CN110468155B - 一种用于拯救猪肠道甲型冠状病毒的***、方法及应用 - Google Patents
一种用于拯救猪肠道甲型冠状病毒的***、方法及应用 Download PDFInfo
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Abstract
本发明公开了一种用于拯救猪肠道甲型冠状病毒的***、方法及应用。所述***包括：包含猪肠道甲型冠状病毒全基因组DNA的重组转录载体；包含SeACoV‑N基因的辅助质粒。本发明成功构建了SADS‑CoV/SeACoV反向遗传操作***，产生的病毒可连续传代，拯救病毒与亲本病毒具有一致生物学特性。建立反向遗传学***受到病毒基因组序列的不稳定性和毒性的阻碍，该拯救方法在猪冠状病毒反向遗传学研究和疫苗创制上具有潜在应用价值，在病毒基因功能研究等方面有广泛的应用前景。
Description
技术领域
本发明涉及生物技术领域，特别是涉及一种用于拯救猪肠道甲型冠状病毒的***、方法及应用。
背景技术
中国是世界第一养猪大国，规模化养殖发展迅速。但随着规模化养猪的快速发展，冠状病毒感染对养猪业的危害越来越大。因此，加强猪冠状病毒的感染与致病机理的认识，将为这些疫病防控提供新的理论与技术支持。
2017年在华南首次发现的猪肠道甲型冠状病毒(swine entericalphacoronavirus，SeACoV)又称猪急性腹泻综合征病毒(Swine acute DiarrhoeaSyndrome Coronavirus，SADS-CoV)引发仔猪严重腹泻，是进化起源为蝙蝠HKU2-CoV的新型冠状病毒。与HKU2相比，SADS-CoV/SeACoV的S蛋白存在高度变异，并主要集中在N端区域(NTD，domain 0)的1-238位氨基酸：该区域有75个氨基酸点突变及2个氨基酸的***。我们推测NTD上的高度变异可能导致了从蝙蝠冠状病毒HKU2变异为猪SeACoV，从而跨种属从蝙蝠传播到猪。但目前对该病毒的研究几乎一片空白。
猪腹泻相关病毒对全球养猪业产生巨大的威胁，因此需要更深入地了解这类病毒。目前面临的主要问题在于缺乏适用于研究猪冠状病毒SADS-CoV/SeACoV的遗传工具。
冠状病毒科的病毒是单股正链RNA病毒，其基因组具有重叠的ORF编码复制酶(ORF1a，1b)，非结构蛋白以及ORF3，纤突蛋白(S)，包膜蛋白(E)，膜蛋白(M)和核衣壳蛋白(N)。研究冠状病毒的发病机制目前很大程度上受到分离培养和缺乏可操纵病毒遗传工具的限制。相较于其他病毒，冠状病毒具有长度约27-30kb的基因组，是RNA病毒中最大的，其在用于产生感染性克隆的工程载体中存在显着的挑战，天然和扩增相关突变之间的区别和测序错误可能极其困难。此外，建立反向遗传学***的尝试受到病毒基因组序列的不稳定性和毒性的阻碍。
反向遗传技术是开展病毒研究的一种有用平台，在阐明病毒致病机制和疫苗研制中能够发挥重要作用。同样，构建猪冠状病毒SADS-CoV/SeACoV感染性分子克隆，以此为基础可以对SADS-CoV/SeACoV的特定基因展开更为有效的研究，也是SADS-CoV/SeACoV研究的一个有用平台。
目前，国内外尚无SADS-CoV/SeACoV的反向遗传***的被成功构建的报道。
发明内容
本发明针对现有技术中存在的不足，提供了一种能够成功拯救出猪肠道甲型冠状病毒的病毒拯救***。
一种用于拯救猪肠道甲型冠状病毒的***，包括：
包含猪肠道甲型冠状病毒全基因组DNA的重组转录载体；
包含SeACoV-N基因的辅助质粒，
其中，所述猪肠道甲型冠状病毒为单股正链RNA病毒，所述猪肠道甲型冠状病毒全基因组DNA是指猪肠道甲型冠状病毒正链RNA序列对应的DNA序列，所述SeACoV-N基因的序列为猪肠道甲型冠状病毒编码该基因的RNA序列对应的DNA序列。
由于猪肠道甲型冠状病毒为单股正链RNA病毒，所以拯救时使用的为病毒的基因组RNA对应的DNA序列，即仅将RNA序列中的碱基U变为T。
SeACoV-N基因的序列大小为1125bp(如SEQ ID NO.1所示)，表达SeACoV-N蛋白，经研究发现能够通过抑制干扰素表达的方式辅助病毒的拯救。
所述重组转录载体使用质粒为pSB，所述辅助质粒使用的质粒为pRK5。这两个质粒均为商业化可购买的质粒。
为了便于检测区分拯救病毒与野生病毒，所述拯救猪肠道甲型冠状病毒全基因组cDNA的第24222-24224位碱基由AGT突变为TCT，这两个密码子均对应编码丝氨酸(Ser)，所以氨基酸Ser保持不变。
本发明还公开了一种拯救猪肠道甲型冠状病毒的方法，包括以下步骤：
(1)将所述***中的重组转录载体和辅助质粒共转染入宿主细胞中；
(2)培养后收集细胞与上清中的拯救所得猪肠道甲型冠状病毒。
步骤(1)中重组转录载体和辅助质粒加入的质量比为2∶1。所述宿主细胞为BHK-21细胞。
本发明还提供了使用所述方法拯救获得的猪肠道甲型冠状病毒。
本发明还提供了所述拯救获得的猪肠道甲型冠状病毒在作为猪肠道甲型冠状病毒研究模型中的应用。
本发明还提供了所述拯救获得的猪肠道甲型冠状病毒在制备预防猪腹泻疫苗中的应用。
本发明成功构建了SADS-CoV/SeACoV反向遗传操作***，产生的病毒可连续传代，拯救病毒与亲本病毒具有一致生物学特性。建立反向遗传学***受到病毒基因组序列的不稳定性和毒性的阻碍，该拯救方法在猪冠状病毒反向遗传学研究和疫苗创制上具有潜在应用价值，在病毒基因功能研究等方面有广泛的应用前景。
附图说明
图1为猪肠道甲型冠状病毒SADS-CoV/SeACoV 16段基因片段结构示意图。
图2为猪肠道甲型冠状病毒SADS-CoV/SeACoV 16段基因片段扩增结果检测图。
图3为猪肠道甲型冠状病毒SADS-CoV/SeACoV感染性克隆质粒pSB-SeACoV构建示意图。
图4为猪肠道甲型冠状病毒SADS-CoV/SeACoV感染性克隆质粒23段测序片段示意图。
图5为猪肠道甲型冠状病毒SADS-CoV/SeACoV感染性克隆质粒23段片段扩增鉴定结果图。
图6为猪肠道甲型冠状病毒SADS-CoV/SeACoV感染性克隆拯救病毒利用SeACoV-N蛋白抗体免疫荧光检测结果图，图A为单独转染感染性克隆质粒pSB-SeACoV拯救病毒组，图B为感染性克隆质粒pSB-SeACoV和pRK5-SeACoV-N质粒共转染拯救病毒组。
图7为拯救病毒rSeACoV的ORF3基因中分子标记位于TF21片段PCR检测结果图，其中泳道1为对照，泳道2为病毒拯救时上清，泳道3为病毒拯救时细胞内，泳道M为标准分子量Marker。
图8为拯救病毒rSeACoV位于ORF3上Ser分子标记测序结果图。
图9为检测拯救病毒与亲本病毒生长曲线比较结果图。
图10为拯救病毒的病毒粒子在电子显微镜下观察结果图。
具体实施方式
实施例1
1.SADS-CoV/SeACoV全长感染性克隆目的片段的扩增
根据Genbank上公布的猪肠道甲型冠状病毒SeACoV CH/GD-01/2017/P2株基因序列(GenBank accession no.MF370205，图1)，病毒基因组全长27155nt，与10年前同一地区发现的感染中华菊头蝠的蝙蝠肠道甲型冠状病毒HKU2在全基因组核酸水平上的同源性为94.9％。细胞稳定传代毒株SADS-CoV/SeACoV-P10的细胞感染上清样品，Trizol法提取RNA后反转录得到cDNA，使用本发明提供的16对引物(引物序列见表1)，扩增得到相应的16个基因组全长片段，通过pCR-Blunt载体克隆，挑取3个单克隆菌落，提取质粒测序拼接，确定SADS-CoV/SeACoV-P10的基因组全长序列。使用相对应的16对引物，分别以保存的带有SADS-CoV/SeACoV 16个片段的的pCR-Blunt质粒为模板，扩增相应全基因组全长的分截断片段(图2)。
表1
为能将SADS-CoV/SeACoV全长各片段连接进载体上，即要求SeACoV-F1片段的5’端与载体片段3’端、SeACoV-F15片段3’端与载体片段5‘端’至少有20bp的同源区，以便于同源重组。设计引物对SeACoV-F1和SeACoV-F15两片段分别加上5’和3’同源序列。引物序列如下：
SeACoV-F1上游引物SeACoV-F1-F：
5’-ATAAGCAGAGCTCGTTTAGTGAACCGTGACTTAAAGATATAA-3’；
SeACoV-F1下游引物SeACoV-F1-R：
5’-GTCATCACAGAGGGCAGTAAAGC-3’；
SeACoV-F15上游引物SeACoV-F15-F：
5’-ATGGCATCAGAATTGCTACTGGTGT-3’；
SeACoV-F15下游引物SeACoV-F15-R：
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTGTGTATCACTGTCAA-3’。
同时在ORF3基因中引入一个氨基酸的同义突变作为拯救病毒的检测分子标记，即24222-24224位碱基由AGT突变为TCT，氨基酸Ser保持不变(图3)，扩增猪肠道甲型冠状病毒SeACoV-F14第23403-24239位碱基和第24207-25298位碱基之间的基因片段，通过重叠PCR将所扩增的2个基因片段利用重叠PCR方法融合，获得融合片段SeACoV-F14-Marker，在位于SeACoV-F14片段的ORF3基因中引入分子标记，引物序列如下：
SeACoV-F14-Marker-a段上游引物SeACoV-F14-F：
5’-ATTTGCTAATGTCATTGCCGTTTCC-3’；
SeACoV-F14-Marker-a段下游引物SeACoV-Marker-a-R：
5’-GAGAACAAAAGCAAAAGACCTG-3’；
SeACoV-F14-Marker-b段上游引物SeACoV-Marker-b-F：
5’-GCTTTTTGTTACAGGTCTTTTGC-3’；
SeACoV-F14-Marker-b段下游引物SeACoV-F14-R：
5’-GGCGACAGTCACAAATTGCGGTA-3’。
2.SADS-CoV/SeACoV全长感染性克隆的拼接
通过Gibson装配方法进行组装，用GBclonart无缝克隆试剂盒将SADS-CoV/SeACoV各个DNA片段及线性化的pSB载体(***位点在CMV启动子下游)通过体外同源重组的方式连接成全长感染性克隆重组质粒。通过转化连接产物至DH10B筛选并扩增阳性重组质粒克隆。
将扩增好的SeACoV-F1～F15总共16个片段与线性化载体片段等比例加入离心管中，按照GBclonart无缝克隆试剂盒的步骤要求操作，反应体系如下：
混匀并45℃孵育2h后，转移到冰上。立即转化DH10B感受态细胞。
3.SADS-CoV/SeACoV全长感染性克隆的验证
随机挑取单个克隆，分别接种到5mL含有氯霉素(30mg/mL)抗性的LB液体培养基中，37℃摇床培养，按照AxyPrep质粒DNA小量试剂盒说明书提取所克隆获得的SADS-CoV/SeACoV全长感染性克隆质粒。为对全长感染性克隆进行PCR扩增验证及后续测序验证，本研究将全长基因组序列分成23段，每段1400bp左右，以便于测序时只需上下游两个反应便可测通(图4)。
23段PCR测序引物见表2对提取的全长感染性克隆质粒进行PCR验证。取5μL PCR产物于1％琼脂糖凝胶电泳检测，选取阳性克隆，将该克隆1～23各段PCR产物送往尚亚生物技术有限公司测序，保存测序正确的全长感染性克隆甘油菌(图5)。将获得的重组BAC，命名为pSB-SeACoV，得到分子标记的猪肠道甲型冠状病毒SADS-CoV/SeACoV全长感染性克隆质粒。
表2
4.病毒拯救
4.1SADS-CoV/SeACoV感染性克隆质粒大提
配备1L 2×YT液体培养基，按照菌液与培养基1∶100的比例将甘油菌接于含有氯霉素(30mg/mL)抗性的2×YT液体培养基，37℃ 200rpm摇床培养12h。按照BAC/PAC DNAIsolation Maxi Kit说明书提取SeACoV感染性克隆质粒，-20℃保存备用。
4.2pRK5-SeACoV-N质粒构建
根据上游引物SeACoV-N-F：
5’-ACCTCGGTTCTATCGATTGGCCACCATGGCCACTGTTAATTGG-3’；
下游引物SeACoV-N-myc-R：
5’-CAGATCCTCTTCAGAGATGAGTTTCTGCTCATTAATAATCTCATC-3’；
以pSB-SeACoV质粒为模板，扩增片段SeACoV-N，使用EcoR I和Xba I两个酶切位点，对pRK5质粒通过双酶切反应进行线性化。使用无缝克隆试剂盒，连接线性化pRK5质粒及片段SeACoV-N，将连接产物转化进入Top10感受态，挑取单克隆菌落，提取质粒测序验证，成功构建pRK5-SeACoV-N质粒。
4.3SADS-CoV/SeACoV转染拯救
转染前在12孔板铺BHK-21细胞，置于5％ CO2，37℃培养箱中培养，待细胞密度达到70％左右进行转染。转染时将细胞培养液弃掉，用Opti-MEM洗两遍，转染试剂作阴性对照，感染性克隆质粒与表达SADS-CoV/SeACoV-N蛋白质粒按照
3000转染试剂盒说明书进行共转染，其中感染性克隆质粒pSB-SeACoV按照2μg/孔添加，pRK5-SeACoV-N质粒按照1μg/孔添加，转染7h后换液，逐日观察，直到出现细胞病变为止，反复冻融收集拯救病毒上清液，命名为rSeACoV。同时另一转染实验组单独添加2μg/孔的感染性克隆质粒pSB-SeACoV，不添加pRK5-SeACoV-N质粒作为对照。
在12孔板铺Vero细胞，置于5％CO2，37℃培养箱中培养，待细胞密度达到70％左右进行感染，感染3天后使用SADS-CoV/SeACoV-N蛋白兔多克隆抗体，通过间接免疫荧光检测(IFA)检测病毒拯救情况(图6)。单独转染感染性克隆质粒pSB-SeACoV拯救病毒组的拯救病毒上清液感染Vero细胞，并未检测到SADS-CoV/SeACoV-N蛋白的表达，而感染性克隆质粒pSB-SeACoV和pRK5-SeACoV-N质粒共转染组的拯救病毒上清液用于感染Vero细胞，间接免疫荧光检测能够明显的观察到SADS-CoV/SeACoV-N蛋白表达的绿色荧光信号，证明rSeACoV病毒的成功拯救。
5.rSeACoV的RT-PCR鉴定结果及测序验证
对rSeACoV感染Vero细胞的上清液和细胞样品提取总RNA，反转录成cDNA。根据引入一个氨基酸的同义突变作为拯救病毒的检测分子标记的ORF3基因位于全长基因组序列分成23段的TF21片段，使用TF21片段的引物扩增片段，
其中，上游引物SeACoV-TF21：5’-TACTGGATGTTGTGGCATGT-3’；
下游引物SeACoV-TR21：5’-TTCCACTTAAAATCGTCAGA-3’，
PCR产物送往尚亚生物技术有限公司测序(图7，图8)。根据测序结果，成功拯救获得能够稳定传代的带有分子标记的猪肠道甲型冠状病毒SADS-CoV/SeACoV。
通过TCID50检测原始病毒SADS-CoV/SeACoV及拯救病毒rSeACoV的生长曲线，二者具有相同的生长动力学特性，保证产生的病毒可连续传代，拯救病毒与亲本病毒具有一致生物学特性(图9)。对拯救病毒扩大培养分离纯化后，在电子显微镜下观察病毒粒子，可以观察到典型的完整冠状病毒粒子(图10)。
序列表
<110> 浙江大学
<120> 一种用于拯救猪肠道甲型冠状病毒的***、方法及应用
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
atttgctaat gtcattgccg tttcc 25
<210> 31
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
ggcgacagtc acaaattgcg gta 23
<210> 32
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
atggcatcag aattgctact ggtgt 25
<210> 33
<211> 42
<212> DNA
<213> 人工序列(artificial sequence)
<400> 33
tttttttttt tttttttttt tttttttgtg tatcactgtc aa 42
<210> 34
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gagaacaaaa gcaaaagacc tg 22
<210> 35
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
gctttttgtt acaggtcttt tgc 23
<210> 36
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
tccaaaatgt cgtaacaact 20
<210> 37
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
tccaagctca taacatgatt 20
<210> 38
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
tccactggta aagttacgac 20
<210> 39
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
agtaccctta agctcaccaa 20
<210> 40
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
tggttcagac tgttgccaat 20
<210> 41
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
taacaagatt atgtgtgcca 20
<210> 42
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
tcattgaggt aaacaaggct 20
<210> 43
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
acatgtcaga taacaagcca 20
<210> 44
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ttgaaacacc tgtggttgaa 20
<210> 45
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
accattacgg ataacaactg 20
<210> 46
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
cagcggtagc ttattaaacg 20
<210> 47
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
agataaacac acgcattctg 20
<210> 48
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
agggttgagt ttagtgatgg 20
<210> 49
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
tataccatcc acctgtctgc 20
<210> 50
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
ggtgctatga cttatggtga 20
<210> 51
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
tgaccattca taacaaaacc 20
<210> 52
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
ctgtgtcaca ggctaatgtt 20
<210> 53
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
gagtcaaaag gacctcttgg 20
<210> 54
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
tggacttatc tcctttgttg 20
<210> 55
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
catgctagct cagtctgttt 20
<210> 56
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
ttaaaggttg tcaagtggga 20
<210> 57
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
ccgtaaaatc atattcaagc 20
<210> 58
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
gcgtcttaac attggacaac 20
<210> 59
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
ccgttagaat gcacaacctc 20
<210> 60
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
tgaatgatgt cgataatggt 20
<210> 61
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
gacttaacac tctcctccct 20
<210> 62
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
ccacccatag gtttatcttg 20
<210> 63
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
gctccattta gggttctttg 20
<210> 64
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
taggacctga tgtgtttttg 20
<210> 65
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
tgagactgaa ctattcgtcc 20
<210> 66
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
gcgactcatg cgtactattg 20
<210> 67
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
agcattacga ccctgtgagt 20
<210> 68
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
gaggacgtgt gtacttgctt 20
<210> 69
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
agcactagaa aagtcaccga 20
<210> 70
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
ttatacgtga aaaacttgcg 20
<210> 71
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
atcaacaatt ctgttcaacc 20
<210> 72
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
gtggttctaa ttcgtgccct 20
<210> 73
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
ccatagcaac agaccatgtg 20
<210> 74
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
ggcttttacg gtgactatta 20
<210> 75
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 75
ttcgtcatta gggtcaagtt 20
<210> 76
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
tactggatgt tgtggcatgt 20
<210> 77
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
ttccacttaa aatcgtcaga 20
<210> 78
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 78
gttcttctcc ttcagcattc 20
<210> 79
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 79
ttatcagtct tggggacctt 20
<210> 80
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 80
tggtcctcgc tcagtttcta 20
<210> 81
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 81
tagaaggcac agtcgagtcc 20
<210> 82
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 82
acctcggttc tatcgattgg ccaccatggc cactgttaat tgg 43
<210> 83
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 83
cagatcctct tcagagatga gtttctgctc attaataatc tcatc 45
Claims (9)
1.一种用于拯救猪肠道甲型冠状病毒的***，其特征在于，包括：
包含猪肠道甲型冠状病毒全基因组DNA的重组转录载体；
包含SeACoV-N基因的辅助质粒，
其中，所述猪肠道甲型冠状病毒为单股正链RNA病毒，所述猪肠道甲型冠状病毒全基因组DNA是指猪肠道甲型冠状病毒正链RNA序列对应的DNA序列，所述SeACoV-N基因的序列为猪肠道甲型冠状病毒编码该基因的RNA序列对应的DNA序列，SeACoV-N基因的序列如SEQ IDNO.1所示。
2.如权利要求1所述的***，其特征在于，所述重组转录载体使用质粒为pSB，所述辅助质粒使用的质粒为pRK5。
3.如权利要求1所述的***，其特征在于，所述猪肠道甲型冠状病毒全基因组DNA的第24222-24224位碱基由AGT突变为TCT。
4.一种拯救猪肠道甲型冠状病毒的方法，其特征在于，包括以下步骤：
(1)将权利要求1～3任一所述***中的重组转录载体和辅助质粒共转染入宿主细胞中；
(2)培养后收集细胞与上清中的拯救所得猪肠道甲型冠状病毒。
5.如权利要求4所述的方法，其特征在于，步骤(1)中重组转录载体和辅助质粒加入的质量比为2∶1。
6.如权利要求4所述的方法，其特征在于，所述宿主细胞为BHK-21细胞。
7.使用拯救猪肠道甲型冠状病毒的方法拯救获得的猪肠道甲型冠状病毒，其中方法包括以下步骤：
(1)将权利要求3所述***中的重组转录载体和辅助质粒共转染入宿主细胞中；
(2)培养后收集细胞与上清中的拯救所得猪肠道甲型冠状病毒。
8.如权利要求7所述拯救获得的猪肠道甲型冠状病毒在作为猪肠道甲型冠状病毒研究模型中的应用。
9.如权利要求7所述拯救获得的猪肠道甲型冠状病毒在制备预防猪腹泻疫苗中的应用。
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