CN113817068A - 一种以人复制缺陷型重组腺病毒为载体的o型口蹄疫疫苗 - Google Patents
一种以人复制缺陷型重组腺病毒为载体的o型口蹄疫疫苗 Download PDFInfo
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Abstract
本发明提供一种以人5型复制缺陷腺病毒为载体的新型O型口蹄疫疫苗。所述疫苗以E1、E3联合缺失的复制缺陷型人5型腺病毒为载体，以整合腺病毒E1基因的HEK293细胞为包装细胞系，插入的抗原基因为经过优化设计的O型口蹄疫结构及非结构基因(FMDV‑O)，最终形成人复制缺陷型腺病毒为载体的重组O型口蹄疫病毒(Ad5‑FMDV‑O)。该病毒制备的疫苗在小鼠模型上表现出良好的免疫原性，在免疫后7天，即可诱导小鼠产生明显的细胞免疫。免疫后7天，在中高剂量疫苗注射组，已产生中和抗体，免疫21天后，所有剂量疫苗注射组，均产生显著的中和抗体，且中高剂量组抗体滴度明显高于低剂量组且中高剂量组无差异。
Description
技术领域
本发明涉及生物工程领域，具体涉及一种以人复制缺陷型重组腺病毒为载体的O型口蹄疫疫苗。
背景技术
口蹄疫是由口蹄疫病毒(foot-and-mouth disease virus,FMDV)引起的,发生于牛、羊、猪等偶蹄动物的一种急性、热性、高度接触性传染病。世界动物卫生组织将口蹄疫列为动物A类烈性传染病,我国农业农村部将口蹄疫病毒列为一类动物病原微生物，口蹄疫为一类疫病。口蹄疫严重危害畜牧业的健康发展以及相关产品的对外贸易,对国家的政治、经济具有深远的影响。口蹄疫病毒(FMDV)有O、A、C、SAT1、SAT2、SAT3(即南非口蹄疫病毒1、2、3型)和Asia1(亚洲1型)7个血清型。各型之间几乎没有交叉免疫保护。我国主要流行O型、A型。我国农业农村部于2019年12月印发了《2020年国家动物疫病强制免疫计划》，对于口蹄疫明确要求：对全国所有猪、牛、羊、骆驼、鹿进行O型口蹄疫免疫；对全国所有奶牛和种公牛进行A型口蹄疫免疫。此外,内蒙古、云南、西藏、新疆和新疆生产建设兵团对所有牛和边境地区的羊、骆驼、鹿进行A型口蹄疫免疫,广西对边境地区牛羊进行A型口蹄疫免疫,吉林、青海、宁夏对所有牛进行A型口蹄疫免疫,辽宁、四川对重点地区的牛进行A型口蹄疫免疫。这些措施及计划，强调了口蹄疫疫苗对于口蹄疫病毒在我国的净化的重要性。目前，我国的经济动物所使用的O型口蹄疫疫苗，主要以O型口蹄疫病毒制备的灭活疫苗为主，一些新型的疫苗，如多肽疫苗，也有一定的使用量。
发明内容
本发明提供如下任一所述蛋白：
(a1)蛋白，其氨基酸序列自N端至C端依次包括：O型口蹄疫病毒的结构蛋白P1的氨基酸序列、O型口蹄疫病毒的非结构蛋白2A的氨基酸序列、O型口蹄疫病毒的非结构蛋白2B的氨基酸序列、O型口蹄疫病毒或A型口蹄疫病毒非结构蛋白3B部分蛋白的氨基酸序列、O型口蹄疫病毒或A型口蹄疫病毒非结构蛋白3C蛋白的氨基酸序列；
(a2)蛋白，其氨基酸序列自N端至C端依次包括：甲硫氨酸、O型口蹄疫病毒的结构蛋白P1的氨基酸序列、O型口蹄疫病毒的非结构蛋白2A的氨基酸序列、O型口蹄疫病毒的非结构蛋白2B的氨基酸序列、O型口蹄疫病毒或A型口蹄疫病毒非结构蛋白3B部分蛋白的氨基酸序列、O型口蹄疫病毒或A型口蹄疫病毒非结构蛋白3C蛋白的氨基酸序列、标签蛋白的氨基酸序列。
可选的，所述O型口蹄疫病毒的结构蛋白P1为O/SEA/Mya98株的结构蛋白P1；所述O型口蹄疫病毒的非结构蛋白2A为O/SEA/Mya98株的非结构蛋白2A；所述O型口蹄疫病毒的非结构蛋白2B为O/SEA/Mya98株的非结构蛋白2B；
所述O型口蹄疫病毒3B部分蛋白为O/SEA/Mya98株的3B部分蛋白；
所述A型口蹄疫病毒非结构蛋白3B部分蛋白为A/A24株或A/A12株的3B部分蛋白；
所述A型口蹄疫病毒非结构蛋白3C蛋白为A/A24株或A/A12株的3C蛋白；
所述O型口蹄疫病毒非结构蛋白3C蛋白为O/SEA/Mya98株的3C蛋白；
可选的，所述O/SEA/Mya98株的结构蛋白P1的氨基酸序列如SEQ ID NO：2第2位至735位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：1第4-2205位所示；
可选的，所述O/SEA/Mya98株的非结构蛋白2A的氨基酸序列如SEQ ID NO：2第736位至753位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：1第2206-2259位所示；
可选的，所述O/SEA/Mya98株的非结构蛋白2B的氨基酸序列如SEQ ID NO：2第754位至907位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：1第2260-2721位所示；
可选的，所述A/A24株的非结构蛋白3B部分蛋白，其氨基酸序列如SEQ ID NO：2自N端起第908位至1009位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：1第2722-3027位所示；
可选的，所述A/A24株的非结构蛋白3C蛋白的氨基酸序列如SEQ ID NO：2自N端起第1010位至1196位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：1第3028-3588位所示；
可选的，A/A12株的非结构蛋白3B部分蛋白，其氨基酸序列如SEQ ID NO：7第1位-92位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：8第1位-276位所示；
可选的，O/SEA/Mya98株的非结构蛋白3B部分蛋白，其氨基酸序列如SEQ ID NO：9第1位-92位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：10第1位-276位所示；
可选的，A/A12株的3C蛋白，其氨基酸序列如如SEQ ID NO：7第93位-279位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：8第277位-837位所示；
可选的，O/SEA/Mya98株的3C蛋白，其氨基酸序列如SEQ ID NO：9第93位-279位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：10第277位-837位所示。
本发明还提供如下任一所述生物材料：
1)编码上述蛋白的DNA分子；
2)含有1)所述DNA分子的表达盒、重组载体或重组微生物；
可选的，所述DNA分子为如下任一一种：
1)核苷酸序列(5’-3’)如SEQ ID NO：1所示DNA分子；
2)SEQ ID NO：1的第2722-3588位核苷酸替换为SEQ ID NO：8后得到的DNA分子；
3)SEQ ID NO：1的第2722-3588位核苷酸替换为SEQ ID NO：10后得到的DNA分子。
可选的，所述重组微生物为重组病毒；可选的，所述重组病毒为重组腺病毒；可选的，所述重组腺病毒为人复制缺陷型重组腺病毒；可选的，人复制缺陷型重组腺病毒为人5型复制缺陷型腺病毒(Ad5)。
一种口蹄疫病毒疫苗，其活性成分为上述重组腺病毒。
可选的，所述口蹄疫病毒为O型口蹄疫病毒；可选的，所述O型口蹄疫病毒为O/SEA/Mya98株。
上述蛋白、或上述生物材料在制备预防口蹄疫病毒的疫苗中应用。
可选的，所述口蹄疫病毒为O型口蹄疫病毒；
可选的，所述疫苗的剂型为注射剂、滴鼻剂或喷雾剂；可选的，所述重组腺病毒被制备为肌肉注射剂；所述疫苗为单价疫苗、双价疫苗或三价疫苗；单价疫苗为O型口蹄疫单价疫苗；双价疫苗为O型、A型口蹄疫双价疫苗；三价疫苗为O型、A型和亚洲1型口蹄疫三价疫苗；
可选的，上述单价疫苗、双价疫苗或三价疫苗中的O型口蹄疫疫苗活性成分为上述重组腺病毒。
一种制备重组腺病毒的方法，所述方法包括以下步骤：
(1)构建含有编码权利要求1或2所述蛋白的DNA分子的重组穿梭质粒载体；
(2)将步骤(1)所述重组穿梭质粒载体与腺病毒骨架质粒一起转染入宿主细胞；
(3)培养步骤(2)所述宿主细胞；
(4)收获从步骤(3)所述细胞中释放的重组腺病毒；
(5)对步聚(4)中的重组腺病毒进行扩大培养；
(6)对步聚(5)中的培养产物进行纯化。
可选的，步骤(1)所述穿梭质粒载体为pADV-mCMV-MCS-3xFLAG；重组穿梭质粒载体为在pADV-mCMV-MCS-3xFLAG的BamHI和XbaI之间插入DNA分子得到的；DNA分子为如下任一一种：
1)核苷酸序列(5’-3’)如SEQ ID NO：1所示DNA分子；
2)SEQ ID NO：1的第2722-3588位核苷酸替换为SEQ ID NO：8后得到的DNA分子；
3)SEQ ID NO：1的第2722-3588位核苷酸替换为SEQ ID NO：10后得到的DNA分子。
可选的，步骤(2)所述骨架质粒为pBHGloxdelE13cre。
所述重组腺病毒为人5型复制缺陷型病毒；具体为Ad5-FMDV–O。
本发明技术方案，具有如下优点：
本发明得到了可正确表达O型口蹄疫病毒(O/SEA/Mya98)结构基因P1及部分非结构基因的以人复制缺陷型腺病毒为载体的重组O型(O/SEA/Mya98)口蹄疫载体病毒株。用此毒株制备的疫苗在体外及动物体内的实验均证明了其携带的结构基因P1能被有效切割，且形成O型口蹄疫(O/SEA/Mya98)病毒样颗粒(VLP)。小鼠实验结果(BALB/C、C57BL/6)显示此疫苗具有良好的免疫原性，能在短期内诱导动物产生特异性的体液及细胞免疫。
本发明提供的以人复制缺陷型重组腺病毒为载体的O型口蹄疫疫苗有如下特点：
(1)具有良好的免疫原性，肌肉注射后，能同时诱导动物产生体液及细胞免疫。
(2)本疫苗是用人5型复制缺陷型腺病毒为载体，免疫动物后在体内不复制，有良好的安全性。
(3)本疫苗无需添加任何佐剂成分。
(4)一针免疫后7天，已开始产生中和抗体和明显的细胞免疫反应。免疫后14天可产生高水平的细胞免疫；免疫21天后，能诱导动物产生高滴度的中和抗体，不需要加强免疫。
(5)本疫苗在生物安全2级条件下，可进行规模化生产，避免了使用口蹄疫强毒株生产可能造成的生物安全风险。
基于以上特点，本发明可为国内口蹄疫的防控及净化，提供一种安全、有效的手段，为广大的养殖户对于O型口蹄疫的防治，提供一种新的选择。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为实施例1双抗夹心ELISA检测特异性抗体检测结果；
图2为实施例1中Western-Blot检测FMDV-O蛋白表达结果；
图3为实施例2中中和抗体效价检测结果；图中NS表示无显著性差异；
图4为实施例3中CD3+，CD107a+T细胞比率、CD3+,CD4+,CD107a+T细胞比率、CD3+,CD8+,CD107a+T细胞比率结果；
图5为实施例3中CD3+，IL-2+T细胞比率、CD3+，CD4+，IL-2+T细胞比率、CD3+，CD8+，IL-2+T细胞比率结果；
图6为CD3+，IFN-γ+T细胞比率、CD3+，CD4+，IFN-γ+T细胞比率、CD3+，CD8+，IFN-γ+T细胞比率结果；
图7为重组穿梭载体pADV-mCMV-FMDV-O-3xFLAG示意图。
具体实施方式
1)0.25％胰蛋白酶配方:100mL PBS,0.25g胰酶；
2)透析缓冲液配制方法：50g蔗糖，10mL 1M pH为8.0的Tris-HCl液，2mL 1M MgCl2溶液定容至1000mL；
3)病毒稀释液为:0.01MPBS。
实施例1以人复制缺陷型重组腺病毒(Ad5-FMDV-O)为载体的O型口蹄疫疫苗的制备
(一)基因的获得
O/SEA/Mya98株的P1基因、2A基因、2B基因、A/A24株3B基因和A/A24株3C基因分别进行密码子优化，使其更适合在哺乳动物细胞中进行表达，优化后的P1基因的核苷酸序列如SEQ ID NO：1第4-2205位所示；2A基因的核苷酸序列如SEQ ID NO：1第2206-2259位所示；2B基因的核苷酸序列如SEQ ID NO：1第2260-2721位所示；通过多次截取3B基因片段，最终选择了核苷酸序列如SEQ ID NO：1第2722-3027位所示的3B基因片段；3C基因的核苷酸序列如SEQ ID NO：1第3028-3588所示。优化后的各个基因序列，按照P1-2A-2B-3B-3C顺序加起始密码子和终止密码子后形成的融合基因的核苷酸序列(5’-3’)如SEQ ID NO：1所示，命名为FMDV-O，该基因编码的蛋白的氨基酸序列如SEQ ID NO：2所示。由生物公司直接合成pMD18-T-FMDV-O。
(二)重组穿梭载体的构建
用BamHI和XbaI双酶切pMD18-T-FMDV-O，回收酶切产物(TaKaRa MiniBESTAgarose Gel DNA Extraction Kit Ver.4.0)，将其与BamHI和XbaI双酶切的穿梭质粒pADV-mCMV-MCS-3xFLAG(和元生物技术(上海)股份有限公司，H225)连接。pADV-mCMV-MCS-3xFLAG穿梭质粒为AdMax腺病毒系统的穿梭质粒。连接产物转化大肠杆菌DH5α感受态，涂布Amp抗性LB平板，挑单克隆进行菌液PCR鉴定，对于鉴定为阳性的克隆送测序公司测序，PCR和测序引物均为MCMV-F ggtataagaggcgcgaccag(SEQ ID NO：3)和SV40-pArevgaaatttgtgatgctattgc(SEQ ID NO：4)。测序正确的重组穿梭载体命名为pADV-mCMV-FMDV-O-3xFLAG，图谱见图7，该载体为在pADV-mCMV-MCS-3xFLAG的BamHI和XbaI之间插入FMDV-O基因得到的。
(三)重组腺病毒Ad5-FMDV-O包装与鉴定
a)转染前一天，将HEK293细胞接种到6孔板中，控制细胞转染时的密度为70-80％；
b)转染前一小时取出细胞培养板，去除原有细胞培养基，加入1.5mL的Opti-MEM培养基，将细胞放回培养箱；
c)转染：
·将待转染的病毒载体质粒4μg溶于Opti-MEM培养基，总体积为250μL，轻轻混匀，得到质粒稀释液；待转染的病毒载体质粒由AdMax腺病毒系统的骨架质粒pBHGloxdelE13cre和重组穿梭质粒组成；AdMax腺病毒系统的骨架质粒pBHGloxdelE13cre：重组穿梭质粒质量比为＝1:1。
·将转染试剂2000TM(life science)8μL溶于Opti-MEM培养基，总体积为250μL，轻轻混匀，得到转染试剂稀释液；
·将转染试剂稀释液滴加到质粒稀释液中，边加边轻轻混匀后在室温放置20min，使DNA和转染试剂充分结合形成稳定的DNA-转染试剂复合体。
·取出细胞培养板，将上面配好的DNA-转染试剂复合体加入到细胞培养板中，做好标记，放回培养箱。
·6h后吸去培养基，PBS洗一次，加入2mL新鲜完全培养基培养；
每三天换液一次，7-15天出现病毒空斑，待完全病变后收集上清液，上清液中的病毒即为表达FMDV-O的重组病毒，命名为Ad5-FMDV-O。
Ad5-FMDV-O病毒鉴定过程为：
用基因组提取试剂盒(生工，B511371)提取Ad5-FMDV-O基因组。以FW:5’-aagaccagattaacgtgctt-3’9(SEQ ID NO：5)；RE:5’-ctgtagggtttgtcgtgtt-3’(SEQ ID NO：6)为引物进行PCR扩增。
PCR反应体系为：模板1μL，引物上下游各0.5uL(引物浓度为5uM)，dNTP 1uL，
High-Fidelity DNA polymerase(NEB)0.5uL，5X Q 5reaction Buffer 10uL，ddH2O32.5uL。
PCR反应条件：94℃5min；94℃30s，56℃30s，72℃50s，30个循环；72℃5min。
PCR扩增阳性的Ad5-FMDV-O进行测序，结果正确，进行后续试验。
(四)重组腺病毒Ad5-FMDV-O大量扩增与纯化
将HEK293细胞铺于40个直径为10cm细胞盘中，待细胞长至70-80％，每块板加入上一步骤中制备得到的上清液(其中含有表达FMDV-O的重组病毒的滴度为107-108IFU/mL)10μL，感染细胞,待细胞全部病变后(2-3天)，每块板中加入约500μL 10％(v/v)Nonidet P 40(碧云天)以裂解细胞。收集细胞裂解物，12000rpm离心10min，弃细胞碎片收集上清。每100mL上清加入50mL病毒沉淀液(20％(v/v)PEG8000，2.5M NaCl)，冰上放置1h以沉淀病毒。12000rpm离心上述混合物20min，弃上清，将沉淀物悬浮在10mL密度为1.10g/mL的CsCl溶液中4℃7000rpm离心5min，收集病毒悬浮液。
在Beckman超速离心管中加入2.0mL的1.40g/mL CsCl溶液。再加入3.0mL 1.30g/mL的CsCl溶液。最后加入5mL的病毒悬浮液。22800rpm，4℃离心2.5h。收集密度在1.30-1.40g/mL之间的病毒条带至1mL透析袋中(透析袋使用前用10mM的EDTA Na2煮沸10min)。在透析缓冲液中，4℃透析过夜，中间更换透析液一次。收集病毒，于-80℃保存。
(五)重组腺病毒Ad5-FMDV-O滴度测定
滴度测定所用的试剂盒为腺病毒感染性滴度快速测定试剂盒(上海美吉，AD-010)。
选取状态良好的HEK293细胞，使用完全培养基重悬细胞，制备成5.0×105个/mL的细胞悬液，24孔板每个孔中种入1mL细胞，37℃、5％CO2培养。
1.准备好的用病毒稀释液进行了10倍梯度稀释的病毒样品，然后将10-7(可以选10-5至10-8)稀释的病毒液加入24孔板中，每孔加入100μL。
2.37℃、5％CO2感染48h。轻轻的去除培养液，沿着24孔板侧壁缓缓加入预冷的甲醇500μL，-20℃固定20min。
4.使用PBS轻轻的冲洗细胞3次，每次5min。
5.加入200μL PBS+1％BSA37℃封闭1h。
6.加入200μL的一抗(试剂盒自带)溶液至每个孔中，37℃孵育1h。
7.使用PBS轻轻的冲洗细胞3次，每次5min。
8.加入200μL的二抗(试剂盒自带)至每孔，37℃孵育1h。
9.使用PBS轻轻的冲洗细胞3次，每次5min。
10.加入200μL新配置的工作液(试剂盒自带)至每孔，室温孵育5-10min。
11.弃工作液，使用PBS清洗2次，每孔加入1000μL PBS。
12.每孔随机选择5个视野，使用光学显微镜10×物镜下计算阳性细胞个数。计算每孔阳性细胞的平均个数和病毒滴度。
滴度检测
(1)计算显微镜下视野中阳性细胞的平均个数。选择一个梯度，此梯度视野中有5-50个阳性细胞，随机选择至少5个区域计数。
(2)计算24孔板中每孔视野的个数。对于多数显微镜(本实施例所用显微镜符合该计算方式)，标准10×目镜与10×物镜所观察到的视野直径为1.8mm，因此每个视野的面积＝3.14x(D/2)2＝3.14x0.92＝2.54mm2
对于一个标准24孔板，培养面积为2.0cm2，因此每孔视野数＝2.0cm2/2.54mm2＝2.0cm2/2.54x10-2cm2＝79，
本实施例一次实验在显微镜下5个视野中计算的阳性细胞平均数为4，此孔病毒稀释了107倍，根据以上公式得出：
实施例中其他滴度的计算均按上述方法。
(六)重组腺病毒Ad5 FMDV-O表达FMDV-O蛋白的鉴定
1、双抗夹心ELISA检测特异性抗体实验流程及结果
实验步骤：
(1)HEK293细胞的复苏：HEK293(1573株)37℃水浴解冻后，以800g，离心5min，弃上清，沉淀细胞用DMEM(10％FBS)重悬，接种于T225(therom)方瓶中。补加DMEM(10％FBS)至70mL，37℃，5％CO2静置培养。
(2)HEK293细胞的传代培养：72h后，待细胞长满T225方瓶后，弃去培养液，加入0.25％胰蛋白酶消化5min，弃去胰蛋白酶，DMEM(10％FBS)吹悬贴壁HEK293细胞，平均分配转入3个T225(therom)方瓶培养，每个T225方瓶补加DMEM(10％FBS)至70mL，37℃，5％CO2静置培养。72h后，相同步骤消化贴壁HEK293细胞，在T225(therom)方瓶1:3传代培养。
(3)Ad5-FMDV-O及Ad5-空载体(即pBHGloxdelE13cre)接种：待HEK293细胞平行扩增至12个T225(therom)方瓶时，将活化的Ad5-FMDV-O及Ad5-空载体分别以MOI＝3接种T225方瓶，Ad5-FMDV-O接种9个T225(therom)方瓶，Ad5-空载体接种3个T225(therom)方瓶。接种液为DMEM，接种体积为70mL/个T225方瓶。37℃，5％CO2静置培养。
(4)Ad5-FMDV-O及Ad5-空载体(即pBHGloxdelE13cre)收获：接种后40h后，将3个T225方瓶(Ad5-FMDV-O)分别离心收集细胞，分别使用10mL病毒稀释液重悬每个T225方瓶内细胞，反复冻融2次后，3000g离心10min收集上清，进行双抗夹心ELISA检测，即下表中的冻融2次(40h)。Ad5-空载体收获步骤同上。接种55h后，将余下6个T225方瓶内细胞同上步骤离心收集，其中3个T225方瓶内细胞冻融2次分别收获上清，双抗夹心ELISA检测，即下表中的冻融2次(55h)；其余3个分别加入10mL细胞裂解液Solarbio R0030，裂解后离心，分别收集上清，双抗夹心ELISA检测，即即下表中的裂解液处理(55h)。
(5)双抗夹心ELISA检测特异性抗体
按照口蹄疫O型液相阻断ELISA检测试剂盒(中国农业科学院兰州兽医研究所，20200108101-1)说明书操作。96孔ELISA板，包被抗体为兔抗FMDV-O多抗，二抗体为豚鼠抗FMDV-O多抗，酶标抗体为兔抗豚鼠IgG-HRP。阳性对照为口蹄疫O型液相阻断ELISA检测试剂盒中O型口蹄疫灭活病毒按照体积比为1:4进行稀释，阴性对照为Ad5-空载体(即pBHGloxdelE13cre)，空白为PBS缓冲液。将样品组(即步骤4中的上清)及空白，阳性，阴性对照品分别加入96孔ELISA板，每个样品3个平行，每孔100μL。37℃，水浴1h，弃去板中液体，PBST洗3次，每次5min。加入酶标抗体，每孔各100μL，37℃，水浴1h，弃去板中液体，PBST洗3次，每次5min。加入显色A，B液(口蹄疫O型液相阻断ELISA检测试剂盒中)，各50μL，37℃，显色10min，加入终止液，OD450检测吸光值。
实验结果见下表2。
表2
将上表数据制作为柱状图，见图1，从上表及图1可以看出，40h及55h收获后冻融2次，OD450无显著性差异(P>0.05)，说明当感染时间超过40小时后，Ad5-FMDV-O外源蛋白的表达无明显提升；裂解液处理后的样品OD450明显高于冻融2次收获液组(P<0.05)，说明FMDV-O蛋白的稳定性在反复冻融下可能有影响；不论是冻融组及裂解液组，对于PBST及空载体组，OD450均有显著性差异，且裂解组与阳性对照近似，说明在双抗夹心ELISA检测中，检测到了特异性抗体。
2、Western-Blot检测FMDV-O蛋白表达。
实验步骤：
(1)样品预处理：将步骤(一)中40h及55h收获后处理得到的上清各取100μL，阳性对照(口蹄疫灭活病毒)100μL，分别与2X的Western上样Buffer混合，沸水浴10min，8000rpm离心5min，混匀样品。
(2)制胶：配置12％的SDS-PAGE电泳胶(bio-rad)及电泳buffer。
(3)上样及SDS-PAGE电泳：分别在泳道内上样，每个样品15μL，上层胶电压80V，下层胶电压120V，SDS-PAGE电泳。
(4)Western-blot转印：将SDS-PAGE电泳后的胶小心取出，按说明书置于预制Western-blot转印膜中(bio-rad)，200V电压转印4h。
(5)封闭及标记，显色：转印膜小心取出，PBST(4％脱脂奶)封闭2h，PBST洗3次，加入兔抗口蹄疫VP1酶标抗体(Bioss：Rabbit Anti-FMDV VP1 polyclonal Antibody bs-4521R)，37℃，孵育1h，PBST洗3次。按说明书加入显色液(北京索莱宝科技有限公司)X光胶片曝光显色。肌动蛋白作为内参，肌动蛋白抗体：Solarbio K200058M。
实验结果见图2：阳性对照(即图3中FMDV(Mya98)-VP1)在30KD处有明显显色，条带清晰，大小与分子量相符合。肌动蛋白内部参考样品在42KD处有明显显色，条带清晰，大小与分子量相符合。40h(Ad5-FMDV(Mya98)-40h)及55h(Ad5-FMDV(Mya98)-55h)收获后Ad5-FMDV-O样品在30KD附近有明显显色，条带清晰。
实施例2、以人复制缺陷型重组腺病毒(Ad5-FMDV-O)为载体的O型口蹄疫疫苗诱发机体体液免疫反应检测
在小鼠模型上，研究重组病毒载体FMDV疫苗的免疫剂量，评价疫苗所诱导的体液免疫的平。
实验步骤：
1、取5-8周龄雌性BALB/C小鼠45只，5只/组，分9组，分别设为空白组、阴性对照组(day7，day21)、高剂量组、中剂量组(day7，day21)、低剂量组(day7，day21)。高剂量组为：1.25×109IFU/mL Ad5-FMDV-O，注射剂量为100μL，注射方式为小鼠大腿外侧肌肉，双侧注射，每侧50μL。中剂量组为：2.5×108IFU/mL Ad5-FMDV-O，注射剂量为100μL，注射方式为小鼠大腿外侧肌肉，双侧注射，每侧50μL。低剂量组为：5×107IFU/mL Ad5-FMDV-O，注射剂量为100μL，注射方式为小鼠大腿外侧肌肉，双侧注射，每侧50μL。阴性对照组为病毒稀释液，注射剂量为100μL，注射方式为小鼠大腿外侧肌肉，双侧注射，每侧50μL。空白组不免疫，于免疫当天采集外周血。阴性对照组、高剂量组和中剂量组分别于免疫后7天(day7)，21天(day21)采集外周血。
2、所有采集得到的外周血，37℃静置50min，8000rpm离心15min，收集血清，分装后-80℃保存。
3、中和抗体效价采用液相阻断ELISA方法进行测定，试剂盒(购自兰州兽医研究所，20200108101-1)
将供试血清分管在稀释板上用PBS缓冲液梯度稀释。起始稀释倍数为4X，后续倍比稀释，稀释后体积为每稀释梯度50μL/孔，加入50μL/孔抗原工作液，震荡混匀后4℃过夜。将过夜的抗原、抗体混合液转移至ELISA板对应孔上，50μL/孔，37℃孵育60分钟。PBST洗板3次，加入豚鼠抗O型口蹄疫工作液，50μL/孔，37℃孵育30分钟。PBST洗板3次，加入兔抗豚鼠工作液，50μL/孔，37℃孵育30分钟。PBST洗板3次，A,B液，50μL/孔，37℃显色15分钟。加入终止液50μL/孔，酶标仪读取OD450值。阳性对照及阴性对照按说明书操作。
实验结果见图3，结果显示，免疫后7天，中、高剂量组即可诱导产生中和抗体。免疫21天后，低剂量组、中剂量组及高剂量组均能诱导产生高水平抗体效价；中、高剂量组之间无显著性差异(P>0.05)。中、高剂量组抗体效价明显高于低剂量组(P<0.05)。结果表明：一针免疫后，即可诱导产生强烈的中和抗体水平且抗体效价呈现明显的剂量相关性。
实施例3、以人复制缺陷型重组腺病毒(Ad5-FMDV-O)为载体的O型口蹄疫疫苗所诱导的细胞免疫反应检测
在小鼠模型上，研究重组病毒载体FMDV疫苗的免疫剂量，评价疫苗所诱导的细胞免疫的平。
实验步骤：
1、取C57BL/6 5-8周龄小鼠15只，5只/组，共三组，分别为阴性对照组，实验组(7day)，实验组(14day)。阴性对照组：阴性对照组为病毒稀释液，注射剂量为100μL，注射方式为小鼠大腿外侧肌肉，双侧注射，每侧50μL。实验组为：2.5×108IFU/mL Ad5-FMDV-O，注射剂量为100μL，注射方式为小鼠大腿外侧肌肉，双侧注射，每侧50μL。
2、实验组(7day)于免疫后7天处死小鼠，实验组(14day)于免疫后14天处死小鼠，阴性对照组于免疫后14天处死小鼠；取阴性对照组，实验组小鼠脾脏，分离脾淋巴细胞，淋巴细胞表面染色抗体孵育，使用细胞刺激用溶液进行刺激后孵育6小时，孵育后裂解红细胞，阻断Fc受体，表面抗体染色，细胞固定和破膜，胞内因子抗体染色。检测CD3+，CD107a+T细胞；CD3+,CD4+,CD107a+T细胞；CD3+,CD8+,CD107a+T细胞；CD3+，IL-2+T细胞，CD3+，CD4+，IL-2+T细胞；CD3+，CD8+，IL-2+T细胞；CD3+，IFN-γ+T细胞，CD3+，CD4+，IFN-γ+T细胞；CD3+，CD8+，IFN-γ+T细胞；所有实验步骤均按BD流式细胞抗体说明书执行。具体步骤为：
a.获取淋巴细胞悬液
剥离小鼠脾脏，置于1mL 1640细胞培养基(gibco，货号：C11875500BT)中。用两个载玻片的粗糙面对脾脏进行研磨，直至脾脏解离，用Stain Buffer(BD，货号：554656)缓慢冲洗细胞至离心管中，经200目尼龙网过滤后，200g(北京白洋医疗器械有限公司，型号：BY-320C)离心5min。用Stain Buffer重悬细胞，计数。
b.配制细胞刺激用溶液
配制含10％FBS的1640细胞培养基(BI，货号：04-001-1ACS)。按1*10^6个细胞加入2μL激活剂(BD，货号：550583),计算所需激活剂体积，加入含10％FBS的1640溶液中，即得细胞刺激用溶液。
c.淋巴细胞表面染色抗体孵育
每1×106个细胞，加入2μL CD107a IgG(BD，货号：553930)和CD107a抗体(BD，货号：558661),涡旋混匀后，放入37℃含5％CO2的培养箱(Thermo，型号：SERIES8000 PH)中孵育4h。
d.淋巴细胞激活和孵育
按每3×106个细胞，加入2mL刺激用溶液，涡旋混匀后，放入37℃含5％CO2的培养箱中孵育6h。
e.裂解红细胞
孵育结束，按1×106个细胞加入1mL红细胞裂解液(BD，货号：555899)，室温避光裂解15min。200g离心5min，弃上清。加入1mL Stain Buffer洗涤1次。用Stain Buffer重悬细胞，按1*10^6细胞100μL。
f.阻断Fc受体
按1×106个细胞加入2μL Fc Block(BD，货号：553141)，4℃孵育15分钟。用StainBuffer洗涤细胞1次。
g.表面抗体染色
按1×106个细胞加入2μL CD3(BD，货号：553066)、1μL CD4(BD，货号：557307)和3μL CD8(BD，货号：553036)抗体,涡旋混匀后,4℃孵育30min。用Stain Buffer洗涤细胞2次。
h.细胞固定和破膜
按1×106个细胞加入100μL Staining Buffer重悬细胞，加入250μL固定破膜溶液(BD，货号：554714)，4℃孵育20min。用洗液(BD，货号：554714)洗细胞2次。
i.胞内因子抗体染色
按1×106个细胞加入3μL IFN-γ(BD，货号：554412)和同型抗体(BD，货号：554685)；按1×106个细胞加入3μL IL-2(BD，货号：554428)和同型抗体(BD，货号：556925),涡旋混匀后,4℃孵育30min。用Stain Buffer洗涤细胞2次。
g.上机检测。
结果表明免疫7天后(图4中D7疫苗组)，实验组脾细胞内CD3+，CD107a+T细胞比率明显上升，CD3+,CD4+,CD107a+T细胞及CD3+,CD8+,CD107a+T细胞比率上调明显(P<0.05)；免疫14天后(图4中D14疫苗组)，CD3+,CD8+,CD107a+T细胞比率仍然维持较高水平，与阴性对照组有明显差异。见图4。
免疫14天后(图5中D14疫苗组)，实验组脾细胞内CD3+，IL-2+T细胞比率明显上升，CD3+,CD4+,IL-2+T细胞及CD3+,CD8+,IL-2+T细胞比率上调明显(P<0.05)；免疫7天后(图5中D7疫苗组)，CD3+,CD4+,IL-2+T细胞及CD3+,CD8+,IL-2+T细胞比率与阴性对照组无显著性差异(P>0.05)，见图5。
免疫14天后(图6中D14疫苗组)，实验组脾细胞内CD3+，IFN-γ+T细胞比率明显上升，CD3+,CD4+,IFN-γ+T细胞及CD3+,CD8+,IFN-γ+T细胞比率上调明显(P<0.05)；免疫7天后(图6中D7疫苗组)，CD3+,CD4+,IFN-γ+T细胞及CD3+,CD8+,IFN-γ+T细胞比率与阴性对照组相比，已开始上调(P>0.05)。见图6。
实施例4
将实施例1中的核苷酸序列如SEQ ID NO：1第2722-3027位所示的3B基因片段(A/A24株的3B部分蛋白编码基因)，替换为核苷酸序列SEQ ID NO：10第1位-276位所示的基因片段(O/SEA/Mya98株的非结构蛋白3B部分蛋白的编码基因)；同时将实施例1中的核苷酸序列如SEQ ID NO：1第3028-3588所示的3C基因(A/A24株的3C蛋白的编码基因)，替换为核苷酸序列如SEQ ID NO：10第277位-837位所示的基因(O/SEA/Mya98株的3C蛋白的编码基因)，其他序列不变，按照实施例1-3的实验步骤进行实验，结果显示，形成的以人复制缺陷型重组腺病毒为载体的O型口蹄疫疫苗的细胞免疫和体液免疫效果均与实施例1中的O型口蹄疫疫苗效果相当。
实施例5
将实施例1中的核苷酸序列如SEQ ID NO：1第2722-3027位所示的3B基因片段(A/A24株的3B部分蛋白编码基因)，替换为核苷酸序列如SEQ ID NO：8第1位-276位所示基因片段(A/A12株的3B部分蛋白编码基因)；同时将实施例1中的核苷酸序列如SEQ ID NO：1第3028-3588所示的3C基因(A/A24株的3C蛋白的编码基因)，替换为核苷酸序列如SEQ ID NO：8第277位-837位所示的基因(A/A12株的3C蛋白的编码基因)，其他序列不变，按照实施例1-3的实验步骤进行实验，结果显示，形成的以人复制缺陷型重组腺病毒为载体的O型口蹄疫疫苗的细胞免疫和体液免疫效果均与实施例1中的O型口蹄疫疫苗效果相当。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
序列表
<110> 北京微佰生物科技有限公司
<120> 一种以人复制缺陷型重组腺病毒为载体的O型口蹄疫疫苗
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3591
<212> DNA
<213> 人工序列
<400> 1
atgggagccg gccagagctc ccccgccacc ggctcccaga accagtctgg caacaccggc 60
agcatcatta acaactacta catgcagcag taccagaact ccatggacac ccagctgggc 120
gataacgcca tttccggcgg atctaatgag ggctccaccg acacgacaag cacccacaca 180
acaaataccc agaataatga ctggttcagt aagctggcct cctccgcctt tagcggcctg 240
ttcggcgccc tgctggctga caagaaaacc gaggagacca ccctgctgga ggatagaatt 300
ctgacaaccc gcaacggcca cacaacctcc accacccaga gctccgtggg aatcacctac 360
ggctacgcca cagccgagga tttcgtgtct ggccccaaca cctccgggct ggagaccagg 420
gtggtgcagg ccgagcggtt ctttaagacc catttgtttg attgggtgac caatgatccc 480
ttcggacgct gccacctgct ggagctgccc accgaccata agggcgtgta cggctcactg 540
accgagagct acgcctacat gaggaacgga tgggacgtgg aggtgaccgc cgtgggcaac 600
cagttcaacg ggggctgcct gctggtcgca atggtgcccg agctgtgcag cattcagaag 660
agggagctgt accagctgac cctgttccca caccagttca tcaaccctcg cactaacatg 720
accgctcata tcaaagtgcc cttcgtgggc gtgaatagat atgaccagta taaagtgcac 780
aagccttgga cactggtcgt gatggtggtg gctccactga ccgtgaacac cgaaggcgcc 840
cctcagatca aggtctacgc taacatcgct cccaccaacg tgcacgtggc tggagagttc 900
cccagtaagg agggcatctt ccccgtggcc tgtagcgatg gctacggcgg cctggtcacc 960
accgatccca aaaccgctga cccagcctac ggaaaggtgt tcaacccccc ccggaacatg 1020
ttgccgggac gcttcaccaa cctcctggac gtcgccgagg cctgtcccac cttcctgcac 1080
tttgaagggg gcgtgcccta tgtcaccacc aagacagaca gcgaccgggt gctcgcccag 1140
ttcgacctga gcctggccgc caagcacatg tccaatacat tcctcgccgg cctggcccag 1200
tactacaccc agtatagcgg caccattaat ctgcatttta tgttcactgg acccactgac 1260
gccaaggctc ggtatatgat cgcctacgct cctcctggca tggagccccc caagacccct 1320
gaggccgccg cccactgcat ccacgctgag tgggatactg gcctgaactc caagttcacc 1380
ttctctatcc cctatctgag cgccgccgat tatgcctaca ccgccagcga cgccgctgaa 1440
accactaacg tgcagggatg ggtgtgcctg tttcagatca cccatggcaa ggccgatggc 1500
gacgcactgg tcgtgctggc ctccgcaggt aaagatttcg agctccgcct gcctgtggat 1560
gcccggcagc agaccacaag caccggagag tccgctgacc ccgtgacagc caccgtggag 1620
aactacggcg gcgagaccca agtgcagcga cgccaccaca ccgatgtgag cttcattctg 1680
gacaggtttg tgaaggtgac gcctaaagac cagattaacg tgcttgacct gatgcagacc 1740
cccccccata ccctggtggg ggctctgctg cggacagcca cgtactattt cgccgacctt 1800
gaagtggccg tgaagcatga gggggacctg acttgggtcc ctaacggcgc ccccgaggcc 1860
gccctcaaca acacgacaaa ccctacagcc taccacaagg ccccgctgac ccggctggcc 1920
ctgccataca ccgcaccaca ccgggtgctg gccaccgtgt ataacggcaa ctgcaagtac 1980
gccgagggta gtttgaccaa cgtccgcggc gacctgcagg tcctggccca gaaggccgcc 2040
aggcccctgc cgacatcctt taactacggg gccatcaaag ccaccagggt gaccgagctg 2100
ctgtatcgca tgaagcgggc cgagacctac tgcccccgcc ccctgctggc agtgcacccc 2160
gacgaggccc ggcacaagca gaagatcgtg gcccctgtga agcagctgct gaatttcgac 2220
ctgctgaagc tggctggaga tgtggagagc aatcccggac catttttctt ctctgatgtg 2280
cgctccaact ttagcaagct ggtggagacc atcaatcaga tgcaggagga catgtccacc 2340
aagcacggac ccgactttaa ccggctggtg tccgctttcg aagagctggc caccggagtg 2400
aaagccattc ggaccgggct tgacgaggct aagccctggt ataagctgat caagctgctg 2460
tcccgtctga gctgcatggc cgccgtggcc gccaggtcta aagatcccgt cctggtggcc 2520
atcatgctgg ccgacaccgg cctggaaatc ctggactcca cctttgtcgt gaagaagatc 2580
tccgatagcc tgagcagcct ctttcacgtg cctgcccccg tgttcagctt cggcgcccca 2640
atcctcctcg ccgggctcgt taaggtggcc agtagcttct tccgcagcac tcccgaggac 2700
ctggagcgcg ccgagaaaca gctgaaggcc agggacatca atgatatttt taaggtgggc 2760
gacgacgtga acagcgaacc cgcccacccc ggcgacgagc agccacaggc cgagggacct 2820
tacgccgggc cgctggagag acagcggccc ctcaaggtgc gcgccaaact gccccagcag 2880
gaggggcctt acgccggacc catggagcgc cagaagcctc tcaaggtgaa agccaaggcc 2940
cccgtggtgc gcgaggggcc ctacgaaggc cctgtgaaga agcccgtcgc actgaaggtg 3000
aaggccaaga acctgattgt caccgagagc ggggctcccc ctaccgacct ccagaagatg 3060
gtcatgggca atactaagcc cgtggagctg atcctggacg gcaagaccgt ggccatctgc 3120
tgcgccaccg gcgtgtttgg cacggcctac ctggtgccta gacacctgtt cgccgagaag 3180
tacgacaaga tcatgctgga cggccgcgcc atgacagact ccgactaccg ggtgttcgag 3240
ttcgaaatca aggtgaaggg ccaggacatg ctgtccgatg ccgccctgat ggtgctgcac 3300
aggggcaatc gcgtgagaga catcactaag cacttccggg acactgcccg catgaagaag 3360
ggcacccccg tggtgggggt cgtgaataac gccgatgtgg gcagactgat cttctctggg 3420
gaggccctga cctacaagga catcgtggtg tgcatggacg gcgacaccat gcctggcctg 3480
ttcgcctata aggccgccac caaggccggc tactgcggcg gggccgtcct ggccaaggac 3540
ggcgccgata ccttcatcgt gggcacccat agcgccggcg gcaacggctg a 3591
<210> 2
<211> 1196
<212> PRT
<213> 人工序列
<400> 2
Met Gly Ala Gly Gly Ser Ser Pro Ala Thr Gly Ser Gly Ala Gly Ser
1 5 10 15
Gly Ala Thr Gly Ser Ile Ile Ala Ala Thr Thr Met Gly Gly Thr Gly
20 25 30
Ala Ser Met Ala Thr Gly Leu Gly Ala Ala Ala Ile Ser Gly Gly Ser
35 40 45
Ala Gly Gly Ser Thr Ala Thr Thr Ser Thr His Thr Thr Ala Thr Gly
50 55 60
Ala Ala Ala Thr Pro Ser Leu Leu Ala Ser Ser Ala Pro Ser Gly Leu
65 70 75 80
Pro Gly Ala Leu Leu Ala Ala Leu Leu Thr Gly Gly Thr Thr Leu Leu
85 90 95
Gly Ala Ala Ile Leu Thr Thr Ala Ala Gly His Thr Thr Ser Thr Thr
100 105 110
Gly Ser Ser Val Gly Ile Thr Thr Gly Thr Ala Thr Ala Gly Ala Pro
115 120 125
Val Ser Gly Pro Ala Thr Ser Gly Leu Gly Thr Ala Val Val Gly Ala
130 135 140
Gly Ala Pro Pro Leu Thr His Leu Pro Ala Thr Val Thr Ala Ala Pro
145 150 155 160
Pro Gly Ala Cys His Leu Leu Gly Leu Pro Thr Ala His Leu Gly Val
165 170 175
Thr Gly Ser Leu Thr Gly Ser Thr Ala Thr Met Ala Ala Gly Thr Ala
180 185 190
Val Gly Val Thr Ala Val Gly Ala Gly Pro Ala Gly Gly Cys Leu Leu
195 200 205
Val Ala Met Val Pro Gly Leu Cys Ser Ile Gly Leu Ala Gly Leu Thr
210 215 220
Gly Leu Thr Leu Pro Pro His Gly Pro Ile Ala Pro Ala Thr Ala Met
225 230 235 240
Thr Ala His Ile Leu Val Pro Pro Val Gly Val Ala Ala Thr Ala Gly
245 250 255
Thr Leu Val His Leu Pro Thr Thr Leu Val Val Met Val Val Ala Pro
260 265 270
Leu Thr Val Ala Thr Gly Gly Ala Pro Gly Ile Leu Val Thr Ala Ala
275 280 285
Ile Ala Pro Thr Ala Val His Val Ala Gly Gly Pro Pro Ser Leu Gly
290 295 300
Gly Ile Pro Pro Val Ala Cys Ser Ala Gly Thr Gly Gly Leu Val Thr
305 310 315 320
Thr Ala Pro Leu Thr Ala Ala Pro Ala Thr Gly Leu Val Pro Ala Pro
325 330 335
Pro Ala Ala Met Leu Pro Gly Ala Pro Thr Ala Leu Leu Ala Val Ala
340 345 350
Gly Ala Cys Pro Thr Pro Leu His Pro Gly Gly Gly Val Pro Thr Val
355 360 365
Thr Thr Leu Thr Ala Ser Ala Ala Val Leu Ala Gly Pro Ala Leu Ser
370 375 380
Leu Ala Ala Leu His Met Ser Ala Thr Pro Leu Ala Gly Leu Ala Gly
385 390 395 400
Thr Thr Thr Gly Thr Ser Gly Thr Ile Ala Leu His Pro Met Pro Thr
405 410 415
Gly Pro Thr Ala Ala Leu Ala Ala Thr Met Ile Ala Thr Ala Pro Pro
420 425 430
Gly Met Gly Pro Pro Leu Thr Pro Gly Ala Ala Ala His Cys Ile His
435 440 445
Ala Gly Thr Ala Thr Gly Leu Ala Ser Leu Pro Thr Pro Ser Ile Pro
450 455 460
Thr Leu Ser Ala Ala Ala Thr Ala Thr Thr Ala Ser Ala Ala Ala Gly
465 470 475 480
Thr Thr Ala Val Gly Gly Thr Val Cys Leu Pro Gly Ile Thr His Gly
485 490 495
Leu Ala Ala Gly Ala Ala Leu Val Val Leu Ala Ser Ala Gly Leu Ala
500 505 510
Pro Gly Leu Ala Leu Pro Val Ala Ala Ala Gly Gly Thr Thr Ser Thr
515 520 525
Gly Gly Ser Ala Ala Pro Val Thr Ala Thr Val Gly Ala Thr Gly Gly
530 535 540
Gly Thr Gly Val Gly Ala Ala His His Thr Ala Val Ser Pro Ile Leu
545 550 555 560
Ala Ala Pro Val Leu Val Thr Pro Leu Ala Gly Ile Ala Val Leu Ala
565 570 575
Leu Met Gly Thr Pro Pro His Thr Leu Val Gly Ala Leu Leu Ala Thr
580 585 590
Ala Thr Thr Thr Pro Ala Ala Leu Gly Val Ala Val Leu His Gly Gly
595 600 605
Ala Leu Thr Thr Val Pro Ala Gly Ala Pro Gly Ala Ala Leu Ala Ala
610 615 620
Thr Thr Ala Pro Thr Ala Thr His Leu Ala Pro Leu Thr Ala Leu Ala
625 630 635 640
Leu Pro Thr Thr Ala Pro His Ala Val Leu Ala Thr Val Thr Ala Gly
645 650 655
Ala Cys Leu Thr Ala Gly Gly Ser Leu Thr Ala Val Ala Gly Ala Leu
660 665 670
Gly Val Leu Ala Gly Leu Ala Ala Ala Pro Leu Pro Thr Ser Pro Ala
675 680 685
Thr Gly Ala Ile Leu Ala Thr Ala Val Thr Gly Leu Leu Thr Ala Met
690 695 700
Leu Ala Ala Gly Thr Thr Cys Pro Ala Pro Leu Leu Ala Val His Pro
705 710 715 720
Ala Gly Ala Ala His Leu Gly Leu Ile Val Ala Pro Val Leu Gly Leu
725 730 735
Leu Ala Pro Ala Leu Leu Leu Leu Ala Gly Ala Val Gly Ser Ala Pro
740 745 750
Gly Pro Pro Pro Pro Ser Ala Val Ala Ser Ala Pro Ser Leu Leu Val
755 760 765
Gly Thr Ile Ala Gly Met Gly Gly Ala Met Ser Thr Leu His Gly Pro
770 775 780
Ala Pro Ala Ala Leu Val Ser Ala Pro Gly Gly Leu Ala Thr Gly Val
785 790 795 800
Leu Ala Ile Ala Thr Gly Leu Ala Gly Ala Leu Pro Thr Thr Leu Leu
805 810 815
Ile Leu Leu Leu Ser Ala Leu Ser Cys Met Ala Ala Val Ala Ala Ala
820 825 830
Ser Leu Ala Pro Val Leu Val Ala Ile Met Leu Ala Ala Thr Gly Leu
835 840 845
Gly Ile Leu Ala Ser Thr Pro Val Val Leu Leu Ile Ser Ala Ser Leu
850 855 860
Ser Ser Leu Pro His Val Pro Ala Pro Val Pro Ser Pro Gly Ala Pro
865 870 875 880
Ile Leu Leu Ala Gly Leu Val Leu Val Ala Ser Ser Pro Pro Ala Ser
885 890 895
Thr Pro Gly Ala Leu Gly Ala Ala Gly Leu Gly Leu Leu Ala Ala Ala
900 905 910
Ile Ala Ala Ile Pro Leu Val Gly Ala Ala Val Ala Ser Gly Pro Ala
915 920 925
His Pro Gly Ala Gly Gly Pro Gly Ala Gly Gly Pro Thr Ala Gly Pro
930 935 940
Leu Gly Ala Gly Ala Pro Leu Leu Val Ala Ala Leu Leu Pro Gly Gly
945 950 955 960
Gly Gly Pro Thr Ala Gly Pro Met Gly Ala Gly Leu Pro Leu Leu Val
965 970 975
Leu Ala Leu Ala Pro Val Val Ala Gly Gly Pro Thr Gly Gly Pro Val
980 985 990
Leu Leu Pro Val Ala Leu Leu Val Leu Ala Leu Ala Leu Ile Val Thr
995 1000 1005
Gly Ser Gly Ala Pro Pro Thr Ala Leu Gly Leu Met Val Met Gly Ala
1010 1015 1020
Thr Leu Pro Val Gly Leu Ile Leu Ala Gly Leu Thr Val Ala Ile Cys
1025 1030 1035 1040
Cys Ala Thr Gly Val Pro Gly Thr Ala Thr Leu Val Pro Ala His Leu
1045 1050 1055
Pro Ala Gly Leu Thr Ala Leu Ile Met Leu Ala Gly Ala Ala Met Thr
1060 1065 1070
Ala Ser Ala Thr Ala Val Pro Gly Pro Gly Ile Leu Val Leu Gly Gly
1075 1080 1085
Ala Met Leu Ser Ala Ala Ala Leu Met Val Leu His Ala Gly Ala Ala
1090 1095 1100
Val Ala Ala Ile Thr Leu His Pro Ala Ala Thr Ala Ala Met Leu Leu
1105 1110 1115 1120
Gly Thr Pro Val Val Gly Val Val Ala Ala Ala Ala Val Gly Ala Leu
1125 1130 1135
Ile Pro Ser Gly Gly Ala Leu Thr Thr Leu Ala Ile Val Val Cys Met
1140 1145 1150
Ala Gly Ala Thr Met Pro Gly Leu Pro Ala Thr Leu Ala Ala Thr Leu
1155 1160 1165
Ala Gly Thr Cys Gly Gly Ala Val Leu Ala Leu Ala Gly Ala Ala Thr
1170 1175 1180
Pro Ile Val Gly Thr His Ser Ala Gly Gly Ala Gly
1185 1190 1195
<210> 3
<211> 20
<212> DNA
<213> 人工序列
<400> 3
ggtataagag gcgcgaccag 20
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<400> 4
gaaatttgtg atgctattgc 20
<210> 5
<211> 20
<212> DNA
<213> 人工序列
<400> 5
aagaccagat taacgtgctt 20
<210> 6
<211> 19
<212> DNA
<213> 人工序列
<400> 6
ctgtagggtt tgtcgtgtt 19
<210> 7
<211> 279
<212> PRT
<213> 人工序列
<400> 7
Arg Ala Cys Asn Asp Val Asn Ser Glu Pro Ala Arg Pro Ala Glu Glu
1 5 10 15
Gln Pro Gln Ala Glu Gly Pro Tyr Thr Gly Pro Leu Glu Arg Gln Arg
20 25 30
Pro Leu Lys Val Arg Ala Lys Leu Pro Gln Gln Glu Gly Pro Tyr Ala
35 40 45
Gly Pro Leu Glu Arg Gln Lys Pro Leu Lys Val Lys Ala Lys Ala Pro
50 55 60
Val Val Lys Glu Gly Pro Tyr Glu Gly Pro Val Lys Lys Pro Val Ala
65 70 75 80
Leu Lys Val Lys Ala Lys Asn Leu Ile Val Thr Glu Ser Gly Ala Pro
85 90 95
Pro Thr Asp Leu Gln Lys Met Val Met Gly Asn Thr Lys Pro Val Glu
100 105 110
Leu Ile Leu Asp Gly Lys Thr Val Ala Ile Cys Cys Ala Thr Gly Val
115 120 125
Phe Gly Thr Ala Tyr Leu Val Pro Arg His Leu Phe Ala Glu Lys Tyr
130 135 140
Asp Lys Ile Met Leu Asp Gly Arg Ala Met Thr Asp Ser Asp Tyr Arg
145 150 155 160
Val Phe Glu Phe Glu Ile Lys Val Lys Gly Gln Asp Met Leu Ser Asp
165 170 175
Ala Ala Leu Met Val Leu His Arg Gly Asn Arg Val Arg Asp Ile Thr
180 185 190
Lys His Phe Arg Asp Thr Ala Arg Met Lys Lys Gly Thr Pro Val Val
195 200 205
Gly Val Val Asn Asn Ala Asp Val Gly Arg Leu Ile Phe Ser Gly Glu
210 215 220
Ala Leu Thr Tyr Lys Asp Ile Val Val Cys Met Asp Gly Asp Thr Met
225 230 235 240
Pro Gly Leu Phe Ala Tyr Lys Ala Ala Thr Lys Ala Gly Tyr Cys Gly
245 250 255
Gly Ala Val Leu Ala Lys Asp Gly Ala Asp Thr Phe Ile Val Gly Thr
260 265 270
His Ser Ala Gly Gly Asn Gly
275
<210> 8
<211> 837
<212> DNA
<213> 人工序列
<400> 8
agggcctgca acgacgtgaa cagcgagccc gccaggcccg ccgaggagca gccccaggcc 60
gagggcccct acaccggccc cctggagagg cagaggcccc tgaaggtgag ggccaagctg 120
ccccagcagg agggccccta cgccggcccc ctggagaggc agaagcccct gaaggtgaag 180
gccaaggccc ccgtggtgaa ggagggcccc tacgagggcc ccgtgaagaa gcccgtggcc 240
ctgaaggtga aggccaagaa cctgatcgtg accgagagcg gcgccccccc caccgacctg 300
cagaagatgg tgatgggcaa caccaagccc gtggagctga tcctggacgg caagaccgtg 360
gccatctgct gcgccaccgg cgtgttcggc accgcctacc tggtgcccag gcacctgttc 420
gccgagaagt acgacaagat catgctggac ggcagggcca tgaccgacag cgactacagg 480
gtgttcgagt tcgagatcaa ggtgaagggc caggacatgc tgagcgacgc cgccctgatg 540
gtgctgcaca ggggcaacag ggtgagggac atcaccaagc acttcaggga caccgccagg 600
atgaagaagg gcacccccgt ggtgggcgtg gtgaacaacg ccgacgtggg caggctgatc 660
ttcagcggcg aggccctgac ctacaaggac atcgtggtgt gcatggacgg cgacaccatg 720
cccggcctgt tcgcctacaa ggccgccacc aaggccggct actgcggcgg cgccgtgctg 780
gccaaggacg gcgccgacac cttcatcgtg ggcacccaca gcgccggcgg caacggc 837
<210> 9
<211> 279
<212> PRT
<213> 人工序列
<400> 9
Lys Val Gly Asp Asp Val Asn Ser Glu Pro Ala His Pro Gly Asp Glu
1 5 10 15
Gln Pro Gln Ala Glu Gly Pro Tyr Ala Gly Pro Leu Glu Arg Gln Arg
20 25 30
Pro Leu Lys Val Arg Ala Lys Leu Pro Gln Gln Glu Gly Pro Tyr Ala
35 40 45
Gly Pro Met Glu Arg Gln Lys Pro Leu Lys Val Lys Ala Lys Ala Pro
50 55 60
Val Val Lys Glu Gly Pro Tyr Glu Gly Pro Val Lys Lys Pro Val Ala
65 70 75 80
Leu Lys Val Lys Ala Lys Asn Leu Ile Val Thr Glu Ser Gly Ala Pro
85 90 95
Pro Thr Asp Leu Gln Lys Met Val Met Gly Asn Thr Lys Pro Val Glu
100 105 110
Leu Ile Leu Asp Gly Lys Thr Val Ala Ile Cys Cys Ala Thr Gly Val
115 120 125
Phe Gly Thr Ala Tyr Leu Val Pro Arg His Leu Phe Ala Glu Lys Tyr
130 135 140
Asp Lys Ile Met Leu Asp Gly Arg Thr Met Thr Asp Ser Asp Tyr Arg
145 150 155 160
Val Phe Glu Phe Glu Ile Lys Val Lys Gly Gln Asp Met Leu Ser Asp
165 170 175
Ala Ala Leu Met Val Leu His Arg Gly Asn Arg Val Arg Asp Ile Thr
180 185 190
Lys His Phe Arg Asp Thr Ala Arg Met Lys Lys Gly Thr Pro Val Val
195 200 205
Gly Val Ile Asn Asn Ala Asp Val Gly Arg Leu Ile Phe Ser Gly Glu
210 215 220
Ala Leu Thr Tyr Lys Asp Ile Val Val Cys Met Asp Gly Asp Thr Met
225 230 235 240
Pro Gly Leu Phe Ala Tyr Lys Ala Ala Thr Lys Ala Gly Tyr Cys Gly
245 250 255
Gly Ala Val Leu Ala Lys Asp Gly Ala Asp Thr Phe Ile Val Gly Thr
260 265 270
His Ser Ala Gly Gly Asn Gly
275
<210> 10
<211> 837
<212> DNA
<213> 人工序列
<400> 10
aaggtgggcg acgacgtgaa cagcgagccc gcccaccccg gcgacgagca gccccaggcc 60
gagggcccct acgccggccc cctggagagg cagaggcccc tgaaggtgag ggccaagctg 120
ccccagcagg agggccccta cgccggcccc atggagaggc agaagcccct gaaggtgaag 180
gccaaggccc ccgtggtgaa ggagggcccc tacgagggcc ccgtgaagaa gcccgtggcc 240
ctgaaggtga aggccaagaa cctgatcgtg accgagagcg gcgccccccc caccgacctg 300
cagaagatgg tgatgggcaa caccaagccc gtggagctga tcctggacgg caagaccgtg 360
gccatctgct gcgccaccgg cgtgttcggc accgcctacc tggtgcccag gcacctgttc 420
gccgagaagt acgacaagat catgctggac ggcaggacca tgaccgacag cgactacagg 480
gtgttcgagt tcgagatcaa ggtgaagggc caggacatgc tgagcgacgc cgccctgatg 540
gtgctgcaca ggggcaacag ggtgagggac atcaccaagc acttcaggga caccgccagg 600
atgaagaagg gcacccccgt ggtgggcgtg atcaacaacg ccgacgtggg caggctgatc 660
ttcagcggcg aggccctgac ctacaaggac atcgtggtgt gcatggacgg cgacaccatg 720
cccggcctgt tcgcctacaa ggccgccacc aaggccggct actgcggcgg cgccgtgctg 780
gccaaggacg gcgccgacac cttcatcgtg ggcacccaca gcgccggcgg caacggc 837
Claims (10)
1.如下任一所述蛋白：
(a1)蛋白，其氨基酸序列自N端至C端依次包括：O型口蹄疫病毒的结构蛋白P1的氨基酸序列、O型口蹄疫病毒的非结构蛋白2A的氨基酸序列、O型口蹄疫病毒的非结构蛋白2B的氨基酸序列、O型口蹄疫病毒或A型口蹄疫病毒非结构蛋白3B部分蛋白的氨基酸序列、O型口蹄疫病毒或A型口蹄疫病毒非结构蛋白3C蛋白的氨基酸序列；
(a2)蛋白，其氨基酸序列自N端至C端依次包括：甲硫氨酸、O型口蹄疫病毒的结构蛋白P1的氨基酸序列、O型口蹄疫病毒的非结构蛋白2A的氨基酸序列、O型口蹄疫病毒的非结构蛋白2B的氨基酸序列、O型口蹄疫病毒或A型口蹄疫病毒非结构蛋白3B部分蛋白的氨基酸序列、O型口蹄疫病毒或A型口蹄疫病毒非结构蛋白3C蛋白的氨基酸序列、标签蛋白的氨基酸序列。
2.如权利要求1所述蛋白，其特征在于，
所述O型口蹄疫病毒的结构蛋白P1为O/SEA/Mya98株的结构蛋白P1；所述O型口蹄疫病毒的非结构蛋白2A为O/SEA/Mya98株的非结构蛋白2A；所述O型口蹄疫病毒的非结构蛋白2B为O/SEA/Mya98株的非结构蛋白2B；
所述O型口蹄疫病毒3B部分蛋白为O/SEA/Mya98株的3B部分蛋白；
所述A型口蹄疫病毒非结构蛋白3B部分蛋白为A/A24株或A/A12株的3B部分蛋白；
所述A型口蹄疫病毒非结构蛋白3C蛋白为A/A24株或A/A12株的3C蛋白；
所述O型口蹄疫病毒非结构蛋白3C蛋白为O/SEA/Mya98株的3C蛋白；
可选的，所述O/SEA/Mya98株的结构蛋白P1的氨基酸序列如SEQ ID NO：2第2位至735位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：1第4-2205位所示；
可选的，所述O/SEA/Mya98株的非结构蛋白2A的氨基酸序列如SEQ ID NO：2第736位至753位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：1第2206-2259位所示；
可选的，所述O/SEA/Mya98株的非结构蛋白2B的氨基酸序列如SEQ ID NO：2第754位至907位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：1第2260-2721位所示；
可选的，所述A/A24株的非结构蛋白3B部分蛋白，其氨基酸序列如SEQ ID NO：2自N端起第908位至1009位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：1第2722-3027位所示；
可选的，所述A/A24株的非结构蛋白3C蛋白的氨基酸序列如SEQ ID NO：2自N端起第1010位至1196位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：1第3028-3588位所示；
可选的，A/A12株的非结构蛋白3B部分蛋白，其氨基酸序列如SEQ ID NO：7第1位-92位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：8第1位-276位所示；
可选的，O/SEA/Mya98株的非结构蛋白3B部分蛋白，其氨基酸序列如SEQ ID NO：9第1位-92位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：10第1位-276位所示；
可选的，A/A12株的3C蛋白，其氨基酸序列如如SEQ ID NO：7第93位-279位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：8第277位-837位所示；
可选的，O/SEA/Mya98株的3C蛋白，其氨基酸序列如SEQ ID NO：9第93位-279位所示；其编码DNA分子的核苷酸序列如SEQ ID NO：10第277位-837位所示。
3.如下任一所述生物材料：
1)编码权利要求1或2所述蛋白的DNA分子；
2)含有1)所述DNA分子的表达盒、重组载体或重组微生物。
4.根据权利要求3所述的生物材料，其特征在于，所述DNA分子为如下任一一种：
1)核苷酸序列为SEQ ID NO：1所示DNA分子；
2)SEQ ID NO：1的第2722-3588位核苷酸替换为SEQ ID NO：8后得到的DNA分子；
3)SEQ ID NO：1的第2722-3588位核苷酸替换为SEQ ID NO：10后得到的DNA分子。
5.根据权利要求3或4所述生物材料，其特征在于，所述重组微生物为重组病毒；可选的，所述重组病毒为重组腺病毒；可选的，所述重组腺病毒为人复制缺陷型重组腺病毒；可选的，人复制缺陷型重组腺病毒为人5型复制缺陷型腺病毒。
6.一种口蹄疫病毒疫苗，其活性成分为权利要求5中所述重组腺病毒。
7.根据权利要求1所述蛋白，或权利要求6所述疫苗，其特征在于，所述口蹄疫病毒为O型口蹄疫病毒；可选的，所述O型口蹄疫病毒为O/SEA/Mya98株。
8.权利要求1所述蛋白、或权利要求2-4任一所述生物材料在制备预防口蹄疫病毒的疫苗中应用。
9.根据权利要求8所述的应用，其特征在于，所述口蹄疫病毒为O型口蹄疫病毒；
或，所述疫苗的剂型为注射剂、滴鼻剂或喷雾剂；
可选的，所述疫苗为单价疫苗、双价疫苗或三价疫苗；单价疫苗为O型口蹄疫单价疫苗；双价疫苗为O型、A型口蹄疫双价疫苗；三价疫苗为O型、A型和亚洲1型口蹄疫三价疫苗。
10.一种制备重组腺病毒的方法，其特征在于，所述方法包括以下步骤：
(1)构建含有编码权利要求1或2所述蛋白的DNA分子的重组穿梭质粒载体；
(2)将步骤(1)所述重组穿梭质粒载体与腺病毒骨架质粒一起转染入宿主细胞；
(3)培养步骤(2)所述宿主细胞；
(4)收获从步骤(3)所述细胞中释放的重组腺病毒；
(5)对步聚(4)中的重组腺病毒进行扩大培养；
(6)对步聚(5)中的培养产物进行纯化。
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