgcccacctcctttaac3720

tatggtgccattaaggcaacccgggtgactgagttactctaccgaatgaagagagccgag3780

acatactgccccaggccccttcttgccattcaaccgagtgacgctagacacaagcagaag3840

atcgtggcacccgcaaaacagcttctgaacttcgacctcctcaagctggcgggagacgtc3900

gagtccaaccctggacccttcttcttctccgacgtcaggtcgaacttcgcaaaactggtg3960

gacaccatcaaccagatgcaggaggatatgtcaacaaagcacggacccgactttaatcga4020

ctggtgtccgcgtttgaggaactggccactggagtgaaggccatcaggactggtctcgac4080

gaggccaagccctggtacaagctcatcaaactcctaagccgcttgtcgtgcatggccgct4140

gtagcagcacggtctaaggacccagtccttgtggccatcatgctagctgacaccggtctt4200

gagattctggacagcacctttgtagtgaagaagatatctgactcgctctccagtctcttc4260

cacgtgccggcccccgccttcagtttcggagccccgatcctattggctgggttggtcaaa4320

gtcgcctcgagtttcttccggtctacgcccgaagaccttgagagagcagaaaagcagctc4380

aaagcacgtgacattaacgatatatttgccattctcaagaatggtgagtggttggtcaag4440

ctgatcctcgccatccgcgactggattaaggcgtggatcgcctctgaagaaaagtttgtc4500

actatgacagacttggtacctggtatccttgaaaaacagcgggatctcaacgaccccggc4560

aagtacaaggaggccaaggagtggcttgacaacgcgcgtcaagcgtgtttgaagagcggg4620

aacgttcacattgccaacctgtgcaaagtgatcgctccagcgcccagcaagtcgagacct4680

gaaccagtggtcgtttgcctccgcggcaaatctggccaggggaaaagtttccttgcgaac4740

gtgctcgcgcaagcaatttcctcacacttcactggcaggaccgactcggtctggtactgc4800

ccgcccgatcctgaccacttcgacggttacaatcagcagaccgttgttgtgatggacgac4860

ttgggccaaaaccctgacggcaaggatttcaagtactttgcccaaatggtttcaaccacg4920

gggttcatcccgcccatggcctcgcttgaggacaaaggcaaacctttcaacagcaaagtc4980

atcatagccactaccaacttgtactcgggtttcaccccgagaaccatggtgtgtcccgac5040

gcactgaaccggaggttccactttgacatcgatgtgagcgccaaggacggatacaagatc5100

gacaacaaactggacatagtcaaagccctcgaggacacccacactaacccagtggcgatg5160

ttccaatacgattgcgcccttctcaacggcatggctgttgaaatgaagagaatgcaacaa5220

gacatgttcaagcctcagccacctcttcagaacgtctaccaacttgttcaagaagtgatt5280

gagcgggtggaactccacgaaaaggtgtcgagtcacccgatttttaaacagatctcaatc5340

ccttcccaaaagtctgtgctgtactttctcattgagaaagggcagcacgaagcagcaatc5400

gagttctttgaggggatggttcacgattctatcaaggaggagctccgacccctcattcaa5460

cagacctcatttgtgaagcgcgccttcaagcgcctgaaggagaattttgagattgtagcc5520

ctgtgtttaaccctcttggcaaacatagtgatcatgctacgcgaagcgcgcaagaggcgc5580

cagtcagtggatgactcactggatgacgacgcggctcttgacgatgcggaaaagaaccct5640

ctagaggcgagtggcgccagcgccgttggtttcagagagagatcccccaccgagcaaaag5700

acgtgcgacgacgtgaacactgagcccgttgtgcccgggagggaacaaccgcgagctgaa5760

ggaccctacaccgcggcactcgaacgtcagaaacctcttaaagtgaaagccaggttgcca5820

caacaagagggaccttacaccgctgcgatggagcggcagaaaccgctgaaagtgaaagca5880

aaagcccccgtcgtgaaggaaggaccctacgaggggccggtgaaaaagcctgtcgctttg5940

aaagtgagagcaaagaacttgatcgtcactgagagtggagcaccaccgaccgacttgcaa6000

aagatggtcatgggcaacactaaacccgtcgagctcatcctcgatggcaagacggtggct6060

atctgctgtgctactggagtgtttggcactgcctacctcgtgcctcgtcatctcttcgca6120

gagaggtatgacaagatcatgttggacggcagagccttgacagacagtgactacagagtg6180

tttgagtttgagattaaagtaaaaggacaggacatgctctcagacgctgctctcatggtg6240

ttacaccgtgggaaccgcgtgcgagatatcacgaagcattttcgcgatgtagcgagaatg6300

aagaagggaacccccgtcgtcggcgtgatcaacaacgctgatgtcgggagactcatattc6360

tctggtgaagccctcacttacaaggacattgtcgtgtgcatggacggagacaccatgcct6420

gggctctttgcctacagagcttccaccaaggcgggctactgtggaggagccgtcctggca6480

aaagacggggccgagacgttcatcgtcggcacccactctgcaggtggcaacggtgtggga6540

tattgttcatgcgtttcccgctcaatgcttctgaaaatgaaggcacacatcgatcccgaa6600

ccacaccacgaggggttgattgtcgacaccagagacgtggaagagcgcgtgcacgtgatg6660

cgcaaaaccaagctcgcgcccaccgtagcgcacggtgtgttcaaccccgaattcgggcct6720

gccgctctgtccaacaaggacccacgcctgaatgagggggttgtcctcgacgatgtcatt6780

ttctccaaacacaaaggagacacaaagatgtctgaagaggacaaagtgctgttccggcgc6840

tgtgctgctgactacgcgtcacgcttacacagcgtgttggggacggcaaatgccccactg6900

agcatttacgaggctatcaaaggcgtcgacggactcgacgccatggaaccggataccgcg6960

cccggtctcccctgggctctccaggggaaacgccgcggtgccctgatcgactttgaaaac7020

ggcaccgtcgggcccgaggtcgaggcagccctcaagctcatggagagacgtgagtacaag7080

ttcgtctgccagaccttcctgaaggacgagattcgcccgctggagaaggtgcgcgctggc7140

aagacacgcattgtcgacgtcctgcctgttgaacacatcctctacaccaggatgatgatt7200

ggtagattctgcgcccaaatgcactcaaacaacggaccgcaaattggctcggcggtcggt7260

tgcaaccctgacgttgattggcaaagatttggcacacatttcgcccagtacaaaaacgtg7320

tgggatgtggactattcggcctttgatgctaaccactgcagtgatgcgatgaacatcatg7380

ttcgaggaggtgttccgcacggagtttggcttccacccgaacgccgagtggattctgaag7440

actctagtgaacacggagcacgcctatgagaacaagcgtatcaccgtcgagggtggaatg7500

ccatctggttgttccgcaacaagcattatcaacacaattttgaacaacatctacgtgctc7560

tacgccctgcgcagacactatgagggagtcgagctggacacttacaccatgatctcctac7620

ggagacgacatcgtggtggcgagtgattacgacctggactttgaggcccttaagcctcac7680

ttcaagtcccttggtcaaaccattactccagccgacaaaagcgacaaaggttttgttctt7740

ggtcactccattaccgatgtcactttcctcaaaagacacttccacatggattacggaact7800

gggttttacaaacctgtgatggcctcgaagaccctcgaggccatcctctcctttgcacgc7860

cgtgggaccatacaggagaagttgatctccgtggcaggactcgccgtccattctggaccc7920

gacgagtaccggcgtctctttgagcccttccaaggcctctttgagattccaagctacaga7980

tcactttacctgcgttgggtgaacgccgtgtgcggtgacgcataatccctcagatgtcac8040

tactggcaaaaagaccctgaggcgcgcgacgccgtaggagtgaaaaaccgcaaaggtttt8100

tcccacttcctatttcaaaaaaaaaaaaaaaaaaaaa8137

<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

metasnthrthraspcyspheilealaleuleuhisvalleuargglu

151015

ilelysalaleupheleuserargthrglnglylysmetgluphethr

202530

leuhisasnglyglulyslysvalphetyralaargproasnasnhis

354045

aspasncystrpleuasnalaileleuglnleupheargtyrvalasp

505560

gluprophepheasptrpvaltyraspserprogluasnleuthrleu

65707580

glualaileargargleuglugluilethrglyleugluleuhisglu

859095

glyglyproproalaleuvalvaltrpasnilelyshisleuleucys

100105110

thrglyileglythralaserargprosergluvalcysmetvalasp

115120125

glythraspmetcysleualaaspphehisalaglyilepheleulys

130135140

glyglnasphisalavalphealacysvalthrseraspglytrptyr

145150155160

alaileaspaspgluaspphetyrprotrpthrproaspproalaasp

165170175

valleuvalphevalprotyraspglngluproleuasnglyglutrp

180185190

lysalalysvalglnlysargleulysglyalaglyglnserserpro

195200205

thrthrglyserglnasnglnserglyasnthrglyserileileasn

210215220

asntyrtyrmetglnglntyrglnasnsermetaspthrglnleugly

225230235240

aspasnalaileserglyglyserasngluglyserthraspthrthr

245250255

serthrhisthrasnasnthrglnasnasnasptrppheserlysleu

260265270

alaasnseralaleuserglyleupheglyalaleuleualaasplys

275280285

lysthrglugluthrthrleuleugluaspargileleuthrthrarg

290295300

asnglyhisthrthrserthrthrglnserservalglyvalthrtyr

305310315320

glytyralathralagluaspphevalserglyproasnthrsergly

325330335

leugluthrargvalvalglnalagluargphephelysthrhisleu

340345350

pheasptrpvalthrserasppropheglyargcyshismetleuglu

355360365

leuprothrasphislysglyvaltyrglyserleuthraspsertyr

370375380

alatyrmetargasnglytrpaspvalgluvalthralavalglyasn

385390395400

glnpheasnglyglycysleuleuvalalametvalprogluleucys

405410415

serileasnlysarggluleutyrglnleuthrleupheprohisgln

420425430

pheileasnproargthrasnmetthralahisilethrvalprotyr

435440445

valglyvalasnargtyraspglntyrlysvalhislysprotrpthr

450455460

leuvalvalmetvalvalalaproleuthrvalasnasngluglyala

465470475480

proglnilelysvaltyralaasnilealaprothrasnvaltyrval

485490495

alaglyglupheproserlysgluglyilepheprovalalacysser

500505510

aspglytyrglyglyleuvalthrthraspprolysthralaasppro

515520525

valtyrglylysvalpheasnproproargasnleuleuproglyarg

530535540

phethrasnleuleuaspvalalaglualacysprothrpheleuhis

545550555560

phegluglyaspvalprotyrvalthrthrlysthraspserasparg

565570575

valleualaglnpheaspleuserleualaalalyshismetserasn

580585590

thrpheleualaglyleualaglntyrtyrthrglntyrserglythr

595600605

ileasnleuhisphemetphethrglyprothraspalalysalaarg

610615620

tyrmetilealatyralaproproglymetgluproprolysthrpro

625630635640

glualaalaalahiscysilehisalaglutrpaspthrglyleuasn

645650655

serlysphethrpheserileprotyrleuseralaalaasptyrala

660665670

tyrthralaseraspvalalagluthrthrasnvalglnglytrpval

675680685

cysleupheglnilethrhisglylysalaaspglyaspalaleuile

690695700

valleualaseralaglylysasppheaspleuargleuprovalasp

705710715720

alaargthrglnthrthrseralaglygluseralaaspprovalthr

725730735

alathrvalgluasntyrglyglygluthrglnvalglnargarggln

740745750

histhraspileserpheileleuaspargphevallysvalthrpro

755760765

lysaspglnileasnvalleuaspleumetglnileproalahisthr

770775780

leuvalglyalaleuleuargthralathrtyrtyrpheseraspleu

785790795800

gluleualavallyshislysglyaspleuthrtrpvalproasngly

805810815

alaprogluthralaleuaspasnthrthrasnprothralatyrhis

820825830

lysalaproleuthrargleualaleuprotyrthralaprohisarg

835840845

valleualathrvaltyrasnglysercyslystyrserglyalaarg

850855860

valserasnvalargglyaspleuglnvalleualaglnlysalaglu

865870875880

argalaleuprothrserpheasntyrglyalailelysalathrarg

885890895

valthrgluleuleutyrargmetlysargalagluthrtyrcyspro

900905910

argproleuleualaileglnproseraspalaarghislysglnlys

915920925

ilevalalaproalalysglnleuleuasnpheaspleuleulysleu

930935940

alaglyaspvalgluserasnproglyprophephepheseraspval

945950955960

argserasnphealalysleuvalaspthrileasnglnmetglnglu

965970975

aspmetserthrlyshisglyproasppheasnargleuvalserala

980985990

pheglugluleualathrglyvallysalaileargthrglyleuasp

99510001005

glualalysprotrptyrlysleuilelysleuleuserargleu

101010151020

sercysmetalaalavalalaalaargserlysaspprovalleu

102510301035

valalailemetleualaaspthrglyleugluileleuaspser

104010451050

thrphevalvallyslysileseraspserleuserserleuphe

105510601065

hisvalproalaproalapheserpheglyalaproileleuleu

107010751080

alaglyleuvallysvalalaserserphepheargserthrpro

108510901095

gluaspleugluargalaglulysglnleulysalaargaspile

110011051110

asnaspilephealaileleulysasnglyglutrpleuvallys

111511201125

leuileleualaileargasptrpilelysalatrpilealaser

113011351140

gluglulysphevalthrmetthraspleuvalproglyileleu

114511501155

glulysglnargaspleuasnaspproglylystyrlysgluala

116011651170

lysglutrpleuaspasnalaargglnalacysleulyssergly

117511801185

asnvalhisilealaasnleucyslysvalilealaproalapro

119011951200

serlysserargprogluprovalvalvalcysleuargglylys

120512101215

serglyglnglylysserpheleualaasnvalleualaglnala

122012251230

ileserserhisphethrglyargthraspservaltrptyrcys

123512401245

proproaspproasphispheaspglytyrasnglnglnthrval

125012551260

valvalmetaspaspleuglyglnasnproaspglylysaspphe

126512701275

lystyrphealaglnmetvalserthrthrglypheilepropro

128012851290

metalaserleugluasplysglylyspropheasnserlysval

129513001305

ileilealathrthrasnleutyrserglyphethrproargthr

131013151320

metvalcysproaspalaleuasnargargphehispheaspile

132513301335

aspvalseralalysaspglytyrlysileaspasnlysleuasp

134013451350

ilevallysalaleugluaspthrhisthrasnprovalalamet

135513601365

pheglntyraspcysalaleuleuasnglymetalavalglumet

137013751380

lysargmetglnglnaspmetphelysproglnproproleugln

138513901395

asnvaltyrglnleuvalglngluvalilegluargvalgluleu

140014051410

hisglulysvalserserhisproilephelysglnileserile

141514201425

proserglnlysservalleutyrpheleuileglulysglygln

143014351440

hisglualaalailegluphephegluglymetvalhisaspser

144514501455

ilelysglugluleuargproleuileglnglnthrserpheval

146014651470

lysargalaphelysargleulysgluasnphegluilevalala

147514801485

leucysleuthrleuleualaasnilevalilemetleuargglu

149014951500

alaarglysargargglnservalaspaspserleuaspaspasp

150515101515

alaalaleuaspaspalaglulysasnproleuglualasergly

152015251530

alaseralavalglyphearggluargserprothrgluglnlys

153515401545

thrcysaspaspvalasnthrgluprovalvalproglyargglu

155015551560

glnproargalagluglyprotyrthralaalaleugluarggln

156515701575

lysproleulysvallysalaargleuproglnglngluglypro

158015851590

tyrthralaalametgluargglnlysproleulysvallysala

159516001605

lysalaprovalvallysgluglyprotyrgluglyprovallys

161016151620

lysprovalalaleulysvalargalalysasnleuilevalthr

162516301635

gluserglyalaproprothraspleuglnlysmetvalmetgly

164016451650

asnthrlysprovalgluleuileleuaspglylysthrvalala

165516601665

ilecyscysalathrglyvalpheglythralatyrleuvalpro

167016751680

arghisleuphealagluargtyrasplysilemetleuaspgly

168516901695

argalaleuthraspserasptyrargvalphegluphegluile

170017051710

lysvallysglyglnaspmetleuseraspalaalaleumetval

171517201725

leuhisargglyasnargvalargaspilethrlyshisphearg

173017351740

aspvalalaargmetlyslysglythrprovalvalglyvalile

174517501755

asnasnalaaspvalglyargleuilepheserglyglualaleu

176017651770

thrtyrlysaspilevalvalcysmetaspglyaspthrmetpro

177517801785

glyleuphealatyrargalaserthrlysalaglytyrcysgly

179017951800

glyalavalleualalysaspglyalagluthrpheilevalgly

180518101815

thrhisseralaglyglyasnglyvalglytyrcyssercysval

182018251830

serargsermetleuleulysmetlysalahisileaspproglu

183518401845

prohishisgluglyleuilevalaspthrargaspvalgluglu

185018551860

argvalhisvalmetarglysthrlysleualaprothrvalala

186518701875

hisglyvalpheasnproglupheglyproalaalaleuserasn

188018851890

lysaspproargleuasngluglyvalvalleuaspaspvalile

189519001905

pheserlyshislysglyaspthrlysmetserglugluasplys

191019151920

valleupheargargcysalaalaasptyralaserargleuhis

192519301935

servalleuglythralaasnalaproleuseriletyrgluala

194019451950

ilelysglyvalaspglyleuaspalametgluproaspthrala

195519601965

proglyleuprotrpalaleuglnglylysargargglyalaleu

197019751980

ileaspphegluasnglythrvalglyprogluvalglualaala

198519901995

leulysleumetgluargargglutyrlysphevalcysglnthr

200020052010

pheleulysaspgluileargproleuglulysvalargalagly

201520202025

lysthrargilevalaspvalleuprovalgluhisileleutyr

203020352040

thrargmetmetileglyargphecysalaglnmethisserasn

204520502055

asnglyproglnileglyseralavalglycysasnproaspval

206020652070

asptrpglnargpheglythrhisphealaglntyrlysasnval

207520802085

trpaspvalasptyrseralapheaspalaasnhiscysserasp

209020952100

alametasnilemetpheglugluvalpheargthrgluphegly

210521102115

phehisproasnalaglutrpileleulysthrleuvalasnthr

212021252130

gluhisalatyrgluasnlysargilethrvalgluglyglymet

213521402145

proserglycysseralathrserileileasnthrileleuasn

215021552160

asniletyrvalleutyralaleuargarghistyrgluglyval

216521702175

gluleuaspthrtyrthrmetilesertyrglyaspaspileval

218021852190

valalaserasptyraspleuasppheglualaleulysprohis

219522002205

phelysserleuglyglnthrilethrproalaasplysserasp

221022152220

lysglyphevalleuglyhisserilethraspvalthrpheleu

222522302235

lysarghisphehismetasptyrglythrglyphetyrlyspro

224022452250

valmetalaserlysthrleuglualaileleuserphealaarg

225522602265

argglythrileglnglulysleuileservalalaglyleuala

227022752280

valhisserglyproaspglutyrargargleuphegluprophe

228522902295

glnglyleuphegluileprosertyrargserleutyrleuarg

230023052310

trpvalasnalavalcysglyaspala

23152320

<210>7

<211>141

<212>dna

<213>重组病毒o/rv-1/3bm3b1和3b2蛋白的核苷酸序列

<400>7

ggaccctacaccgcggcactcgaacgtcagaaacctcttaaagtgaaagccaggttgcca60

caacaagagggaccttacaccgctgcgatggagcggcagaaaccgctgaaagtgaaagca120

aaagcccccgtcgtgaaggaa141

<210>8

<211>47

<212>prt

<213>重组病毒o/rv-1/3bm3b1和3b2蛋白编码的氨基酸序列

<400>8

glyprotyrthralaalaleugluargglnlysproleulysvallys

151015

alaargleuproglnglngluglyprotyrthralaalametgluarg

202530

glnlysproleulysvallysalalysalaprovalvallysglu

354045