CN115521942B - 一种bvdv表位基因疫苗的构建方法及其应用 - Google Patents
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Abstract
本发明公开了一种BVDV表位基因疫苗的构建方法及其应用。它是从NCBI上获得BVDV‑1四种具有免疫原性的C、E0、E2、NS3基因序列，利用生物学方法预测细胞表位；连接获得新肽段（命名为AKK），通过生物学软件分析AKK序列的二级结构、亲水性、抗原性、三级结构；PCR扩增基因序列，构建重组表达载体，经PCR、双酶切和测序等方法检测目的基因。并将构建成功的重组质粒转染至MDBK细胞，通过蛋白免疫印迹和RT‑PCR检测目的基因在细胞水平的表达；通过流式细胞术与ELISA检测IgG抗体水平评价重组质粒免疫效果。
Description
技术领域
本发明属于基因生物工程领域，涉及BVDV C、NS3、E0及E2基因的优势B/T细胞表位的预测与设计合成，构建重组真核质粒GV658载体的方法，具体说，是一种利用生物信息学获得BVDV基因表位预测序列AKK与E0、E2基因重组真核载体构建的方法及应用。
背景技术
牛病毒性腹泻病毒(BVDV)是黄病毒科瘟病毒属单股正链RNA病毒，可引起猪、牛、羊等多种动物的腹泻/黏膜病，给世界各地的肉牛和乳制品行业造成了重大的经济损失。目前，疫苗接种是预防该病的主要措施，国内外大多数的灭活苗和弱毒苗均存在排散病毒、病毒返强的风险。而表位疫苗具有安全性好、保护力强和应答类型可调控等优点。E2基因是主要的保护性抗原，能够诱导机体产生中和抗体并保护牛免受BVDV攻击，而且是BVDV的受体结合基因，负责病毒进入宿主细胞。E0基因含有BVDV的主要抗原表位，可以诱导保护性免疫应答，其基因序列高度保守，可以作为BVDV基因工程疫苗和免疫诊断的合适候选抗原；C基因和NS3基因均为含有免疫原性的基因质，可用于疫苗研发和抗体制备。基于以上种种原因，表位疫苗的设计及研发迫在眉睫。
发明内容
本发明所要解决的技术问题是，通过各种前沿的在线生物预测软件对C、E0、E2和NS3基因进行表位筛选，连接肽将所筛选的表位进行连接，获得一条具有高免疫原性的肽段（命名为AKK，表位预测基因）。使其进入机体后首先启动自身的细胞和体液免疫功能。将AKK、E0和E2基因分别克隆至载体GV658，使AKK与E0、E2蛋白分别发挥各自免疫功效，实现多基因的协同免疫效应，实现对BVDV的免疫保护作用，籍此为BVD表位疫苗研究提供新的研究思路和方法。
为了解决上述技术问题，本发明采用的技术方案是：一种对牛病毒性腹泻病毒BVDV具有免疫保护作用的表位基因疫苗，命名为AKK，属于表位预测基因。
本发明进一步公开了利用生物信息学获得BVDV基因表位预测序列AKK与E0、E2基因重组真核载体构建的方法，包括以下步骤：
1）从NCBI上查找BVDV-1的E0、E2、C、NS3氨基酸序列，登录号为NCBI参考序列：C蛋白序列NP_776260.1，E0蛋白序列NP_776261.1，E2蛋白序列NP_776263.1，NS3蛋白NP_776267.1，选取蛋白CDS区的氨基酸序列。使用在线预测工具BepiPred、ABCPred分别预测E0、E2、C、NS3蛋白的B细胞表位；使用在线预测工具IEBD分别预测E0、E2、C、NS3蛋白的CTL细胞表位、TH细胞表位。将上述获得的B细胞表位和T细胞表位序列串联，相同类型细胞表位间选择柔性肽GGGGS连接，不同类型细胞表位间选择刚性短肽GPLS连接，串联序列末端添加Flag标签以便蛋白检测，连接获得新肽段命名为AKK。利用生物学在线软件SOPMA、VaxiJen、ExPASY、I-TASSER分析其二级结构、抗原性、稳定性及三级结构。
2）AKK-E0-E2基因、AKK基因、E0基因、E2基因PCR扩增：以AKK-F SEQ ID NO.1和E2-R ID NO.6序列扩增AKK-E0-E2基因；以AKK-F SEQ ID NO.1和AKK-R SEQ ID NO.2序列扩增AKK基因；以E0-F SEQ ID NO.3和E0-R SEQ ID NO.4序列扩增E0基因；以E2-F SEQ ID NO.5和E2-R SEQ ID NO.6序列扩增E2基因。
3）将获得的目的基因克隆到pMD-19-T载体中，再利用限制性内切酶EcoR I和BamHI分别对质粒pMD-AKK-E0-E2SEQ ID NO.7、pMD-AKKSEQ ID NO.8、pMD-E0SEQ ID NO.9、pMD-E2SEQ ID NO.10及GV658载体进行双酶切，T4 DNA连接酶16℃连接过夜后分别获得重组质粒GV658-AKK-E0-E2、GV658-AKK、GV658-E0及GV658-E2。将上述重组质粒和GV658分别转化Top10感受态细胞，通过氨苄霉素抗性培养基筛选阳性菌株。
4）按照天根质粒大提（无内毒素）试剂盒提取质粒，重组真核质粒转染MDBK细胞可见绿色荧光；经Western blot验证真核表达载体在细胞水平上获得E0、E2融合表达基因，实现了AKK与E0-E2基因共表达；RT-PCR验证成功扩增目的基因片段；CCK-8验证重组质粒对细胞有无毒性及损伤作用。
5）重组质粒免疫后，大鼠生长和脏器进行称重，病理切片观察各组大鼠脏器组织结构有无明显病理变化；ELISA检测血清抗体水平；流式细胞术检测CD3+、CD4+和CD8+细胞比例。
本发明更进一步公开了对牛病毒性腹泻病毒BVDV具有免疫保护作用的表位基因疫苗在制备用于疫苗研发和抗体制备方面的应用。实验结果显示：ELISA检测AKK-E0-E2、AKK、E0、E2和PBS组抗体滴度分别为152.58μg/mL、133.13μg/mL、131.32μg/mL、127.22μg/mL、117.21μg/mL。各组疫苗AKK-E0-E2极显著高于PBS组（P<0.01），其中，AKK、E2显著高于PBS组（0.05<P<0.01），AKK-E0-E2组高于E0组（0.05<P<0.01），能引起机体体液免疫反应；流式细胞术检测CD3+、CD4+和CD8+细胞比例AKK-E0-E2、AKK、E0、E2组均极显著高于PBS组（P<0.01），AKK-E0-E2显著高于E0组（0.05<P<0.01），能引起机体细胞免疫反应；重组质粒疫苗能够显著激发大鼠免疫力。
上述利用生物信息学预测BVDV E0、E2、C、NS3基因表位，获得AKK基因序列。上述表位预测序列AKK与E0、E2基因重组真核载体GV658的构建及大鼠免疫水平评价。
本发明公开的SEQ ID NO.1-SEQ ID NO.11基因序列如下：
SEQ ID NO.1：
GAATTCGAGGAGGGCGCTACAAAGAA
SEQ ID NO.2：
GGATCCGGTCACGCTGCCAGCAGG
SEQ ID NO.3：
GAATTCGAGAACATCACCCAGTGGAACCTCCA
SEQ ID NO.4：
GGATCCGGCGTAGGCGCCGAACC
SEQ ID NO.5：
GAATTCCACCTCGACTGCAAGCCTG
SEQ ID NO.6：
GGATCCGCCCAGAGCCTTCTGCTC
SEQ ID NO.7：
GAATTCGCCACCATGGAGGAGGGCGCTACAAAGAAAAAGACACAGAAGCCAGGCGGCGGC
GGCTCTGGCAAGATGAAGATCGTGCCTAAGGAGTCCGAGAAGGGCGGCGGCGGCTCCGAC
TCTAAGACAAAGCCTCCCGACGCTGGCCCACTGTCTCCTTGCAACTTCGAGATCGCCGCC
TCCGACGTGCTGGGCGGCGGCGGCAGCGCTCGGGACTCTCCAACACCACTCACAGGCTGC
AAGAAGGGCAAGAACGGCCCCCTGTCTAGCATGTTCCAGGACACAACCCTGTACGGCGGC
GGCGGCTCCATCGCCGCCTCGGACGTGCTGTTCAAGGGCCCTCTGAGCACAAAGCTGGGC
CCAATGCCTTGCAGACCTTACGAGATCATCTCTTCTGGCGGCGGCGGCAGCTTCAAGGAG
TCTGAGGGCCTGCCTCACTACCCTATCGGCAAGTGCAAGGGCCCACTGTCCATGCTGAAG
GGCGAGTACCAGTACTGGGGCGGCGGCGGCTCTTACCAGTTCAAGGAGTCCGAGGGCCTG
GGCCCACTCAGCCAGAGATGCACAAGAGAGACACGGTATCTCGCCATCCTCCACACAGGC
GGCGGCGGCAGCCGGGAGACCCGTTATCTCGCCATCCTGCACACCAGGGCACTGCCAGG
CCCACTGTCTAACGGCGAGGTGACCGACACATACGAGAACTACTCTTTCCTCAACGCTGG
CGGCGGCGGCAGCACCGCTACAGGCTCTAAGGACTACCACTACGACCTCTTACAAGCTCA
GGGCCCCCTGTCTGGCCAGAAGCACCCAATCGAGGAGTTCGGCGGCGGCGGCTCGCGTCT
GAAGCACCCTTCTATCAGCTTCGGCCCACTGTCCATCCGGGTGGTGGCTATGACCGCTAC
ACCTGCTGGCAGCGTGACCGATTACAAGGATGACGACGATAAGGAGGGCAGGGGAAGTCT
TCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCCGAGAACATCACCCAGTGGAACCT
CCAAGACAACGGCACCGAGGGCATCCAGAGAGCCATGTTCCAGAGAGGCGTGAACAGGTC
CCTCCACGGCATCTGGCCCGAGAAAATTTGCACCGGCGTGCCTAGCCACCTGGCCACCGA
CATCGAGCTTAAGACCATCCACGGCATGATGGACGCCAGCGAGAAAACCAACTACACCTG
TTGTAGACTCCAGAGACACGAGTGGAACAAGCACGGCTGGTGCAACTGGTACAACATCGA
GCCTTGGATACTCGTGATGAACAGAACCCAGGCCAACCTGACCGAGGGCCAGCCCCCTAG
GGAGTGCGCCGTGACATGTAGATACGACAGAGCCTCCGACCTCAACGTGGTGACCCAGGC
CAGGGACTCCCCTACCCCTCTGACCGGCTGCAAGAAGGGCAAGAACTTCAGCTTCGCCGG
CATCCTCATGAGGGGCCCTTGCAACTTCGAGATCGCCGCCTCCGACGTGCTCTTCAAGGA
GCACGAGAGGATCAGCATGTTCCAGGACACCACCCTGTACCTCGTGGACGGCCTGACCAA
CTCCCTTGAGGGCGCCAGACAGGGCACCGCCAAGCTCACCACCTGGCTCGGCAAGCAGCT
CGGCATCCTGGGCAAGAAGCTGGAGAACAAGTCCAAGACCTGGTTCGGCGCCTACGCCGA
TTACAAGGATGACGACGATAAGGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGA
GGAAAATCCCGGCCCCCACCTCGACTGCAAGCCTGAGTTCTCTTACGCCATCGCTAAGGA
CGAGCGGATCGGCCAGCTCGGCGCCGAGGGCCTGACAACCACATGGAAGGAGTACTCCCC
AGGCATGAAGCTAGAGGACACAATGGTGATCGCTTGGTGCGAGGACGGCAAGCTCATGTA
CCTCCAGAGATGCACACGCGAGACCCGTTATCTCGCCATCCTCCACACCCGTGCTCTCCC
TACATCTGTGGTGTTCAAGAAGCTGTTCGACGGCCGGAAGCAGGAGGACGTGGTGGAGAT
GAACGACAACTTCGAGTTCGGCCTGTGCCCTTGCGACGCTAAGCCAATCGTGAGAGGCAA
GTTCAACACCACACTGCTGAACGGCCCCGCTTTCCAGATGGTGTGCCCTATCGGCTGGAC
AGGCACAGTGTCTTGCACATCTTTCAACATGGACACACTCGCTACAACAGTGGTGAGAAC
ATACCGGAGGTCTAAGCCATTCCCTCACAGGCAGGGCTGCATCACCCAGAAGAACCTGGG
CGAGGACCTCCACAACTGCATCCTGGGCGGCAACTGGACATGCGTGCCTGGCGACCAGCT
GCTGTACAAGGGCGGCTCTATCGAGTCTTGCAAGTGGTGCGGCTACCAGTTCAAGGAGTC
TGAGGGCCTCCCTCACTACCCAATCGGCAAGTGCAAGTTAGAAAACGAGACAGGCTACAG
ACTCGTGGACTCTACATCTTGCAACCGGGAGGGCGTGGCTATCGTGCCTCAGGGCACACT
GAAGTGCAAGATCGGCAAGACAACCGTGCAGGTGATCGCTATGGACACAAAGCTGGGCCC
AATGCCTTGCCGGCCATACGAGATCATCTCTTCTGAGGGCCCCGTGGAAAAGACCGCTTG
CACCTTCAACTACACAAAGACCCTGAAGAACAAGTACTTCGAGCCTAGAGACTCTTACTT
CCAGCAGTACATGCTGAAGGGCGAGTACCAGTACTGGTTCGACCTGGAGGTGACCGACCA
CCACCGGGACTACTTCGCCGAGTCTATCCTCGTGGTGGTGGTGGCTCTCCTGGGCGGCAG
ATACGTGCTGTGGCTGCTCGTGACATACATGGTGCTCAGCGAGCAGAAGGCTCTGGGCGA
TTACAAGGATGACGACGATAAGTGAGGATCC
SEQ ID NO.8：
GAATTCGCCACCATGGAGGAGGGCGCTACAAAGAAAAAGACACAGAAGCCAGGCGGCGGC
GGCTCTGGCAAGATGAAGATCGTGCCTAAGGAGTCCGAGAAGGGCGGCGGCGGCTCCGAC
TCTAAGACAAAGCCTCCCGACGCTGGCCCACTGTCTCCTTGCAACTTCGAGATCGCCGCC
TCCGACGTGCTGGGCGGCGGCGGCAGCGCTCGGGACTCTCCAACACCACTCACAGGCTGC
AAGAAGGGCAAGAACGGCCCCCTGTCTAGCATGTTCCAGGACACAACCCTGTACGGCGGC
GGCGGCTCCATCGCCGCCTCGGACGTGCTGTTCAAGGGCCCTCTGAGCACAAAGCTGGGC
CCAATGCCTTGCAGACCTTACGAGATCATCTCTTCTGGCGGCGGCGGCAGCTTCAAGGAG
TCTGAGGGCCTGCCTCACTACCCTATCGGCAAGTGCAAGGGCCCACTGTCCATGCTGAAG
GGCGAGTACCAGTACTGGGGCGGCGGCGGCTCTTACCAGTTCAAGGAGTCCGAGGGCCTG
GGCCCACTCAGCCAGAGATGCACAAGAGAGACACGGTATCTCGCCATCCTCCACACAGGC
GGCGGCGGCAGCCGGGAGACCCGTTATCTCGCCATCCTGCACACCAGGGCACTGCCAGGC
CCACTGTCTAACGGCGAGGTGACCGACACATACGAGAACTACTCTTTCCTCAACGCTGGC
GGCGGCGGCAGCACCGCTACAGGCTCTAAGGACTACCACTACGACCTCTTACAAGCTCAG
GGCCCCCTGTCTGGCCAGAAGCACCCAATCGAGGAGTTCGGCGGCGGCGGCTCGCGTCTG
AAGCACCCTTCTATCAGCTTCGGCCCACTGTCCATCCGGGTGGTGGCTATGACCGCTACA
CCTGCTGGCAGCGTGACCGATTACAAGGATGACGACGATAAGTGAGGATCC
SEQ ID NO.9：
GAATTCGCCACCATGGAGAACATCACCCAGTGGAACCTCCAAGACAACGGCACCGAGGGC
ATCCAGAGAGCCATGTTCCAGAGAGGCGTGAACAGGTCCCTCCACGGCATCTGGCCCGAG
AAAATTTGCACCGGCGTGCCTAGCCACCTGGCCACCGACATCGAGCTTAAGACCATCCAC
GGCATGATGGACGCCAGCGAGAAAACCAACTACACCTGTTGTAGACTCCAGAGACACGAG
TGGAACAAGCACGGCTGGTGCAACTGGTACAACATCGAGCCTTGGATACTCGTGATGAAC
AGAACCCAGGCCAACCTGACCGAGGGCCAGCCCCCTAGGGAGTGCGCCGTGACATGTAGA
TACGACAGAGCCTCCGACCTCAACGTGGTGACCCAGGCCAGGGACTCCCCTACCCCTCTG
ACCGGCTGCAAGAAGGGCAAGAACTTCAGCTTCGCCGGCATCCTCATGAGGGGCCCTTGC
AACTTCGAGATCGCCGCCTCCGACGTGCTCTTCAAGGAGCACGAGAGGATCAGCATGTTC
CAGGACACCACCCTGTACCTCGTGGACGGCCTGACCAACTCCCTTGAGGGCGCCAGACAG
GGCACCGCCAAGCTCACCACCTGGCTCGGCAAGCAGCTCGGCATCCTGGGCAAGAAGCTG
GAGAACAAGTCCAAGACCTGGTTCGGCGCCTACGCCGATTACAAGGATGACGACGATAAGTGAGGATCC
SEQ ID NO.10：
AATTCGCCACCATGCACCTCGACTGCAAGCCTGAGTTCTCTTACGCCATCGCTAAGGACGAGCGGATCGGCCAGCTCGGCGCCGAGGGCCTGACAACCACATGGAAGGAGTACTCCCCAGGCATGAAGCTAGAGGACACAATGGTGATCGCTTGGTGCGAGGACGGCAAGCTCATGTACCTCCAGAGATGCACACGCGAGACCCGTTATCTCGCCATCCTCCACACCCGTGCTCTCCCTACATCTGTGGTGTTCAAGAAGCTGTTCGACGGCCGGAAGCAGGAGGACGTGGTGGAGATGAACGACAACTTCGAGTTCGGCCTGTGCCCTTGCGACGCTAAGCCAATCGTGAGAGGCAAGTTCAACACCACACTGCTGAACGGCCCCGCTTTCCAGATGGTGTGCCCTATCGGCTGGACAGGCACAGTGTCTTGCACATCTTTCAACATGGACACACTCGCTACAACAGTGGTGAGAACATACCGGAGGTCTAAGCCATTCCCTCACAGGCAGGGCTGCATCACCCAGAAGAACCTGGGCGAGGACCTCCACAACTGCATCCTGGGCGGCAACTGGACATGCGTGCCTGGCGACCAGCTGCTGTACAAGGGCGGCTCTATCGAGTCTTGCAAGTGGTGCGGCTACCAGTTCAAGGAGTCTGAGGGCCTCCCTCACTACCCAATCGGCAAGTGCAAGTTAGAAAACGAGACAGGCTACAGACTCGTGGACTCTACATCTTGCAACCGGGAGGGCGTGGCTATCGTGCCTCAGGGCACACTGAAGTGCAAGATCGGCAAGACAACCGTGCAGGTGATCGCTATGGACACAAAGCTGGGCCCAATGCCTTGCCGGCCATACGAGATCATCTCTTCTGAGGGCCCCGTGGAAAAGACCGCTTGCACCTTCAACTACACAAAGACCCTGAAGAACAAGTACTTCGAGCCTAGAGACTCTTACTTCCAGCAGTACATGCTGAAGGGCGAGTACCAGTACTGGTTCGACCTGGAGGTGACCGACCACCACCGGGACTACTTCGCCGAGTCTATCCTCGTGGTGGTGGTGGCTCTCCTGGGCGGCAGATACGTGCTGTGGCTGCTCGTGACATACATGGTGCTCAGCGAGCAGAAGGCTCTGGGCGATTACAAGGATGACGACGATAAGTGAGGATCC
氨基酸序列SEQ ID NO.11
EEGATKKKTQKPGGGGSGKMKIVPKESEKGGGGSDSKTKPPDAGPLSPCNFEIAASDVLGGGGSARDSPTPLTGCKKGKNGPLSSMFQDTTLYGGGGSIAASDVLFKGPLSTKLGPMPCRPYEIISSGGGGSFKESEGLPHYPIGKCKGPLSMLKGEYQYWGGGGSYQFKESEGLGPLSQRCTRETRYLAILHTGGGGSRETRYLAILHTRALPGPLSNGEVTDTYENYSFLNAGGGGSTATGSKDYHYDLLQAQGPLSGQKHPIEEFGGGGSRLKHPSISFGPLSIRVVAMTATPAGSVDYKDDDDK（flag）。
牛病毒性腹泻病毒(BVDV)是黄病毒科瘟病毒属单股正链RNA病毒，可引起猪、牛、羊等多种动物的腹泻/黏膜病，给世界各地的肉牛和乳制品行业造成了重大的经济损失。目前，疫苗接种是预防该病的主要措施，国内外大多数的灭活苗和弱毒苗均存在排散病毒、病毒返强的风险。而表位疫苗具有安全性好、保护力强和应答类型可调控等优点。E2基因是主要的保护性抗原，能够诱导机体产生中和抗体并保护牛免受BVDV攻击，而且是BVDV的受体结合基因，负责病毒进入宿主细胞。E0基因含有BVDV的主要抗原表位，可以诱导保护性免疫应答，其基因序列高度保守，可以作为BVDV基因工程疫苗和免疫诊断的合适候选抗原；C基因和NS3基因均为含有免疫原性的基因质，可用于疫苗研发和抗体制备。基于以上种种原因，表位疫苗的设计及研发迫在眉睫。
本发明利用表位预测序列AKK与E0、E2基因重组真核表达载体GV658构建的方法，包括以下步骤：
1）AKK基因序列的设计与合成；
2）构建重组质粒GV658-AKK-E0-E2、GV658-AKK、GV658-E0、GV658-E2；
3）在MDBK细胞中转染重组质粒GV658-AKK-E0-E2、GV658-AKK、GV658-E0、GV658-E2；
4）重组质粒GV658-AKK-E0-E2、GV658-AKK、GV658-E0、GV658-E2免疫大鼠，评价其免疫效果。
具体包括步骤如下：
1)从NCBI上查找BVDV-1的E0、E2、C、NS3氨基酸序列，登录号为NCBI参考序列：C蛋白序列NP_776260.1，E0蛋白序列NP_776261.1，E2蛋白序列NP_776263.1，NS3蛋白NP_776267.1，选取蛋白CDS区的氨基酸序列。使用在线预测工具BepiPred、ABCPred分别预测E0、E2、C、NS3蛋白的B细胞表位；使用在线预测工具IEBD分别预测E0、E2、C、NS3蛋白的CTL细胞表位、TH细胞表位。将上述获得的B细胞表位和T细胞表位序列串联，相同类型细胞表位间选择柔性肽GGGGS连接，不同类型细胞表位间选择刚性短肽GPLS连接，串联序列末端添加Flag标签以便蛋白检测，连接获得新肽段命名为AKK。利用生物学在线软件SOPMA、VaxiJen、ExPASY、I-TASSER分析其二级结构、抗原性、稳定性及三级结构。
2)AKK-E0-E2基因、AKK基因、E0基因、E2基因PCR扩增：以AKK-F SEQ ID NO.1和E2-R ID NO.6序列扩增AKK-E0-E2基因；以AKK-F SEQ ID NO.1和AKK-R SEQ ID NO.2序列扩增AKK基因；以E0-F SEQ ID NO.3和E0-R SEQ ID NO.4序列扩增E0基因；以E2-F SEQ ID NO.5和E2-R SEQ ID NO.6序列扩增E2基因。
3)将获得的目的基因克隆到pMD-19-T载体中，再利用限制性内切酶EcoR I和BamHI分别对质粒pMD-AKK-E0-E2、pMD-AKK、pMD-E0、pMD-E2及GV658载体进行双酶切，T4 DNA连接酶16℃连接过夜后分别获得重组质粒GV658-AKK-E0-E2、GV658-AKK、GV658-E0及GV658-E2。将上述重组质粒和GV658分别转化Top10感受态细胞，通过氨苄霉素抗性培养基筛选阳性菌株。
4)按照天根质粒大提（无内毒素）试剂盒提取质粒，重组真核质粒转染MDBK细胞可见绿色荧光；经Western blot验证真核表达载体在细胞水平上获得E0、E2融合表达基因，实现了AKK与E0-E2基因共表达；RT-PCR验证成功扩增目的基因片段；CCK-8验证重组质粒对细胞无毒性，无损伤作用。
5) 重组质粒免疫后，大鼠生长和脏器无显著差异，病理切片观察各组大鼠脏器组织结构无明显病理变化，表明疫苗安全性较高；ELISA检测血清抗体水平GV658-AKK-E0-E2、GV658-AKK、GV658-E0、GV658-E2组均极显著高于PBS组，GV658-AKK-E0-E2显著高于GV658-E0组；流式细胞术检测CD3+、CD4+和CD8+细胞比例GV658-AKK-E0-E2、GV658-AKK、GV658-E0、GV658-E2组均极显著高于PBS组，GV658-AKK-E0-E2显著高于GV658-E0组；重组质粒疫苗能够显著激发大鼠免疫力。
本发明公开的BVDV多表位基因疫苗的构建方法及其应用所具有的积极效果在于：
本发明成功构建表达载体GV658-AKK、GV658-E0、GV658-E2及GV658-AKK-E0-E2，在表达载体AKK-E0-E2中利用自剪切肽T2A进行连接。重组质粒免疫后，大鼠生长和脏器无显著差异，病理切片观察各组大鼠脏器组织结构无明显病理变化，表明疫苗安全性较高。重组质粒免疫大鼠后，ELISA检测技术评估AKK-E0-E2、AKK、E0、E2和PBS组抗体滴度分别为152.58μg/mL、133.13μg/mL、131.32μg/mL、127.22μg/mL、117.21μg/mL。AKK-E0-E2极显著高于PBS组（P<0.01），AKK、E2显著高于PBS组（0.05<P<0.01），AKK-E0-E2组高于E0组（0.05<P<0.01）。流式细胞术检测CD3+、CD4+和CD8+细胞比例，AKK-E0-E2组CD4+/CD8+比值为1.71，PBS组CD4+/CD8+比值为1.11，差异极显著（P＜0.01），各组疫苗免疫之后均可诱发免疫反应，引起CD3+、CD4+/CD8+细胞增加，发挥细胞与体液免疫效应。重组质粒免疫后，大鼠生长和脏器无显著差异，病理切片观察各组大鼠脏器组织结构无明显病理变化，表明疫苗安全性较高。本发明最终研制一种针对BVD的表位疫苗，其安全性较高，能促进动物产生免疫应答，为我国畜牧业BVD的预防提供策略。
附图说明
图1是AKK-E0-E2基因、AKK基因、E0基因、E2基因PCR扩增图（左）（M：DNA Marker；1：AKK-E0-E2 gene；2：E2 gene；3：AKKgene；4：E0 gene）及重组质粒双酶切结果图（右）（M：DNAMarker；1：AKK-E0-E2 gene；2：E2 gene；3：AKKgene；4：E0 gene）；
图2是重组质粒转染MDBK细胞结果图，可看到细胞中绿色荧光蛋白得到表达（A:GV658-AKK-E0-E2、B:GV658-AKK、C:GV658-E0、D:GV658-E2、E:GV658、F：PBS）；
图3是RT-PCR检测目的基因图，将重组质粒GV658-AKK-E0-E2、GV658-AKK、GV658-E0、GV658-E2转染细胞48h后，提取细胞总RNA，逆转录为cDNA后，RT-PCR扩增目的基因（M：DNA Marker；1：AKK-E0-E2 gene；2：E2 gene；3：AKKgene；4：E0 gene）；
图4是Western blot检测AKK-E0-E2蛋白、AKK蛋白、E0蛋白及E2蛋白的表达情况图（1: GV658-AKK-E0-E2； 2: GV658-AKK； 3: GV658-E0；4: GV658-E2； 5: GV658； 6:PBS）；
图5是BVDV IgG抗体水平检测结果图；
图6是流式细胞术检测CD3+结果图（A:GV658-AKK-E0-E2；B:GV658-AKK；C:GV658-E2；D:GV658-E0；E:PBS）；
图7是流式细胞术检测CD4+、CD8+结果图（Q1.CD8+细胞；Q2.CD8+、CD4+细胞；Q3.CD4+细胞；Q4.CD8+、CD4+细胞）。
具体实施方式
下面通过具体的实施方案叙述本发明。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。另外，实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。本发明所用原料及试剂均有市售。下面结合具体实例对本发明作进一步详细说明：
实施例1
1 材料与方法
1.1 表位基因预测
使用在线预测工具BepiPred、ABCPred分别预测E0、E2、C、NS3蛋白的B细胞表位；使用在线预测工具IEBD分别预测E0、E2、C、NS3蛋白的CTL细胞表位、TH细胞表位。将上述获得的B细胞表位和T细胞表位序列串联，相同类型细胞表位间选择柔性肽GGGGS连接，不同类型细胞表位间选择刚性短肽GPLS连接，连接获得新肽段命名为AKK。
1.2 目的基因扩增及载体的构建
利用限制性内切酶EcoR I和BamH I分别对质粒pMD-AKK-E0-E2、pMD-AKK、pMD-E0、pMD-E2及GV658载体进行双酶切，T4 DNA连接酶16℃连接过夜后分别获得重组质粒GV658-AKK-E0-E2、GV658-AKK、GV658-E0、及GV658-E2。将上述重组质粒和GV658分别转化Top10感受态细胞，通过氨苄霉素抗性培养基筛选阳性菌株。提取质粒进行PCR扩增及双酶切（EcoRI／BamH I）鉴定，送生工生物工程（上海）股份有限公司测序。并将测序正确的质粒阳性菌于-80℃保存备用。（PCR反应体系25μL：2×PCR Taq Mix Buffer 12.5μL，上、下游引物各1.0μL，模版1.0μL，ddH2O 9.5μL。反应程序：95 ℃，预变性5 min；{95 ℃，30 s；52 ℃，30s；72 ℃，1 min 20 s}32个循环；72℃延伸10 min。4℃保存备用
表1 PCR反应体系
表2 酶切体系
表3 连接体系
表4 引物序列
1.3 重组质粒转染MDBK细胞
按照天根质粒小提试剂盒（无内毒素）操作提取质粒。转染具体步骤如下：取两个无菌离心管，其中一个管中加入2 µg质粒DNA+50 µL 0.15M的NaCl，制备DNA稀释液；另一管中加入5 µL GeneTwin+50 µL 0.15M的NaCl，制备GeneTwin稀释液；将50 µLGeneTwin稀释液和50 µL DNA稀释液混匀，室温静置5-10min。
1.4 Westron blot检测
细胞总蛋白提取：预冷PBS清洗转染后细胞，加入细胞裂解液（PMSFːRIPA=1ː1000），冰上裂解30 min后，4℃，12,000g离心10min，收集上清，每40 µL 细胞裂解上清加10µL 5×蛋白上样缓冲液，混匀后100℃煮沸10min，冷却后电泳。将提取的细胞总蛋白经12%SDS-PAGE分离后，半干转膜仪转移至PVDF膜上，5% 脱脂乳室温封闭2h；TBST洗涤3次，每次10min，以鼠源单抗Anti-Glag标签抗体作为一抗(1ː3 000稀释)，4℃孵育过夜；TBST洗涤3次，每次10min，加入HPR标记的羊抗鼠IgG作为二抗(1ː5000稀释)，室温孵育1h；TBST洗涤3次，每次10min，DAB显色。
1.5 流式细胞术检测
将30只大鼠随机分为5组：AKK-E0-E2、AKK、E0、E2及PBS组，每组6只。免疫间隔2周，免疫剂量150μg/只，免疫3次后，称重处死解剖后，取脾用研磨棒研磨，经200目筛过滤至新的离心管中，细胞表面染色，向样本管中分别加入适量的CD3-FITC、CD4-APC、CD8-PE特异性表面抗体，4℃孵育3-5h上机检测抗体水平。
1.6 ELISA检测抗体水平
同上方法免疫大鼠，采血分离血清，3500r/min，5min。取淡黄色血清，并标记。依照ELISA试剂盒操作说明书，进行样品稀释、加样、孵育、洗涤、加酶标二抗、洗涤、再孵育、加显色剂、加终止液及酶标仪读数等步骤进行试验检测血清中IgG抗体水平。
2 结果与分析
2.1 B细胞筛选结果
通过生物在线软件Bepipred、ABCpred分别预测E0、E2、C、NS3蛋白的线性表位，筛选出两种软件预测结果重叠的片段，预测得分越高表示作为潜在表位的可能性越好（见表5）
表 5 B细胞表位筛选结果
2.2 T细胞筛选结果
通过在线软件IEBD、NetCTLpan，预测E0、E2、C、NS3蛋白的TH表位和CTL表位，选取不同等位基因重叠的肽段且评分较高的肽段，同时用ToxinPred软件预测是否具有细胞毒性（见表6）
表 6 T细胞表位筛选结果
将上述预测后的表位片段通过linkGLPS和linkGGGGS连接，序列末端添加Flag标签便于后续蛋白的表达鉴定；氨基酸序列为：SEQ ID NO.11。
氨基酸序列SEQ ID NO.11：
2.3 目的片段的扩增及酶切验证
分别以AKK-E0-E2、AKK、E0、E2菌液为模板，用特异性引物扩增目的片段，用EcoRⅠ和BamHⅠ双酶切验证，经1.0%琼脂糖凝胶电泳检测，结果显示，在2910bp、1170bp、951bp、729bp处有清晰的特异性目的条带，大小与目的基因序列一致（见图1）。
2.4 重组质粒转染BVDV细胞
将重组质粒GV658-AKK-E0-E2、GV658-AKK、GV658-E0及GV658-E2转染MDBK细胞48h，经荧光显微镜观察，有明显的绿色荧光（见图2）。
2.5 Western blot鉴定
将重组质粒GV658-AKK-E0-E2、GV658-AKK、GV658-E0及GV658-E2转染MDBK细胞，提取蛋白，经Western blot鉴定，结果显示，在43kd、35kd、26kd及68kd处有明显的蛋白条带（见图3）。
2.6 重组质粒转录与表达
将重组质粒GV658-AKK-E0-E2、GV658-AKK、GV658-E0、GV658-E2转染细胞48h后，提取细胞总RNA，逆转录为cDNA后，RT-PCR扩增目的基因。结果如图4-2所示，在2910bp、1170bp、951bp、729bp处有清晰的特异性条带（见图4）。
2.7流式细胞术检测
疫苗免疫后，脾细胞中CD3+细胞均呈上升趋势，大鼠各组经过3次免疫之后，通过流式细胞仪分析，结果如图，GV658-AKK-E0-E2、GV658-AKK、GV658-E0及GV658-E2均极显著高于PBS组（P＜0.01），GV658-AKK-E0-E2、GV658-E2极显著高于GV658-E0组（P＜0.01）（见图5）。
流式细胞术分析CD4+细胞、CD8+细胞占淋巴细胞百分比情况，结果显示，细胞分群明显，CD4+细胞显著大于CD8+细胞（P＜0.01），经spss分析GV658-AKK-E0-E2组CD4+/CD8+比值为1.71，PBS组CD4+/CD8+比值为1.11，差异极显著（P＜0.01）（见图6）。
2.8 ELISA检测抗体水平
重组质粒免疫后，实验动物采外周血，血清抗体通过Elisa试剂盒检测结果显示，AKK-E0-E2组、E0组、AKK组、E2组及PBS组血清抗体滴度为156μg/mL、119μg/mL、134μg/mL、123μg/mL、89μg/mL。经SPSS分析AKK-E0-E2显著高于PBS组，AKK、E2高于PBS组（0.05<P<0.01），AKK-E0-E2组高于E0组（0.05<P<0.01）。
3 结论
3.1本试验成功构建表达载体GV658-AKK、GV658-E0、GV658-E2及GV658-AKK-E0-E2。
3.3 成功将重组质粒转染MDBK细胞。
3.4 经Western blot验证真核表达载体在细胞水平上获得E0、E2融合表达蛋白，实现了AKK与E0和E2蛋白共表达。RT-PCR验证成功扩增目的基因片段。
3.5 重组质粒免疫大鼠后，ELISA检测血清抗体水平GV658-AKK-E0-E2、GV658-AKK、GV658-E0、GV658-E2组均极显著高于PBS组（P＜0.01），GV658-AKK-E0-E2显著高于GV658-E0组（0.05<P<0.01）。
3.6 重组质粒免疫大鼠后，流式细胞术检测CD3+、CD4+和CD8+细胞比例GV658-AKK-E0-E2、GV658-AKK、GV658-E0、GV658-E2组均极显著高于PBS组，GV658-AKK-E0-E2显著高于GV658-E0组（0.05<P<0.01）。
最终验证表明：
本发明最终研制一种针对BVD的表位疫苗，其安全性较高，能促进动物产生免疫应答，为我国畜牧业BVD的预防提供策略。
以上所述的实施例仅用于说明本发明的技术思想及特点，其目的在于使本领域内的技术人员能够理解本发明的内容并据以实施，不能仅以本实施例来限定本发明的专利范围，即凡本发明所揭示的精神所作的同等变化或修饰，仍落在本发明的专利范围内。
SEQUENCE LISTING
<110> 天津市农业科学院
<120> 一种BVDV表位基因疫苗的构建方法及其应用
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 26
<212> DNA
<213> 人工序列
<400> 1
gaattcgagg agggcgctac aaagaa 26
<210> 2
<211> 24
<212> DNA
<213> 人工序列
<400> 2
ggatccggtc acgctgccag cagg 24
<210> 3
<211> 32
<212> DNA
<213> 人工序列
<400> 3
gaattcgaga acatcaccca gtggaacctc ca 32
<210> 4
<211> 23
<212> DNA
<213> 人工序列
<400> 4
ggatccggcg taggcgccga acc 23
<210> 5
<211> 25
<212> DNA
<213> 人工序列
<400> 5
gaattccacc tcgactgcaa gcctg 25
<210> 6
<211> 24
<212> DNA
<213> 人工序列
<400> 6
ggatccgccc agagccttct gctc 24
<210> 7
<211> 2910
<212> DNA
<213> 人工序列
<400> 7
gaattcgcca ccatggagga gggcgctaca aagaaaaaga cacagaagcc aggcggcggc 60
ggctctggca agatgaagat cgtgcctaag gagtccgaga agggcggcgg cggctccgac 120
tctaagacaa agcctcccga cgctggccca ctgtctcctt gcaacttcga gatcgccgcc 180
tccgacgtgc tgggcggcgg cggcagcgct cgggactctc caacaccact cacaggctgc 240
aagaagggca agaacggccc cctgtctagc atgttccagg acacaaccct gtacggcggc 300
ggcggctcca tcgccgcctc ggacgtgctg ttcaagggcc ctctgagcac aaagctgggc 360
ccaatgcctt gcagacctta cgagatcatc tcttctggcg gcggcggcag cttcaaggag 420
tctgagggcc tgcctcacta ccctatcggc aagtgcaagg gcccactgtc catgctgaag 480
ggcgagtacc agtactgggg cggcggcggc tcttaccagt tcaaggagtc cgagggcctg 540
ggcccactca gccagagatg cacaagagag acacggtatc tcgccatcct ccacacaggc 600
ggcggcggca gccgggagac ccgttatctc gccatcctgc acaccagggc actgccaggc 660
ccactgtcta acggcgaggt gaccgacaca tacgagaact actctttcct caacgctggc 720
ggcggcggca gcaccgctac aggctctaag gactaccact acgacctctt acaagctcag 780
ggccccctgt ctggccagaa gcacccaatc gaggagttcg gcggcggcgg ctcgcgtctg 840
aagcaccctt ctatcagctt cggcccactg tccatccggg tggtggctat gaccgctaca 900
cctgctggca gcgtgaccga ttacaaggat gacgacgata aggagggcag gggaagtctt 960
ctaacatgcg gggacgtgga ggaaaatccc ggccccgaga acatcaccca gtggaacctc 1020
caagacaacg gcaccgaggg catccagaga gccatgttcc agagaggcgt gaacaggtcc 1080
ctccacggca tctggcccga gaaaatttgc accggcgtgc ctagccacct ggccaccgac 1140
atcgagctta agaccatcca cggcatgatg gacgccagcg agaaaaccaa ctacacctgt 1200
tgtagactcc agagacacga gtggaacaag cacggctggt gcaactggta caacatcgag 1260
ccttggatac tcgtgatgaa cagaacccag gccaacctga ccgagggcca gccccctagg 1320
gagtgcgccg tgacatgtag atacgacaga gcctccgacc tcaacgtggt gacccaggcc 1380
agggactccc ctacccctct gaccggctgc aagaagggca agaacttcag cttcgccggc 1440
atcctcatga ggggcccttg caacttcgag atcgccgcct ccgacgtgct cttcaaggag 1500
cacgagagga tcagcatgtt ccaggacacc accctgtacc tcgtggacgg cctgaccaac 1560
tcccttgagg gcgccagaca gggcaccgcc aagctcacca cctggctcgg caagcagctc 1620
ggcatcctgg gcaagaagct ggagaacaag tccaagacct ggttcggcgc ctacgccgat 1680
tacaaggatg acgacgataa ggagggcagg ggaagtcttc taacatgcgg ggacgtggag 1740
gaaaatcccg gcccccacct cgactgcaag cctgagttct cttacgccat cgctaaggac 1800
gagcggatcg gccagctcgg cgccgagggc ctgacaacca catggaagga gtactcccca 1860
ggcatgaagc tagaggacac aatggtgatc gcttggtgcg aggacggcaa gctcatgtac 1920
ctccagagat gcacacgcga gacccgttat ctcgccatcc tccacacccg tgctctccct 1980
acatctgtgg tgttcaagaa gctgttcgac ggccggaagc aggaggacgt ggtggagatg 2040
aacgacaact tcgagttcgg cctgtgccct tgcgacgcta agccaatcgt gagaggcaag 2100
ttcaacacca cactgctgaa cggccccgct ttccagatgg tgtgccctat cggctggaca 2160
ggcacagtgt cttgcacatc tttcaacatg gacacactcg ctacaacagt ggtgagaaca 2220
taccggaggt ctaagccatt ccctcacagg cagggctgca tcacccagaa gaacctgggc 2280
gaggacctcc acaactgcat cctgggcggc aactggacat gcgtgcctgg cgaccagctg 2340
ctgtacaagg gcggctctat cgagtcttgc aagtggtgcg gctaccagtt caaggagtct 2400
gagggcctcc ctcactaccc aatcggcaag tgcaagttag aaaacgagac aggctacaga 2460
ctcgtggact ctacatcttg caaccgggag ggcgtggcta tcgtgcctca gggcacactg 2520
aagtgcaaga tcggcaagac aaccgtgcag gtgatcgcta tggacacaaa gctgggccca 2580
atgccttgcc ggccatacga gatcatctct tctgagggcc ccgtggaaaa gaccgcttgc 2640
accttcaact acacaaagac cctgaagaac aagtacttcg agcctagaga ctcttacttc 2700
cagcagtaca tgctgaaggg cgagtaccag tactggttcg acctggaggt gaccgaccac 2760
caccgggact acttcgccga gtctatcctc gtggtggtgg tggctctcct gggcggcaga 2820
tacgtgctgt ggctgctcgt gacatacatg gtgctcagcg agcagaaggc tctgggcgat 2880
tacaaggatg acgacgataa gtgaggatcc 2910
<210> 8
<211> 951
<212> DNA
<213> 人工序列
<400> 8
gaattcgcca ccatggagga gggcgctaca aagaaaaaga cacagaagcc aggcggcggc 60
ggctctggca agatgaagat cgtgcctaag gagtccgaga agggcggcgg cggctccgac 120
tctaagacaa agcctcccga cgctggccca ctgtctcctt gcaacttcga gatcgccgcc 180
tccgacgtgc tgggcggcgg cggcagcgct cgggactctc caacaccact cacaggctgc 240
aagaagggca agaacggccc cctgtctagc atgttccagg acacaaccct gtacggcggc 300
ggcggctcca tcgccgcctc ggacgtgctg ttcaagggcc ctctgagcac aaagctgggc 360
ccaatgcctt gcagacctta cgagatcatc tcttctggcg gcggcggcag cttcaaggag 420
tctgagggcc tgcctcacta ccctatcggc aagtgcaagg gcccactgtc catgctgaag 480
ggcgagtacc agtactgggg cggcggcggc tcttaccagt tcaaggagtc cgagggcctg 540
ggcccactca gccagagatg cacaagagag acacggtatc tcgccatcct ccacacaggc 600
ggcggcggca gccgggagac ccgttatctc gccatcctgc acaccagggc actgccaggc 660
ccactgtcta acggcgaggt gaccgacaca tacgagaact actctttcct caacgctggc 720
ggcggcggca gcaccgctac aggctctaag gactaccact acgacctctt acaagctcag 780
ggccccctgt ctggccagaa gcacccaatc gaggagttcg gcggcggcgg ctcgcgtctg 840
aagcaccctt ctatcagctt cggcccactg tccatccggg tggtggctat gaccgctaca 900
cctgctggca gcgtgaccga ttacaaggat gacgacgata agtgaggatc c 951
<210> 9
<211> 729
<212> DNA
<213> 人工序列
<400> 9
gaattcgcca ccatggagaa catcacccag tggaacctcc aagacaacgg caccgagggc 60
atccagagag ccatgttcca gagaggcgtg aacaggtccc tccacggcat ctggcccgag 120
aaaatttgca ccggcgtgcc tagccacctg gccaccgaca tcgagcttaa gaccatccac 180
ggcatgatgg acgccagcga gaaaaccaac tacacctgtt gtagactcca gagacacgag 240
tggaacaagc acggctggtg caactggtac aacatcgagc cttggatact cgtgatgaac 300
agaacccagg ccaacctgac cgagggccag ccccctaggg agtgcgccgt gacatgtaga 360
tacgacagag cctccgacct caacgtggtg acccaggcca gggactcccc tacccctctg 420
accggctgca agaagggcaa gaacttcagc ttcgccggca tcctcatgag gggcccttgc 480
aacttcgaga tcgccgcctc cgacgtgctc ttcaaggagc acgagaggat cagcatgttc 540
caggacacca ccctgtacct cgtggacggc ctgaccaact cccttgaggg cgccagacag 600
ggcaccgcca agctcaccac ctggctcggc aagcagctcg gcatcctggg caagaagctg 660
gagaacaagt ccaagacctg gttcggcgcc tacgccgatt acaaggatga cgacgataag 720
tgaggatcc 729
<210> 10
<211> 1169
<212> DNA
<213> 人工序列
<400> 10
aattcgccac catgcacctc gactgcaagc ctgagttctc ttacgccatc gctaaggacg 60
agcggatcgg ccagctcggc gccgagggcc tgacaaccac atggaaggag tactccccag 120
gcatgaagct agaggacaca atggtgatcg cttggtgcga ggacggcaag ctcatgtacc 180
tccagagatg cacacgcgag acccgttatc tcgccatcct ccacacccgt gctctcccta 240
catctgtggt gttcaagaag ctgttcgacg gccggaagca ggaggacgtg gtggagatga 300
acgacaactt cgagttcggc ctgtgccctt gcgacgctaa gccaatcgtg agaggcaagt 360
tcaacaccac actgctgaac ggccccgctt tccagatggt gtgccctatc ggctggacag 420
gcacagtgtc ttgcacatct ttcaacatgg acacactcgc tacaacagtg gtgagaacat 480
accggaggtc taagccattc cctcacaggc agggctgcat cacccagaag aacctgggcg 540
aggacctcca caactgcatc ctgggcggca actggacatg cgtgcctggc gaccagctgc 600
tgtacaaggg cggctctatc gagtcttgca agtggtgcgg ctaccagttc aaggagtctg 660
agggcctccc tcactaccca atcggcaagt gcaagttaga aaacgagaca ggctacagac 720
tcgtggactc tacatcttgc aaccgggagg gcgtggctat cgtgcctcag ggcacactga 780
agtgcaagat cggcaagaca accgtgcagg tgatcgctat ggacacaaag ctgggcccaa 840
tgccttgccg gccatacgag atcatctctt ctgagggccc cgtggaaaag accgcttgca 900
ccttcaacta cacaaagacc ctgaagaaca agtacttcga gcctagagac tcttacttcc 960
agcagtacat gctgaagggc gagtaccagt actggttcga cctggaggtg accgaccacc 1020
accgggacta cttcgccgag tctatcctcg tggtggtggt ggctctcctg ggcggcagat 1080
acgtgctgtg gctgctcgtg acatacatgg tgctcagcga gcagaaggct ctgggcgatt 1140
acaaggatga cgacgataag tgaggatcc 1169
<210> 11
<211> 308
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma texanum
<400> 11
Glu Glu Gly Ala Thr Lys Lys Lys Thr Gln Lys Pro Gly Gly Gly Gly
1 5 10 15
Ser Gly Lys Met Lys Ile Val Pro Lys Glu Ser Glu Lys Gly Gly Gly
20 25 30
Gly Ser Asp Ser Lys Thr Lys Pro Pro Asp Ala Gly Pro Leu Ser Pro
35 40 45
Cys Asn Phe Glu Ile Ala Ala Ser Asp Val Leu Gly Gly Gly Gly Ser
50 55 60
Ala Arg Asp Ser Pro Thr Pro Leu Thr Gly Cys Lys Lys Gly Lys Asn
65 70 75 80
Gly Pro Leu Ser Ser Met Phe Gln Asp Thr Thr Leu Tyr Gly Gly Gly
85 90 95
Gly Ser Ile Ala Ala Ser Asp Val Leu Phe Lys Gly Pro Leu Ser Thr
100 105 110
Lys Leu Gly Pro Met Pro Cys Arg Pro Tyr Glu Ile Ile Ser Ser Gly
115 120 125
Gly Gly Gly Ser Phe Lys Glu Ser Glu Gly Leu Pro His Tyr Pro Ile
130 135 140
Gly Lys Cys Lys Gly Pro Leu Ser Met Leu Lys Gly Glu Tyr Gln Tyr
145 150 155 160
Trp Gly Gly Gly Gly Ser Tyr Gln Phe Lys Glu Ser Glu Gly Leu Gly
165 170 175
Pro Leu Ser Gln Arg Cys Thr Arg Glu Thr Arg Tyr Leu Ala Ile Leu
180 185 190
His Thr Gly Gly Gly Gly Ser Arg Glu Thr Arg Tyr Leu Ala Ile Leu
195 200 205
His Thr Arg Ala Leu Pro Gly Pro Leu Ser Asn Gly Glu Val Thr Asp
210 215 220
Thr Tyr Glu Asn Tyr Ser Phe Leu Asn Ala Gly Gly Gly Gly Ser Thr
225 230 235 240
Ala Thr Gly Ser Lys Asp Tyr His Tyr Asp Leu Leu Gln Ala Gln Gly
245 250 255
Pro Leu Ser Gly Gln Lys His Pro Ile Glu Glu Phe Gly Gly Gly Gly
260 265 270
Ser Arg Leu Lys His Pro Ser Ile Ser Phe Gly Pro Leu Ser Ile Arg
275 280 285
Val Val Ala Met Thr Ala Thr Pro Ala Gly Ser Val Asp Tyr Lys Asp
290 295 300
Asp Asp Asp Lys
305
Claims (3)
1.一种AKK肽段，所述AKK肽段的氨基酸序列如SEQ ID NO.11所示。
2.一种对牛病毒性腹泻病毒具有免疫保护作用的表位基因疫苗，所述表位基因疫苗通过以下步骤构建：
利用限制性内切酶EcoR I和BamH I分别对质粒pMD-AKK-E0-E2、pMD-AKK和GV658载体进行双酶切，使用T4 DNA连接酶于16℃连接过夜，获得重组质粒GV658-AKK-E0-E2和GV658-AKK，即为表位基因疫苗；其中，质粒pMD-AKK-E0-E2包含如SEQ ID NO.7所示的核苷酸序列，质粒pMD-AKK包含如SEQ ID NO.8所示的核苷酸序列。
3.权利要求1所述的AKK肽段在制备牛病毒性腹泻病毒表位基因疫苗中的应用。
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