caaacttcagcagtgcagagagagcaccaagggaatgaaatagtggatgtgatgtgccacgccactctgacccata gactgatgtcaccgaacagagtgcccaactacaacctatttgtcatggatgaagctcatttcaccgacccagccagt atagccgcacgaggatacattgctaccaaggtggaattaggggaggcagcagccatctttatgacagcgaccccgcc tggaaccacggatccttttcctgactcaaatgccccaatccatgatttgcaagatgagataccagacagggcatgga gcagtggatacgaatggatcacagaatatgcgggtaaaaccgtgtggtttgtggcgagcgtaaaaatggggaatgag attgcaatgtgcctccaaagagcggggaaaaaggtcatccaactcaaccgcaagtcctatgacacagaatacccaaa atgtaagaatggagactgggattttgtcattaccaccgacatctctgaaatgggggccaacttcggtgcgagcaggg tcatcgactgtagaaagagcgtgaaacccaccatcttagaagagggagaaggcagagtcatcctcggaaacccatct cccataaccagtgcaagcgcagctcaacggaggggcagagtaggcagaaaccccaatcaagttggagatgaatacca ctatgggggggctaccagtgaagatgacagtaacctagcccattggacagaggcaaagatcatgttagacaacatac acatgcccaatggactggtggcccagctctatggaccagagagggaaaaggctttcacaatggatggcgaataccgt ctcagaggtgaagaaaagaaaaacttcttagagctgcttaggacggctgacctcccggtgtggctggcctacaaggt ggcgtccaatggcattcagtacaccgacagaaagtggtgttttgatgggccgcgtacgaatgccatactggaggaca acaccgaggtagagatagtcacccggatgggtgagaggaaaatcctcaagccgagatggcttgatgcaagagtttat gcagatcaccaggccctcaagtggttcaaagactttgcagcagggaagagatcagccgttagcttcatagaggtgct cggtcgcatgcctgagcatttcatgggaaagacgcgggaagctttagacaccatgtacttggttgcaacggctgaga aaggtgggaaagcacaccgaatggctctcgaagagctgccagatgcactggaaaccatcacacttattgtcgccatt actgtgatgacaggaggattcttcctactaatgatgcagcgaaagggtatagggaagatgggtcttggagctctagt gctcacactagctaccttcttcctgtgggcggcagaggttcctggaaccaaaatagcagggaccctgctgatcgccc tgctgctgatggtggttctcatcccagaaccggaaaaacagaggtcacagacagataaccaactggcggtgtttctc atctgtgtcttgaccgtggttggagtggtggcagcaaacgagtacgggatgctagaaaaaaccaaagcggatctcaa gagcatgtttggcggaaagacgcaggcatcaggactgactggattgccaagcatggcactggacctgcgtccagcca cagcctgggcactgtatggggggagcacagtcgtgctaacccctcttctgaagcacctgatcacgtcggaatacgtc accacatcgctagcttcaattaactcacaagctggctcattattcgtcttgccacgaggcgtgccttttaccgacct agacttgactgttggcctcgtcttccttggctgttggggtcaagtcaccctcacaacgtttctgacagccatggttc tggcgacacttcactatgggtacatgctccctggatggcaagcagaagcactcagggctgcccagagaaggacagcg gctggaataatgaagaatgccgttgttgacggaatggtcgccactgatgtgcctgaactggaaaggactactcctct gatgcaaaagaaagtcggacaggtgctcctcataggggtaagcgtggcagcgttcctcgtcaaccctaatgtcacca ctgtgagagaagcaggggtgttggtgacggcggctacgcttactttgtgggacaatggagccagtgccgtttggaat tccaccacagccacgggactctgccatgtcatgcgaggtagctacctggctggaggctccattgcttggactctcat caagaacgctgataagccctccttgaaaaggggaaggcctgggggcaggacgctaggggagcagtggaaggaaaaac taaatgccatgagtagagaagagttttttaaataccggagagaggccataatcgaggtggaccgcactgaagcacgc agggccagacgtgaaaataacatagtgggaggacatccggtttcgcgaggctcagcaaaactccgttggctcgtgga gaaaggatttgtctcgccaataggaaaagtcattgatctagggtgtgggcgtggaggatggagctactacgcagcaa ccctgaagaaggtccaggaagtcagaggatacacgaaaggtggggcgggacatgaagaaccgatgctcatgcagagc tacggctggaacctggtctccctgaagagtggagtggacgtgttttacaaaccttcagagcccagtgataccctgtt ctgtgacataggggaatcctccccaagtccagaagtagaagaacaacgcacactacgcgtcctagagatgacatctg actggttgcaccgaggacctagagagttctgcattaaagttctctgcccttacatgcccaaggttatagaaaaaatg gaagttctgcagcgtcgcttcggaggtgggctagtgcgtctccccctgtcccgaaactccaatcacgagatgtattg ggttagtggagccgctggcaatgtggtgcacgctgtgaacatgaccagccaggtattactggggcgaatggatcgca cagtgtggagagggccaaagtatgaggaagatgtcaacctagggagcggaacaagagccgtgggaaagggagaagtc catagcaatcaggagaaaatcaagaagagaatccagaagcttaaagaagaattcgccacaacgtggcacaaagaccc tgagcatccataccgcacttggacataccacggaagctatgaagtgaaggctactggctcagccagctctctcgtca acggagtggtgaagctcatgagcaaaccttgggacgccattgccaacgtcaccaccatggccatgactgacaccacc ccttttggacagcaaagagttttcaaggagaaagttgacacgaaggctcctgagccaccagctggagccaaggaagt gctcaacgagaccaccaactggctgtgggcctacttgtcacgggaaaaaagaccccgcttgtgcaccaaggaagaat tcattaagaaagttaacagcaacgcggctcttggagcagtgttcgctgaacagaatcaatggagcacggcgcgtgag gctgtggatgacccgcggttttgggagatggttgatgaagagagggaaaaccatctgcgaggagagtgtcacacatg tatctacaacatgatgggaaaaagagagaagaagcctggagagtttggaaaagctaaaggaagcagggccatttggt tcatgtggcttggagcacggtatctagagtttgaagctttggggttcctgaatgaagaccattggctgagccgagag aattcaggaggtggagtggaaggctcaggcgtccaaaagctgggatacatcctccgtgacatagcaggaaagcaagg agggaaaatgtacgctgatgataccgccgggtgggacactagaattaccagaactgatttagaaaatgaagctaagg tactggagctcctagacggtgaacaccgcatgctcgcccgagccataattgaactgacttacaggcacaaagtggtc aaggtcatgagacctgcagcagaaggaaagaccgtgatggacgtgatatcaagagaagatcaaagggggagtggaca ggtggtcacttatgctcttaacactttcacgaacatcgctgtccagctcgtcaggctgatggaggctgagggggtca ttggaccacaacacttggaacatctacctaggaaaaacaagatagctgtcaggacctggctctttgagaatggagag gagagagtgaccaggatggcgatcagcggagacgactgtgccgtcaaaccgctggacgacagattcgccacagccct ccacttcctcaacgcaatgtcaaaggtcagaaaagacatccaggaatggaagccttcgcatggctggcacgattggc agcaagttcccttctgttctaaccattttcaggagattgtgatgaaagatggaaggagtatagttgtcccgtgcaga ggacaggatgagctgataggcagggctcgcatctctccaggagctggatggaatgtgaaggacacagcttgcctggc caaagcatatgcacagatgtggctactcctatacttccatcgcagggacttgcgtctcatggcaaatgcgatttgct cagcagtgccagtagattgggtgcccacaggcaggacatcctggtcaatacactcgaaaggagagtggatgaccacg gaagacatgctgcaggtctggaacagagtttggattgaagaaaatgaatggatgatggacaagactccaatcacaag ctggacagacgttccgtatgtgggaaagcgcgaggacatctggtgtggcagcctcatcggaacgcgatccagagcaa cctgggctgagaacatctatgcggcgataaaccaggttagagctgtcattgggaaagaaaattatgttgactacatg acctcactcaggagatacgaagacgtcttgatccaggaagacagggtcatctagtgtgatttaaggtagaaaagtag actatgtaaacaatgtaaatgagaaaatgcatgcatatggagtcaggccagcaaaagctgccaccggatactgggta gacggtgctgcctgcgtctcagtcccaggaggactgggttaacaaatctgacaacagaaagtgagaaagccctcaga accgtctcggaagtaggtccctgctcactggaagttgaaagaccaacgtcaggccacaaatttgtgccactccgcta gggagtgcggcctgcgcagccccaggaggactgggttaccaaagccgttgaggcccccacggcccaagcctcgtcta ggatgcaatagacgaggtgtaaggactagaggttagaggagaccccgtggaaacaacaacatgcggcccaagccccc tcgaagctgtagaggaggtggaaggactagaggttagaggagaccccgcatttgcatcaaacagcatattgacacct gggaatagactgggagatcttctgctctatctcaacatcagctactaggcacagagcgccgaagtatgtagctggtg gtgaggaagaacacaggatct
[0082] 4、病毒RNA的体外转录、转染及嵌合病毒的获得及传代
[0083] 于37°C用Xhol消化pJYF-FC 3h后，加入绿豆核酸酶于30°C作用30min，加入终浓 度为lg/L的SDS灭活绿豆核酸酶，PCR产物回收试剂盒回收线性化的酶切片段，以回收片 段为模板，用体外转录试剂盒体外转录RNA，并用RNA回收试剂盒回收RNA，电转染原代地 鼠肾（PHK)细胞（电转染条件为140伏电压）后，转移至T25培养瓶中，于37°C、5%的CO 2 培养5d，用反复冻融的方法收集培养液上清，将获得的病毒命名为Chimeri-JYF，即本发明 乙脑/黄热嵌合病毒，其核苷酸序列（SEQ ID NO. 2)为：
[0084] agaaguuuaucugugugaacuucuuggcuuaguaucguagagaagaaucgagagauuagugcaguuuaa acaguuuuuuagaacggaagauaaccaugacuaaaaaaccaggagggcccgguaaaaaccgggcuaucaauaugcug aaacgcggccuaccccgcguauucccacuagugggagugaagaggguaguaaugagcuuguuggacggcagagggcc aguacguuucgugcuggcucuuaucacguucuucaaguuuacagcauuagccccgaccaaggcgcuuucaggccgau ggaaagcaguggaaaagaguguggcaaugaaacaucuuacuaguuucaaacgagaacuuggaacacucauugacgcc gugaacaagcggggcagaaagcaaaacaaaagaggaggaaaugaaggcucaaucauguggcucgcgagcuuggcagu ugucauagcuugugcaggcgccgugaccuuggugcggaaaaacagaugguugcuccuaaaugugacaucugaggacc ucgggaaaacauucucugugggcacaggcaacugcacaacaaacauuuuggaagccaaguacuggugcccagacuca auggaauacaacugucccaaucucaguccaagagaggagccagaugacauugauugcuggugcuaugggguggaaaa cguuagagucgcauaugguaagugugacucagcaggcaggucuaggaggucaagaagggccauugacuugccuacgc augaaaaccaugguuugaagacccggcaagaaaaauggaugacuggaagaaugggugaaaggcaacuccaaaagauu gagagaugguucgugaggaaccccuuuuuugcagugacggcucugaccauugccuaccuugugggaagcaacaugac gcaa