本发明涉及生物技术领域,具体地,本发明涉及分离的核酸、转移质粒、病毒包装试剂盒以及包装病毒的方法。
背景技术:
目前,慢病毒作为一种基因递送载体,被广泛的应用于对各种疾病进行基因治疗,如car-t制备、造血干细胞基因转导。
慢病毒的生产工艺流程一般包括:①质粒(包括1个转移质粒和辅助质粒)提取和纯化→②质粒共转染293t或293f细胞→③收集病毒上清→④纯化、浓缩、无菌→⑤得到可供基因治疗使用的慢病毒。
现有的技术中,步骤③中得到的慢病毒生产初始滴度低、经步骤⑤得到的可供使用的慢病毒收率也极低(仅20%左右);而步骤②中是采用瞬时转染的方式,生产成本高,且体系扩大难度极大。这些都导致单批次慢病毒产量十分有限,无法满足高病毒载体用量适应症的治疗需求。
因此,迫切需要开发出新的提高慢病毒产量的技术。
技术实现要素:
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。
为此,在本发明的第一方面,本发明提出了一种分离的核酸。根据本发明的实施例,所述核酸具有x1x2tax5x6x7tx9所示的核苷酸序列,其中,x1为a或g;x2为t或c;x5为a或g;x6为c或t;x7为t或缺失;x9为t或g;但不具有gttaacttt所示的核苷酸序列。根据本发明实施例的分离的核酸位于慢病毒包装转移质粒的cppt/cts元件的5’端及5’上游,具有上述分离的核酸的转移质粒进入细胞核效率大幅提高,所包装出的病毒的滴度大幅提高。
根据本发明的实施例,上述核酸还可以进一步包括如下附加技术特征至少之一:
根据本发明的实施例,具有actagttg所示的核苷酸序列。具有actagttg所示的核苷酸序列的核酸的转移质粒所包装出的病毒滴度提高显著。
在本发明的第二方面,本发明提出了一种转移质粒。根据本发明的实施例,所述转移质粒携带前面所述的核酸。根据本发明实施例的转移质粒所包装出的病毒的滴度得到显著提高,相比于现有技术,病毒滴度至少可提高1.3倍。
根据本发明的实施例,上述转移质粒还可以包括如下附加技术特征至少之一:
根据本发明的实施例,位于所述转移质粒中cppt/cts元件的5’端及5’端上游。
根据本发明的实施例,所述核酸位于所述转移质粒中cppt/cts元件的5’端2~5bp及5’端上游4~8bp范围内,优选地,所述核酸位于所述转移质粒中cppt/cts元件的5’端3bp及5’端上游6bp范围内。
根据本发明的实施例,所述转移质粒具有seqidno:1所示的核苷酸序列。
gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgactagttgtaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattcaaaattttatcgataagcttgggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgactctagaggatccgccgccaccatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaac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根据本发明实施例的上述具有seqidno:1所示核苷酸序列的转移质粒所包装出的病毒,相比于现有技术,病毒滴度可提高1.3~1.5倍。
根据本发明的实施例,所述转移质粒包含rsv启动子区和hiv-15’ltr区,所述启动子区与hiv-15’ltr区可操作地连接,所述hiv-15’ltr区的5’端具有单个鸟甘酸。根据本发明实施例的转移质粒的hiv-15’ltr区位于hiv-1基因组的5’端,而单个鸟嘌呤rna更加倾向于形成二聚体grna结构,被包裹入病毒颗粒中,形成成熟的病毒颗粒,2个和3个鸟嘌呤的rna则更倾向于作为mrna招募核糖体等进行翻译。根据本发明实施例的转移质粒,相比于现有技术,会产生更多的包装有grna的完整病毒颗粒,进一步提高慢病毒初始产物的生物滴度。
根据本发明的实施例,所述转移质粒具有seqidno:2所示的核苷酸序列。
gtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgactagttgtaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattcaaaattttatcgataagcttgggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgactctagaggatccgccgccaccatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaaccc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根据本发明实施例的上述具有seqidno:2所示核苷酸序列的转移质粒所包装出的病毒,相比于现有技术,病毒滴度可提高2-3倍。
在发明的第三方面,本发明提出了一种转移质粒。根据本发明的实施例,相比于野生型plv-mcherry(简称wt),所述转移质粒具有下列突变的至少之一:第1569位碱基由g突变为a;第1570位碱基由t突变为c;第1573位碱基由a突变为g;第1574位碱基由c突变为t;第1575位碱基缺失;第1577位碱基由t突变为g。
根据本发明的实施例,所述plv-mcherry的质粒图谱如图1所示,所述野生型plv-mcherry质粒的核苷酸序列如seqidno:3所示。
gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattcaaaattttatcgataagcttgggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgactctagaggatccgccgccaccatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttccactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaac(seqidno:3)。
其中,在seqidno:3中,gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaaga为第一hiv-1基因组序列(其中,gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcag为截短的hiv-15’ltr(i型人免疫缺陷病毒5’端长末端重复序列longterminalrepeatfromhumanimmunodeficiencyvirus-1)区序列,ctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtc为病毒包装信号功能域ψ序列,),gatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatc为第二hiv-1基因组序列(其中,aggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctg为rre功能域(revresponseelement)序列),gttaac为hpai酶切位点,ttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattcaaaatttt为第三hiv-1基因组序列,称为cppt\cts(centralpolypurinetractandcentralterminationsequence)区序列,cgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatg为cmv(hunancytomegalovirus,人巨细胞病毒)增强子区序列,gtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctc为cmv启动子区序列,atggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtaa为mcherry区序列,aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgc为woodchuckhepatitisvirusposttranscriptionalregulatoryelement(wpre)区序列,tggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcag为第四hiv-1基因组序列,是缺失u3区域的hiv-13’ltr(3’长末端重复序列区序列)序列,aacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatctta为sv40polya信号序列,tgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaac为rsv(roussarcomavirus肉瘤病毒)启动子区序列。
根据本发明的实施例,相比于野生型plv-mcherry,所述转移质粒的hiv-15’ltr区的5’端缺失2个gg。进而进一步提高了包装出病毒的滴度。
根据本发明的实施例,所述转移质粒具有seqidno:1或2所述的核苷酸序列。
在本发明的第四方面,本发明提出了一种病毒包装试剂盒。根据本发明的实施例,所述试剂盒包括前面所述的转移质粒。利用根据本发明实施例的试剂盒包装病毒,病毒的滴度显著提高。
根据本发明的实施例,所述试剂盒进一步包括辅助质粒。
在本发明的第五方面,本发明提出了一种包装病毒的方法。根据本发明的实施例,所述方法包括将病毒包装质粒对病毒包装工具细胞进行转染;将转染后的病毒包装工具细胞进行孵育,以便获得病毒,其中,所述病毒包装质粒包括前面所述的转移质粒和辅助质粒。根据本发明实施例的方法,显著提高了包装出的病毒的滴度。
根据本发明的实施例,所述病毒包装工具细胞为293t或293f。293t或293f方便培养,复制迅速,易于转染,进而进一步提高了病毒滴度。
需要说明的是,如无特殊说明,本申请所述的“转移质粒”是指携带待转导目的基因和hiv-1调控顺式元件序列信息的质粒,如plv-mcherry,该质粒在病毒包装细胞中转录生成的rna能够被包装进慢病毒载体中;本申请所述的“辅助质粒”是指除转移质粒外,携带hiv-1的gag/pol,rev或包膜蛋白序列信息的质粒,如pspax2、pmdlg-prre、prsv-rev或pmd2.g,该类质粒在病毒包装细胞中经转录、翻译后,以蛋白的形式参与到慢病毒载体的生命周期中。
附图说明
图1为根据本发明实施例的plv-mcherry的质粒图谱;
图2为根据本发明实施例的plv-mcherry转移质粒及其突变体模式图;
图3为根据本发明实施例的三质粒系统包装慢病毒滴度比较图;以及
图4为根据本发明实施例的四质粒系统包装慢病毒滴度比较图。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
需要说明的是,本申请所述的seqidno:1~4所示的转移质粒的序列是以hiv-15’ltr区的5’端为起点展示的序列,与seqidno:1~4具有同一性的转移质粒,均在本申请的保护范围内。同时,本领域技术人员所理解的是,质粒为环状结构,以任意位置的5’端为起点均可展示质粒的序列。以其它5’端为起点展示的转移质粒的序列,如果与seqidno:1~4仅是展示起点不同,但序列相同,或展示起点不同,但序列与seqidno:1~4具有同一性,也均在本申请的保护范围内。
需要说明的是,如无特别说明,本申请所述的“同一性”是指具有与本申请的序列具有至少70%、75%、80%、85%、90%、95%或99%同一性的序列。术语百分比(%)“序列同一性”描述了相对于构成模板核酸总长度,比对中的两个以上核酸序列中相一致的核苷酸的匹配(“命中”)数。换言之,通过使用比对,对于两个以上的序列或子序列,在对这些(子)序列进行比较和比对以在比较视窗或指定区域上获得用本领域已知的序列比较算法测得的最大对应时,或者在进行人工比对和目视检查时,可以确定其相同的核苷酸的百分比(例如,80%、85%、90%或95%的同一性)。此定义同样适用于测试序列的互补序列。本发明的目的在于提供三种新型生产高滴度的基于hiv-1的慢病毒载体的转移质粒。此三种质粒分别为:
一种新型生产高滴度的基于hiv-1的慢病毒载体的转移质粒(本申请中命名为△gg或plv-△gg(1-2)-mcherry质粒)(序列如seqidno:4所示),在慢病毒hiv-1基因组编码区的5’末端进行缺失突变,仅保留一个鸟苷酸,使得慢病毒生物滴度提高至原来的1.5~2倍。
一种新型生产高滴度的基于hiv-1的慢病毒载体的转移质粒(本申请中命名为mut或plv-mut(1569-1577)-mcherry质粒)(序列如seqidno:1所示),将慢病毒cppt/cts元件的5’端及5’端附近的9个碱基(gttaacttt)突变为8个碱基(actagttg),使得慢病毒载体生物滴度提高至原来的1.3-1.5倍。
一种新型生产高滴度的基于hiv-1的慢病毒载体的转移质粒(本申请中命名为△gg&mut或plv-△gg(1-2)-mut(1569-1577)-mcherry质粒)(序列如seqidno:2所示),同时在慢病毒基因组编码区中引入两个突变,一是hiv-1基因组5’末端引入2个碱基的缺失突变,二是将cppt/cts元件的5’端及其附近的9个碱基(gttaacttt)突变为8个碱基(actagttg),使得慢病毒生物滴度提高至原来的2-3倍。
gtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattcaaaattttatcgataagcttgggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgactctagaggatccgccgccaccatggtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttccactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaac(seqidno:4)。
本发明获得的新型转移质粒相比于plv-mcherry转移质粒,在生产慢病毒载体能力上具有先天优势,从根本上提高了慢病毒载体的产量。
△gg突变体中的突变是将文献报道中hiv-1的复制机理应用到慢病毒载体生产中,提高慢病载体产量。所使用的基于hiv-1的慢病毒载体保留了hiv-1基因组中的包装、逆转录和整合的顺式作用元件,用这些元件调控外源目的基因的转导。因此,病毒的包装、逆转录和整合方式仍同hiv-1一致。
hiv-1的基因组为正链rna。研究表明,在hiv-1包装的过程中,全长的正链rna可以单体形式用作信使rna(mrna)进行翻译,也可形成二聚体作为基因组rna(grna)被包装进入病毒颗粒中。病毒通过调控单体和二聚体的比例,来调控病毒的复制周期进程。siarhei等发现hiv-1全长rna5’端的鸟嘌呤数量为1-3个,单个鸟嘌呤rna更加倾向于形成二聚体grna结构,被包裹入病毒颗粒中,形成成熟的病毒颗粒;而2个和3个鸟嘌呤的rna则更倾向于作为mrna招募核糖体等进行翻译。目前使用的基于hiv-1的慢病毒载体转移质粒中所编码的rna起始位置均为3个鸟嘌呤,倾向于翻译,却不利于组装到病毒颗粒中。的确,慢病毒的包装过程中,会产生大量没有包装到grna的空壳病毒。但是,慢病毒载体包装的目的是产生更多的包装有grna的完整病毒颗粒,说明可以通过提高二聚体grna比例的方式来获得更多完整病毒颗粒,提高慢病毒初始产物的生物滴度。
mut突变体是本申请的发明人在科学研究中意外发现地可以显著提高包装的慢病毒滴度的转移质粒。
△gg&mut突变体是将上述两个突变同时引入慢病毒基因组中,两个突变可协同提高慢病毒滴度,获得的新型的生产高滴度的基于hiv-1的慢病毒载体的转移质粒。
下面将结合具体实施例对本发明进行进一步解释说明。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
构建一种突变型转移质粒:含有gg(1-2)缺失突变的plv-△gg(1-2)-mcherry质粒,此处△gg(1-2)表示转移质粒plv-mcherry(简称wt,附图2a)中hiv基因组编码区第1至第2位碱基对缺失(附图2b)。
首先根据plv-mcherry序列,设计overlappingpcr引物:
gg(1-2)-forward-primer-1:(5’-3’)
tgcatgccgattggtgg(seqidno:5)。
gg(1-2)-forward-primer-2:(5’-3’)
gatacataaacgtctctctggttagaccag(seqidno:6)。
gg(1-2)-rerward-primer-1:(5’-3’)
tcagcggccgcttgctgt(seqidno:7)。
gg(1-2)-rerward-primer-2:(5’-3’)
ccagagagacgtttatgtatcgagctaggc(seqidno:8)。
由基因合成服务公司合成上述序列。
分别以gg(1-2)-forward-primer-1和gg(1-2)-rerward-primer-2、gg(1-2)-forward-primer-2和gg(1-2)-rerward-primer-1为引物,以plv-mcherry为模板,通过pcr方式获得产物片段a和b。回收片段a和b产物,再以片段a和b混合物为模板,gg(1-2)-forward-primer-1和gg(1-2)-rerward-primer-1为引物,通过over-lappingpcr方式获得产物片段c。通过酶切,将片段c连接至plv-mcherry质粒载体的对应酶切位点之间,将该载体转化大肠杆菌stbl3感受态,经酶切和测序验证得到正确plv-△gg(1-2)-mcherry质粒(简称△gg,如附图2b)。
实施例2
构建一种突变型转移质粒:含有1569-1577突变的plv-mut(1569-1577)-mcherry质粒,此处含有1569-1577突变表示将转移质粒plv-mcherry中hiv基因组编码区第1569-1577位碱基对gttaacttt突变为actagttg(附图2c)。
首先根据plv--mcherry序列,设计overlappingpcr引物:
mut(1569-1577)-forward-primer-1:(5’-3’)
aagcggccgctgatcttca(seqidno:9)。
mut(1569-1577)-forward-primer-2:(5’-3’)
gtatcgactagttgtaaaagaaaaggggggattg(seqidno:10)。
mut(1569-1577)-rerward-primer-1:(5’-3’)
gcttatcgataaaattttg(seqidno:11)。
mut(1569-1577)-rerward-primer-2:(5’-3’)
tttacaactagtcgataccgtcgagatcc(seqidno:12)。
由基因合成服务公司合成上述序列。
分别以mut(1569-1577)-forward-primer-1和mut(1569-1577)-rerward-primer-2、mut(1569-1577)-forward-primer-2和mut(1569-1577)-rerward-primer-1为引物,以plv-mcherry为模板,通过pcr方式获得产物片段d和e。回收片段d和e产物,再以片段d和e混合物为模板,mut(1569-1577)-forward-primer-1和mut(1569-1577)-rerward-primer-1为引物,通过over-lappingpcr方式获得产物片段f。通过酶切,将片段f连接至plv-mcherry质粒载体的对应酶切位点之间,将该载体转化大肠杆菌stbl3感受态,经酶切和测序验证得到正确plv-mut(1569-1577)-mcherry质粒(简称mut,如附图2c)。
实施例3
构建一种突变型转移质粒:同时含有gg(1-2)缺失和1569-1577突变的plv-△gg(1-2)-mut(1569-1577)-mcherry质粒,此处△gg(1-2)表示转移质粒plv-mcherry中hiv基因组编码区第1至第2位碱基对缺失,mut(1569-1577)表示转移质粒plv-mcherry中hiv基因组编码区第1569-1577位碱基对gttaacttt突变为actagttg(附图2d)。
取实施例1和实施例2中构建好的质粒,分别用sphi和noti进行酶切,分别回收△gg的小片段和mut的大片段。用t4连接酶将回收的两片段进行连接,转化大肠杆菌stbl3感受态,经酶切和测序验证得到正确plv-△gg(1-2)-mut(1569-1577)-mcherry质粒(简称△gg&mut,如附图2d)。
实施例4
取实施例1和实施例2中构建好的质粒,以plv-mcherry转移质粒为对照组,以pspax2和pmd2.g为辅助质粒,用三质粒系统进行慢病毒包装。按转移质粒:pspax2:pmd2.g=2:1:1比例,用lipofect3000将质粒转染到293t细胞中,转染后50小时收集细胞培养物中病毒上清,经0.45μm滤头过滤后,进行滴度测定。
将293t细胞按3.6e+04cells/孔接种至24孔板内,培养过夜,以使细胞贴壁。通过10倍梯度稀释法,将过滤后的病毒上清进行稀释,稀释病毒液中添加8μg/ml的polybrene。弃293t孔内原培养基,加入病毒稀释液,室温,1000g,离心40分钟后,37℃、5%co2静置培养72h后,流式检测mcherry阳性率,计算对应样品滴度和同plv-mcherry所获慢病毒滴度的比值。其中,数值>1则提高病毒滴度;数值<1,则降低病毒滴度;数值=1,则无影响。结果如图3所示,mut和△gg&mut组获得的慢病毒的滴度相比于plv-mcherry所获得的慢病毒滴度显著提高。
实施例5
取实施例1和实施例2中构建好的质粒,以plv-mcherry转移质粒为对照组,以pmdlg-prre、prsv-rev和pmd2.g为辅助质粒,用四质粒系统进行慢病毒包装。按转移质粒:pmdlg-prre:prsv-rev:pmd2.g=1:1:1:1比例,用lipofect3000将质粒转染到293t细胞中,转染后50小时收集细胞培养上清,经0.45μm滤头过滤后,进行滴度测定。
将293t细胞按3.6e+04cells/孔接种至24孔板内,培养过夜,以使细胞贴壁。通过10倍梯度稀释法,将过滤后的病毒上清进行稀释,稀释病毒液中添加8μg/ml的polybrene。弃293t孔内原培养基,加入病毒稀释液,室温,1000g,离心40分钟后,37℃、5%co2静置培养72h后,流式检测mcherry阳性率,计算对应样品滴度和同plv-mcherry所获慢病毒滴度的比值。其中,数值>1则提高病毒滴度;数值<1,则降低病毒滴度;数值=1,则无影响。结果如图4所示,mut和△gg&mut组获得的慢病毒的滴度相比于plv-mcherry所获得的慢病毒滴度显著提高。
此外,术语“第一”、“第二”仅用于描述目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此,限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中,“多个”的含义是至少两个,例如两个,三个等,除非另有明确具体的限定。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
sequencelisting
<110>东莞市东阳光生物药研发有限公司
广东东阳光药业有限公司
<120>慢病毒的滴度提高型转移质粒
<130>pidc4200114
<160>12
<170>patentinversion3.5
<210>1
<211>7412
<212>dna
<213>artificialsequence
<220>
<223>转移质粒的核苷酸序列
<400>1
gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaaccca60
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tgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagc180
agtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgca240
ggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgc300
caaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtatta360
agcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaa420
aatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatc480
ctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatccc540
ttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtg600
tgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagc660
aaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggagga720
gatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaacca780
ttaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtg840
ggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcg900
tcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaac960
aatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatc1020
aagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctg1080
gggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagt1140
tggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacaga1200
gaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaa1260
gaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggttt1320
aacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggta1380
ggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattca1440
ccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaata1500
gaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcga1560
cggtatcgactagttgtaaaagaaaaggggggattggggggtacagtgcaggggaaagaa1620
tagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaat1680
tcaaaattttatcgataagcttgggagttccgcgttacataacttacggtaaatggcccg1740
cctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccata1800
gtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcc1860
cacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgac1920
ggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttgg1980
cagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatc2040
aatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtc2100
aatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactcc2160
gccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagct2220
cgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccataga2280
agacaccgactctagaggatccgccgccaccatggtgagcaagggcgaggaggataacat2340
ggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggcca2400
cgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaa2460
gctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagtt2520
catgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagct2580
gtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggt2640
gaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcg2700
cggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggc2760
ctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggct2820
gaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaa2880
gcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaa2940
cgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcgg3000
catggacgagctgtacaagtaagtcgacaatcaacctctggattacaaaatttgtgaaag3060
attgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaat3120
gcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatc3180
ctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtg3240
cactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcct3300
ttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgcct3360
tgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggg3420
gaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggac3480
gtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgct3540
gccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccct3600
ttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaag3660
gcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcac3720
tcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatc3780
tgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttg3840
ccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatcc3900
ctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttatt3960
attcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttatt4020
gcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattt4080
ttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgg4140
ctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgccc4200
attctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcgg4260
cctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaa4320
ttcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtga4380
ctgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccag4440
ctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaa4500
tggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcg4560
cagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttc4620
ctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagg4680
gttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttc4740
acgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgtt4800
ctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattc4860
ttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgattta4920
acaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcactttt4980
cggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtat5040
ccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatg5100
agtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtt5160
tttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacga5220
gtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaa5280
gaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgt5340
attgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggtt5400
gagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgc5460
agtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcgga5520
ggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgat5580
cgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcct5640
gtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcc5700
cggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcg5760
gcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgc5820
ggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacg5880
acggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctca5940
ctgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgattta6000
aaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgacc6060
aaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaa6120
ggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca6180
ccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggta6240
actggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggc6300
caccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacca6360
gtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagtta6420
ccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggag6480
cgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgctt6540
cccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgc6600
acgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccac6660
ctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaac6720
gccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttc6780
tttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgat6840
accgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagag6900
cgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcac6960
gacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctc7020
actcattaggcaccccaggctttccactttatgcttccggctcgtatgttgtgtggaatt7080
gtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgca7140
attaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaa7200
tactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagaga7260
aaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaa7320
ggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagata7380
ttgtatttaagtgcctagctcgatacataaac7412
<210>2
<211>7410
<212>dna
<213>artificialsequence
<220>
<223>转移质粒的核苷酸序列
<400>2
gtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccact60
gcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtg120
tgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcag180
tggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcagg240
actcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgcca300
aaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaag360
cgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaa420
tataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcct480
ggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatccctt540
cagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtg600
catcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaa660
aacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggaga720
tatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccatt780
aggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtggg840
aataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtc900
aatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaa960
tttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaa1020
gcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggg1080
gatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttg1140
gagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagaga1200
aattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaaga1260
aaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaa1320
cataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagg1380
tttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcacc1440
attatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaataga1500
agaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacg1560
gtatcgactagttgtaaaagaaaaggggggattggggggtacagtgcaggggaaagaata1620
gtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaattc1680
aaaattttatcgataagcttgggagttccgcgttacataacttacggtaaatggcccgcc1740
tggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagt1800
aacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgccca1860
cttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacgg1920
taaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggca1980
gtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaa2040
tgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaa2100
tgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgc2160
cccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcg2220
tttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaag2280
acaccgactctagaggatccgccgccaccatggtgagcaagggcgaggaggataacatgg2340
ccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacg2400
agttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagc2460
tgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttca2520
tgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgt2580
ccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtga2640
ccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcg2700
gcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcct2760
cctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctga2820
agctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagc2880
ccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacg2940
aggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggca3000
tggacgagctgtacaagtaagtcgacaatcaacctctggattacaaaatttgtgaaagat3060
tgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgc3120
ctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcct3180
ggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgca3240
ctgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctccttt3300
ccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttg3360
cccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcgggga3420
agctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgt3480
ccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgc3540
cggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctcccttt3600
gggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggc3660
agctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactc3720
ccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctg3780
agcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgcc3840
ttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccct3900
cagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattat3960
tcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgc4020
agcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattttt4080
ttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggct4140
ctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccat4200
tctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcc4260
tctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaatt4320
cgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgact4380
gggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagct4440
ggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatg4500
gcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgca4560
gcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcct4620
ttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggt4680
tccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcac4740
gtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttct4800
ttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattctt4860
ttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaac4920
aaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcg4980
gggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatcc5040
gctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgag5100
tattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttt5160
tgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagt5220
gggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaaga5280
acgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtat5340
tgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttga5400
gtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcag5460
tgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggagg5520
accgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcg5580
ttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgt5640
agcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccg5700
gcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggc5760
ccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcgg5820
tatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgac5880
ggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcact5940
gattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaa6000
acttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaa6060
aatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaagg6120
atcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccacc6180
gctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaac6240
tggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggcca6300
ccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagt6360
ggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttacc6420
ggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcg6480
aacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcc6540
cgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcac6600
gagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacct6660
ctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgc6720
cagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctt6780
tcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgatac6840
cgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcg6900
cccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacga6960
caggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcac7020
tcattaggcaccccaggctttccactttatgcttccggctcgtatgttgtgtggaattgt7080
gagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaat7140
taaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaata7200
ctcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaa7260
aagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaagg7320
caacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatatt7380
gtatttaagtgcctagctcgatacataaac7410
<210>3
<211>7413
<212>dna
<213>artificialsequence
<220>
<223>野生型plv-mcherry质粒的核苷酸序列
<400>3
gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaaccca60
ctgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttg120
tgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagc180
agtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgca240
ggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgc300
caaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtatta360
agcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaa420
aatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatc480
ctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatccc540
ttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtg600
tgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagc660
aaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggagga720
gatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaacca780
ttaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtg840
ggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcg900
tcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaac960
aatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatc1020
aagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctg1080
gggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagt1140
tggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacaga1200
gaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaa1260
gaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggttt1320
aacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggta1380
ggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattca1440
ccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaata1500
gaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcga1560
cggtatcggttaacttttaaaagaaaaggggggattggggggtacagtgcaggggaaaga1620
atagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaa1680
ttcaaaattttatcgataagcttgggagttccgcgttacataacttacggtaaatggccc1740
gcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccat1800
agtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgc1860
ccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatga1920
cggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttg1980
gcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacat2040
caatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgt2100
caatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactc2160
cgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagc2220
tcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatag2280
aagacaccgactctagaggatccgccgccaccatggtgagcaagggcgaggaggataaca2340
tggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggcc2400
acgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgcca2460
agctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagt2520
tcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagc2580
tgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtgg2640
tgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgc2700
gcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggagg2760
cctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggc2820
tgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaaga2880
agcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccaca2940
acgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcg3000
gcatggacgagctgtacaagtaagtcgacaatcaacctctggattacaaaatttgtgaaa3060
gattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaa3120
tgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaat3180
cctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgt3240
gcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcc3300
tttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgcc3360
ttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcgg3420
ggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcggga3480
cgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgc3540
tgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccc3600
tttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaa3660
ggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattca3720
ctcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagat3780
ctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagctt3840
gccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatc3900
cctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttat3960
tattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttat4020
tgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatt4080
tttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctg4140
gctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcc4200
cattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcg4260
gcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtaccca4320
attcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtg4380
actgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgcca4440
gctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctga4500
atggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgc4560
gcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttccctt4620
cctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttag4680
ggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggtt4740
cacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgt4800
tctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctatt4860
cttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgattt4920
aacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttt4980
tcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgta5040
tccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtat5100
gagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgt5160
ttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacg5220
agtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccga5280
agaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccg5340
tattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggt5400
tgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatg5460
cagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcgg5520
aggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttga5580
tcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcc5640
tgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttc5700
ccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctc5760
ggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcg5820
cggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacac5880
gacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctc5940
actgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgattt6000
aaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgac6060
caaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaa6120
aggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacc6180
accgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggt6240
aactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttagg6300
ccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacc6360
agtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagtt6420
accggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttgga6480
gcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgct6540
tcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcg6600
cacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgcca6660
cctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaa6720
cgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtt6780
ctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctga6840
taccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaaga6900
gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggca6960
cgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagct7020
cactcattaggcaccccaggctttccactttatgcttccggctcgtatgttgtgtggaat7080
tgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgc7140
aattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgca7200
atactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagag7260
aaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattagga7320
aggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagat7380
attgtatttaagtgcctagctcgatacataaac7413
<210>4
<211>7411
<212>dna
<213>artificialsequence
<220>
<223>plv-△gg(1-2)-mcherry质粒序列
<400>4
gtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccact60
gcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtg120
tgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcag180
tggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcagg240
actcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgcca300
aaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaag360
cgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaa420
tataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcct480
ggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatccctt540
cagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtg600
catcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaa660
aacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggaga720
tatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccatt780
aggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtggg840
aataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtc900
aatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaa960
tttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaa1020
gcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggg1080
gatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttg1140
gagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagaga1200
aattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaaga1260
aaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaa1320
cataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagg1380
tttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcacc1440
attatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaataga1500
agaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacg1560
gtatcggttaacttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaat1620
agtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaatt1680
caaaattttatcgataagcttgggagttccgcgttacataacttacggtaaatggcccgc1740
ctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatag1800
taacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgccc1860
acttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacg1920
gtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggc1980
agtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatca2040
atgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtca2100
atgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccg2160
ccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctc2220
gtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaa2280
gacaccgactctagaggatccgccgccaccatggtgagcaagggcgaggaggataacatg2340
gccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccac2400
gagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaag2460
ctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctcagttc2520
atgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctg2580
tccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtg2640
accgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgc2700
ggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcc2760
tcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaagcagaggctg2820
aagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaag2880
cccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaac2940
gaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggc3000
atggacgagctgtacaagtaagtcgacaatcaacctctggattacaaaatttgtgaaaga3060
ttgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatg3120
cctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcc3180
tggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgc3240
actgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctt3300
tccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgcctt3360
gcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcgggg3420
aagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacg3480
tccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctg3540
ccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctt3600
tgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaagg3660
cagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcact3720
cccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatct3780
gagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgc3840
cttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccc3900
tcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttatta3960
ttcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattg4020
cagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttt4080
tttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggc4140
tctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgccca4200
ttctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggc4260
ctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaat4320
tcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgac4380
tgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagc4440
tggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaat4500
ggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgc4560
agcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcc4620
tttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttaggg4680
ttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttca4740
cgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttc4800
tttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattct4860
tttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaa4920
caaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttc4980
ggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatc5040
cgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatga5100
gtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttt5160
ttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgag5220
tgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaag5280
aacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgta5340
ttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttg5400
agtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgca5460
gtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggag5520
gaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatc5580
gttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctg5640
tagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttccc5700
ggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcgg5760
cccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcg5820
gtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacga5880
cggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcac5940
tgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaa6000
aacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgacca6060
aaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaag6120
gatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccac6180
cgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaa6240
ctggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggcc6300
accacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccag6360
tggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttac6420
cggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagc6480
gaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttc6540
ccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgca6600
cgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacc6660
tctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacg6720
ccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttct6780
ttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgata6840
ccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagc6900
gcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacg6960
acaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctca7020
ctcattaggcaccccaggctttccactttatgcttccggctcgtatgttgtgtggaattg7080
tgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaa7140
ttaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagtcttatgcaat7200
actcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaa7260
aaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaag7320
gcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatat7380
tgtatttaagtgcctagctcgatacataaac7411
<210>5
<211>17
<212>dna
<213>artificialsequence
<220>
<223>gg(1-2)-forward-primer-1
<400>5
tgcatgccgattggtgg17
<210>6
<211>30
<212>dna
<213>artificialsequence
<220>
<223>gg(1-2)-forward-primer-2
<400>6
gatacataaacgtctctctggttagaccag30
<210>7
<211>18
<212>dna
<213>artificialsequence
<220>
<223>gg(1-2)-rerward-primer-1
<400>7
tcagcggccgcttgctgt18
<210>8
<211>30
<212>dna
<213>artificialsequence
<220>
<223>gg(1-2)-rerward-primer-2
<400>8
ccagagagacgtttatgtatcgagctaggc30
<210>9
<211>19
<212>dna
<213>artificialsequence
<220>
<223>mut(1569-1577)-forward-primer-1
<400>9
aagcggccgctgatcttca19
<210>10
<211>34
<212>dna
<213>artificialsequence
<220>
<223>mut(1569-1577)-forward-primer-2
<400>10
gtatcgactagttgtaaaagaaaaggggggattg34
<210>11
<211>19
<212>dna
<213>artificialsequence
<220>
<223>mut(1569-1577)-rerward-primer-1
<400>11
gcttatcgataaaattttg19
<210>12
<211>29
<212>dna
<213>artificialsequence
<220>
<223>mut(1569-1577)-rerward-primer-2
<400>12
tttacaactagtcgataccgtcgagatcc29