一种广谱抗牛科病毒性腹泻病毒的mRNA疫苗及其应用转让专利
申请号 : CN202310541256.2
文献号 : CN116284272B
文献日 : 2023-08-25
发明人 : 韩悌云 , 徐实 , 费才溢
申请人 : 南京澄实生物医药科技有限公司
摘要 :
权利要求 :
1.一种BVDV抗原融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如SEQ ID NO.1所示。
2.一种重组核酸,其特征在于,所述重组核酸为编码权利要求1所述BVDV抗原融合蛋白的核酸。
3.根据权利要求2所述的重组核酸,其特征在于,所述重组核酸的核酸序列如SEQ ID NO.2所示。
4.一种重组mRNA,其特征在于,所述重组mRNA为编码权利要求1所述BVDV抗原融合蛋白的mRNA。
5.根据权利要求4所述的重组mRNA,其特征在于,所述mRNA还包含5`‑UTR,3`‑UTR,polyA序列,IRES序列中的一种或多种。
6.根据权利要求5所述的重组mRNA,其特征在于,所述5`‑UTR的序列如SEQ ID NO.3所示,所述3`‑UTR的序列如SEQ ID NO.4所示,所述polyA的序列如SEQ ID NO.5所示,所述IRES序列如SEQ ID NO.8所示。
7.一种表达盒,其特征在于,包含权利要求2或3所述重组核酸、或权利要求4‑6任一项所述重组mRNA。
8.一种载体,其特征在于,包含权利要求2或3所述重组核酸、或权利要求4‑6任一项所述重组mRNA、或权利要求7所述表达盒。
9.一种细胞,其特征在于,包含权利要求2或3所述重组核酸、或权利要求4‑6任一项所述重组mRNA、或权利要求7所述表达盒、或权利要求8所述载体。
10.一种重组菌,其特征在于,包含权利要求2或3所述重组核酸、或权利要求4‑6任一项所述重组mRNA、或权利要求7所述表达盒、或权利要求8所述载体。
11.一种组合物,其特征在于,包含权利要求1所述BVDV抗原融合蛋白、或权利要求2或3所述重组核酸、或权利要求4‑6任一项所述重组mRNA、或权利要求7所述表达盒、或权利要求
8所述载体、或权利要求9所述细胞、或权利要求10所述重组菌。
12.一种试剂盒,其特征在于,包含权利要求1所述BVDV抗原融合蛋白、或权利要求2或3所述重组核酸、或权利要求4‑6任一项所述重组mRNA、或权利要求7所述表达盒、或权利要求
8所述载体、或权利要求9所述细胞、或权利要求10所述重组菌、或权利要求11所述组合物。
13.一种三价BVDV mRNA疫苗,包含:
(a) 权利要求4‑6任一项所述重组mRNA;和
(b) 药学上可接受的载体。
14.一种表达重组三价BVDV mRNA的加帽mRNA重组表达载体、非加帽mRNA重组表达载体或自复制RNA重组表达载体的制备方法,其特征在于,包括如下步骤:步骤a:合成权利要求2或3所述重组核酸的基因片段;
步骤b:构建加帽mRNA架构载体、构建非加帽mRNA架构载体、或构建VEE自复制载体;
所述加帽mRNA架构载体包含5`‑UTR,3`‑UTR和polyA序列以及固定加帽mRNA架构的载体,序列分别如SEQ ID NO.3‑6所示;或者,所述非加帽mRNA架构载体包含IRES序列,3`‑UTR和polyA序列,以及固定非加帽mRNA架构的载体,其中IRES的序列如SEQ ID No.8所示;或者,构建VEE自复制载体:根据α病毒家族的基因组获得自复制RNA序列;所述自复制RNA序列包含编码α病毒自复制组件的基因,缺乏制造具有传染性的α病毒颗粒的结构蛋白的基因;将自复制RNA序列构建获得的质粒作为模版扩增,通过合成获得所述VEE自复制载体;所述VEE自复制载体的序列如SEQ ID NO.10所示;
步骤c:制备加帽mRNA、非加帽mRNA或自复制RNA重组表达载体;
将步骤a合成的基因插入加帽mRNA架构载体中,得到加帽mRNA重组表达载体;或者,将步骤a合成的基因插入非加帽mRNA架构载体中,得到非加帽mRNA重组表达载体;或者,将步骤a合成的基因插入到VEE自复制载体的指定位置,得到自复制RNA重组表达载体。
15.一种三价BVDV mRNA疫苗的制备方法,其特征在于,包括:(a)提供权利要求4‑6任一项所述重组mRNA;
(b)将所述mRNA与脂质颗粒进行混合并孵育,从而形成包裹有mRNA的脂质纳米颗粒。
16.一种三价BVDV加帽mRNA架构载体疫苗、非加帽mRNA架构载体疫苗、或基于自复制RNA结构载体疫苗的制备方法,其特征在于,包含如下步骤:步骤a:将权利要求14中的所述加帽mRNA重组表达载体、非加帽mRNA重组表达载体或自复制RNA重组表达载体用限制性内切酶酶切线性化;
步骤b:将线性化的所述加帽mRNA重组表达载体进行体外共转录加帽反应,将7‑甲基化鸟苷酸帽结构加至转录的mRNA的5`端,并对模板DNA进行降解;或者,将线性化的所述非加帽mRNA重组表达载体进行体外非加帽的转录反应,并对模板DNA进行降解;或者,将线性化的所述自复制RNA重组表达载体进行体外共转录加帽反应,将7‑甲基化鸟苷酸帽结构加至转录的RNA的5`端,并对模板DNA进行降解;
步骤c:将步骤b中各自制备的加帽、非加帽、自复制mRNA分别与脂类混合,mRNA包裹进入LNP内,得到mRNA‑LNP复合物,通过浓缩及换液制得制剂溶液,从而获得所述疫苗。
17.权利要求1所述BVDV抗原融合蛋白、或权利要求4‑6任一项所述重组mRNA在以下任一项中的用途:(I)制备治疗或预防BVDV感染引起的疾病的药物;或者,(II)制备治疗或预防牛病毒性腹泻的药物;或者(III)制备在施用对象体内诱导产生针对BVDV的特异性免疫反应的药物。
说明书 :
一种广谱抗牛科病毒性腹泻病毒的mRNA疫苗及其应用
技术领域
背景技术
然而,未检索到有效用于BVDV防治的多价mRNA疫苗。本领域亟需一种可同时表达多种BVDV基因型的融合蛋白多价抗原BVDV mRNA疫苗,具有强免疫原性,且能够预防或治疗由多种BVDV基因型毒株所造成的感染,从而有效防控牛病毒性腹泻疾病,降低农牧业的损失。
发明内容
附图说明
具体实施方式
优选地,授乳或妊娠母牛及其胎牛或哺乳的牛犊。
3’‑非翻译区可以包含一个或更多个反向重复,其可以折叠以产生茎环结构,所述结构充当核糖核酸外切酶的屏障或与已知提高RNA稳定性的蛋白质(例如RNA结合蛋白)相互作用。
融合蛋白 EVTCVVVDVGHDDPEVKFSWFVDDVEVNTATTKPREEQFNSTYRVVSALRIQHQDWTG
GKEFKCKVHNEGLPAPIVRTISRTKGPAREPQVYVLAPPQEELSKSTVSLTCMVTSFY
PDYIAVEWQRNGQPESEDKYGTTPPQLDADGSYFLYSRLRVDRNSWQEGDTYTCVVMH
EALHNHYTQKSTSKSAGKGGSGGSGGSGGSGGSGGSGGHLDCKPEFSYAIAKSDRIGP
LGAEGLTTTWKDYSHEMKLEDTMVIAWCKDGKFVYLQRCTRETRYLAILHSRALPTSV
VFKKLFDGRRQEDTVEMDDNFEFGLCPCDAKPIVRGKFNTTLLNGPAFQMVCPIGWTG
TVSCMLANRDTLDTAVVRTYRRSKPFPYRQGCGGSGGGGSGGLPDCKPDFSYAIAKND
EIGPLGATGLTTQWYEYSDGMRLQDTEVVVWCKDGEFKYLIRCEREARYLAILHTRAL
PTSVVFEKILNGKEQEDVVEMDDNFEFGLCPCDAKPLVRGKFNTTLLNGPAFQMVCPI
GWTGTVSCTLANKDTLATTVVRTYKRHRPFPYRQGCGGSGGGGSGGFPECKEGFQYAI
SKDKKMGLLGPESLTTTWHLPTKKIVDSMVQVWCEGKDLKILRTCTKEERYLVAVHER
ALSTSAEFLQISDGTTGPEVIDMPDDFEFGLCPCDSKPVIKGKFNTSLLNGPAFQMVC
PQGWTGTIECILANQDTLDTTVIRTYRRTTPFQRRKWCGSG (SEQ ID NO.1)
编码BVDV ATGAACCCTTTATGGACATTGTTGTTCGTACTCAGCGCCCCGCGGGGGGTGCTGAGCG
抗原融合 ACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCC
蛋白的DNA GTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCG
序列 GAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCT
GGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTT
TAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGA
GGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGA
CCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCC
TCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTAT
CCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGT
ACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTT
GAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCAC
GAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAAGGCG
GATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGGTTCCGGCGGATCTGGAGGTCA
TCTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCGAAAAGCGATCGCATAGGACCG
CTCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATTACAGCCACGAGATGAAGTTGG
AGGACACCATGGTCATCGCGTGGTGTAAGGACGGGAAGTTCGTCTACCTTCAGCGGTG
CACGCGGGAGACGCGGTACCTGGCCATCTTACATTCTCGAGCACTACCGACCTCGGTA
GTGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGGACACAGTCGAGATGGACGATA
ACTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCCCATTGTGCGGGGTAAGTTCAA
TACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTGTGTCCCATAGGGTGGACCGGT
ACGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAGACACAGCAGTGGTGCGCACCT
ATAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTGCGGGGGGTCAGGGGGAGGTGG
TAGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCCTATGCGATCGCGAAGAACGAT
GAAATCGGACCGCTCGGTGCCACTGGGTTAACGACCCAGTGGTACGAGTATTCCGATG
GCATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTGCAAGGACGGCGAGTTCAAGTA
CCTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCCATCCTCCACACCCGTGCCCTT
CCCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGAAGGAGCAGGAAGATGTCGTCG
AGATGGACGACAACTTCGAGTTCGGGCTATGCCCATGTGACGCGAAGCCGCTCGTTCG
GGGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCTTTTCAGATGGTATGCCCGATA
GGGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACAAGGACACGCTCGCCACTACCG
TTGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTACCGGCAGGGTTGCGGGGGCTC
GGGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAGGAAGGCTTCCAGTACGCGATC
AGCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGTCCCTCACTACGACCTGGCACC
TGCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGTCTGGTGCGAGGGGAAGGATCT
GAAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTACTTGGTGGCAGTGCACGAGCGT
GCACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGGACGGGACGACCGGACCCGAAG
TCATCGACATGCCCGATGACTTCGAGTTCGGTCTGTGCCCGTGCGATAGCAAGCCCGT
TATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGTCCCGCCTTCCAGATGGTCTGC
CCGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGGCCAACCAGGATACACTCGATA
CGACGGTGATCCGGACCTACCGCCGCACTACGCCGTTCCAGCGGCGTAAGTGGTGTGG
GTCCGGATAATGA (SEQ ID NO.2)
5`‑UTR aggactcactatttgttttcgcgcccagttgcaaaaagtgtcgccacc(SEQ ID
NO.3)
3`‑UTR cccgggctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccc
cgagtctcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcac
cacctctgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgctta
gcctagccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagt
ttaactaagctatactaaccccagggttggtcaatttcgtgccagccacacc (SEQ
ID NO.4)
polyA aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaaaa (SEQ ID NO.5)
载体 ggaagagctcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggt
pUC57 atcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcagga
aagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgc
tggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaag
tcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagc
tccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttc
tcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggt
gtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgc
tgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc
cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctac
agagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatc
tgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggca
aacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcag
aaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtgg
aacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacct
agatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac
ttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtcta
tttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagg
gcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctcc
agatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgca
actttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagtt
cgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacg
ctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttaca
tgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtca
gaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctct
tactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtca
ttctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggata
ataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggg
gcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgt
gcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaa
caggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatact
catactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagc
ggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttc
cccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataa
aaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaac
ctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccggga
gcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaa
ctatgcggcatcagagcagattgtactgagagtgcaccatatg (SEQ ID NO.6)
CS‑BVDV‑ tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggt
210 cacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcg
ggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactga
gagtgcaccatatgtaatacgactcactataAggactcactatttgttttcgcgccca
gttgcaaaaagtgtcgccaccATGAACCCTTTATGGACATTGTTGTTCGTACTCAGCG
CCCCGCGGGGGGTGCTGAGCGACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCC
CCCGGAGCTTCCGGGGGGGCCGTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACA
CTGACGATCTCGGGGACTCCGGAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACG
ACCCGGAGGTGAAGTTCTCCTGGTTCGTGGACGACGTCGAGGTCAACACCGCGACCAC
GAAGCCCCGAGAGGAGCAGTTTAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATC
CAGCACCAGGACTGGACCGGAGGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCC
TGCCGGCTCCCATCGTCCGGACCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCA
GGTCTACGTTCTAGCTCCTCCTCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACC
TGCATGGTGACTAGCTTCTATCCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTC
AGCCGGAGTCGGAGGACAAGTACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAG
CTATTTTCTCTATAGCAGGTTGAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACA
TACACGTGTGTCGTCATGCACGAGGCACTTCACAACCACTATACTCAGAAGAGTACCT
CCAAGTCCGCCGGAAAAGGCGGATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGG
TTCCGGCGGATCTGGAGGTCATCTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCG
AAAAGCGATCGCATAGGACCGCTCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATT
ACAGCCACGAGATGAAGTTGGAGGACACCATGGTCATCGCGTGGTGTAAGGACGGGAA
GTTCGTCTACCTTCAGCGGTGCACGCGGGAGACGCGGTACCTGGCCATCTTACATTCT
CGAGCACTACCGACCTCGGTAGTGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGG
ACACAGTCGAGATGGACGATAACTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCC
CATTGTGCGGGGTAAGTTCAATACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTG
TGTCCCATAGGGTGGACCGGTACGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAG
ACACAGCAGTGGTGCGCACCTATAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTG
CGGGGGGTCAGGGGGAGGTGGTAGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCC
TATGCGATCGCGAAGAACGATGAAATCGGACCGCTCGGTGCCACTGGGTTAACGACCC
AGTGGTACGAGTATTCCGATGGCATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTG
CAAGGACGGCGAGTTCAAGTACCTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCC
ATCCTCCACACCCGTGCCCTTCCCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGA
AGGAGCAGGAAGATGTCGTCGAGATGGACGACAACTTCGAGTTCGGGCTATGCCCATG
TGACGCGAAGCCGCTCGTTCGGGGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCT
TTTCAGATGGTATGCCCGATAGGGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACA
AGGACACGCTCGCCACTACCGTTGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTA
CCGGCAGGGTTGCGGGGGCTCGGGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAG
GAAGGCTTCCAGTACGCGATCAGCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGT
CCCTCACTACGACCTGGCACCTGCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGT
CTGGTGCGAGGGGAAGGATCTGAAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTAC
TTGGTGGCAGTGCACGAGCGTGCACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGG
ACGGGACGACCGGACCCGAAGTCATCGACATGCCCGATGACTTCGAGTTCGGTCTGTG
CCCGTGCGATAGCAAGCCCGTTATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGT
CCCGCCTTCCAGATGGTCTGCCCGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGG
CCAACCAGGATACACTCGATACGACGGTGATCCGGACCTACCGCCGCACTACGCCGTT
CCAGCGGCGTAAGTGGTGTGGGTCCGGACATCACCATCACCACCACTAATGAcccggg
ctggtactgcatgcacgcaatgctagctgcccctttcccgtcctgggtaccccgagtc
tcccccgacctcgggtcccaggtatgctcccacctccacctgccccactcaccacctc
tgctagttccagacacctcccaagcacgcagcaatgcagctcaaaacgcttagcctag
ccacacccccacgggaaacagcagtgattaacctttagcaataaacgaaagtttaact
aagctatactaaccccagggttggtcaatttcgtgccagccacaccaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaggaagagctcctcgctcactgactcgctgcgctcggtcgttcggct
gcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggg
gataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaa
aggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaa
tcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgttt
ccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacc
tgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggta
tctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgtt
cagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagac
acgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt
aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaaca
gtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagct
cttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca
gattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtct
gacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaa
ggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtat
atatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctca
gcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataacta
cgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacg
ctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcaga
agtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagcta
gagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcat
cgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatca
aggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctc
cgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcact
gcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtac
tcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgt
caatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaa
acgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatg
taacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctg
ggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaa
atgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttat
tgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttc
cgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgac
attaacctataaaaataggcgtatcacgaggccctttcgtc(SEQ ID NO.7)
IRES TTAAAACAGCCTGTGGGTTGATCCCACCCACAGGCCCATTGGGCGCTAGCACTCTGGT
ATCACGGTACCTTTGTGCGCCTGTTTTATACCCCCTCCCCCAACTGTAACTTAGAAGT
AACACACACCGATCAACAGTCAGCGTGGCACACCAGCCACGTTTTGATCAAGCACTTC
TGTTACCCCGGACTGAGTATCAATAGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCG
TTATCCGGCCAACTACTTCGAAAAACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTC
GCTCAGCACTACCCCAGTGTAGATCAGGTCGATGAGTCACCGCATTCCCCACGGGCGA
CCGTGGCGGTGGCTGCGTTGGCGGCCTGCCCATGGGACTCACTATTTGTTTTCGCGCC
CAGTTGCAAAAAGTGTCGCCCATGGGACGCTCTAATACAGACATGGTGCGAAGAGTCT
ATTGAGCTAGTTGGTAGTCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCA
CACACCCTCAAGCCAGAGGGCAGTGTGTCGTAACGGGCAACTCTGCAGCGGAACCGAC
TACTTTGGGTGTCCGTGTTTCATTTTATTCCTATACTGGCTGCTTATGGTGACAATTG
AGAGATCGTTACCATATAGCTATTGGATTGGCCATCCGGTGACTAATAGAGCTATTAT
ATATCCCTTTGTTGGGTTTATACCACTTAGCTTGAAAGAGGTTAAAACATTACAATTC
ATTGTTAAGTTGAATACAGCAAA (SEQ ID NO.8)
CS‑BVDV‑ tcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggt
220 cacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcg
ggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactga
gagtgcaccatatgtaatacgactcactataAggTTAAAACAGCCTGTGGGTTGATCC
CACCCACAGGCCCATTGGGCGCTAGCACTCTGGTATCACGGTACCTTTGTGCGCCTGT
TTTATACCCCCTCCCCCAACTGTAACTTAGAAGTAACACACACCGATCAACAGTCAGC
GTGGCACACCAGCCACGTTTTGATCAAGCACTTCTGTTACCCCGGACTGAGTATCAAT
AGACTGCTCACGCGGTTGAAGGAGAAAGCGTTCGTTATCCGGCCAACTACTTCGAAAA
ACCTAGTAACACCGTGGAAGTTGCAGAGTGTTTCGCTCAGCACTACCCCAGTGTAGAT
CAGGTCGATGAGTCACCGCATTCCCCACGGGCGACCGTGGCGGTGGCTGCGTTGGCGG
CCTGCCCATGGGACTCACTATTTGTTTTCGCGCCCAGTTGCAAAAAGTGTCGCCCATG
GGACGCTCTAATACAGACATGGTGCGAAGAGTCTATTGAGCTAGTTGGTAGTCCTCCG
GCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACACACCCTCAAGCCAGAGGGCAGT
GTGTCGTAACGGGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCATT
TTATTCCTATACTGGCTGCTTATGGTGACAATTGAGAGATCGTTACCATATAGCTATT
GGATTGGCCATCCGGTGACTAATAGAGCTATTATATATCCCTTTGTTGGGTTTATACC
ACTTAGCTTGAAAGAGGTTAAAACATTACAATTCATTGTTAAGTTGAATACAGCAAAA
TGAACCCTTTATGGACATTGTTGTTCGTACTCAGCGCCCCGCGGGGGGTGCTGAGCGA
CCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCCG
TCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCGG
AGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCTG
GTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTTT
AACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGAG
GGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGAC
CATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCCT
CAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTATC
CGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGTA
CGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTTG
AGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCACG
AGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAAGGCGG
ATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGGTTCCGGCGGATCTGGAGGTCAT
CTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCGAAAAGCGATCGCATAGGACCGC
TCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATTACAGCCACGAGATGAAGTTGGA
GGACACCATGGTCATCGCGTGGTGTAAGGACGGGAAGTTCGTCTACCTTCAGCGGTGC
ACGCGGGAGACGCGGTACCTGGCCATCTTACATTCTCGAGCACTACCGACCTCGGTAG
TGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGGACACAGTCGAGATGGACGATAA
CTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCCCATTGTGCGGGGTAAGTTCAAT
ACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTGTGTCCCATAGGGTGGACCGGTA
CGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAGACACAGCAGTGGTGCGCACCTA
TAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTGCGGGGGGTCAGGGGGAGGTGGT
AGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCCTATGCGATCGCGAAGAACGATG
AAATCGGACCGCTCGGTGCCACTGGGTTAACGACCCAGTGGTACGAGTATTCCGATGG
CATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTGCAAGGACGGCGAGTTCAAGTAC
CTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCCATCCTCCACACCCGTGCCCTTC
CCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGAAGGAGCAGGAAGATGTCGTCGA
GATGGACGACAACTTCGAGTTCGGGCTATGCCCATGTGACGCGAAGCCGCTCGTTCGG
GGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCTTTTCAGATGGTATGCCCGATAG
GGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACAAGGACACGCTCGCCACTACCGT
TGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTACCGGCAGGGTTGCGGGGGCTCG
GGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAGGAAGGCTTCCAGTACGCGATCA
GCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGTCCCTCACTACGACCTGGCACCT
GCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGTCTGGTGCGAGGGGAAGGATCTG
AAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTACTTGGTGGCAGTGCACGAGCGTG
CACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGGACGGGACGACCGGACCCGAAGT
CATCGACATGCCCGATGACTTCGAGTTCGGTCTGTGCCCGTGCGATAGCAAGCCCGTT
ATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGTCCCGCCTTCCAGATGGTCTGCC
CGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGGCCAACCAGGATACACTCGATAC
GACGGTGATCCGGACCTACCGCCGCACTACGCCGTTCCAGCGGCGTAAGTGGTGTGGG
TCCGGACATCACCATCACCACCACTAATGAcccgggctggtactgcatgcacgcaatg
ctagctgcccctttcccgtcctgggtaccccgagtctcccccgacctcgggtcccagg
tatgctcccacctccacctgccccactcaccacctctgctagttccagacacctccca
agcacgcagcaatgcagctcaaaacgcttagcctagccacacccccacgggaaacagc
agtgattaacctttagcaataaacgaaagtttaactaagctatactaaccccagggtt
ggtcaatttcgtgccagccacaccaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaagagctc
ctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcac
tcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgt
gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttttt
ccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtgg
cgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgc
gctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcggg
aagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgtt
cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttat
ccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagc
agccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttg
aagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgc
tgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccac
cgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaagga
tctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaact
cacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcctttt
aaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgac
agttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcat
ccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatc
tggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatca
gcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccg
cctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaa
tagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgttt
ggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatccccca
tgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagtt
ggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatg
ccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaat
agtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgcc
acatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactc
tcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaact
gatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggca
aaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttc
ctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatat
ttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagt
gccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgt
atcacgaggccctttcgtc (SEQ ID NO.9)
VEE自复制 atgggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttca
载体 cgttgacatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcag
tttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgtttt
cgcatctggcttcaaaactgatcgaaacggaggtggacccatccgacacgatccttga
cattggaagtgcgcccgcccgcagaatgtattctaagcacaagtatcattgtatctgt
ccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaaga
aaaactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctcgccgc
cgtcatgagcgaccctgacctggaaactgagactatgtgcctccacgacgacgagtcg
tgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggttgacggaccga
caagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttga
caccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaac
tgggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgtta
tggagcggtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaa
caatgttctattctctgttggctcgaccatctaccacgagaagagggacttactgagg
agctggcacctgccgtctgtatttcacttacgtggcaagcaaaattacacatgtcggt
gtgagactatagttagttgcgacgggtacgtcgttaaaagaatagctatcagtccagg
cctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgc
tgcaaagtgacagacacattgaacggggagagggtctcttttcccgtgtgcacgtatg
tgccagctacattgtgtgaccaaatgactggcatactggcaacagatgtcagtgcgga
cgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcacc
cagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttg
ctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacg
agatagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatct
atttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcat
tcgtgctgcccaggataggcagtaacacattggagatcgggctgagaacaagaatcag
gaaaatgttagaggagcacaaggagccgtcacctctcattaccgccgaggacgtacaa
gaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaagccgaggagttgcgcg
cagctctaccacctttggcagctgatgttgaggagcccactctggaagccgatgtcga
cttgatgttacaagaggctggggccggctcagtggagacacctcgtggcttgataaag
gttaccagctacgatggcgaggacaagatcggctcttacgctgtgctttctccgcagg
ctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagt
gataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta
gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtg
ccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccac
acatggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgag
cacgacggcgaatacctgtacgacatcgacaggaaacagtgcgtcaagaaagaactag
tcactgggctagggctcacaggcgagctggtggatcctcccttccatgaattcgccta
cgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggggtgtat
ggcgtgccaggatcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatc
tagtggtgagcgccaagaaagaaaactgtgcagaaattataagggacgtcaagaaaat
gaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaatggatgcaaa
caccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctca
gagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaaca
gtgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcaca
caagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtct
caaccttgttttacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgt
gattgacactaccggcagtaccaaacctaagcaggacgatctcattctcacttgtttc
agagggtgggtgaagcagttgcaaatagattacaaaggcaacgaaataatgacggcag
ctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatga
aaatcctctgtacgcacccacctcagaacatgtgaacgtcctactgacccgcacggag
gaccgcatcgtgtggaaaacactagccggcgacccatggataaaaacactgactgcca
agtaccctgggaatttcactgccacgatagaggagtggcaagcagagcatgatgccat
catgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaaac
gtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgacca
ctgaacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagat
agtattgaaccaactatgcgtgaggttctttggactcgatctggactccggtctattt
tctgcacccactgttccgttatccattaggaataatcactgggataactccccgtcgc
ctaacatgtacgggctgaataaagaagtggtccgtcagctctctcgcaggtacccaca
actgcctcgggcagttgccactggaagagtctatgacatgaacactggtacactgcgc
aattatgatccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttag
tcctccaccataatgaacacccacagagtgacttttcttcattcgtcagcaaattgaa
gggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggcaaaatggttgac
tggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccag
gtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca
tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagct
tgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgaca
gggccagcgaaagcatcattggtgctatagcgcggcagttcaagttttcccgggtatg
caaaccgaaatcctcacttgaagagacggaagttctgtttgtattcattcggtacgat
cgcaaggcccgtacgcacaattcttacaagctttcatcaaccttgaccaacatttata
caggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgagggga
tattgccacggccaccgaaggagtgattataaatgctgctaacagcaaaggacaacct
ggcggaggggtgtgcggagcgctgtataagaaattcccggaaagcttcgatttacagc
cgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcattcatgc
cgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagag
gcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattc
cactgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaa
ccatttgctgacagctttagacaccactgatgcagatgtagccatatactgcagggac
aagaaatgggaaatgactctcaaggaagcagtggctaggagagaagcagtggaggaga
tatgcatatccgacgactcttcagtgacagaacctgatgcagagctggtgagggtgca
tccgaagagttctttggctggaaggaagggctacagcacaagcgatggcaaaactttc
tcatatttggaagggaccaagtttcaccaggcggccaaggatatagcagaaattaatg
ccatgtggcccgttgcaacggaggccaatgagcaggtatgcatgtatatcctcggaga
aagcatgagcagtattaggtcgaaatgccccgtcgaagagtcggaagcctccacacca
cctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcgcc
taaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaagta
tagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaa
gtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgaga
ctccggagccatcggcagagaaccaatccacagaggggacacctgaacaaccaccact
tataaccgaggatgagaccaggactagaacgcctgagccgatcatcatcgaagaggaa
gaagaggatagcataagtttgctgtcagatggcccgacccaccaggtgctgcaagtcg
aggcagacattcacgggccgccctctgtatctagctcatcctggtccattcctcatgc
atccgactttgatgtggacagtttatccatacttgacaccctggagggagctagcgtg
accagcggggcaacgtcagccgagactaactcttacttcgcaaagagtatggagtttc
tggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctccacatcccgctcc
gcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtt
tccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc
cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccagg
cgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatga
cggtttgatgcgggtgcatacatcttttcctccgacaccggtcaagggcatttacaac
aaaaatcagtaaggcaaacggtgctatccgaagtggtgttggagaggaccgaattgga
gatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaagaaatta
cagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggagaaca
tgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggcaga
aggaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtg
aaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaag
agaactttccgactgtggcttcttactgtattattccagagtacgatgcctatttgga
catggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctg
cgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgcctt
cagcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaa
tgtcacgcaaatgagagaattgcccgtattggattcggcggcctttaatgtggaatgc
ttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaaagaaaaccccatca
ggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgc
tgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggacagg
tttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaag
aacggcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtg
cggaatccaccgagagctggttaggagattaaatgcggtcctgcttccgaacattcat
acactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttccagc
ctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgc
catggctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttg
acgctgattgaggcggctttcggcgaaatttcatcaatacatttgcccactaaaacta
aatttaaattcggagccatgatgaaatctggaatgttcctcacactgtttgtgaacac
agtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccggatcacca
tgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggacaaattaa
tggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggt
gggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggc
acagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctgg
cagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtcaacacg
ctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa
accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaat
cattcagctacctgagaggggcccctataactctctacggctaacctgaatggactac
gacatagtctagtccgccaagtctagaacaagtttgtacaaaaaagcaggctgccacc
acccagctttcttgtacaaagtggtggcgcgcctatgttacgtgcaaaggtgattgtc
accccccgaaagaccatattgtgacacaccctcagtatcacgcccaaacatttacagc
cgcggtgtcaaaaaccgcgtggacgtggttaacatccctgctgggaggatcagccgta
attattataattggcttggtgctggctactattgtggccatgtacgtgctgaccaacc
agaaacataattgaatacagcagcaattggcaagctgcttacatagaactcgcggcga
ttggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattt
tgtttttaatatttcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaggaagagctagggataacagg
gtaat(SEQ ID NO.10)
CS‑BVDV‑ atgggcggcgcatgagagaagcccagaccaattacctacccaaaatggagaaagttca
230 cgttgacatcgaggaagacagcccattcctcagagctttgcagcggagcttcccgcag
tttgaggtagaagccaagcaggtcactgataatgaccatgctaatgccagagcgtttt
cgcatctggcttcaaaactgatcgaaacggaggtggacccatccgacacgatccttga
cattggaagtgcgcccgcccgcagaatgtattctaagcacaagtatcattgtatctgt
ccgatgagatgtgcggaagatccggacagattgtataagtatgcaactaagctgaaga
aaaactgtaaggaaataactgataaggaattggacaagaaaatgaaggagctcgccgc
cgtcatgagcgaccctgacctggaaactgagactatgtgcctccacgacgacgagtcg
tgtcgctacgaagggcaagtcgctgtttaccaggatgtatacgcggttgacggaccga
caagtctctatcaccaagccaataagggagttagagtcgcctactggataggctttga
caccaccccttttatgtttaagaacttggctggagcatatccatcatactctaccaac
tgggccgacgaaaccgtgttaacggctcgtaacataggcctatgcagctctgacgtta
tggagcggtcacgtagagggatgtccattcttagaaagaagtatttgaaaccatccaa
caatgttctattctctgttggctcgaccatctaccacgagaagagggacttactgagg
agctggcacctgccgtctgtatttcacttacgtggcaagcaaaattacacatgtcggt
gtgagactatagttagttgcgacgggtacgtcgttaaaagaatagctatcagtccagg
cctgtatgggaagccttcaggctatgctgctacgatgcaccgcgagggattcttgtgc
tgcaaagtgacagacacattgaacggggagagggtctcttttcccgtgtgcacgtatg
tgccagctacattgtgtgaccaaatgactggcatactggcaacagatgtcagtgcgga
cgacgcgcaaaaactgctggttgggctcaaccagcgtatagtcgtcaacggtcgcacc
cagagaaacaccaataccatgaaaaattaccttttgcccgtagtggcccaggcatttg
ctaggtgggcaaaggaatataaggaagatcaagaagatgaaaggccactaggactacg
agatagacagttagtcatggggtgttgttgggcttttagaaggcacaagataacatct
atttataagcgcccggatacccaaaccatcatcaaagtgaacagcgatttccactcat
tcgtgctgcccaggataggcagtaacacattggagatcgggctgagaacaagaatcag
gaaaatgttagaggagcacaaggagccgtcacctctcattaccgccgaggacgtacaa
gaagctaagtgcgcagccgatgaggctaaggaggtgcgtgaagccgaggagttgcgcg
cagctctaccacctttggcagctgatgttgaggagcccactctggaagccgatgtcga
cttgatgttacaagaggctggggccggctcagtggagacacctcgtggcttgataaag
gttaccagctacgatggcgaggacaagatcggctcttacgctgtgctttctccgcagg
ctgtactcaagagtgaaaaattatcttgcatccaccctctcgctgaacaagtcatagt
gataacacactctggccgaaaagggcgttatgccgtggaaccataccatggtaaagta
gtggtgccagagggacatgcaatacccgtccaggactttcaagctctgagtgaaagtg
ccaccattgtgtacaacgaacgtgagttcgtaaacaggtacctgcaccatattgccac
acatggaggagcgctgaacactgatgaagaatattacaaaactgtcaagcccagcgag
cacgacggcgaatacctgtacgacatcgacaggaaacagtgcgtcaagaaagaactag
tcactgggctagggctcacaggcgagctggtggatcctcccttccatgaattcgccta
cgagagtctgagaacacgaccagccgctccttaccaagtaccaaccataggggtgtat
ggcgtgccaggatcaggcaagtctggcatcattaaaagcgcagtcaccaaaaaagatc
tagtggtgagcgccaagaaagaaaactgtgcagaaattataagggacgtcaagaaaat
gaaagggctggacgtcaatgccagaactgtggactcagtgctcttgaatggatgcaaa
caccccgtagagaccctgtatattgacgaagcttttgcttgtcatgcaggtactctca
gagcgctcatagccattataagacctaaaaaggcagtgctctgcggggatcccaaaca
gtgcggtttttttaacatgatgtgcctgaaagtgcattttaaccacgagatttgcaca
caagtcttccacaaaagcatctctcgccgttgcactaaatctgtgacttcggtcgtct
caaccttgttttacgacaaaaaaatgagaacgacgaatccgaaagagactaagattgt
gattgacactaccggcagtaccaaacctaagcaggacgatctcattctcacttgtttc
agagggtgggtgaagcagttgcaaatagattacaaaggcaacgaaataatgacggcag
ctgcctctcaagggctgacccgtaaaggtgtgtatgccgttcggtacaaggtgaatga
aaatcctctgtacgcacccacctcagaacatgtgaacgtcctactgacccgcacggag
gaccgcatcgtgtggaaaacactagccggcgacccatggataaaaacactgactgcca
agtaccctgggaatttcactgccacgatagaggagtggcaagcagagcatgatgccat
catgaggcacatcttggagagaccggaccctaccgacgtcttccagaataaggcaaac
gtgtgttgggccaaggctttagtgccggtgctgaagaccgctggcatagacatgacca
ctgaacaatggaacactgtggattattttgaaacggacaaagctcactcagcagagat
agtattgaaccaactatgcgtgaggttctttggactcgatctggactccggtctattt
tctgcacccactgttccgttatccattaggaataatcactgggataactccccgtcgc
ctaacatgtacgggctgaataaagaagtggtccgtcagctctctcgcaggtacccaca
actgcctcgggcagttgccactggaagagtctatgacatgaacactggtacactgcgc
aattatgatccgcgcataaacctagtacctgtaaacagaagactgcctcatgctttag
tcctccaccataatgaacacccacagagtgacttttcttcattcgtcagcaaattgaa
gggcagaactgtcctggtggtcggggaaaagttgtccgtcccaggcaaaatggttgac
tggttgtcagaccggcctgaggctaccttcagagctcggctggatttaggcatcccag
gtgatgtgcccaaatatgacataatatttgttaatgtgaggaccccatataaatacca
tcactatcagcagtgtgaagaccatgccattaagcttagcatgttgaccaagaaagct
tgtctgcatctgaatcccggcggaacctgtgtcagcataggttatggttacgctgaca
gggccagcgaaagcatcattggtgctatagcgcggcagttcaagttttcccgggtatg
caaaccgaaatcctcacttgaagagacggaagttctgtttgtattcattcggtacgat
cgcaaggcccgtacgcacaattcttacaagctttcatcaaccttgaccaacatttata
caggttccagactccacgaagccggatgtgcaccctcatatcatgtggtgcgagggga
tattgccacggccaccgaaggagtgattataaatgctgctaacagcaaaggacaacct
ggcggaggggtgtgcggagcgctgtataagaaattcccggaaagcttcgatttacagc
cgatcgaagtaggaaaagcgcgactggtcaaaggtgcagctaaacatatcattcatgc
cgtaggaccaaacttcaacaaagtttcggaggttgaaggtgacaaacagttggcagag
gcttatgagtccatcgctaagattgtcaacgataacaattacaagtcagtagcgattc
cactgttgtccaccggcatcttttccgggaacaaagatcgactaacccaatcattgaa
ccatttgctgacagctttagacaccactgatgcagatgtagccatatactgcagggac
aagaaatgggaaatgactctcaaggaagcagtggctaggagagaagcagtggaggaga
tatgcatatccgacgactcttcagtgacagaacctgatgcagagctggtgagggtgca
tccgaagagttctttggctggaaggaagggctacagcacaagcgatggcaaaactttc
tcatatttggaagggaccaagtttcaccaggcggccaaggatatagcagaaattaatg
ccatgtggcccgttgcaacggaggccaatgagcaggtatgcatgtatatcctcggaga
aagcatgagcagtattaggtcgaaatgccccgtcgaagagtcggaagcctccacacca
cctagcacgctgccttgcttgtgcatccatgccatgactccagaaagagtacagcgcc
taaaagcctcacgtccagaacaaattactgtgtgctcatcctttccattgccgaagta
tagaatcactggtgtgcagaagatccaatgctcccagcctatattgttctcaccgaaa
gtgcctgcgtatattcatccaaggaagtatctcgtggaaacaccaccggtagacgaga
ctccggagccatcggcagagaaccaatccacagaggggacacctgaacaaccaccact
tataaccgaggatgagaccaggactagaacgcctgagccgatcatcatcgaagaggaa
gaagaggatagcataagtttgctgtcagatggcccgacccaccaggtgctgcaagtcg
aggcagacattcacgggccgccctctgtatctagctcatcctggtccattcctcatgc
atccgactttgatgtggacagtttatccatacttgacaccctggagggagctagcgtg
accagcggggcaacgtcagccgagactaactcttacttcgcaaagagtatggagtttc
tggcgcgaccggtgcctgcgcctcgaacagtattcaggaaccctccacatcccgctcc
gcgcacaagaacaccgtcacttgcacccagcagggcctgctcgagaaccagcctagtt
tccaccccgccaggcgtgaatagggtgatcactagagaggagctcgaggcgcttaccc
cgtcacgcactcctagcaggtcggtctcgagaaccagcctggtctccaacccgccagg
cgtaaatagggtgattacaagagaggagtttgaggcgttcgtagcacaacaacaatga
cggtttgatgcgggtgcatacatcttttcctccgacaccggtcaagggcatttacaac
aaaaatcagtaaggcaaacggtgctatccgaagtggtgttggagaggaccgaattgga
gatttcgtatgccccgcgcctcgaccaagaaaaagaagaattactacgcaagaaatta
cagttaaatcccacacctgctaacagaagcagataccagtccaggaaggtggagaaca
tgaaagccataacagctagacgtattctgcaaggcctagggcattatttgaaggcaga
aggaaaagtggagtgctaccgaaccctgcatcctgttcctttgtattcatctagtgtg
aaccgtgccttttcaagccccaaggtcgcagtggaagcctgtaacgccatgttgaaag
agaactttccgactgtggcttcttactgtattattccagagtacgatgcctatttgga
catggttgacggagcttcatgctgcttagacactgccagtttttgccctgcaaagctg
cgcagctttccaaagaaacactcctatttggaacccacaatacgatcggcagtgcctt
cagcgatccagaacacgctccagaacgtcctggcagctgccacaaaaagaaattgcaa
tgtcacgcaaatgagagaattgcccgtattggattcggcggcctttaatgtggaatgc
ttcaagaaatatgcgtgtaataatgaatattgggaaacgtttaaagaaaaccccatca
ggcttactgaagaaaacgtggtaaattacattaccaaattaaaaggaccaaaagctgc
tgctctttttgcgaagacacataatttgaatatgttgcaggacataccaatggacagg
tttgtaatggacttaaagagagacgtgaaagtgactccaggaacaaaacatactgaag
aacggcccaaggtacaggtgatccaggctgccgatccgctagcaacagcgtatctgtg
cggaatccaccgagagctggttaggagattaaatgcggtcctgcttccgaacattcat
acactgtttgatatgtcggctgaagactttgacgctattatagccgagcacttccagc
ctggggattgtgttctggaaactgacatcgcgtcgtttgataaaagtgaggacgacgc
catggctctgaccgcgttaatgattctggaagacttaggtgtggacgcagagctgttg
acgctgattgaggcggctttcggcgaaatttcatcaatacatttgcccactaaaacta
aatttaaattcggagccatgatgaaatctggaatgttcctcacactgtttgtgaacac
agtcattaacattgtaatcgcaagcagagtgttgagagaacggctaaccggatcacca
tgtgcagcattcattggagatgacaatatcgtgaaaggagtcaaatcggacaaattaa
tggcagacaggtgcgccacctggttgaatatggaagtcaagattatagatgctgtggt
gggcgagaaagcgccttatttctgtggagggtttattttgtgtgactccgtgaccggc
acagcgtgccgtgtggcagaccccctaaaaaggctgtttaagcttggcaaacctctgg
cagcagacgatgaacatgatgatgacaggagaagggcattgcatgaagagtcaacacg
ctggaaccgagtgggtattctttcagagctgtgcaaggcagtagaatcaaggtatgaa
accgtaggaacttccatcatagttatggccatgactactctagctagcagtgttaaat
cattcagctacctgagaggggcccctataactctctacggctaacctgaatggactac
gacatagtctagtccgccaagtctagaacaagtttgtacaaaaaagcaggctgccacc
ATGAACCCTTTATGGACATTGTTGTTCGTACTCAGCGCCCCGCGGGGGGTGCTGAGCG
ACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGCC
GTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCCG
GAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCCT
GGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGTT
TAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGGA
GGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGGA
CCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTCC
TCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTAT
CCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAGT
ACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGTT
GAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCAC
GAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAAGGCG
GATCCGGCGGATCCGGTGGATCCGGCGGATCCGGCGGTTCCGGCGGATCTGGAGGTCA
TCTGGATTGCAAGCCCGAGTTTTCCTATGCGATCGCGAAAAGCGATCGCATAGGACCG
CTCGGTGCTGAAGGTTTGACGACTACCTGGAAGGATTACAGCCACGAGATGAAGTTGG
AGGACACCATGGTCATCGCGTGGTGTAAGGACGGGAAGTTCGTCTACCTTCAGCGGTG
CACGCGGGAGACGCGGTACCTGGCCATCTTACATTCTCGAGCACTACCGACCTCGGTA
GTGTTCAAGAAGTTGTTTGATGGCCGGCGTCAGGAGGACACAGTCGAGATGGACGATA
ACTTCGAGTTTGGACTGTGTCCTTGTGACGCCAAGCCCATTGTGCGGGGTAAGTTCAA
TACTACCCTGCTCAATGGGCCTGCGTTCCAGATGGTGTGTCCCATAGGGTGGACCGGT
ACGGTGAGCTGCATGCTCGCCAACCGGGACACCCTAGACACAGCAGTGGTGCGCACCT
ATAGGCGGAGCAAGCCGTTCCCCTATAGGCAGGGGTGCGGGGGGTCAGGGGGAGGTGG
TAGTGGGGGACTCCCGGACTGCAAGCCCGACTTCTCCTATGCGATCGCGAAGAACGAT
GAAATCGGACCGCTCGGTGCCACTGGGTTAACGACCCAGTGGTACGAGTATTCCGATG
GCATGCGACTTCAGGACACTGAAGTCGTGGTGTGGTGCAAGGACGGCGAGTTCAAGTA
CCTCATCCGGTGTGAGAGGGAGGCCAGATATCTGGCCATCCTCCACACCCGTGCCCTT
CCCACTTCAGTGGTCTTTGAGAAGATCCTGAATGGGAAGGAGCAGGAAGATGTCGTCG
AGATGGACGACAACTTCGAGTTCGGGCTATGCCCATGTGACGCGAAGCCGCTCGTTCG
GGGGAAGTTTAACACGACTCTCCTGAACGGGCCGGCTTTTCAGATGGTATGCCCGATA
GGGTGGACCGGTACGGTGAGCTGTACCTTGGCCAACAAGGACACGCTCGCCACTACCG
TTGTCCGCACCTATAAGCGCCACCGCCCGTTCCCGTACCGGCAGGGTTGCGGGGGCTC
GGGCGGTGGCGGTTCTGGAGGCTTTCCTGAGTGCAAGGAAGGCTTCCAGTACGCGATC
AGCAAGGACAAGAAGATGGGCTTGCTTGGTCCGGAGTCCCTCACTACGACCTGGCACC
TGCCCACGAAGAAGATCGTGGACAGCATGGTCCAGGTCTGGTGCGAGGGGAAGGATCT
GAAGATCCTTCGCACTTGCACCAAGGAGGAGCGGTACTTGGTGGCAGTGCACGAGCGT
GCACTGTCCACCAGTGCCGAGTTCCTCCAGATATCGGACGGGACGACCGGACCCGAAG
TCATCGACATGCCCGATGACTTCGAGTTCGGTCTGTGCCCGTGCGATAGCAAGCCCGT
TATCAAGGGGAAGTTCAATACTTCCCTCTTGAACGGTCCCGCCTTCCAGATGGTCTGC
CCGCAGGGGTGGACCGGGACCATCGAGTGTATCCTGGCCAACCAGGATACACTCGATA
CGACGGTGATCCGGACCTACCGCCGCACTACGCCGTTCCAGCGGCGTAAGTGGTGTGG
GTCCGGACATCACCATCACCACCACTAATGAacccagctttcttgtacaaagtggtgg
cgcgcctatgttacgtgcaaaggtgattgtcaccccccgaaagaccatattgtgacac
accctcagtatcacgcccaaacatttacagccgcggtgtcaaaaaccgcgtggacgtg
gttaacatccctgctgggaggatcagccgtaattattataattggcttggtgctggct
actattgtggccatgtacgtgctgaccaaccagaaacataattgaatacagcagcaat
tggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttatt
ttatttttcttttcttttccgaatcggattttgtttttaatatttcaaaaaaaaaaaa
aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa
aaaaaaaaaaggaagagctagggataacagggtaat (SEQ ID NO.11)
编码牛 GACCCACGCTGTAAGACGACGTGTGACTGCTGCCCCCCCCCGGAGCTTCCGGGGGGGC
IgG1 Fc的 CGTCAGTGTTTATCTTTCCGCCCAAGCCGAAAGATACACTGACGATCTCGGGGACTCC
DNA序列 GGAGGTCACGTGTGTTGTCGTCGACGTCGGCCACGACGACCCGGAGGTGAAGTTCTCC
TGGTTCGTGGACGACGTCGAGGTCAACACCGCGACCACGAAGCCCCGAGAGGAGCAGT
TTAACTCGACTTACAGGGTGGTGTCGGCCCTCCGGATCCAGCACCAGGACTGGACCGG
AGGGAAGGAGTTCAAGTGCAAGGTGCATAACGAAGGCCTGCCGGCTCCCATCGTCCGG
ACCATTTCGCGGACGAAAGGTCCGGCGAGGGAGCCGCAGGTCTACGTTCTAGCTCCTC
CTCAGGAGGAGCTATCCAAGAGTACGGTCTCCCTCACCTGCATGGTGACTAGCTTCTA
TCCGGATTATATTGCGGTGGAGTGGCAGCGTAACGGTCAGCCGGAGTCGGAGGACAAG
TACGGTACCACTCCACCGCAGTTAGATGCGGATGGAAGCTATTTTCTCTATAGCAGGT
TGAGGGTAGACCGTAACTCTTGGCAGGAGGGCGACACATACACGTGTGTCGTCATGCA
CGAGGCACTTCACAACCACTATACTCAGAAGAGTACCTCCAAGTCCGCCGGAAAA
(SEQ ID NO.12)
AJ133738.1),BVDV 1b:JS2201株(GenBank ID:OP856581),BVDV2:C201602(GenBankID:
MG420995))按照100TCID50/50μl的接种量用含2%胎牛血清的DMEM稀释。将等体积的病毒液与不同稀释度的待检血清混合,37℃孵育1h。孵育完成的混合液加入96孔细胞培养板的MDBK细胞中,每孔100ul,每份样品做4个重复。同时设置阳性、阴性血清对照(1:8稀释)、空白对照和接毒细胞对照。放入细胞培养箱继续培养5天。