CN113493775B - 一种猪德尔塔冠状病毒毒株及其应用 - Google Patents
一种猪德尔塔冠状病毒毒株及其应用 Download PDFInfo
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Abstract
本发明提供了一种猪德尔塔冠状病毒毒株，该毒株为PDCoV/GX/2021株，其保藏编号为CCTCCC NO:V202151，该毒株免疫原性好，制得的疫苗具有抗猪德尔塔冠状病毒作用，对怀孕母猪接种PDCoV/GX/2021株制得的灭活疫苗后，可以使新生哺乳仔猪获得PDCoV免疫力，免受PDCoV感染。
Description
技术领域
本发明涉及兽用疫苗制备技术，具体涉及一种猪德尔塔冠状病毒毒株及其在疫苗制备方面的应用。
背景技术
猪德尔塔冠状病毒(Porcine Deltacoronavirus，以下简称PDCoV)是一种新发的猪冠状病毒，为有囊膜的、单股正链RNA病毒，属于尼多目(Nidovrales)冠状病毒科(Coronavirilae)冠状病毒亚科(Coronavirinae)德尔塔冠状病毒属(Deltacoronavirusgenus)。PDCoV能够感染各年龄段的猪群，引起仔猪出现严重的肠炎、水样腹泻和呕吐，仔猪死亡率约30％～40％，与猪流行性腹泻和猪传染性胃肠炎症状十分相似。PDCoV基因组结构与其他冠状病毒类似，全长约25.4kb，从5’-3’端依次编码复制酶ORF1ab、S糖蛋白(S)、小分子膜蛋白(E)、膜蛋白(M)以及he衣壳蛋白(N)。
发明内容
本发明的目的是提供一种猪德尔塔冠状病毒毒株以及其在制备疫苗方面的应用。
本发明通过以下技术方案实现：
一种猪德尔塔冠状病毒毒株，为PDCoV/GX/2021株，其保藏信息如下：
保藏编号：CCTCCC NO:V202151
保藏日期：2021/5/26
保藏单位：中国典型培养物保藏中心(简称：CCTCCC)
保藏单位地址：中国武汉武汉大学
猪德尔塔冠状病毒PDCoV/GX/2021株在制备猪德尔塔冠状病毒疫苗中的应用，以免疫量的PDCoV/GX/2021株作为猪德尔塔冠状病毒疫苗的免疫原。
本发明提供的猪德尔塔冠状病毒毒株PDCoV/GX/2021株为最新流行毒株，具有增值能力高、免疫原性好的特点，其灭活制备的疫苗，可以有效预防PDCoV，通过对怀孕母猪接种PDCoV/GX/2021株疫苗，可有效保护新生哺乳仔猪免受PDCoV感染。
附图说明
图1为实施例1中的PDCoV致细胞病变图，其中图A为PDCoV/GX/2021株接种于ST细胞后致细胞病变情况，图B为未接种PDCoV/GX/2021株的ST细胞的培养情况。
图2为实施例1中的PDCoV/GX/2021株病毒S基因PCR鉴定电泳图，其中，条带1为PDCoV-S1引物扩增产物；条带2为PDCoV-S2引物扩增产物。
图3为实施例4中不同试验组仔猪攻毒后的存活数量情况。
图4为实施例4中不同试验组仔猪攻毒后的腹泻得分情况。
具体实施方式
下面结合具体实施例对本发明进行详细的描述。
实施例1：猪德尔塔冠状病毒分离与鉴定
于2021年广西省某患有腹泻疾病的猪场中的发病仔猪的小肠组织中分离得到一株病毒，将发现病毒的小肠组织经充分研磨、双抗(青霉素、链霉素)处理后，12000rpm/min4℃离心10min处理，再经0.22μm微孔滤膜过滤除菌后，获得病毒样品，将其分别与猪德尔塔冠状病毒(PDCoV)、猪圆环病毒2型(PCV2)、猪伪狂犬病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪流行性腹泻病毒(PEDV)等病毒进行PCR/RT-PCR对照检测，最终检测其为PDCoV单纯阳性。
将样品接种于ST细胞进行连续传代至第3代，获得一株高增殖能力的PDCoV毒株，命名为PDCoV/GX/2021株，该病毒毒株已送至湖北省武汉市武汉大学中国典型培养物保藏中心保藏，保藏编号为NO:V202151。
通过对PDCoV/GX/2021株进行毒力回归试验、致病试验以及PCR扩增测序分析，鉴定其分类。
(1)毒力回归试验
取3日龄的猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪轮状病毒(PoRV)和PDCoV抗原阴性的哺乳仔猪10头，随机分成2组，每组5头，分别为攻毒组和对照组。攻毒组经口服接种病毒含量为106.00TCID50/mL的PDCoV/GX/2021株2mL，对照组经口服接种ST细胞培养液2mL，隔离饲养，观察仔猪生长情况。
接种后24h，攻毒组有5/5头仔猪在口服接种PDCoV/GX/2021株后出现腹泻情况，排出黄色水样粪便；而对照组无异常表现。将出现腹泻情况后12h的仔猪全部剖杀检测，剖检可见攻毒组全部仔猪出现十二指肠、空肠、回肠肠壁变薄情况，且十二指肠、空肠、回肠内充满气体和黄色水样稀便。对攻毒组和空白对照组中的全部仔猪的十二指肠、空肠、回肠组织进行PDCoV荧光定量PCR检测，结果显示，在攻毒组仔猪的空肠和回肠中检测到大量的PDCoV抗原，而对照组仔猪的空肠和回肠中PDCoV抗原为阴性。
(2)细胞致病试验
将生长至单层的ST细胞用磷酸盐缓冲液(PBS)清洗3～4次，加入1％(V/V)浓度为1‰～3‰的胰酶作用30min～1h，弃去胰酶液，将PDCoV/GX/2021株毒液按照5％(V/V)接种于ST细胞上，加入含有1‰～3‰胰酶的细胞维持液，置37℃、5％CO2的细胞培养箱中进行培养，观察细胞病变情况。接种PDCoV/GX/2021株24h后和未接种PDCoV/GX/2021株24h后的ST细胞显微观察结果分别如图1A、图1B所示。
由图1可知，接种PDCoV/GX/2021株24h后，ST细胞出现细胞病变，表现为细胞变大、变圆，聚集，最后脱落，PDCoV/GX/2021株可致ST细胞产生明显的特征性细胞病变，表明，该毒株的增殖性能良好。
(3)PCR扩增检测
参照GenBank中发表的PDCoV基因序列，使用Primier5设计了PDCoV S1和S2基因的特异性扩增引物，引物由擎科生物合成。所述的特异性引物的核苷酸序列如下：
PDCoV-S1-F：5’-CTCGGCTCGTGAGTTAGAGAAG-3’；
PDCoV-S1-R:5’-CAACAGTGCCAAGGCCACTAGT-3’
PDCoV-S2-F:5’-CCAACTTCAAGGGTGACTACAA-3’
PDCoV-S2-R:5’-CGCCAAGCGCCCATATGATC-3’
用商品化的病毒DNA/RNA基因组提取试剂盒提取病毒RNA，按照说明书操作。
PDCoV-S1的PCR扩增总体系为50μL，其中上游PDCoV-S1-F 2.5μL，下游引物PDCoV-S1-R 2.5μL，Q5高保真PCR预混酶25μL，无菌蒸馏水20μL。PCR反应程序为98℃预变性30秒；98℃变性10秒，56℃退火30秒，72℃延伸1分钟，共34个循环；72℃再延伸2分钟。
PDCoV-S2的PCR扩增总体系为50μL，其中上游PDCoV-S2-F 2.5μL，下游引物PDCoV-S2-R 2.5μL，Q5高保真PCR预混酶25μL，无菌蒸馏水20μL。PCR反应程序为98℃预变性30秒；98℃变性10秒，56℃退火30秒，72℃延伸1分钟，共34个循环；72℃再延伸2分钟。
PCR产物于1％琼脂糖凝胶上电泳检测(结果见图2)。由图2可见，PDCoV-S1和PDCoV-S2均扩增出一条约2000bp的条带，与预期扩增片段大小相符。
对PDCoV/GX/2021株的S基因进行测定，发现该毒株S基因全长为3480nt，其核苷酸序列如SEQ ID No.1所示，该毒株S基因翻译出的蛋白的氨基酸序列如SEQ ID No.2所示，其与国内PDCoV流行毒株S基因氨基酸同源性在98.4％左右，因此，PDCoV/GX/2021株属于PDCoV。
(4)病毒效价测定
将PDCoV/GX/2021株用含浓度为1‰～3‰胰酶的细胞维持液进行10倍系列稀释，稀释至10-4、10-5、10-6、10-7、10-8 5个稀释梯度，分别接种于生长至单层的96孔ST细胞培养板中，将培养板置于37℃、5％CO2的细胞培养箱中进行培养，每天观察细胞的CPE情况，记录CPE细胞孔，按照Reed-Muench法计算TCID50。
经计算，该培养方法下，PDCoV/GX/2021株的病毒滴度可达106.00TCID50/mL。
实施例2：PDCoV灭活疫苗制备
1、细胞制备：从液氮罐中取出ST种子细胞管，置37℃水浴锅中快速融化，将细胞转移入无菌的10mL离心管中，1000rpm/min常温离心5min，弃去上清液，细胞沉淀用含10％新生牛血清的DMEM细胞培养液重悬，移入一个新的细胞培养瓶中，置37℃、5％CO2培养箱中进行培养，48h后细胞生长成良好的单层，用胰酶-EDTA消化细胞进行传代培养。
2、种毒制备：ST细胞生长至单层时，接种PDCoV/GX/2021株，弃去细胞生长液，用磷酸盐缓冲液(PBS)清洗细胞3～4次，加入1％～2％(V/V)的浓度为1‰～3‰胰酶作用30min～1h，弃去胰酶液，将PDCoV/GX/2021株种毒液按照5％(V/V)接种ST细胞，加入含有1‰～3‰胰酶的DMEM细胞维持液，置37℃、5％CO2的细胞培养箱中进行培养；接毒后，每日观察2次，记录细胞病变情况，当细胞病变达80％以上时收集病毒，将收集的病毒在-80℃反复冻融三次，6000rpm离心20min分钟，收集上清液，即获得病毒液。
3、病毒灭活：将检验合格的病毒液导入灭活灌中，边搅拌边加入甲醛溶液，使甲醛的最终浓度为1‰(V/V)，在37℃下搅拌灭活24～28h。灭活后取样接种ST细胞进行灭活检验，连续盲传3代无细胞病变，即为灭活彻底，灭活后的抗原液置于2～8℃保存。
4、疫苗制备：采用畜禽用水包油包水佐剂，佐剂配比为40％，抗原体积为60％；混合后磁力搅拌(转速400转/min，搅拌时间为40～60min)，即获得猪德尔塔冠状病毒灭活疫苗(病毒含量为106.00TCID50/mL)。
实施例3：疫苗安全试验
选择预产前6周、预产前4周的怀孕母猪各5头，通过后海穴注射双倍剂量(6mL/头份)的106.00TCID50/mL PDCoV/GX/2021株灭活疫苗，接种疫苗后连续14天观察母猪的精神状态、行为活动、眼睛和鼻孔有无分泌物、采食状况、流产、局部不良反应和全身反应、，其中全身反应主要包括过敏反应和应急反应，局部不良反应主要包括红、肿、热、痛等。
观察得到，免疫后母猪没有观察到任何临床异常，采食饮水、体温正常，健康状况良好、疫苗刚接种后，后海穴有轻微肿大，半天后基本消失，未发现任何局部不良反应和全身反应，没有流产症状。
因此，PDCoV/GX/2021株灭活疫苗双倍剂量对怀孕母猪是安全的。
实施例4：疫苗免疫原性试验
取实施例2中制备的三批次PDCoV/GX/2021株灭活疫苗进行攻毒试验。
(1)怀孕母猪免疫试验
将40头怀孕母猪随机均分成4组，分别为试验1组、试验2组、试验3组、试验4组，其中试验1-3组分别接种实施例2中制备的三批次PDCoV/GX/2021株灭活疫苗，作为免疫组，试验4组不作处理，作为对照组，隔离饲养，自由采食。其中，接种方法为：在怀孕母猪预产前6周、预产前4周分别经后海穴注射接种1头份(3mL/头份)106.00TCID50/mL PDCoV/GX/2021株灭活疫苗。具体免疫试验处理情况如表1所示。
表1.免疫试验处理情况
(2)仔猪攻毒保护试验
从上述试验1组、试验2组、试验3组、试验4组中的母猪所产仔猪中，每组随机选择20头体重相近、精神状况及食欲良好的5日龄仔猪为研究对象，继续对应分为试验1组、试验2组、试验3组、试验4组，每组仔猪口服1.0mL1.0×106.00TCID50/mL PDCoV/GX/2021强毒株进行攻毒测试，攻毒后隔离饲养，各仔猪采用人工奶粉饲养。具体攻毒处理情况如表2所示。在攻毒前后每日观察记录仔猪的精神食欲状况，对仔猪的腹泻对仔猪的腹泻情况和死亡情况进行统计，“正常不拉稀”为0分，“轻微拉稀”为1分，“中度拉稀”为2分，“严重拉稀”为3分，“死亡”为4分。攻毒后7天内，各组仔猪存活及腹泻程度情况分别如图3、4所示。
表2.仔猪攻毒处理情况
| 分组 | 攻毒毒株 | 仔猪数量 |
| 试验1组 | PDCoV/GX/2021 | 20 |
| 试验2组 | PDCoV/GX/2021 | 20 |
| 试验3组 | PDCoV/GX/2021 | 20 |
| 试验4组 | PDCoV/GX/2021 | 20 |
试验1组攻毒后有4头仔猪出现轻微腹泻，采食量略有下降，第3天自行恢复，攻毒后第2天有3头仔猪死亡，其余仔猪攻毒后7天均康复，采食良好；试验2组攻毒后所有仔猪未出现腹泻情况，攻毒后第2天有2头仔猪死亡，其余仔猪均存活，采食良好；试验3组攻毒后所有仔猪也未出现腹泻情况，攻毒后3天有1头仔猪死亡，其余仔猪均存活，采食良好；试验4组攻毒后24h开始出现腹泻情况，48-96h出现严重腹泻，仔猪倒地不起，随后开始死亡，至攻毒结束全部死亡。对各试验组仔猪进行剖检，可见，试验4组仔猪十二指肠、空肠、回肠肠壁变薄，其内充满气体和黄色水样稀便，荧光定量PCR检测结果显示，在感染猪的空肠和回肠中检测到大量的PDCoV抗原。
攻毒后每日定期采集仔猪肛门拭子，采用荧光定量PCR检测拭子样品中PDCoV的含量，结果显示，攻毒后72h，试验1组、试验2组和试验3组的各仔猪肛门拭子均为PDCoV阴性，而试验4组仔猪肛门拭子为PDCoV强阳性。
表明，通过对怀孕母猪接种PDCoV/GX/2021株灭活疫苗，可以使仔猪获得较好的免疫保护，使仔猪免受PDCoV感染。
综上所述，本发明通过筛选获得的高增殖力的PDCoV/GX/2021株，将其制备为PDCoV灭活疫苗，该疫苗具有良好的安全性和免疫原性，可以使免疫仔猪获得良好的保护。
最后应说明的是：以上实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述实施例对本申请进行了详细的说明，本领域的普通技术人员当理解：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；而这些修改或者替换，并不驱使相应技术方案的本质脱离本申请各实施例技术方案的精神和范围。
序列表
<110> 江西正邦科技股份有限公司
江西正邦农业科学院有限公司
<120> 一种猪德尔塔冠状病毒毒株及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3480
<212> DNA
<213> 猪德尔塔冠状病毒(Porcine Deltacoronavirus)
<400> 1
atgcagagag ctctattgat tatgacctta ctttgtctcg ttcgagcaaa gtttgctgat 60
gatctactcg atttgctcac cttcccgggt gcacatcgat tcttacataa actcacgagt 120
aattccagca ctctccactc gcgggctaat aattttaatg ttggcgttct tcctggctat 180
cccactaaga acgttaacct cttctcacca cttactaact ctactttgcc cattaatggc 240
cttcatcgga gttatcaacc actcatgctg aattgtttta ctaaaataac taaccacact 300
ctcagcatgt atctcctacc tagtgagata caaacttata gctgcggcgg tgccatggtt 360
aaataccaga cacatgatgc agttcgtatc attttagacc tcactgccac tgaccacatc 420
tctgttgaag tcgttggcca gcatggtgaa aattatgtgt ttgtttgtag tgagcagtct 480
acctacacca ctgcattaca caactctacc tttttctcac ttaattcaga gctttattgc 540
tttactaata acacctactt aggtattctt ccacctgatt taactgactt tacggtctac 600
cgtactggtc agttctatgc taatggttac cttttaggta ctttacctat tacggttaac 660
tatgttaggt tgtatcgggg tcaattggcg gccaatagtg cccactttgc cctagcaaac 720
ctaaccgata cactcataac acttaccaat accactatat cgcaaatcac ttattgtgat 780
aagtcagtag ttgattcaat agcatgccag cgctcttctc acgaagtgga ggatgggttt 840
tactccgacc ctaaatctgc cgttagagct aggcaacgta ctattgtcac actacctaag 900
ctccccgagc ttgaagtagt gcagttaaat atttctgcac acatggattt tggcgaagcc 960
agacttgaca gcattaccat caatggtaac acatcctatt gtgtcactaa gccttacttc 1020
aggcttgaaa ctaactttat gtgtacaggt tgcactatga atctgcgcac tgatacttgt 1080
agttttgacc tgtcagcagt aaacaatggc atgtcattct ctcaattctg tctaagcact 1140
gaagctggtg cttgtgagat gaaaattatt gttacctacg tatggaatta cttgctaagg 1200
cagcgtttgt atgttactgc tgtagagggc cagactcaca ctggaaccac ttcagtacat 1260
gcaacagaca cttctagtgt aatcactgat atttgcactg actacactat ctatggcgtc 1320
tctggtactg gcattattaa gccatcagat ctcttattgc acaatggcat agcattcacc 1380
tctccaacag gtgagcttta tgcatttaaa aatataacca ctggcaaagc cctccaggtc 1440
ttaccgtgtg aaaccccttc tcaactgatt gtgataaaca acaccgtcgt cggtgctatc 1500
acatccagta actcaactga aaataatagg tttactacta ctattgtcac acctactttc 1560
ttttattcca caaatgccac cattttcaac tgcactaagc ctgttttgtc ctatggacct 1620
atcagtgtgt gtagtgatgg tgcaattgtt ggaacatcca cattacagaa tactcgacca 1680
tctatagttt cactatacga tggcgaagtt gaaataccat ctgcattttc tctttctgtt 1740
cagacggagt acttgcaagt tcaagcagag caagttatag ttgattgtcc ccagtatgta 1800
tgcaatggca acagccgttg tctacaatta ctggcacaat acacctcagc ttgctctaac 1860
attgaagtag ctctgcattc ctctgcacag tttgaaagca gagagattat aaatatgttt 1920
caaacatcaa cacagtcctt gcagttggct aatattacca acttcaaggg tgactacaat 1980
tttagcagca taataacccc cagaattggt ggcagatctg ctattgaaga ccttcttttt 2040
aataaagttg ttactagtgg ccttggcact gttgatcagg actacaaagc ctgctctaga 2100
gacatggcca tcgctgactt agtttgttcc cagtattaca atggcatcat ggttctacct 2160
ggtgttgttg atgctgagaa aatggcaatg tatactggct ctcttactgg agctatggta 2220
tttggaggac tgactgctgc agcggcaata ccattcgcca cggcagtaca agcccgcctc 2280
aattatgtcg cactgcaaac aaatgtacta caagaaaacc agaaaatact tgcagaatca 2340
tttaaccaag cagttggcaa tatatcactt gcactatctt ctgttaatga tgccatccag 2400
caaacttctg aggctcttag caccgtagct attgctatta aaaagattca aacagttgtc 2460
aaccagcagg gtgaggcatt atcacacctg actgcacagc tgtcaaacaa ttttcaagca 2520
atttcgactt ctattcaaga catttacaac cgtcttgagg aagtagaggc taaccagcaa 2580
gttgaccgtc tcattacagg acggttggct gcacttaatg catatgttac tcagttactc 2640
aatcagatgt ctcagattag acaatctcga ttgttagctc agcaaaagat taatgagtgt 2700
gtcaaatctc agtcgtctag atacggtttc tgtggaaatg gcacacacat cttctcactt 2760
acacagactg caccaaatgg catatttttc atgcatgcag tactcgtacc caacaaattc 2820
acacgtgtca acgcttctgc gggtatttgt gtggataata cgagaggcta ctcattgcag 2880
cctcaactta tactctacca gtttaataac tcctggagag ttacacctag aaatatgtat 2940
gaacccagac tgccccggca agctgatttc atacaattaa ctgattgcag cgttaatttt 3000
tacaatacca ccgctgctaa tcttcccaat attatccctg acgttataga tgtcaatcaa 3060
acagtcagtg atattattga caatttacct acagcaacac ctcctcagtg ggatgttggt 3120
atctataaca acactattct caacctcacc gttgagatta atgatctaca agagcggtct 3180
aaaaacctct cacagattgc agatcgttta caaaattata ttgacaatct caacaatact 3240
ctagttgacc ttgaatggct caacaaagtg gaaacttacc ttaaatggcc gtggtatata 3300
tggcttgcca tagccctggc tcttattgca tttgtgacaa tcctcataac aatctttctc 3360
tgtactggtt gttgtggtgg ttgttttggt tgttgtggcg gttgttttgg ccttttctct 3420
aagaagaaaa ggtataccga cgaccaacca acaccgtcct ttaaatttaa ggaatggtag 3480
<210> 2
<211> 1159
<212> PRT
<213> 猪德尔塔冠状病毒(Porcine Deltacoronavirus)
<400> 2
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Arg Ile Ile Leu Asp Leu Thr Ala Thr Asp His Ile Ser Val Glu Val
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Val Gly Gln His Gly Glu Asn Tyr Val Phe Val Cys Ser Glu Gln Ser
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Asp Leu Thr Asp Phe Thr Val Tyr Arg Thr Gly Gln Phe Tyr Ala Asn
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Thr Tyr Cys Asp Lys Ser Val Val Asp Ser Ile Ala Cys Gln Arg Ser
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Ser His Glu Val Glu Asp Gly Phe Tyr Ser Asp Pro Lys Ser Ala Val
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Glu Val Val Gln Leu Asn Ile Ser Ala His Met Asp Phe Gly Glu Ala
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Arg Leu Asp Ser Ile Thr Ile Asn Gly Asn Thr Ser Tyr Cys Val Thr
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Lys Pro Tyr Phe Arg Leu Glu Thr Asn Phe Met Cys Thr Gly Cys Thr
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Met Asn Leu Arg Thr Asp Thr Cys Ser Phe Asp Leu Ser Ala Val Asn
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Asn Gly Met Ser Phe Ser Gln Phe Cys Leu Ser Thr Glu Ala Gly Ala
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Cys Glu Met Lys Ile Ile Val Thr Tyr Val Trp Asn Tyr Leu Leu Arg
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Gln Arg Leu Tyr Val Thr Ala Val Glu Gly Gln Thr His Thr Gly Thr
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Thr Ser Val His Ala Thr Asp Thr Ser Ser Val Ile Thr Asp Ile Cys
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Thr Asp Tyr Thr Ile Tyr Gly Val Ser Gly Thr Gly Ile Ile Lys Pro
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Ser Asp Leu Leu Leu His Asn Gly Ile Ala Phe Thr Ser Pro Thr Gly
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Gly Ala Met Val Phe Gly Gly Leu Thr Ala Ala Ala Ala Ile Pro Phe
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Ala Thr Ala Val Gln Ala Arg Leu Asn Tyr Val Ala Leu Gln Thr Asn
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Val Leu Gln Glu Asn Gln Lys Ile Leu Ala Glu Ser Phe Asn Gln Ala
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Val Gly Asn Ile Ser Leu Ala Leu Ser Ser Val Asn Asp Ala Ile Gln
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Gln Thr Ser Glu Ala Leu Ser Thr Val Ala Ile Ala Ile Lys Lys Ile
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Gln Thr Val Val Asn Gln Gln Gly Glu Ala Leu Ser His Leu Thr Ala
820 825 830
Gln Leu Ser Asn Asn Phe Gln Ala Ile Ser Thr Ser Ile Gln Asp Ile
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Tyr Asn Arg Leu Glu Glu Val Glu Ala Asn Gln Gln Val Asp Arg Leu
850 855 860
Ile Thr Gly Arg Leu Ala Ala Leu Asn Ala Tyr Val Thr Gln Leu Leu
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Asn Gln Met Ser Gln Ile Arg Gln Ser Arg Leu Leu Ala Gln Gln Lys
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Ile Asn Glu Cys Val Lys Ser Gln Ser Ser Arg Tyr Gly Phe Cys Gly
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Phe Phe Met His Ala Val Leu Val Pro Asn Lys Phe Thr Arg Val Asn
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Ala Ser Ala Gly Ile Cys Val Asp Asn Thr Arg Gly Tyr Ser Leu Gln
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Pro Gln Leu Ile Leu Tyr Gln Phe Asn Asn Ser Trp Arg Val Thr Pro
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Pro Asn Ile Ile Pro Asp Val Ile Asp Val Asn Gln Thr Val Ser Asp
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Ile Ile Asp Asn Leu Pro Thr Ala Thr Pro Pro Gln Trp Asp Val Gly
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Ile Tyr Asn Asn Thr Ile Leu Asn Leu Thr Val Glu Ile Asn Asp Leu
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Ala Leu Ala Leu Ile Ala Phe Val Thr Ile Leu Ile Thr Ile Phe Leu
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Cys Thr Gly Cys Cys Gly Gly Cys Phe Gly Cys Cys Gly Gly Cys Phe
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Gly Leu Phe Ser Lys Lys Lys Arg Tyr Thr Asp Asp Gln Pro Thr Pro
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Ser Phe Lys Phe Lys Glu Trp
1155
Claims (4)
1.一种猪德尔塔冠状病毒毒株，其特征在于，该毒株为PDCoV/GX/2021株，其保藏编号为CCTCC NO:V202151。
2.一种猪德尔塔冠状病毒毒株在制备猪德尔塔冠状病毒疫苗中的应用，其特征在于，以免疫量的权利要求1所述的猪德尔塔冠状病毒毒株作为疫苗的抗原。
3.一种猪德尔塔冠状病毒疫苗，其特征在于，所述疫苗为灭活疫苗，包括抗原和疫苗佐剂，其中抗原为权利要求1所述的猪德尔塔冠状病毒毒株。
4.如权利要求3所述的疫苗，其特征在于，所述灭活疫苗是毒株经甲醛灭活得到。
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| CN110144332A (zh) * | 2019-05-13 | 2019-08-20 | 中国农业科学院兰州兽医研究所 | 一株猪德尔塔冠状病毒毒株及其应用 |
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