CN114736885A - 一种携带bcr-abl1基因激酶区双位点突变的细胞株及其构建方法 - Google Patents

一种携带bcr-abl1基因激酶区双位点突变的细胞株及其构建方法 Download PDF

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CN114736885A
CN114736885A CN202210584242.4A CN202210584242A CN114736885A CN 114736885 A CN114736885 A CN 114736885A CN 202210584242 A CN202210584242 A CN 202210584242A CN 114736885 A CN114736885 A CN 114736885A
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野五
龚玉萍
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West China Hospital of Sichuan University
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Abstract

本发明涉及发明属于生物技术领域，具体的涉及一种携带BCR‑ABL1基因激酶区双位点突变的细胞株及其构建方法。本构建过程得到的携带BCR‑ABL1基因激酶区双位点突变的细胞株，可以避免现有技术的缺陷，不需要扩增质粒全长，只需扩增涵盖突变位点在内的部分区域，可以避开高GC含量区域，同时避免了扩增序列太长导致的高错配率，最重要是现有技术在构建BCR‑ABL1突变质粒中难以适用。本发明构建携带BCR‑ABL1基因激酶区突变的细胞株为探索治疗携带BCR‑ABL1复合突变的慢性髓细胞白血病的治疗方法提供了基础。

Description

一种携带BCR-ABL1基因激酶区双位点突变的细胞株及其构建 方法
技术领域
本发明属于生物技术领域，具体的涉及一种携带BCR-ABL1基因激酶区双位点突变的细胞株及其构建方法。
背景技术
慢性髓细胞白血病(CML)是一种骨髓增殖性造血细胞癌，以9号染色体和22号染色体之间的遗传易位形成BCR-ABL1融合基因为特点。BCR-ABL1融合癌蛋白具有酪氨酸激酶活性，可以激活不同的下游信号通路，从而促进了肿瘤的发生和发展。第一代酪氨酸激酶抑制剂(TKI)伊马替尼极大程度的改善了CML患者的预后，但是治疗过程中BCR-ABL1基因激酶区突变的出现使患者对伊马替尼产生耐药，导致疾病进展。第二代和第三代TKIs，如达沙替尼、尼罗替尼、伯舒替尼和普纳替尼给BCR-ABL1激酶区点突变导致的耐药提供了有效的控制，但是这些TKIs不能对所有位点突变造成的耐药提供有效的控制,尤其是BCR-ABL1激酶区复合突变位点的出现对所有批准的靶向BCR-ABL1的TKIs产生耐药性。为了探索治疗携带BCR-ABL1复合突变的慢性髓系白血病的治疗方法，我们需要进行基础研究，此时构建携带BCR-ABL1基因激酶区突变的细胞株便是开始的第一步。
目前，构建突变细胞株最常用的方法是用通过设计带有突变碱基的引物，利用高保真DNA聚合酶对野生型质粒直接进行PCR扩增，扩增产物经DpnI消化(去除甲基化模板质粒)，然后进行重组反应(正反向引物5’端包含完全反向互补的一段序列)，反应产物进行转化、涂板、克隆鉴定等
现有的技术存在某些缺陷，当质粒中某段序列GC含量高时，要想完全把质粒扩增一圈难度是很高的，最后很难成功构建细胞株。
发明内容
本发明为了解决上述背景技术中当质粒中某段序列GC含量高时，要想完全把质粒扩增一圈难度是很高的，最后很难成功构建细胞株的技术问题，提供一种携带BCR-ABL1基因激酶区双位点突变的细胞株及其构建方法。
本发明采用的技术方案如下：
本发明一方面提供了一种BCR-ABL1基因的单位点或双位点突变，所述BCR-ABL1基因的双位点突变的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2所示；
所述BCR-ABL1基因的单位点突变的核苷酸序列如SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5所示。
SEQ ID NO:1的核苷酸序列为：
TACGAGGACGCCGAGTTGAACCCCCGCTTCCTGAAGGACAACCTGATCGACGCCAATGGCGGTAGCAGGCCCCCTTGGCCGCCCCTGGAGTACCAGCCCTACCAGAGCATCTACGTCGGGGGCATGATGGAAGGGGAGGGCAAGGGCCCGCTCCTGCGCAGCCAGAGCACCTCTGAGCAGGAGAAGCGCCTTACCTGGCCCCGCAGGTCCTACTCCCCCCGGAGTTTTGAGGATTGCGGAGGCGGCTATACCCCGGACTGCAGCTCCAATGAGAACCTCACCTCCAGCGAGGAGGACTTCTCCTCTGGCCAGTCCAGCCGCGTGTCCCCAAGCCCCACCACCTACCGCATGTTCCGGGACAAAAGCCGCTCTCCCTCGCAGAACTCGCAACAGTCCTTCGACAGCAGCAGTCCCCCCACGCCGCAGTGCCATAAGCGGCACCGGCACTGCCCGGTTGTCGTGTCCGAGGCCACCATCGTGGGCGTCCGCAAGACCGGGCAGATCTGGCCCAACGATGGCGAGGGCGCCTTCCATGGAGACGCAGATGGCTCGTTCGGAACACCACCTGGATACGGCTGCGCTGCAGACCGGGCAGAGGAGCAGCGCCGGCACCAAGATGGGCTGCCCTACATTGATGACTCGCCCTCCTCATCGCCCCACCTCAGCAGCAAGGGCAGGGGCAGCCGGGATGCGCTGGTCTCGGGAGCCCTGGAGTCCACTAAAGCGAGTGAGCTGGACTTGGAAAAGGGCTTGGAGATGAGAAAATGGGTCCTGTCGGGAATCCTGGCTAGCGAGGAGACTTACCTGAGCCACCTGGAGGCACTGCTGCTGCCCATGAAGCCTTTGAAAGCCGCTGCCACCACCTCTCAGCCGGTGCTGACGAGTCAGCAGATCGAGACCATCTTCTTCAAAGTGCCTGAGCTCTACGAGATCCACAAGGAGTTCTATGATGGGCTCTTCCCCCGCGTGCAGCAGTGGAGCCACCAGCAGCGGGTGGGCGACCTCTTCCAGAAGCTGGCCAGCCAGCTGGGTGTGTACCGGGCCTTCGTGGACAACTACGGAGTTGCCATGGAAATGGCTGAGAAGTGCTGTCAGGCCAATGCTCAGTTTGCAGAAATCTCCGAGAACCTGAGAGCCAGAAGCAACAAAGATGCCAAGGATCCAACGACCAAGAACTCTCTGGAAACTCTGCTCTACAAGCCTGTGGACCGTGTGACGAGGAGCACGCTGGTCCTCCATGACTTGCTGAAGCACACTCCTGCCAGCCACCCTGACCACCCCTTGCTGCAGGACGCCCTCCGCATCTCACAGAACTTCCTGTCCAGCATCAATGAGGAGATCACACCCCGACGGCAGTCCATGACGGTGAAGAAGGGAGAGCACCGGCAGCTGCTGAAGGACAGCTTCATGGTGGAGCTGGTGGAGGGGGCCCGCAAGCTGCGCCACGTCTTCCTGTTCACCGACCTGCTTCTCTGCACCAAGCTCAAGAAGCAGAGCGGAGGCAAAACGCAGCAGTATGACTGCAAATGGTACATTCCGCTCACGGATCTCAGCTTCCAGATGGTGGATGAACTGGAGGCAGTGCCCAACATCCCCCTGGTGCCCGATGAGGAGCTGGACGCTTTGAAGATCAAGATCTCCCAGATCAAGAGTGACATCCAGAGAGAGAAGAGGGCGAACAAGGGCAGCAAGGCTACGGAGAGGCTGAAGAAGAAGCTGTCGGAGCAGGAGTCACTGCTGCTGCTTATGTCTCCCAGCATGGCCTTCAGGGTGCACAGCCGCAACGGCAAGAGTTACACGTTCCTGATCTCCTCTGACTATGAGCGTGCAGAGTGGAGGGAGAACATCCGGGAGCAGCAGAAGAAGTGTTTCAGAAGCTTCTCCCTGACATCCGTGGAGCTGCAGATGCTGACCAACTCGTGTGTGAAACTCCAGACTGTCCACAGCATTCCGCTGACCATCAATAAGGAAGATGATGAGTCTCCGGGGCTCTATGGGTTTCTGAATGTCATCGTCCACTCAGCCACTGGATTTAAGCAGAGTTCAAAAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAAAACCTTCTCGCTGGACCCAGTGAAAATGACCCCAACCTTTTCGTTGCACTGTATGATTTTGTGGCCAGTGGAGATAACACTCTAAGCATAACTAAAGGTGAAAAGCTCCGGGTCTTAGGCTATAATCACAATGGGGAATGGTGTGAAGCCCAAACCAAAAATGGCCAAGGCTGGGTCCCAAGCAACTACATCACGCCAGTCAACAGTCTGGAGAAACACTCCTGGTACCATGGGCCTGTGTCCCGCAATGCCGCTGAGTATCTGCTGAGCAGCGGGATCAATGGCAGCTTCTTGGTGCGTGAGAGTGAGAGCAGTCCTGGCCAGAGGTCCATCTCGCTGAGATACGAAGGGAGGGTGTACCATTACAGGATCAACACTGCTTCTGATGGCAAGCTCTACGTCTCCTCCGAGAGCCGCTTCAACACCCTGGCCGAGTTGGTTCATCATCATTCAACGGTGGCCGACGGGCTCATCACCACGCTCCATTATCCAGCCCCAAAGCGCAACAAGCCCACTGTCTATGGTGTGTCCCCCAACTACGACAAGTGGGAGATGGAACGCACGGACATCACCATGAAGCACAAGCTGGGCGGGGGCCAGTACGGGGAGGTGTACGAGGGCGTGTGGAAGAAATACAGCCTGACGGTGGCCGTGAAGACCTTGAAGGAGGACACCATGGAGGTGGAAGAGTTCTTGAAAGAAGCTGCAGTCATGAAAGAGATCAAACACCCTAACCTGGTGCAGCTCCTTGGGGTCTGCACCCGGGAGCCCCCGTTCTATATCATCATTGAGTTCATGACCTACGGGAACCTCCTGGACTACCTGAGGGAGTGCAACCGGCAGGAGGTGAACGCCGTGGTGCTGCTGTACATGGCCACTCAGATCTCGTCAGCCATGGAGTACCTGGAGAAGAAAAACTTCATCCACAGAGATCTTGCTGCCCGAAACTGCCTGGTAGGGGAGAACCACTTGGTGAAGGTAGCTGATTTTGGCCTGAGCAGGATGATGACAGGGGACACCTACACAGCCCATGCTGGAGCCAAGTTCCCCATCAAATGGACTGCACCCGAGAGCCTGGCCTACAACAAGTTCTCCATCAAGTCCGACGTCTGGGCATTTGGAGTATTGCTTTGGGAAATTGCTACCTATGGCATGTCCCCTTACCCGGGAATTGACCTGTCCCAGGTGTATGAGCTGCTAGAGAAGGACTACCGCATGGAGCGCCCAGAAGGCTGCCCAGAGAAGGTCTATGAACTCATGCGAGCATGTTGGCAGTGGAATCCCTCTGACCGGCCCTCCTTTGCTGAAATCCACCAAGCCTTTGAAACAATGTTCCAGGAATCCAGTATCTCAGACGAAGTGGAAAAGGAGCTGGGGAAACAAGGCGTCCGTGGGGCTGTGAGTACCTTGCTGCAGGCCCCAGAGCTGCCCACCAAGACGAGGACCTCCAGGAGAGCTGCAGAGCACAGAGACACCACTGACGTGCCTGAGATGCCTCACTCCAAGGGCCAGGGAGAGAGCGATCCTCTGGACCATGAGCCTGCCGTGTCTCCATTGCTCCCTCGAAAAGAGCGAGGTCCCCCGGAGGGCGGCCTGAATGAAGATGAGCGCCTTCTCCCCAAAGACAAAAAGACCAACTTGTTCAGCGCCTTGATCAAGAAGAAGAAGAAGACAGCCCCAACCCCTCCCAAACGCAGCAGCTCCTTCCGGGAGATGGACGGCCAGCCGGAGCGCAGAGGGGCCGGCGAGGAAGAGGGCCGAGACATCAGCAACGGGGCACTGGCTTTCACCCCCTTGGACACAGCTGACCCAGCCAAGTCCCCAAAGCCCAGCAATGGGGCTGGGGTCCCCAATGGAGCCCTCCGGGAGTCCGGGGGCTCAGGCTTCCGGTCTCCCCACCTGTGGAAGAAGTCCAGCACGCTGACCAGCAGCCGCCTAGCCACCGGCGAGGAGGAGGGCGGTGGCAGCTCCAGCAAGCGCTTCCTGCGCTCTTGCTCCGCCTCCTGCGTTCCCCATGGGGCCAAGGACACGGAGTGGAGGTCAGTCACGCTGCCTCGGGACTTGCAGTCCACGGGAAGACAGTTTGACTCGTCCACATTTGGAGGGCACAAAAGTGAGAAGCCGGCTCTGCCTCGGAAGAGGGCAGGGGAGAACAGGTCTGACCAGGTGACCCGAGGCACAGTAA
SEQ ID NO:2的核苷酸序列为：
TACGAGGACGCCGAGTTGAACCCCCGCTTCCTGAAGGACAACCTGATCGACGCCAATGGCGGTAGCAGGCCCCCTTGGCCGCCCCTGGAGTACCAGCCCTACCAGAGCATCTACGTCGGGGGCATGATGGAAGGGGAGGGCAAGGGCCCGCTCCTGCGCAGCCAGAGCACCTCTGAGCAGGAGAAGCGCCTTACCTGGCCCCGCAGGTCCTACTCCCCCCGGAGTTTTGAGGATTGCGGAGGCGGCTATACCCCGGACTGCAGCTCCAATGAGAACCTCACCTCCAGCGAGGAGGACTTCTCCTCTGGCCAGTCCAGCCGCGTGTCCCCAAGCCCCACCACCTACCGCATGTTCCGGGACAAAAGCCGCTCTCCCTCGCAGAACTCGCAACAGTCCTTCGACAGCAGCAGTCCCCCCACGCCGCAGTGCCATAAGCGGCACCGGCACTGCCCGGTTGTCGTGTCCGAGGCCACCATCGTGGGCGTCCGCAAGACCGGGCAGATCTGGCCCAACGATGGCGAGGGCGCCTTCCATGGAGACGCAGATGGCTCGTTCGGAACACCACCTGGATACGGCTGCGCTGCAGACCGGGCAGAGGAGCAGCGCCGGCACCAAGATGGGCTGCCCTACATTGATGACTCGCCCTCCTCATCGCCCCACCTCAGCAGCAAGGGCAGGGGCAGCCGGGATGCGCTGGTCTCGGGAGCCCTGGAGTCCACTAAAGCGAGTGAGCTGGACTTGGAAAAGGGCTTGGAGATGAGAAAATGGGTCCTGTCGGGAATCCTGGCTAGCGAGGAGACTTACCTGAGCCACCTGGAGGCACTGCTGCTGCCCATGAAGCCTTTGAAAGCCGCTGCCACCACCTCTCAGCCGGTGCTGACGAGTCAGCAGATCGAGACCATCTTCTTCAAAGTGCCTGAGCTCTACGAGATCCACAAGGAGTTCTATGATGGGCTCTTCCCCCGCGTGCAGCAGTGGAGCCACCAGCAGCGGGTGGGCGACCTCTTCCAGAAGCTGGCCAGCCAGCTGGGTGTGTACCGGGCCTTCGTGGACAACTACGGAGTTGCCATGGAAATGGCTGAGAAGTGCTGTCAGGCCAATGCTCAGTTTGCAGAAATCTCCGAGAACCTGAGAGCCAGAAGCAACAAAGATGCCAAGGATCCAACGACCAAGAACTCTCTGGAAACTCTGCTCTACAAGCCTGTGGACCGTGTGACGAGGAGCACGCTGGTCCTCCATGACTTGCTGAAGCACACTCCTGCCAGCCACCCTGACCACCCCTTGCTGCAGGACGCCCTCCGCATCTCACAGAACTTCCTGTCCAGCATCAATGAGGAGATCACACCCCGACGGCAGTCCATGACGGTGAAGAAGGGAGAGCACCGGCAGCTGCTGAAGGACAGCTTCATGGTGGAGCTGGTGGAGGGGGCCCGCAAGCTGCGCCACGTCTTCCTGTTCACCGACCTGCTTCTCTGCACCAAGCTCAAGAAGCAGAGCGGAGGCAAAACGCAGCAGTATGACTGCAAATGGTACATTCCGCTCACGGATCTCAGCTTCCAGATGGTGGATGAACTGGAGGCAGTGCCCAACATCCCCCTGGTGCCCGATGAGGAGCTGGACGCTTTGAAGATCAAGATCTCCCAGATCAAGAGTGACATCCAGAGAGAGAAGAGGGCGAACAAGGGCAGCAAGGCTACGGAGAGGCTGAAGAAGAAGCTGTCGGAGCAGGAGTCACTGCTGCTGCTTATGTCTCCCAGCATGGCCTTCAGGGTGCACAGCCGCAACGGCAAGAGTTACACGTTCCTGATCTCCTCTGACTATGAGCGTGCAGAGTGGAGGGAGAACATCCGGGAGCAGCAGAAGAAGTGTTTCAGAAGCTTCTCCCTGACATCCGTGGAGCTGCAGATGCTGACCAACTCGTGTGTGAAACTCCAGACTGTCCACAGCATTCCGCTGACCATCAATAAGGAAGATGATGAGTCTCCGGGGCTCTATGGGTTTCTGAATGTCATCGTCCACTCAGCCACTGGATTTAAGCAGAGTTCAAAAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAAAACCTTCTCGCTGGACCCAGTGAAAATGACCCCAACCTTTTCGTTGCACTGTATGATTTTGTGGCCAGTGGAGATAACACTCTAAGCATAACTAAAGGTGAAAAGCTCCGGGTCTTAGGCTATAATCACAATGGGGAATGGTGTGAAGCCCAAACCAAAAATGGCCAAGGCTGGGTCCCAAGCAACTACATCACGCCAGTCAACAGTCTGGAGAAACACTCCTGGTACCATGGGCCTGTGTCCCGCAATGCCGCTGAGTATCTGCTGAGCAGCGGGATCAATGGCAGCTTCTTGGTGCGTGAGAGTGAGAGCAGTCCTGGCCAGAGGTCCATCTCGCTGAGATACGAAGGGAGGGTGTACCATTACAGGATCAACACTGCTTCTGATGGCAAGCTCTACGTCTCCTCCGAGAGCCGCTTCAACACCCTGGCCGAGTTGGTTCATCATCATTCAACGGTGGCCGACGGGCTCATCACCACGCTCCATTATCCAGCCCCAAAGCGCAACAAGCCCACTGTCTATGGTGTGTCCCCCAACTACGACAAGTGGGAGATGGAACGCACGGACATCACCATGAAGCACAAGCTGGGCGGGGGCCAGTACGGGGAGGTGTACGAGGGCGTGTGGAAGAAATACAGCCTGACGGTGGCCGTGAAGACCTTGAAGGAGGACACCATGGAGGTGGAAGAGTTCTTGAAAGAAGCTGCAGTCATGAAAGAGATCAAACACCCTAACCTGGTGCAGCTCCTTGGGGTCTGCACCCGGGAGCCCCCGTTCTATATCATCATTGAGTTCATGACCTACGGGAACCTCCTGGACTACCTGAGGGAGTGCAACCGGCAGGAGGTGAACGCCGTGGTGCTGCTGTACATGGCCACTCAGATCTCGTCAGCCATGGAGTACCTGGAGAAGAAAAACTTCATCCACAGAGATCTTGCTGCCCGAAACTGCCTGGTAGGGGAGAACCACTTGGTGAAGGTAGCTGATTTTGGCCTGAGCAGGTTGATGACAGGGGACACCTACACAGCCCATGCTGGAGCCAAGTTCCCCATCAAATGGACTGCACCCGAGAGCCTGGCCTACAACAAGTTCTCCATCAAGTCCGACGTCTGGGCATTTGGAGTATTGCTTTGGGAAATTGCTACCTATGGCATGTCCCCTTACCCGGGAATTGACCTGTCCCAGGTGTATGAGCTGCTAGAGAAGGACTACCGCATGGAGCGCCCAGAAGGCTGCCCAGAGAAGGTCTATGAACTCATGCGAGCATGTTGGCAGTGGAATCCCTCTGACCGGCCCTCCTCTGCTGAAATCCACCAAGCCTTTGAAACAATGTTCCAGGAATCCAGTATCTCAGACGAAGTGGAAAAGGAGCTGGGGAAACAAGGCGTCCGTGGGGCTGTGAGTACCTTGCTGCAGGCCCCAGAGCTGCCCACCAAGACGAGGACCTCCAGGAGAGCTGCAGAGCACAGAGACACCACTGACGTGCCTGAGATGCCTCACTCCAAGGGCCAGGGAGAGAGCGATCCTCTGGACCATGAGCCTGCCGTGTCTCCATTGCTCCCTCGAAAAGAGCGAGGTCCCCCGGAGGGCGGCCTGAATGAAGATGAGCGCCTTCTCCCCAAAGACAAAAAGACCAACTTGTTCAGCGCCTTGATCAAGAAGAAGAAGAAGACAGCCCCAACCCCTCCCAAACGCAGCAGCTCCTTCCGGGAGATGGACGGCCAGCCGGAGCGCAGAGGGGCCGGCGAGGAAGAGGGCCGAGACATCAGCAACGGGGCACTGGCTTTCACCCCCTTGGACACAGCTGACCCAGCCAAGTCCCCAAAGCCCAGCAATGGGGCTGGGGTCCCCAATGGAGCCCTCCGGGAGTCCGGGGGCTCAGGCTTCCGGTCTCCCCACCTGTGGAAGAAGTCCAGCACGCTGACCAGCAGCCGCCTAGCCACCGGCGAGGAGGAGGGCGGTGGCAGCTCCAGCAAGCGCTTCCTGCGCTCTTGCTCCGCCTCCTGCGTTCCCCATGGGGCCAAGGACACGGAGTGGAGGTCAGTCACGCTGCCTCGGGACTTGCAGTCCACGGGAAGACAGTTTGACTCGTCCACATTTGGAGGGCACAAAAGTGAGAAGCCGGCTCTGCCTCGGAAGAGGGCAGGGGAGAACAGGTCTGACCAGGTGACCCGAGGCACAGTAA
SEQ ID NO:3的核苷酸序列为：
TACGAGGACGCCGAGTTGAACCCCCGCTTCCTGAAGGACAACCTGATCGACGCCAATGGCGGTAGCAGGCCCCCTTGGCCGCCCCTGGAGTACCAGCCCTACCAGAGCATCTACGTCGGGGGCATGATGGAAGGGGAGGGCAAGGGCCCGCTCCTGCGCAGCCAGAGCACCTCTGAGCAGGAGAAGCGCCTTACCTGGCCCCGCAGGTCCTACTCCCCCCGGAGTTTTGAGGATTGCGGAGGCGGCTATACCCCGGACTGCAGCTCCAATGAGAACCTCACCTCCAGCGAGGAGGACTTCTCCTCTGGCCAGTCCAGCCGCGTGTCCCCAAGCCCCACCACCTACCGCATGTTCCGGGACAAAAGCCGCTCTCCCTCGCAGAACTCGCAACAGTCCTTCGACAGCAGCAGTCCCCCCACGCCGCAGTGCCATAAGCGGCACCGGCACTGCCCGGTTGTCGTGTCCGAGGCCACCATCGTGGGCGTCCGCAAGACCGGGCAGATCTGGCCCAACGATGGCGAGGGCGCCTTCCATGGAGACGCAGATGGCTCGTTCGGAACACCACCTGGATACGGCTGCGCTGCAGACCGGGCAGAGGAGCAGCGCCGGCACCAAGATGGGCTGCCCTACATTGATGACTCGCCCTCCTCATCGCCCCACCTCAGCAGCAAGGGCAGGGGCAGCCGGGATGCGCTGGTCTCGGGAGCCCTGGAGTCCACTAAAGCGAGTGAGCTGGACTTGGAAAAGGGCTTGGAGATGAGAAAATGGGTCCTGTCGGGAATCCTGGCTAGCGAGGAGACTTACCTGAGCCACCTGGAGGCACTGCTGCTGCCCATGAAGCCTTTGAAAGCCGCTGCCACCACCTCTCAGCCGGTGCTGACGAGTCAGCAGATCGAGACCATCTTCTTCAAAGTGCCTGAGCTCTACGAGATCCACAAGGAGTTCTATGATGGGCTCTTCCCCCGCGTGCAGCAGTGGAGCCACCAGCAGCGGGTGGGCGACCTCTTCCAGAAGCTGGCCAGCCAGCTGGGTGTGTACCGGGCCTTCGTGGACAACTACGGAGTTGCCATGGAAATGGCTGAGAAGTGCTGTCAGGCCAATGCTCAGTTTGCAGAAATCTCCGAGAACCTGAGAGCCAGAAGCAACAAAGATGCCAAGGATCCAACGACCAAGAACTCTCTGGAAACTCTGCTCTACAAGCCTGTGGACCGTGTGACGAGGAGCACGCTGGTCCTCCATGACTTGCTGAAGCACACTCCTGCCAGCCACCCTGACCACCCCTTGCTGCAGGACGCCCTCCGCATCTCACAGAACTTCCTGTCCAGCATCAATGAGGAGATCACACCCCGACGGCAGTCCATGACGGTGAAGAAGGGAGAGCACCGGCAGCTGCTGAAGGACAGCTTCATGGTGGAGCTGGTGGAGGGGGCCCGCAAGCTGCGCCACGTCTTCCTGTTCACCGACCTGCTTCTCTGCACCAAGCTCAAGAAGCAGAGCGGAGGCAAAACGCAGCAGTATGACTGCAAATGGTACATTCCGCTCACGGATCTCAGCTTCCAGATGGTGGATGAACTGGAGGCAGTGCCCAACATCCCCCTGGTGCCCGATGAGGAGCTGGACGCTTTGAAGATCAAGATCTCCCAGATCAAGAGTGACATCCAGAGAGAGAAGAGGGCGAACAAGGGCAGCAAGGCTACGGAGAGGCTGAAGAAGAAGCTGTCGGAGCAGGAGTCACTGCTGCTGCTTATGTCTCCCAGCATGGCCTTCAGGGTGCACAGCCGCAACGGCAAGAGTTACACGTTCCTGATCTCCTCTGACTATGAGCGTGCAGAGTGGAGGGAGAACATCCGGGAGCAGCAGAAGAAGTGTTTCAGAAGCTTCTCCCTGACATCCGTGGAGCTGCAGATGCTGACCAACTCGTGTGTGAAACTCCAGACTGTCCACAGCATTCCGCTGACCATCAATAAGGAAGATGATGAGTCTCCGGGGCTCTATGGGTTTCTGAATGTCATCGTCCACTCAGCCACTGGATTTAAGCAGAGTTCAAAAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAAAACCTTCTCGCTGGACCCAGTGAAAATGACCCCAACCTTTTCGTTGCACTGTATGATTTTGTGGCCAGTGGAGATAACACTCTAAGCATAACTAAAGGTGAAAAGCTCCGGGTCTTAGGCTATAATCACAATGGGGAATGGTGTGAAGCCCAAACCAAAAATGGCCAAGGCTGGGTCCCAAGCAACTACATCACGCCAGTCAACAGTCTGGAGAAACACTCCTGGTACCATGGGCCTGTGTCCCGCAATGCCGCTGAGTATCTGCTGAGCAGCGGGATCAATGGCAGCTTCTTGGTGCGTGAGAGTGAGAGCAGTCCTGGCCAGAGGTCCATCTCGCTGAGATACGAAGGGAGGGTGTACCATTACAGGATCAACACTGCTTCTGATGGCAAGCTCTACGTCTCCTCCGAGAGCCGCTTCAACACCCTGGCCGAGTTGGTTCATCATCATTCAACGGTGGCCGACGGGCTCATCACCACGCTCCATTATCCAGCCCCAAAGCGCAACAAGCCCACTGTCTATGGTGTGTCCCCCAACTACGACAAGTGGGAGATGGAACGCACGGACATCACCATGAAGCACAAGCTGGGCGGGGGCCAGTACGGGGAGGTGTACGAGGGCGTGTGGAAGAAATACAGCCTGACGGTGGCCGTGAAGACCTTGAAGGAGGACACCATGGAGGTGGAAGAGTTCTTGAAAGAAGCTGCAGTCATGAAAGAGATCAAACACCCTAACCTGGTGCAGCTCCTTGGGGTCTGCACCCGGGAGCCCCCGTTCTATATCATCATTGAGTTCATGACCTACGGGAACCTCCTGGACTACCTGAGGGAGTGCAACCGGCAGGAGGTGAACGCCGTGGTGCTGCTGTACATGGCCACTCAGATCTCGTCAGCCATGGAGTACCTGGAGAAGAAAAACTTCATCCACAGAGATCTTGCTGCCCGAAACTGCCTGGTAGGGGAGAACCACTTGGTGAAGGTAGCTGATTTTGGCCTGAGCAGGTTGATGACAGGGGACACCTACACAGCCCATGCTGGAGCCAAGTTCCCCATCAAATGGACTGCACCCGAGAGCCTGGCCTACAACAAGTTCTCCATCAAGTCCGACGTCTGGGCATTTGGAGTATTGCTTTGGGAAATTGCTACCTATGGCATGTCCCCTTACCCGGGAATTGACCTGTCCCAGGTGTATGAGCTGCTAGAGAAGGACTACCGCATGGAGCGCCCAGAAGGCTGCCCAGAGAAGGTCTATGAACTCATGCGAGCATGTTGGCAGTGGAATCCCTCTGACCGGCCCTCCTTTGCTGAAATCCACCAAGCCTTTGAAACAATGTTCCAGGAATCCAGTATCTCAGACGAAGTGGAAAAGGAGCTGGGGAAACAAGGCGTCCGTGGGGCTGTGAGTACCTTGCTGCAGGCCCCAGAGCTGCCCACCAAGACGAGGACCTCCAGGAGAGCTGCAGAGCACAGAGACACCACTGACGTGCCTGAGATGCCTCACTCCAAGGGCCAGGGAGAGAGCGATCCTCTGGACCATGAGCCTGCCGTGTCTCCATTGCTCCCTCGAAAAGAGCGAGGTCCCCCGGAGGGCGGCCTGAATGAAGATGAGCGCCTTCTCCCCAAAGACAAAAAGACCAACTTGTTCAGCGCCTTGATCAAGAAGAAGAAGAAGACAGCCCCAACCCCTCCCAAACGCAGCAGCTCCTTCCGGGAGATGGACGGCCAGCCGGAGCGCAGAGGGGCCGGCGAGGAAGAGGGCCGAGACATCAGCAACGGGGCACTGGCTTTCACCCCCTTGGACACAGCTGACCCAGCCAAGTCCCCAAAGCCCAGCAATGGGGCTGGGGTCCCCAATGGAGCCCTCCGGGAGTCCGGGGGCTCAGGCTTCCGGTCTCCCCACCTGTGGAAGAAGTCCAGCACGCTGACCAGCAGCCGCCTAGCCACCGGCGAGGAGGAGGGCGGTGGCAGCTCCAGCAAGCGCTTCCTGCGCTCTTGCTCCGCCTCCTGCGTTCCCCATGGGGCCAAGGACACGGAGTGGAGGTCAGTCACGCTGCCTCGGGACTTGCAGTCCACGGGAAGACAGTTTGACTCGTCCACATTTGGAGGGCACAAAAGTGAGAAGCCGGCTCTGCCTCGGAAGAGGGCAGGGGAGAACAGGTCTGACCAGGTGACCCGAGGCACAGTAA
SEQ ID NO:4的核苷酸序列为：
TACGAGGACGCCGAGTTGAACCCCCGCTTCCTGAAGGACAACCTGATCGACGCCAATGGCGGTAGCAGGCCCCCTTGGCCGCCCCTGGAGTACCAGCCCTACCAGAGCATCTACGTCGGGGGCATGATGGAAGGGGAGGGCAAGGGCCCGCTCCTGCGCAGCCAGAGCACCTCTGAGCAGGAGAAGCGCCTTACCTGGCCCCGCAGGTCCTACTCCCCCCGGAGTTTTGAGGATTGCGGAGGCGGCTATACCCCGGACTGCAGCTCCAATGAGAACCTCACCTCCAGCGAGGAGGACTTCTCCTCTGGCCAGTCCAGCCGCGTGTCCCCAAGCCCCACCACCTACCGCATGTTCCGGGACAAAAGCCGCTCTCCCTCGCAGAACTCGCAACAGTCCTTCGACAGCAGCAGTCCCCCCACGCCGCAGTGCCATAAGCGGCACCGGCACTGCCCGGTTGTCGTGTCCGAGGCCACCATCGTGGGCGTCCGCAAGACCGGGCAGATCTGGCCCAACGATGGCGAGGGCGCCTTCCATGGAGACGCAGATGGCTCGTTCGGAACACCACCTGGATACGGCTGCGCTGCAGACCGGGCAGAGGAGCAGCGCCGGCACCAAGATGGGCTGCCCTACATTGATGACTCGCCCTCCTCATCGCCCCACCTCAGCAGCAAGGGCAGGGGCAGCCGGGATGCGCTGGTCTCGGGAGCCCTGGAGTCCACTAAAGCGAGTGAGCTGGACTTGGAAAAGGGCTTGGAGATGAGAAAATGGGTCCTGTCGGGAATCCTGGCTAGCGAGGAGACTTACCTGAGCCACCTGGAGGCACTGCTGCTGCCCATGAAGCCTTTGAAAGCCGCTGCCACCACCTCTCAGCCGGTGCTGACGAGTCAGCAGATCGAGACCATCTTCTTCAAAGTGCCTGAGCTCTACGAGATCCACAAGGAGTTCTATGATGGGCTCTTCCCCCGCGTGCAGCAGTGGAGCCACCAGCAGCGGGTGGGCGACCTCTTCCAGAAGCTGGCCAGCCAGCTGGGTGTGTACCGGGCCTTCGTGGACAACTACGGAGTTGCCATGGAAATGGCTGAGAAGTGCTGTCAGGCCAATGCTCAGTTTGCAGAAATCTCCGAGAACCTGAGAGCCAGAAGCAACAAAGATGCCAAGGATCCAACGACCAAGAACTCTCTGGAAACTCTGCTCTACAAGCCTGTGGACCGTGTGACGAGGAGCACGCTGGTCCTCCATGACTTGCTGAAGCACACTCCTGCCAGCCACCCTGACCACCCCTTGCTGCAGGACGCCCTCCGCATCTCACAGAACTTCCTGTCCAGCATCAATGAGGAGATCACACCCCGACGGCAGTCCATGACGGTGAAGAAGGGAGAGCACCGGCAGCTGCTGAAGGACAGCTTCATGGTGGAGCTGGTGGAGGGGGCCCGCAAGCTGCGCCACGTCTTCCTGTTCACCGACCTGCTTCTCTGCACCAAGCTCAAGAAGCAGAGCGGAGGCAAAACGCAGCAGTATGACTGCAAATGGTACATTCCGCTCACGGATCTCAGCTTCCAGATGGTGGATGAACTGGAGGCAGTGCCCAACATCCCCCTGGTGCCCGATGAGGAGCTGGACGCTTTGAAGATCAAGATCTCCCAGATCAAGAGTGACATCCAGAGAGAGAAGAGGGCGAACAAGGGCAGCAAGGCTACGGAGAGGCTGAAGAAGAAGCTGTCGGAGCAGGAGTCACTGCTGCTGCTTATGTCTCCCAGCATGGCCTTCAGGGTGCACAGCCGCAACGGCAAGAGTTACACGTTCCTGATCTCCTCTGACTATGAGCGTGCAGAGTGGAGGGAGAACATCCGGGAGCAGCAGAAGAAGTGTTTCAGAAGCTTCTCCCTGACATCCGTGGAGCTGCAGATGCTGACCAACTCGTGTGTGAAACTCCAGACTGTCCACAGCATTCCGCTGACCATCAATAAGGAAGATGATGAGTCTCCGGGGCTCTATGGGTTTCTGAATGTCATCGTCCACTCAGCCACTGGATTTAAGCAGAGTTCAAAAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAAAACCTTCTCGCTGGACCCAGTGAAAATGACCCCAACCTTTTCGTTGCACTGTATGATTTTGTGGCCAGTGGAGATAACACTCTAAGCATAACTAAAGGTGAAAAGCTCCGGGTCTTAGGCTATAATCACAATGGGGAATGGTGTGAAGCCCAAACCAAAAATGGCCAAGGCTGGGTCCCAAGCAACTACATCACGCCAGTCAACAGTCTGGAGAAACACTCCTGGTACCATGGGCCTGTGTCCCGCAATGCCGCTGAGTATCTGCTGAGCAGCGGGATCAATGGCAGCTTCTTGGTGCGTGAGAGTGAGAGCAGTCCTGGCCAGAGGTCCATCTCGCTGAGATACGAAGGGAGGGTGTACCATTACAGGATCAACACTGCTTCTGATGGCAAGCTCTACGTCTCCTCCGAGAGCCGCTTCAACACCCTGGCCGAGTTGGTTCATCATCATTCAACGGTGGCCGACGGGCTCATCACCACGCTCCATTATCCAGCCCCAAAGCGCAACAAGCCCACTGTCTATGGTGTGTCCCCCAACTACGACAAGTGGGAGATGGAACGCACGGACATCACCATGAAGCACAAGCTGGGCGGGGGCCAGTACGGGGAGGTGTACGAGGGCGTGTGGAAGAAATACAGCCTGACGGTGGCCGTGAAGACCTTGAAGGAGGACACCATGGAGGTGGAAGAGTTCTTGAAAGAAGCTGCAGTCATGAAAGAGATCAAACACCCTAACCTGGTGCAGCTCCTTGGGGTCTGCACCCGGGAGCCCCCGTTCTATATCATCACTGAGTTCATGACCTACGGGAACCTCCTGGACTACCTGAGGGAGTGCAACCGGCAGGAGGTGAACGCCGTGGTGCTGCTGTACATGGCCACTCAGATCTCGTCAGCCATGGAGTACCTGGAGAAGAAAAACTTCATCCACAGAGATCTTGCTGCCCGAAACTGCCTGGTAGGGGAGAACCACTTGGTGAAGGTAGCTGATTTTGGCCTGAGCAGGATGATGACAGGGGACACCTACACAGCCCATGCTGGAGCCAAGTTCCCCATCAAATGGACTGCACCCGAGAGCCTGGCCTACAACAAGTTCTCCATCAAGTCCGACGTCTGGGCATTTGGAGTATTGCTTTGGGAAATTGCTACCTATGGCATGTCCCCTTACCCGGGAATTGACCTGTCCCAGGTGTATGAGCTGCTAGAGAAGGACTACCGCATGGAGCGCCCAGAAGGCTGCCCAGAGAAGGTCTATGAACTCATGCGAGCATGTTGGCAGTGGAATCCCTCTGACCGGCCCTCCTTTGCTGAAATCCACCAAGCCTTTGAAACAATGTTCCAGGAATCCAGTATCTCAGACGAAGTGGAAAAGGAGCTGGGGAAACAAGGCGTCCGTGGGGCTGTGAGTACCTTGCTGCAGGCCCCAGAGCTGCCCACCAAGACGAGGACCTCCAGGAGAGCTGCAGAGCACAGAGACACCACTGACGTGCCTGAGATGCCTCACTCCAAGGGCCAGGGAGAGAGCGATCCTCTGGACCATGAGCCTGCCGTGTCTCCATTGCTCCCTCGAAAAGAGCGAGGTCCCCCGGAGGGCGGCCTGAATGAAGATGAGCGCCTTCTCCCCAAAGACAAAAAGACCAACTTGTTCAGCGCCTTGATCAAGAAGAAGAAGAAGACAGCCCCAACCCCTCCCAAACGCAGCAGCTCCTTCCGGGAGATGGACGGCCAGCCGGAGCGCAGAGGGGCCGGCGAGGAAGAGGGCCGAGACATCAGCAACGGGGCACTGGCTTTCACCCCCTTGGACACAGCTGACCCAGCCAAGTCCCCAAAGCCCAGCAATGGGGCTGGGGTCCCCAATGGAGCCCTCCGGGAGTCCGGGGGCTCAGGCTTCCGGTCTCCCCACCTGTGGAAGAAGTCCAGCACGCTGACCAGCAGCCGCCTAGCCACCGGCGAGGAGGAGGGCGGTGGCAGCTCCAGCAAGCGCTTCCTGCGCTCTTGCTCCGCCTCCTGCGTTCCCCATGGGGCCAAGGACACGGAGTGGAGGTCAGTCACGCTGCCTCGGGACTTGCAGTCCACGGGAAGACAGTTTGACTCGTCCACATTTGGAGGGCACAAAAGTGAGAAGCCGGCTCTGCCTCGGAAGAGGGCAGGGGAGAACAGGTCTGACCAGGTGACCCGAGGCACAGTAA
SEQ ID NO:5的核苷酸序列为：
TACGAGGACGCCGAGTTGAACCCCCGCTTCCTGAAGGACAACCTGATCGACGCCAATGGCGGTAGCAGGCCCCCTTGGCCGCCCCTGGAGTACCAGCCCTACCAGAGCATCTACGTCGGGGGCATGATGGAAGGGGAGGGCAAGGGCCCGCTCCTGCGCAGCCAGAGCACCTCTGAGCAGGAGAAGCGCCTTACCTGGCCCCGCAGGTCCTACTCCCCCCGGAGTTTTGAGGATTGCGGAGGCGGCTATACCCCGGACTGCAGCTCCAATGAGAACCTCACCTCCAGCGAGGAGGACTTCTCCTCTGGCCAGTCCAGCCGCGTGTCCCCAAGCCCCACCACCTACCGCATGTTCCGGGACAAAAGCCGCTCTCCCTCGCAGAACTCGCAACAGTCCTTCGACAGCAGCAGTCCCCCCACGCCGCAGTGCCATAAGCGGCACCGGCACTGCCCGGTTGTCGTGTCCGAGGCCACCATCGTGGGCGTCCGCAAGACCGGGCAGATCTGGCCCAACGATGGCGAGGGCGCCTTCCATGGAGACGCAGATGGCTCGTTCGGAACACCACCTGGATACGGCTGCGCTGCAGACCGGGCAGAGGAGCAGCGCCGGCACCAAGATGGGCTGCCCTACATTGATGACTCGCCCTCCTCATCGCCCCACCTCAGCAGCAAGGGCAGGGGCAGCCGGGATGCGCTGGTCTCGGGAGCCCTGGAGTCCACTAAAGCGAGTGAGCTGGACTTGGAAAAGGGCTTGGAGATGAGAAAATGGGTCCTGTCGGGAATCCTGGCTAGCGAGGAGACTTACCTGAGCCACCTGGAGGCACTGCTGCTGCCCATGAAGCCTTTGAAAGCCGCTGCCACCACCTCTCAGCCGGTGCTGACGAGTCAGCAGATCGAGACCATCTTCTTCAAAGTGCCTGAGCTCTACGAGATCCACAAGGAGTTCTATGATGGGCTCTTCCCCCGCGTGCAGCAGTGGAGCCACCAGCAGCGGGTGGGCGACCTCTTCCAGAAGCTGGCCAGCCAGCTGGGTGTGTACCGGGCCTTCGTGGACAACTACGGAGTTGCCATGGAAATGGCTGAGAAGTGCTGTCAGGCCAATGCTCAGTTTGCAGAAATCTCCGAGAACCTGAGAGCCAGAAGCAACAAAGATGCCAAGGATCCAACGACCAAGAACTCTCTGGAAACTCTGCTCTACAAGCCTGTGGACCGTGTGACGAGGAGCACGCTGGTCCTCCATGACTTGCTGAAGCACACTCCTGCCAGCCACCCTGACCACCCCTTGCTGCAGGACGCCCTCCGCATCTCACAGAACTTCCTGTCCAGCATCAATGAGGAGATCACACCCCGACGGCAGTCCATGACGGTGAAGAAGGGAGAGCACCGGCAGCTGCTGAAGGACAGCTTCATGGTGGAGCTGGTGGAGGGGGCCCGCAAGCTGCGCCACGTCTTCCTGTTCACCGACCTGCTTCTCTGCACCAAGCTCAAGAAGCAGAGCGGAGGCAAAACGCAGCAGTATGACTGCAAATGGTACATTCCGCTCACGGATCTCAGCTTCCAGATGGTGGATGAACTGGAGGCAGTGCCCAACATCCCCCTGGTGCCCGATGAGGAGCTGGACGCTTTGAAGATCAAGATCTCCCAGATCAAGAGTGACATCCAGAGAGAGAAGAGGGCGAACAAGGGCAGCAAGGCTACGGAGAGGCTGAAGAAGAAGCTGTCGGAGCAGGAGTCACTGCTGCTGCTTATGTCTCCCAGCATGGCCTTCAGGGTGCACAGCCGCAACGGCAAGAGTTACACGTTCCTGATCTCCTCTGACTATGAGCGTGCAGAGTGGAGGGAGAACATCCGGGAGCAGCAGAAGAAGTGTTTCAGAAGCTTCTCCCTGACATCCGTGGAGCTGCAGATGCTGACCAACTCGTGTGTGAAACTCCAGACTGTCCACAGCATTCCGCTGACCATCAATAAGGAAGATGATGAGTCTCCGGGGCTCTATGGGTTTCTGAATGTCATCGTCCACTCAGCCACTGGATTTAAGCAGAGTTCAAAAGCCCTTCAGCGGCCAGTAGCATCTGACTTTGAGCCTCAGGGTCTGAGTGAAGCCGCTCGTTGGAACTCCAAGGAAAACCTTCTCGCTGGACCCAGTGAAAATGACCCCAACCTTTTCGTTGCACTGTATGATTTTGTGGCCAGTGGAGATAACACTCTAAGCATAACTAAAGGTGAAAAGCTCCGGGTCTTAGGCTATAATCACAATGGGGAATGGTGTGAAGCCCAAACCAAAAATGGCCAAGGCTGGGTCCCAAGCAACTACATCACGCCAGTCAACAGTCTGGAGAAACACTCCTGGTACCATGGGCCTGTGTCCCGCAATGCCGCTGAGTATCTGCTGAGCAGCGGGATCAATGGCAGCTTCTTGGTGCGTGAGAGTGAGAGCAGTCCTGGCCAGAGGTCCATCTCGCTGAGATACGAAGGGAGGGTGTACCATTACAGGATCAACACTGCTTCTGATGGCAAGCTCTACGTCTCCTCCGAGAGCCGCTTCAACACCCTGGCCGAGTTGGTTCATCATCATTCAACGGTGGCCGACGGGCTCATCACCACGCTCCATTATCCAGCCCCAAAGCGCAACAAGCCCACTGTCTATGGTGTGTCCCCCAACTACGACAAGTGGGAGATGGAACGCACGGACATCACCATGAAGCACAAGCTGGGCGGGGGCCAGTACGGGGAGGTGTACGAGGGCGTGTGGAAGAAATACAGCCTGACGGTGGCCGTGAAGACCTTGAAGGAGGACACCATGGAGGTGGAAGAGTTCTTGAAAGAAGCTGCAGTCATGAAAGAGATCAAACACCCTAACCTGGTGCAGCTCCTTGGGGTCTGCACCCGGGAGCCCCCGTTCTATATCATCACTGAGTTCATGACCTACGGGAACCTCCTGGACTACCTGAGGGAGTGCAACCGGCAGGAGGTGAACGCCGTGGTGCTGCTGTACATGGCCACTCAGATCTCGTCAGCCATGGAGTACCTGGAGAAGAAAAACTTCATCCACAGAGATCTTGCTGCCCGAAACTGCCTGGTAGGGGAGAACCACTTGGTGAAGGTAGCTGATTTTGGCCTGAGCAGGTTGATGACAGGGGACACCTACACAGCCCATGCTGGAGCCAAGTTCCCCATCAAATGGACTGCACCCGAGAGCCTGGCCTACAACAAGTTCTCCATCAAGTCCGACGTCTGGGCATTTGGAGTATTGCTTTGGGAAATTGCTACCTATGGCATGTCCCCTTACCCGGGAATTGACCTGTCCCAGGTGTATGAGCTGCTAGAGAAGGACTACCGCATGGAGCGCCCAGAAGGCTGCCCAGAGAAGGTCTATGAACTCATGCGAGCATGTTGGCAGTGGAATCCCTCTGACCGGCCCTCCTCTGCTGAAATCCACCAAGCCTTTGAAACAATGTTCCAGGAATCCAGTATCTCAGACGAAGTGGAAAAGGAGCTGGGGAAACAAGGCGTCCGTGGGGCTGTGAGTACCTTGCTGCAGGCCCCAGAGCTGCCCACCAAGACGAGGACCTCCAGGAGAGCTGCAGAGCACAGAGACACCACTGACGTGCCTGAGATGCCTCACTCCAAGGGCCAGGGAGAGAGCGATCCTCTGGACCATGAGCCTGCCGTGTCTCCATTGCTCCCTCGAAAAGAGCGAGGTCCCCCGGAGGGCGGCCTGAATGAAGATGAGCGCCTTCTCCCCAAAGACAAAAAGACCAACTTGTTCAGCGCCTTGATCAAGAAGAAGAAGAAGACAGCCCCAACCCCTCCCAAACGCAGCAGCTCCTTCCGGGAGATGGACGGCCAGCCGGAGCGCAGAGGGGCCGGCGAGGAAGAGGGCCGAGACATCAGCAACGGGGCACTGGCTTTCACCCCCTTGGACACAGCTGACCCAGCCAAGTCCCCAAAGCCCAGCAATGGGGCTGGGGTCCCCAATGGAGCCCTCCGGGAGTCCGGGGGCTCAGGCTTCCGGTCTCCCCACCTGTGGAAGAAGTCCAGCACGCTGACCAGCAGCCGCCTAGCCACCGGCGAGGAGGAGGGCGGTGGCAGCTCCAGCAAGCGCTTCCTGCGCTCTTGCTCCGCCTCCTGCGTTCCCCATGGGGCCAAGGACACGGAGTGGAGGTCAGTCACGCTGCCTCGGGACTTGCAGTCCACGGGAAGACAGTTTGACTCGTCCACATTTGGAGGGCACAAAAGTGAGAAGCCGGCTCTGCCTCGGAAGAGGGCAGGGGAGAACAGGTCTGACCAGGTGACCCGAGGCACAGTAA
本发明另一方面提供了一种含有如上所述的携带BCR-ABL1基因的双位点突变的质粒。
本发明另一方面提供了一种构建如上所述的携带BCR-ABL1基因的双位点突变的质粒的方法，包括如下步骤：
S1、采用重叠延伸PCR方法进行扩增得到核苷酸序列SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5的片段，包括两个反应体系：PCR1和PCR2，其中：
PCR1以MIG-p210野生型质粒为模板，两端扩增引物为P210-RES-F/R，带有突变位点的引物为T315I-F/R、L387M-F/R和F486S-F/R中的任意一种，同一位点的正反向引物具有15～20bp互补序列，所述两端扩增引物与所述带有突变位点的引物在高保真DNA聚合酶为反应体系的条件进行PCR扩增，得到产物为5’端具有15～20bp反向互补序列的两个DNA片段，所述引物序列为：
Figure BDA0003665221280000081
PCR2以PCR1得到的两个所述DNA片段为模板，互补序列为引物，将两个所述DNA片段连接，得到核苷酸序列SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5的片段；
S2、将MIG-p210野生型质粒在Dam-JM110感受态中转化，接着在培养板上涂板，最后用质粒提取试剂盒提取质粒，得到Dam-MIG-p210野生型质粒；
S3、将步骤S1得到的核苷酸序列SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5的片段和步骤S2得到的Dam-MIG-p210野生型质粒经限制性内切酶SfiⅠ和BclⅠ分别双酶切后得到产物进行连接构建，并筛选出带有单突变位点的MIG-p210质粒；
S4、将步骤S3中得到的所述带有单突变位点的MIG-p210质粒为模板，重复步骤S1至S3的过程，筛选出携带BCR-ABL1基因的双位点突变的质粒。
在上述技术方案的基础上，本发明还可以做如下改进。
进一步，在步骤S3中，连接构建，并筛选出带有单突变位点的MIG-p210质粒具体步骤如下：
(1)、通过琼脂糖凝胶电泳技术对所述产物进行分离，理论上双酶切Dam-MIG-p210质粒反应体系产物可以分离得到9.8kb和3.7kb大小的两个条带，双酶切SEQ ID NO:3、SEQID NO:4或SEQ ID NO:5的DNA片段反应体系可以得到3.7kb大小的条带和0.5kb、70bp两小片段的条带，利用胶回收纯化试剂盒分别纯化回收两个体系中的长片段；
(2)、利用T4-DNA连接酶连接步骤(1)中回收的9.8kb和3.7kb两个长度的DNA片段；
(3)、取步骤(2)中的连接产物加入感受态细胞中孵育后加入培养基孵育，接着摇菌后将感受态悬液涂在含有氨苄西林LB板上过夜后挑单克隆菌落，摇菌，将菌液送测序，筛选后得到带有单突变位点的MIG-p210质粒。
进一步，在步骤(3)中，所述感受态细胞为T1感受态细胞。
进一步，在步骤(3)中，所述培养基为LB培养基。
本发明另一方面提供了一种构建携带BCR-ABL1基因激酶区双位点突变的细胞株的方法，包括如下步骤：
步骤1、以如上所述的携带BCR-ABL1基因的双位点突变的质粒为原料进行病毒包装，病毒包装前一天，接种293T细胞于6孔板中，使第二天下午细胞密度达到80～90％，转染前2小时给细胞换液，更换2mL新的含10％FBS的DMEM高糖培养基。采用磷酸钙法，配制培养体系，将所述携带BCR-ABL1基因的双位点突变的质粒缓慢加入到准备好的293T细胞培养皿中，12～16小时后换液，48小时后收上清得到MIG-p210逆转录病毒；
步骤2、将Ba/F3细胞以每孔10万个接种于6孔板，加入步骤1中的1mL MIG-p210逆转录病毒、1mL含2ng/mL浓度IL-3的1640完全培养基及2uL的浓度为8mg/mL的polybrene,摇匀放置于孵箱中培养48小时后更换为不含IL-3的含10％FBS的1640培养基进行筛选，经过检测验证后得到携带BCR-ABL1基因激酶区双位点突变的细胞株。
在上述技术方案的基础上，本发明还可以做如下改进。
进一步，在步骤1中，所述病毒包装体系如下：
Figure BDA0003665221280000101
进一步，在步骤3中，所述具体的检测验证方法如下：
当用Western blot技术检测到有210KD大小的BCR-ABL1融合蛋白的表达时，证明成功构建了包括BCR-ABL1基因激酶区双位点突变的稳转细胞株。
本发明另一方面提供了一种携带BCR-ABL1基因激酶区双位点突变的细胞株，采用如上所述的构建携带BCR-ABL1基因激酶区双位点突变的细胞株的方法得到。
本发明的有益效果是，现有的技术存在某些缺陷，当质粒中某段序列GC含量高时，要想完全把质粒扩增一圈难度是很高的，除此之外，PCR扩增模板过长，错配率也就会变高，会引入很多额外的突变。以构建BCR-ABL突变质粒为例，以MSCV-IRES-GFP-p210(MIG-p210)野生型质粒为模板，利用上述方法进行PCR扩增时，多次实验均未得到扩增产物，分析原因可能包含两方面：1.质粒部分区域GC含量过高，2.需要扩增的模板太长(13502碱基对)。本构建过程主要针对构建携带BCR-ABL1突变的细胞株，可以避免上述技术的缺陷，不需要扩增质粒全长，只需扩增涵盖突变位点在内的部分区域，可以避开高GC含量区域，同时避免了扩增序列太长导致的高错配率，最重要是现有技术在构建BCR-ABL1突变质粒中难以适用。本发明构建携带BCR-ABL1基因激酶区突变的细胞株为探索治疗携带BCR-ABL1复合突变的慢性髓细胞白血病的治疗方法提供了基础。
附图说明
图1为本发明MIG-p210质粒及引物设计图谱图；
图2为本发明带有单突变位点的MIG-p210质粒与原无突变序列对比结果图；
图3为本发明带有双突变位点的MIG-p210质粒与原无突变序列对比结果图；
图4为本发明双位点突变的(T315I+L387M、T315I+F486S)Ba/F3-MIG-p210稳转细胞株融合蛋白的表达图；
具体实施方式
以下结合附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
实施例1、
构建BCR-ABL1融合基因激酶区突变质粒
1.1、通过设计带有突变位点的引物和两端进行扩增的引物(T315I-F/R，L387M-F/R，F486S-F/R为携带有突变碱基的引物，同一位点的正反向引物具有15-20bp互补序列，P210-RES-F/R为两端扩增引物)，以MSCV-IRES-GFP-P210(MIG-p210)野生型质粒，P210-RES-F和任一突变位点反向引物(或P210-RES-R和任一突变位点正向引物)，高保真DNA聚合酶为反应体系，利用聚合酶链式反应(PCR)技术扩增模板，产物为5’端具有15-20bp反向互补序列的两个DNA片段。反应体系如表1所示，质粒构建使用的引物及序列如表2所示，MIG-p210质粒图谱及引物位置如图1所示。
表1MIG-p210质粒扩增体系
试剂名称 用量(uL)
ddH20 Up to 50
2x Max Buffer 25
dNTP Mix(10mM each) 1
MIG-p210质粒 Optional
P210-RES-F/P210-RES-R 2
突变位点反向引物/正向引物 2
Phanta Max Super-Fidelity DNA Polymerase 1
表2质粒构建使用的引物及序列
引物名称 序列(5'-3')
P210-Res-F TACGAGGACGCCGAGTTGA
P210-Res-R TTACTGTGCCTCGGGTCACC
T315I-F TCATCATTGAGTTCATGACCTACGGGAACC
T315I-R GGTCATGAACTCAATGATGATATAGAACGGGGGCTC
L387M-F CAGGATGATGACAGGGGACACCTACACA
L387M-R CCCTGTCATCATCCTGCTCAGGCCAAAATCAG
F486S-F TCCTCTGCTGAAATCCACCAAGCCTT
F486S-R TGGATTTCAGCAGAGGAGGGCCGGTCAGAG
如图1所示，T315I-F/R，L387M-F/R，F486S-F/R为携带有突变碱基的引物，其中同一位点的正反向引物5’端具有15-20bp反向互补序列，P210-RES-F/R为两端扩增引物，SfiⅠ和BclⅠ*为本所选的内切酶位点，BclⅠ*限制性内切酶可被Dam甲基化阻断。
1.2、以上面两条DNA片段(产物1和产物2)为模板，互补序列为引物，进行重叠PCR，从而将两个DNA片段连接，反应体系见表3，PCR反应条件如表4所示。
表3重叠PCR反应体系
试剂名称 用量(uL)
ddH20 Up to 50
2x Max Buffer 25
dNTP Mix(10mM each) 1
产物1 Optional
产物2 Optional
Phanta Max Super-Fidelity DNA Polymerase 1
注：产物1和产物2由1.1过程所获得，两者具有15-20bp长度的互补序列。
表4PCR反应条件
Figure BDA0003665221280000131
1.3、将MIG-p210野生型质粒在Dam-JM110感受态中转化，在含有氨苄西林的LB培养板上涂板，过夜后挑单克隆菌落，摇菌过夜，用质粒提取试剂盒提取质粒，得到Dam-MIG-p210野生型质粒。
1.4、通过SfiⅠ和BclⅠ限制性内切酶分别双酶切Dam-MIG-p210野生型质粒和SEQID NO:3、SEQ ID NO:4或SEQ ID NO:5扩增得到的DNA片段，反应体系见表5，在37℃条件下，反应2小时。
1.5、通过琼脂糖凝胶电泳技术对1.4中的产物进行分离，理论上双酶切Dam-MIG-p210质粒反应体系产物可以分离得到9.8kb和3.7kb大小的两个条带，双酶切SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5扩增得到的DNA片段反应体系可以得到3.7kb大小的条带和0.5kb、70bp两小片段的条带，利用胶回收纯化试剂盒分别纯化回收两个体系中的长片段。
表5限制性内切酶双酶切体系
Figure BDA0003665221280000141
1.6、利用T4-DNA连接酶连接1.5中回收的两个DNA片段，即9.8kb和3.7kb两个长度的DNA片段，反应体系如表6所示。
表6两DNA片段连接反应体系
试剂 用量
9.8kb DNA片段 10ng
3.7kb DNA片段 11.3ng
10x T4-DNA连接酶缓冲液 2uL
T4-DNA连接酶 1uL
ddH2O Up to 20uL
1.7、取1.6中的连接产物5uL加入到50uL的T1感受态细胞中，用小枪头轻轻混匀，冰上放30分钟后，42℃热激45S，冰水浴孵育2分钟，加入500uL LB培养基，37℃孵育10分钟，37℃、220rmp摇菌45分钟，然后将感受态悬液涂在含有氨苄西林(100ug/mL)LB板上过夜后挑单克隆菌落，摇菌，将菌液送测序，筛选带有单突变位点的MIG-p210质粒，测序结果如图2所示，MIG-p210-WT质粒引入突变后形成的带有单突变位点的MIG-p210质粒与原无突变序列对比结果。
1.8、以构建出的单位点突变MIG-p210质粒为模板，重复上述过程，则可筛选出双位点突变的质粒，测序结果如图3所示，MIG-p210-WT质粒引入突变后形成的带有双突变位点的MIG-p210质粒与原无突变序列对比结果。
构建BCR-ABL1基因激酶区突变的Ba/F3-MIG-p210稳定转染细胞株
2.1、病毒包装：病毒包装前一天，接种293T于6孔板中，使第二天下午细胞密度达到80～90％，转染前2小时给细胞换液，更换2mL新的含10％FBS的DMEM高糖培养基。采用磷酸钙法，配制表7中的体系，缓慢加入到准备好的293T细胞培养皿中，12～16小时后换液，48小时后收上清得到MIG-p210逆转录病毒，离心后用0.45um的滤网过滤后保存于-80℃冰箱备用。
表7病毒包装体系
试剂 用量
MIG-p210 3.2ug
pCL-Eco 1.6ug
CaCl<sub>2</sub>(2.5mol/L) 10uL
ddH<sub>2</sub>O Up to 100uL
2xHBSS 100uL
注：质粒用量+CaCl2+ddH2O＝100uL
2.2、将Ba/F3细胞以每孔10万个接种于6孔板，加入1mL MIG-p210逆转录病毒、1mL含IL-3(2ng/mL)的1640完全培养基及2uL的polybrene(8mg/mL),摇匀放置于孵箱中培养48小时后更换为不含IL-3的含10％FBS的1640培养基进行筛选。
2.3、2周后用Western blot技术检测到有210KD大小的BCR-ABL1融合蛋白的表达，构建包括双位点突变的(T315I+L387M、T315I+F486S)Ba/F3-MIG-p210稳转细胞株，检测结果如图4所示,用Western blot技术检测到转染细胞株均有210KD大小的BCR-ABL1融合蛋白的表达，图上方标记了细胞所携带BCR-ABL1的突变位点。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 野五
龚玉萍
<120> 一种携带BCR-ABL1基因激酶区双位点突变的细胞株及其构建方法
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4309
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tacgaggacg ccgagttgaa cccccgcttc ctgaaggaca acctgatcga cgccaatggc 60
ggtagcaggc ccccttggcc gcccctggag taccagccct accagagcat ctacgtcggg 120
ggcatgatgg aaggggaggg caagggcccg ctcctgcgca gccagagcac ctctgagcag 180
gagaagcgcc ttacctggcc ccgcaggtcc tactcccccc ggagttttga ggattgcgga 240
ggcggctata ccccggactg cagctccaat gagaacctca cctccagcga ggaggacttc 300
tcctctggcc agtccagccg cgtgtcccca agccccacca cctaccgcat gttccgggac 360
aaaagccgct ctccctcgca gaactcgcaa cagtccttcg acagcagcag tccccccacg 420
ccgcagtgcc ataagcggca ccggcactgc ccggttgtcg tgtccgaggc caccatcgtg 480
ggcgtccgca agaccgggca gatctggccc aacgatggcg agggcgcctt ccatggagac 540
gcagatggct cgttcggaac accacctgga tacggctgcg ctgcagaccg ggcagaggag 600
cagcgccggc accaagatgg gctgccctac attgatgact cgccctcctc atcgccccac 660
ctcagcagca agggcagggg cagccgggat gcgctggtct cgggagccct ggagtccact 720
aaagcgagtg agctggactt ggaaaagggc ttggagatga gaaaatgggt cctgtcggga 780
atcctggcta gcgaggagac ttacctgagc cacctggagg cactgctgct gcccatgaag 840
cctttgaaag ccgctgccac cacctctcag ccggtgctga cgagtcagca gatcgagacc 900
atcttcttca aagtgcctga gctctacgag atccacaagg agttctatga tgggctcttc 960
ccccgcgtgc agcagtggag ccaccagcag cgggtgggcg acctcttcca gaagctggcc 1020
agccagctgg gtgtgtaccg ggccttcgtg gacaactacg gagttgccat ggaaatggct 1080
gagaagtgct gtcaggccaa tgctcagttt gcagaaatct ccgagaacct gagagccaga 1140
agcaacaaag atgccaagga tccaacgacc aagaactctc tggaaactct gctctacaag 1200
cctgtggacc gtgtgacgag gagcacgctg gtcctccatg acttgctgaa gcacactcct 1260
gccagccacc ctgaccaccc cttgctgcag gacgccctcc gcatctcaca gaacttcctg 1320
tccagcatca atgaggagat cacaccccga cggcagtcca tgacggtgaa gaagggagag 1380
caccggcagc tgctgaagga cagcttcatg gtggagctgg tggagggggc ccgcaagctg 1440
cgccacgtct tcctgttcac cgacctgctt ctctgcacca agctcaagaa gcagagcgga 1500
ggcaaaacgc agcagtatga ctgcaaatgg tacattccgc tcacggatct cagcttccag 1560
atggtggatg aactggaggc agtgcccaac atccccctgg tgcccgatga ggagctggac 1620
gctttgaaga tcaagatctc ccagatcaag agtgacatcc agagagagaa gagggcgaac 1680
aagggcagca aggctacgga gaggctgaag aagaagctgt cggagcagga gtcactgctg 1740
ctgcttatgt ctcccagcat ggccttcagg gtgcacagcc gcaacggcaa gagttacacg 1800
ttcctgatct cctctgacta tgagcgtgca gagtggaggg agaacatccg ggagcagcag 1860
aagaagtgtt tcagaagctt ctccctgaca tccgtggagc tgcagatgct gaccaactcg 1920
tgtgtgaaac tccagactgt ccacagcatt ccgctgacca tcaataagga agatgatgag 1980
tctccggggc tctatgggtt tctgaatgtc atcgtccact cagccactgg atttaagcag 2040
agttcaaaag cccttcagcg gccagtagca tctgactttg agcctcaggg tctgagtgaa 2100
gccgctcgtt ggaactccaa ggaaaacctt ctcgctggac ccagtgaaaa tgaccccaac 2160
cttttcgttg cactgtatga ttttgtggcc agtggagata acactctaag cataactaaa 2220
ggtgaaaagc tccgggtctt aggctataat cacaatgggg aatggtgtga agcccaaacc 2280
aaaaatggcc aaggctgggt cccaagcaac tacatcacgc cagtcaacag tctggagaaa 2340
cactcctggt accatgggcc tgtgtcccgc aatgccgctg agtatctgct gagcagcggg 2400
atcaatggca gcttcttggt gcgtgagagt gagagcagtc ctggccagag gtccatctcg 2460
ctgagatacg aagggagggt gtaccattac aggatcaaca ctgcttctga tggcaagctc 2520
tacgtctcct ccgagagccg cttcaacacc ctggccgagt tggttcatca tcattcaacg 2580
gtggccgacg ggctcatcac cacgctccat tatccagccc caaagcgcaa caagcccact 2640
gtctatggtg tgtcccccaa ctacgacaag tgggagatgg aacgcacgga catcaccatg 2700
aagcacaagc tgggcggggg ccagtacggg gaggtgtacg agggcgtgtg gaagaaatac 2760
agcctgacgg tggccgtgaa gaccttgaag gaggacacca tggaggtgga agagttcttg 2820
aaagaagctg cagtcatgaa agagatcaaa caccctaacc tggtgcagct ccttggggtc 2880
tgcacccggg agcccccgtt ctatatcatc attgagttca tgacctacgg gaacctcctg 2940
gactacctga gggagtgcaa ccggcaggag gtgaacgccg tggtgctgct gtacatggcc 3000
actcagatct cgtcagccat ggagtacctg gagaagaaaa acttcatcca cagagatctt 3060
gctgcccgaa actgcctggt aggggagaac cacttggtga aggtagctga ttttggcctg 3120
agcaggatga tgacagggga cacctacaca gcccatgctg gagccaagtt ccccatcaaa 3180
tggactgcac ccgagagcct ggcctacaac aagttctcca tcaagtccga cgtctgggca 3240
tttggagtat tgctttggga aattgctacc tatggcatgt ccccttaccc gggaattgac 3300
ctgtcccagg tgtatgagct gctagagaag gactaccgca tggagcgccc agaaggctgc 3360
ccagagaagg tctatgaact catgcgagca tgttggcagt ggaatccctc tgaccggccc 3420
tcctttgctg aaatccacca agcctttgaa acaatgttcc aggaatccag tatctcagac 3480
gaagtggaaa aggagctggg gaaacaaggc gtccgtgggg ctgtgagtac cttgctgcag 3540
gccccagagc tgcccaccaa gacgaggacc tccaggagag ctgcagagca cagagacacc 3600
actgacgtgc ctgagatgcc tcactccaag ggccagggag agagcgatcc tctggaccat 3660
gagcctgccg tgtctccatt gctccctcga aaagagcgag gtcccccgga gggcggcctg 3720
aatgaagatg agcgccttct ccccaaagac aaaaagacca acttgttcag cgccttgatc 3780
aagaagaaga agaagacagc cccaacccct cccaaacgca gcagctcctt ccgggagatg 3840
gacggccagc cggagcgcag aggggccggc gaggaagagg gccgagacat cagcaacggg 3900
gcactggctt tcaccccctt ggacacagct gacccagcca agtccccaaa gcccagcaat 3960
ggggctgggg tccccaatgg agccctccgg gagtccgggg gctcaggctt ccggtctccc 4020
cacctgtgga agaagtccag cacgctgacc agcagccgcc tagccaccgg cgaggaggag 4080
ggcggtggca gctccagcaa gcgcttcctg cgctcttgct ccgcctcctg cgttccccat 4140
ggggccaagg acacggagtg gaggtcagtc acgctgcctc gggacttgca gtccacggga 4200
agacagtttg actcgtccac atttggaggg cacaaaagtg agaagccggc tctgcctcgg 4260
aagagggcag gggagaacag gtctgaccag gtgacccgag gcacagtaa 4309
<210> 2
<211> 4309
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tacgaggacg ccgagttgaa cccccgcttc ctgaaggaca acctgatcga cgccaatggc 60
ggtagcaggc ccccttggcc gcccctggag taccagccct accagagcat ctacgtcggg 120
ggcatgatgg aaggggaggg caagggcccg ctcctgcgca gccagagcac ctctgagcag 180
gagaagcgcc ttacctggcc ccgcaggtcc tactcccccc ggagttttga ggattgcgga 240
ggcggctata ccccggactg cagctccaat gagaacctca cctccagcga ggaggacttc 300
tcctctggcc agtccagccg cgtgtcccca agccccacca cctaccgcat gttccgggac 360
aaaagccgct ctccctcgca gaactcgcaa cagtccttcg acagcagcag tccccccacg 420
ccgcagtgcc ataagcggca ccggcactgc ccggttgtcg tgtccgaggc caccatcgtg 480
ggcgtccgca agaccgggca gatctggccc aacgatggcg agggcgcctt ccatggagac 540
gcagatggct cgttcggaac accacctgga tacggctgcg ctgcagaccg ggcagaggag 600
cagcgccggc accaagatgg gctgccctac attgatgact cgccctcctc atcgccccac 660
ctcagcagca agggcagggg cagccgggat gcgctggtct cgggagccct ggagtccact 720
aaagcgagtg agctggactt ggaaaagggc ttggagatga gaaaatgggt cctgtcggga 780
atcctggcta gcgaggagac ttacctgagc cacctggagg cactgctgct gcccatgaag 840
cctttgaaag ccgctgccac cacctctcag ccggtgctga cgagtcagca gatcgagacc 900
atcttcttca aagtgcctga gctctacgag atccacaagg agttctatga tgggctcttc 960
ccccgcgtgc agcagtggag ccaccagcag cgggtgggcg acctcttcca gaagctggcc 1020
agccagctgg gtgtgtaccg ggccttcgtg gacaactacg gagttgccat ggaaatggct 1080
gagaagtgct gtcaggccaa tgctcagttt gcagaaatct ccgagaacct gagagccaga 1140
agcaacaaag atgccaagga tccaacgacc aagaactctc tggaaactct gctctacaag 1200
cctgtggacc gtgtgacgag gagcacgctg gtcctccatg acttgctgaa gcacactcct 1260
gccagccacc ctgaccaccc cttgctgcag gacgccctcc gcatctcaca gaacttcctg 1320
tccagcatca atgaggagat cacaccccga cggcagtcca tgacggtgaa gaagggagag 1380
caccggcagc tgctgaagga cagcttcatg gtggagctgg tggagggggc ccgcaagctg 1440
cgccacgtct tcctgttcac cgacctgctt ctctgcacca agctcaagaa gcagagcgga 1500
ggcaaaacgc agcagtatga ctgcaaatgg tacattccgc tcacggatct cagcttccag 1560
atggtggatg aactggaggc agtgcccaac atccccctgg tgcccgatga ggagctggac 1620
gctttgaaga tcaagatctc ccagatcaag agtgacatcc agagagagaa gagggcgaac 1680
aagggcagca aggctacgga gaggctgaag aagaagctgt cggagcagga gtcactgctg 1740
ctgcttatgt ctcccagcat ggccttcagg gtgcacagcc gcaacggcaa gagttacacg 1800
ttcctgatct cctctgacta tgagcgtgca gagtggaggg agaacatccg ggagcagcag 1860
aagaagtgtt tcagaagctt ctccctgaca tccgtggagc tgcagatgct gaccaactcg 1920
tgtgtgaaac tccagactgt ccacagcatt ccgctgacca tcaataagga agatgatgag 1980
tctccggggc tctatgggtt tctgaatgtc atcgtccact cagccactgg atttaagcag 2040
agttcaaaag cccttcagcg gccagtagca tctgactttg agcctcaggg tctgagtgaa 2100
gccgctcgtt ggaactccaa ggaaaacctt ctcgctggac ccagtgaaaa tgaccccaac 2160
cttttcgttg cactgtatga ttttgtggcc agtggagata acactctaag cataactaaa 2220
ggtgaaaagc tccgggtctt aggctataat cacaatgggg aatggtgtga agcccaaacc 2280
aaaaatggcc aaggctgggt cccaagcaac tacatcacgc cagtcaacag tctggagaaa 2340
cactcctggt accatgggcc tgtgtcccgc aatgccgctg agtatctgct gagcagcggg 2400
atcaatggca gcttcttggt gcgtgagagt gagagcagtc ctggccagag gtccatctcg 2460
ctgagatacg aagggagggt gtaccattac aggatcaaca ctgcttctga tggcaagctc 2520
tacgtctcct ccgagagccg cttcaacacc ctggccgagt tggttcatca tcattcaacg 2580
gtggccgacg ggctcatcac cacgctccat tatccagccc caaagcgcaa caagcccact 2640
gtctatggtg tgtcccccaa ctacgacaag tgggagatgg aacgcacgga catcaccatg 2700
aagcacaagc tgggcggggg ccagtacggg gaggtgtacg agggcgtgtg gaagaaatac 2760
agcctgacgg tggccgtgaa gaccttgaag gaggacacca tggaggtgga agagttcttg 2820
aaagaagctg cagtcatgaa agagatcaaa caccctaacc tggtgcagct ccttggggtc 2880
tgcacccggg agcccccgtt ctatatcatc attgagttca tgacctacgg gaacctcctg 2940
gactacctga gggagtgcaa ccggcaggag gtgaacgccg tggtgctgct gtacatggcc 3000
actcagatct cgtcagccat ggagtacctg gagaagaaaa acttcatcca cagagatctt 3060
gctgcccgaa actgcctggt aggggagaac cacttggtga aggtagctga ttttggcctg 3120
agcaggttga tgacagggga cacctacaca gcccatgctg gagccaagtt ccccatcaaa 3180
tggactgcac ccgagagcct ggcctacaac aagttctcca tcaagtccga cgtctgggca 3240
tttggagtat tgctttggga aattgctacc tatggcatgt ccccttaccc gggaattgac 3300
ctgtcccagg tgtatgagct gctagagaag gactaccgca tggagcgccc agaaggctgc 3360
ccagagaagg tctatgaact catgcgagca tgttggcagt ggaatccctc tgaccggccc 3420
tcctctgctg aaatccacca agcctttgaa acaatgttcc aggaatccag tatctcagac 3480
gaagtggaaa aggagctggg gaaacaaggc gtccgtgggg ctgtgagtac cttgctgcag 3540
gccccagagc tgcccaccaa gacgaggacc tccaggagag ctgcagagca cagagacacc 3600
actgacgtgc ctgagatgcc tcactccaag ggccagggag agagcgatcc tctggaccat 3660
gagcctgccg tgtctccatt gctccctcga aaagagcgag gtcccccgga gggcggcctg 3720
aatgaagatg agcgccttct ccccaaagac aaaaagacca acttgttcag cgccttgatc 3780
aagaagaaga agaagacagc cccaacccct cccaaacgca gcagctcctt ccgggagatg 3840
gacggccagc cggagcgcag aggggccggc gaggaagagg gccgagacat cagcaacggg 3900
gcactggctt tcaccccctt ggacacagct gacccagcca agtccccaaa gcccagcaat 3960
ggggctgggg tccccaatgg agccctccgg gagtccgggg gctcaggctt ccggtctccc 4020
cacctgtgga agaagtccag cacgctgacc agcagccgcc tagccaccgg cgaggaggag 4080
ggcggtggca gctccagcaa gcgcttcctg cgctcttgct ccgcctcctg cgttccccat 4140
ggggccaagg acacggagtg gaggtcagtc acgctgcctc gggacttgca gtccacggga 4200
agacagtttg actcgtccac atttggaggg cacaaaagtg agaagccggc tctgcctcgg 4260
aagagggcag gggagaacag gtctgaccag gtgacccgag gcacagtaa 4309
<210> 3
<211> 4309
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tacgaggacg ccgagttgaa cccccgcttc ctgaaggaca acctgatcga cgccaatggc 60
ggtagcaggc ccccttggcc gcccctggag taccagccct accagagcat ctacgtcggg 120
ggcatgatgg aaggggaggg caagggcccg ctcctgcgca gccagagcac ctctgagcag 180
gagaagcgcc ttacctggcc ccgcaggtcc tactcccccc ggagttttga ggattgcgga 240
ggcggctata ccccggactg cagctccaat gagaacctca cctccagcga ggaggacttc 300
tcctctggcc agtccagccg cgtgtcccca agccccacca cctaccgcat gttccgggac 360
aaaagccgct ctccctcgca gaactcgcaa cagtccttcg acagcagcag tccccccacg 420
ccgcagtgcc ataagcggca ccggcactgc ccggttgtcg tgtccgaggc caccatcgtg 480
ggcgtccgca agaccgggca gatctggccc aacgatggcg agggcgcctt ccatggagac 540
gcagatggct cgttcggaac accacctgga tacggctgcg ctgcagaccg ggcagaggag 600
cagcgccggc accaagatgg gctgccctac attgatgact cgccctcctc atcgccccac 660
ctcagcagca agggcagggg cagccgggat gcgctggtct cgggagccct ggagtccact 720
aaagcgagtg agctggactt ggaaaagggc ttggagatga gaaaatgggt cctgtcggga 780
atcctggcta gcgaggagac ttacctgagc cacctggagg cactgctgct gcccatgaag 840
cctttgaaag ccgctgccac cacctctcag ccggtgctga cgagtcagca gatcgagacc 900
atcttcttca aagtgcctga gctctacgag atccacaagg agttctatga tgggctcttc 960
ccccgcgtgc agcagtggag ccaccagcag cgggtgggcg acctcttcca gaagctggcc 1020
agccagctgg gtgtgtaccg ggccttcgtg gacaactacg gagttgccat ggaaatggct 1080
gagaagtgct gtcaggccaa tgctcagttt gcagaaatct ccgagaacct gagagccaga 1140
agcaacaaag atgccaagga tccaacgacc aagaactctc tggaaactct gctctacaag 1200
cctgtggacc gtgtgacgag gagcacgctg gtcctccatg acttgctgaa gcacactcct 1260
gccagccacc ctgaccaccc cttgctgcag gacgccctcc gcatctcaca gaacttcctg 1320
tccagcatca atgaggagat cacaccccga cggcagtcca tgacggtgaa gaagggagag 1380
caccggcagc tgctgaagga cagcttcatg gtggagctgg tggagggggc ccgcaagctg 1440
cgccacgtct tcctgttcac cgacctgctt ctctgcacca agctcaagaa gcagagcgga 1500
ggcaaaacgc agcagtatga ctgcaaatgg tacattccgc tcacggatct cagcttccag 1560
atggtggatg aactggaggc agtgcccaac atccccctgg tgcccgatga ggagctggac 1620
gctttgaaga tcaagatctc ccagatcaag agtgacatcc agagagagaa gagggcgaac 1680
aagggcagca aggctacgga gaggctgaag aagaagctgt cggagcagga gtcactgctg 1740
ctgcttatgt ctcccagcat ggccttcagg gtgcacagcc gcaacggcaa gagttacacg 1800
ttcctgatct cctctgacta tgagcgtgca gagtggaggg agaacatccg ggagcagcag 1860
aagaagtgtt tcagaagctt ctccctgaca tccgtggagc tgcagatgct gaccaactcg 1920
tgtgtgaaac tccagactgt ccacagcatt ccgctgacca tcaataagga agatgatgag 1980
tctccggggc tctatgggtt tctgaatgtc atcgtccact cagccactgg atttaagcag 2040
agttcaaaag cccttcagcg gccagtagca tctgactttg agcctcaggg tctgagtgaa 2100
gccgctcgtt ggaactccaa ggaaaacctt ctcgctggac ccagtgaaaa tgaccccaac 2160
cttttcgttg cactgtatga ttttgtggcc agtggagata acactctaag cataactaaa 2220
ggtgaaaagc tccgggtctt aggctataat cacaatgggg aatggtgtga agcccaaacc 2280
aaaaatggcc aaggctgggt cccaagcaac tacatcacgc cagtcaacag tctggagaaa 2340
cactcctggt accatgggcc tgtgtcccgc aatgccgctg agtatctgct gagcagcggg 2400
atcaatggca gcttcttggt gcgtgagagt gagagcagtc ctggccagag gtccatctcg 2460
ctgagatacg aagggagggt gtaccattac aggatcaaca ctgcttctga tggcaagctc 2520
tacgtctcct ccgagagccg cttcaacacc ctggccgagt tggttcatca tcattcaacg 2580
gtggccgacg ggctcatcac cacgctccat tatccagccc caaagcgcaa caagcccact 2640
gtctatggtg tgtcccccaa ctacgacaag tgggagatgg aacgcacgga catcaccatg 2700
aagcacaagc tgggcggggg ccagtacggg gaggtgtacg agggcgtgtg gaagaaatac 2760
agcctgacgg tggccgtgaa gaccttgaag gaggacacca tggaggtgga agagttcttg 2820
aaagaagctg cagtcatgaa agagatcaaa caccctaacc tggtgcagct ccttggggtc 2880
tgcacccggg agcccccgtt ctatatcatc attgagttca tgacctacgg gaacctcctg 2940
gactacctga gggagtgcaa ccggcaggag gtgaacgccg tggtgctgct gtacatggcc 3000
actcagatct cgtcagccat ggagtacctg gagaagaaaa acttcatcca cagagatctt 3060
gctgcccgaa actgcctggt aggggagaac cacttggtga aggtagctga ttttggcctg 3120
agcaggttga tgacagggga cacctacaca gcccatgctg gagccaagtt ccccatcaaa 3180
tggactgcac ccgagagcct ggcctacaac aagttctcca tcaagtccga cgtctgggca 3240
tttggagtat tgctttggga aattgctacc tatggcatgt ccccttaccc gggaattgac 3300
ctgtcccagg tgtatgagct gctagagaag gactaccgca tggagcgccc agaaggctgc 3360
ccagagaagg tctatgaact catgcgagca tgttggcagt ggaatccctc tgaccggccc 3420
tcctttgctg aaatccacca agcctttgaa acaatgttcc aggaatccag tatctcagac 3480
gaagtggaaa aggagctggg gaaacaaggc gtccgtgggg ctgtgagtac cttgctgcag 3540
gccccagagc tgcccaccaa gacgaggacc tccaggagag ctgcagagca cagagacacc 3600
actgacgtgc ctgagatgcc tcactccaag ggccagggag agagcgatcc tctggaccat 3660
gagcctgccg tgtctccatt gctccctcga aaagagcgag gtcccccgga gggcggcctg 3720
aatgaagatg agcgccttct ccccaaagac aaaaagacca acttgttcag cgccttgatc 3780
aagaagaaga agaagacagc cccaacccct cccaaacgca gcagctcctt ccgggagatg 3840
gacggccagc cggagcgcag aggggccggc gaggaagagg gccgagacat cagcaacggg 3900
gcactggctt tcaccccctt ggacacagct gacccagcca agtccccaaa gcccagcaat 3960
ggggctgggg tccccaatgg agccctccgg gagtccgggg gctcaggctt ccggtctccc 4020
cacctgtgga agaagtccag cacgctgacc agcagccgcc tagccaccgg cgaggaggag 4080
ggcggtggca gctccagcaa gcgcttcctg cgctcttgct ccgcctcctg cgttccccat 4140
ggggccaagg acacggagtg gaggtcagtc acgctgcctc gggacttgca gtccacggga 4200
agacagtttg actcgtccac atttggaggg cacaaaagtg agaagccggc tctgcctcgg 4260
aagagggcag gggagaacag gtctgaccag gtgacccgag gcacagtaa 4309
<210> 4
<211> 4309
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tacgaggacg ccgagttgaa cccccgcttc ctgaaggaca acctgatcga cgccaatggc 60
ggtagcaggc ccccttggcc gcccctggag taccagccct accagagcat ctacgtcggg 120
ggcatgatgg aaggggaggg caagggcccg ctcctgcgca gccagagcac ctctgagcag 180
gagaagcgcc ttacctggcc ccgcaggtcc tactcccccc ggagttttga ggattgcgga 240
ggcggctata ccccggactg cagctccaat gagaacctca cctccagcga ggaggacttc 300
tcctctggcc agtccagccg cgtgtcccca agccccacca cctaccgcat gttccgggac 360
aaaagccgct ctccctcgca gaactcgcaa cagtccttcg acagcagcag tccccccacg 420
ccgcagtgcc ataagcggca ccggcactgc ccggttgtcg tgtccgaggc caccatcgtg 480
ggcgtccgca agaccgggca gatctggccc aacgatggcg agggcgcctt ccatggagac 540
gcagatggct cgttcggaac accacctgga tacggctgcg ctgcagaccg ggcagaggag 600
cagcgccggc accaagatgg gctgccctac attgatgact cgccctcctc atcgccccac 660
ctcagcagca agggcagggg cagccgggat gcgctggtct cgggagccct ggagtccact 720
aaagcgagtg agctggactt ggaaaagggc ttggagatga gaaaatgggt cctgtcggga 780
atcctggcta gcgaggagac ttacctgagc cacctggagg cactgctgct gcccatgaag 840
cctttgaaag ccgctgccac cacctctcag ccggtgctga cgagtcagca gatcgagacc 900
atcttcttca aagtgcctga gctctacgag atccacaagg agttctatga tgggctcttc 960
ccccgcgtgc agcagtggag ccaccagcag cgggtgggcg acctcttcca gaagctggcc 1020
agccagctgg gtgtgtaccg ggccttcgtg gacaactacg gagttgccat ggaaatggct 1080
gagaagtgct gtcaggccaa tgctcagttt gcagaaatct ccgagaacct gagagccaga 1140
agcaacaaag atgccaagga tccaacgacc aagaactctc tggaaactct gctctacaag 1200
cctgtggacc gtgtgacgag gagcacgctg gtcctccatg acttgctgaa gcacactcct 1260
gccagccacc ctgaccaccc cttgctgcag gacgccctcc gcatctcaca gaacttcctg 1320
tccagcatca atgaggagat cacaccccga cggcagtcca tgacggtgaa gaagggagag 1380
caccggcagc tgctgaagga cagcttcatg gtggagctgg tggagggggc ccgcaagctg 1440
cgccacgtct tcctgttcac cgacctgctt ctctgcacca agctcaagaa gcagagcgga 1500
ggcaaaacgc agcagtatga ctgcaaatgg tacattccgc tcacggatct cagcttccag 1560
atggtggatg aactggaggc agtgcccaac atccccctgg tgcccgatga ggagctggac 1620
gctttgaaga tcaagatctc ccagatcaag agtgacatcc agagagagaa gagggcgaac 1680
aagggcagca aggctacgga gaggctgaag aagaagctgt cggagcagga gtcactgctg 1740
ctgcttatgt ctcccagcat ggccttcagg gtgcacagcc gcaacggcaa gagttacacg 1800
ttcctgatct cctctgacta tgagcgtgca gagtggaggg agaacatccg ggagcagcag 1860
aagaagtgtt tcagaagctt ctccctgaca tccgtggagc tgcagatgct gaccaactcg 1920
tgtgtgaaac tccagactgt ccacagcatt ccgctgacca tcaataagga agatgatgag 1980
tctccggggc tctatgggtt tctgaatgtc atcgtccact cagccactgg atttaagcag 2040
agttcaaaag cccttcagcg gccagtagca tctgactttg agcctcaggg tctgagtgaa 2100
gccgctcgtt ggaactccaa ggaaaacctt ctcgctggac ccagtgaaaa tgaccccaac 2160
cttttcgttg cactgtatga ttttgtggcc agtggagata acactctaag cataactaaa 2220
ggtgaaaagc tccgggtctt aggctataat cacaatgggg aatggtgtga agcccaaacc 2280
aaaaatggcc aaggctgggt cccaagcaac tacatcacgc cagtcaacag tctggagaaa 2340
cactcctggt accatgggcc tgtgtcccgc aatgccgctg agtatctgct gagcagcggg 2400
atcaatggca gcttcttggt gcgtgagagt gagagcagtc ctggccagag gtccatctcg 2460
ctgagatacg aagggagggt gtaccattac aggatcaaca ctgcttctga tggcaagctc 2520
tacgtctcct ccgagagccg cttcaacacc ctggccgagt tggttcatca tcattcaacg 2580
gtggccgacg ggctcatcac cacgctccat tatccagccc caaagcgcaa caagcccact 2640
gtctatggtg tgtcccccaa ctacgacaag tgggagatgg aacgcacgga catcaccatg 2700
aagcacaagc tgggcggggg ccagtacggg gaggtgtacg agggcgtgtg gaagaaatac 2760
agcctgacgg tggccgtgaa gaccttgaag gaggacacca tggaggtgga agagttcttg 2820
aaagaagctg cagtcatgaa agagatcaaa caccctaacc tggtgcagct ccttggggtc 2880
tgcacccggg agcccccgtt ctatatcatc actgagttca tgacctacgg gaacctcctg 2940
gactacctga gggagtgcaa ccggcaggag gtgaacgccg tggtgctgct gtacatggcc 3000
actcagatct cgtcagccat ggagtacctg gagaagaaaa acttcatcca cagagatctt 3060
gctgcccgaa actgcctggt aggggagaac cacttggtga aggtagctga ttttggcctg 3120
agcaggatga tgacagggga cacctacaca gcccatgctg gagccaagtt ccccatcaaa 3180
tggactgcac ccgagagcct ggcctacaac aagttctcca tcaagtccga cgtctgggca 3240
tttggagtat tgctttggga aattgctacc tatggcatgt ccccttaccc gggaattgac 3300
ctgtcccagg tgtatgagct gctagagaag gactaccgca tggagcgccc agaaggctgc 3360
ccagagaagg tctatgaact catgcgagca tgttggcagt ggaatccctc tgaccggccc 3420
tcctttgctg aaatccacca agcctttgaa acaatgttcc aggaatccag tatctcagac 3480
gaagtggaaa aggagctggg gaaacaaggc gtccgtgggg ctgtgagtac cttgctgcag 3540
gccccagagc tgcccaccaa gacgaggacc tccaggagag ctgcagagca cagagacacc 3600
actgacgtgc ctgagatgcc tcactccaag ggccagggag agagcgatcc tctggaccat 3660
gagcctgccg tgtctccatt gctccctcga aaagagcgag gtcccccgga gggcggcctg 3720
aatgaagatg agcgccttct ccccaaagac aaaaagacca acttgttcag cgccttgatc 3780
aagaagaaga agaagacagc cccaacccct cccaaacgca gcagctcctt ccgggagatg 3840
gacggccagc cggagcgcag aggggccggc gaggaagagg gccgagacat cagcaacggg 3900
gcactggctt tcaccccctt ggacacagct gacccagcca agtccccaaa gcccagcaat 3960
ggggctgggg tccccaatgg agccctccgg gagtccgggg gctcaggctt ccggtctccc 4020
cacctgtgga agaagtccag cacgctgacc agcagccgcc tagccaccgg cgaggaggag 4080
ggcggtggca gctccagcaa gcgcttcctg cgctcttgct ccgcctcctg cgttccccat 4140
ggggccaagg acacggagtg gaggtcagtc acgctgcctc gggacttgca gtccacggga 4200
agacagtttg actcgtccac atttggaggg cacaaaagtg agaagccggc tctgcctcgg 4260
aagagggcag gggagaacag gtctgaccag gtgacccgag gcacagtaa 4309
<210> 5
<211> 4309
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tacgaggacg ccgagttgaa cccccgcttc ctgaaggaca acctgatcga cgccaatggc 60
ggtagcaggc ccccttggcc gcccctggag taccagccct accagagcat ctacgtcggg 120
ggcatgatgg aaggggaggg caagggcccg ctcctgcgca gccagagcac ctctgagcag 180
gagaagcgcc ttacctggcc ccgcaggtcc tactcccccc ggagttttga ggattgcgga 240
ggcggctata ccccggactg cagctccaat gagaacctca cctccagcga ggaggacttc 300
tcctctggcc agtccagccg cgtgtcccca agccccacca cctaccgcat gttccgggac 360
aaaagccgct ctccctcgca gaactcgcaa cagtccttcg acagcagcag tccccccacg 420
ccgcagtgcc ataagcggca ccggcactgc ccggttgtcg tgtccgaggc caccatcgtg 480
ggcgtccgca agaccgggca gatctggccc aacgatggcg agggcgcctt ccatggagac 540
gcagatggct cgttcggaac accacctgga tacggctgcg ctgcagaccg ggcagaggag 600
cagcgccggc accaagatgg gctgccctac attgatgact cgccctcctc atcgccccac 660
ctcagcagca agggcagggg cagccgggat gcgctggtct cgggagccct ggagtccact 720
aaagcgagtg agctggactt ggaaaagggc ttggagatga gaaaatgggt cctgtcggga 780
atcctggcta gcgaggagac ttacctgagc cacctggagg cactgctgct gcccatgaag 840
cctttgaaag ccgctgccac cacctctcag ccggtgctga cgagtcagca gatcgagacc 900
atcttcttca aagtgcctga gctctacgag atccacaagg agttctatga tgggctcttc 960
ccccgcgtgc agcagtggag ccaccagcag cgggtgggcg acctcttcca gaagctggcc 1020
agccagctgg gtgtgtaccg ggccttcgtg gacaactacg gagttgccat ggaaatggct 1080
gagaagtgct gtcaggccaa tgctcagttt gcagaaatct ccgagaacct gagagccaga 1140
agcaacaaag atgccaagga tccaacgacc aagaactctc tggaaactct gctctacaag 1200
cctgtggacc gtgtgacgag gagcacgctg gtcctccatg acttgctgaa gcacactcct 1260
gccagccacc ctgaccaccc cttgctgcag gacgccctcc gcatctcaca gaacttcctg 1320
tccagcatca atgaggagat cacaccccga cggcagtcca tgacggtgaa gaagggagag 1380
caccggcagc tgctgaagga cagcttcatg gtggagctgg tggagggggc ccgcaagctg 1440
cgccacgtct tcctgttcac cgacctgctt ctctgcacca agctcaagaa gcagagcgga 1500
ggcaaaacgc agcagtatga ctgcaaatgg tacattccgc tcacggatct cagcttccag 1560
atggtggatg aactggaggc agtgcccaac atccccctgg tgcccgatga ggagctggac 1620
gctttgaaga tcaagatctc ccagatcaag agtgacatcc agagagagaa gagggcgaac 1680
aagggcagca aggctacgga gaggctgaag aagaagctgt cggagcagga gtcactgctg 1740
ctgcttatgt ctcccagcat ggccttcagg gtgcacagcc gcaacggcaa gagttacacg 1800
ttcctgatct cctctgacta tgagcgtgca gagtggaggg agaacatccg ggagcagcag 1860
aagaagtgtt tcagaagctt ctccctgaca tccgtggagc tgcagatgct gaccaactcg 1920
tgtgtgaaac tccagactgt ccacagcatt ccgctgacca tcaataagga agatgatgag 1980
tctccggggc tctatgggtt tctgaatgtc atcgtccact cagccactgg atttaagcag 2040
agttcaaaag cccttcagcg gccagtagca tctgactttg agcctcaggg tctgagtgaa 2100
gccgctcgtt ggaactccaa ggaaaacctt ctcgctggac ccagtgaaaa tgaccccaac 2160
cttttcgttg cactgtatga ttttgtggcc agtggagata acactctaag cataactaaa 2220
ggtgaaaagc tccgggtctt aggctataat cacaatgggg aatggtgtga agcccaaacc 2280
aaaaatggcc aaggctgggt cccaagcaac tacatcacgc cagtcaacag tctggagaaa 2340
cactcctggt accatgggcc tgtgtcccgc aatgccgctg agtatctgct gagcagcggg 2400
atcaatggca gcttcttggt gcgtgagagt gagagcagtc ctggccagag gtccatctcg 2460
ctgagatacg aagggagggt gtaccattac aggatcaaca ctgcttctga tggcaagctc 2520
tacgtctcct ccgagagccg cttcaacacc ctggccgagt tggttcatca tcattcaacg 2580
gtggccgacg ggctcatcac cacgctccat tatccagccc caaagcgcaa caagcccact 2640
gtctatggtg tgtcccccaa ctacgacaag tgggagatgg aacgcacgga catcaccatg 2700
aagcacaagc tgggcggggg ccagtacggg gaggtgtacg agggcgtgtg gaagaaatac 2760
agcctgacgg tggccgtgaa gaccttgaag gaggacacca tggaggtgga agagttcttg 2820
aaagaagctg cagtcatgaa agagatcaaa caccctaacc tggtgcagct ccttggggtc 2880
tgcacccggg agcccccgtt ctatatcatc actgagttca tgacctacgg gaacctcctg 2940
gactacctga gggagtgcaa ccggcaggag gtgaacgccg tggtgctgct gtacatggcc 3000
actcagatct cgtcagccat ggagtacctg gagaagaaaa acttcatcca cagagatctt 3060
gctgcccgaa actgcctggt aggggagaac cacttggtga aggtagctga ttttggcctg 3120
agcaggttga tgacagggga cacctacaca gcccatgctg gagccaagtt ccccatcaaa 3180
tggactgcac ccgagagcct ggcctacaac aagttctcca tcaagtccga cgtctgggca 3240
tttggagtat tgctttggga aattgctacc tatggcatgt ccccttaccc gggaattgac 3300
ctgtcccagg tgtatgagct gctagagaag gactaccgca tggagcgccc agaaggctgc 3360
ccagagaagg tctatgaact catgcgagca tgttggcagt ggaatccctc tgaccggccc 3420
tcctctgctg aaatccacca agcctttgaa acaatgttcc aggaatccag tatctcagac 3480
gaagtggaaa aggagctggg gaaacaaggc gtccgtgggg ctgtgagtac cttgctgcag 3540
gccccagagc tgcccaccaa gacgaggacc tccaggagag ctgcagagca cagagacacc 3600
actgacgtgc ctgagatgcc tcactccaag ggccagggag agagcgatcc tctggaccat 3660
gagcctgccg tgtctccatt gctccctcga aaagagcgag gtcccccgga gggcggcctg 3720
aatgaagatg agcgccttct ccccaaagac aaaaagacca acttgttcag cgccttgatc 3780
aagaagaaga agaagacagc cccaacccct cccaaacgca gcagctcctt ccgggagatg 3840
gacggccagc cggagcgcag aggggccggc gaggaagagg gccgagacat cagcaacggg 3900
gcactggctt tcaccccctt ggacacagct gacccagcca agtccccaaa gcccagcaat 3960
ggggctgggg tccccaatgg agccctccgg gagtccgggg gctcaggctt ccggtctccc 4020
cacctgtgga agaagtccag cacgctgacc agcagccgcc tagccaccgg cgaggaggag 4080
ggcggtggca gctccagcaa gcgcttcctg cgctcttgct ccgcctcctg cgttccccat 4140
ggggccaagg acacggagtg gaggtcagtc acgctgcctc gggacttgca gtccacggga 4200
agacagtttg actcgtccac atttggaggg cacaaaagtg agaagccggc tctgcctcgg 4260
aagagggcag gggagaacag gtctgaccag gtgacccgag gcacagtaa 4309
<210> 6
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tacgaggacg ccgagttga 19
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttactgtgcc tcgggtcacc 20
<210> 8
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tcatcattga gttcatgacc tacgggaacc 30
<210> 9
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggtcatgaac tcaatgatga tatagaacgg gggctc 36
<210> 10
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caggatgatg acaggggaca cctacaca 28
<210> 11
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ccctgtcatc atcctgctca ggccaaaatc ag 32
<210> 12
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tcctctgctg aaatccacca agcctt 26
<210> 13
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tggatttcag cagaggaggg ccggtcagag 30

Claims (10)

1.一种BCR-ABL1基因的单位点或双位点突变，其特征在于，所述BCR-ABL1基因的双位点突变的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2所示；
所述BCR-ABL1基因的单位点突变的核苷酸序列如SEQ ID NO:3、SEQ ID NO:4或SEQ IDNO:5所示。
2.一种含有权利要求1所述的携带BCR-ABL1基因的双位点突变的质粒。
3.一种构建如权利要求1或2所述的携带BCR-ABL1基因的双位点突变的质粒的方法，其特征在于，包括如下步骤：
S1、采用重叠延伸PCR方法进行扩增得到核苷酸序列SEQ ID NO:3、SEQ ID NO:4或SEQID NO:5的片段，包括两个反应体系：PCR1和PCR2，其中：
所述PCR1以MIG-p210野生型质粒为模板，两端扩增引物为P210-RES-F/R，带有突变位点的引物为T315I-F/R、L387M-F/R和F486S-F/R中的任意一种，同一位点的正反向引物具有15～20bp互补序列，所述两端扩增引物与所述带有突变位点的引物在高保真DNA聚合酶为反应体系的条件进行PCR扩增，得到产物为5’端具有15～20bp反向互补序列的两个DNA片段，所述引物序列为：
Figure FDA0003665221270000011
Figure FDA0003665221270000021
所述PCR2以PCR1得到的两个所述DNA片段为模板，互补序列为引物，将两个所述DNA片段连接，得到核苷酸序列SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5的片段；
S2、将MIG-p210野生型质粒在Dam-JM110感受态中转化，接着在培养板上涂板，最后用质粒提取试剂盒提取质粒，得到Dam-MIG-p210野生型质粒；
S3、将步骤S1得到的核苷酸序列SEQ ID NO:3、SEQ ID NO:4或SEQ ID NO:5的片段和步骤S2得到的Dam-MIG-p210野生型质粒经限制性内切酶SfiⅠ和BclⅠ分别双酶切后得到产物进行连接构建，并筛选出带有单突变位点的MIG-p210质粒；
S4、将步骤S3中得到的所述带有单突变位点的MIG-p210质粒为模板，重复步骤S1至S3的过程，筛选出携带BCR-ABL1基因的双位点突变的质粒。
4.根据权利要求3所述的构建携带BCR-ABL1基因的双位点突变的质粒的方法，其特征在于，在步骤S3中，连接构建，并筛选出带有单突变位点的MIG-p210质粒具体步骤如下：
(1)、通过琼脂糖凝胶电泳技术对所述产物进行分离，理论上双酶切Dam-MIG-p210质粒反应体系产物可以分离得到9.8kb和3.7kb大小的两个条带，双酶切SEQ ID NO:3、SEQ IDNO:4或SEQ ID NO:5的DNA片段反应体系可以得到3.7kb大小的条带和0.5kb、70bp两小片段的条带，利用胶回收纯化试剂盒分别纯化回收两个体系中的长片段；
(2)、利用T4-DNA连接酶连接步骤(1)中回收的9.8kb和3.7kb两个长度的DNA片段；
(3)、取步骤(2)中的连接产物加入感受态细胞中孵育后加入培养基孵育，接着摇菌后将感受态悬液涂在含有氨苄西林LB板上过夜后挑单克隆菌落，摇菌，将菌液送测序，筛选后得到带有单突变位点的MIG-p210质粒。
5.根据权利要求4所述的构建携带BCR-ABL1基因的双位点突变的质粒的方法，其特征在于，在步骤(3)中，所述感受态细胞为T1感受态细胞。
6.根据权利要求4所述的构建携带BCR-ABL1基因的双位点突变的质粒的方法，其特征在于，在步骤(3)中，所述培养基为LB培养基。
7.一种构建携带BCR-ABL1基因激酶区双位点突变的细胞株的方法，其特征在于，包括如下步骤：
步骤1、以权利要求3至6任一项所述的携带BCR-ABL1基因的双位点突变的质粒为原料进行病毒包装，病毒包装前一天，接种293T细胞于6孔板中，使第二天下午细胞密度达到80～90％，转染前2小时给细胞换液，更换2mL新的含10％FBS的DMEM高糖培养基。采用磷酸钙法，配制病毒包装体系，将所述携带BCR-ABL1基因的双位点突变的质粒缓慢加入到准备好的293T细胞培养皿中，12～16小时后换液，48小时后收上清得到MIG-p210逆转录病毒；
步骤2、将Ba/F3细胞以每孔10万个接种于6孔板，加入步骤1中的1mL MIG-p210逆转录病毒、1mL含2ng/mL浓度IL-3的1640完全培养基及2uL的浓度为8mg/mL的polybrene,摇匀放置于孵箱中培养48小时后更换为不含IL-3的含10％FBS的1640培养基进行筛选，经过检测验证后得到携带BCR-ABL1基因激酶区双位点突变的细胞株。
8.根据权利要求7所述的构建携带BCR-ABL1基因激酶区双位点突变的细胞株的方法，其特征在于，在步骤1中，所述病毒包装体系如下：
Figure FDA0003665221270000041
9.根据权利要求7所述的构建携带BCR-ABL1基因激酶区双位点突变的细胞株的方法，其特征在于，在步骤3中，所述具体的检测验证方法如下：
当用Western blot技术检测到有210KD大小的BCR-ABL1融合蛋白的表达时，证明成功构建了包括BCR-ABL1基因激酶区双位点突变的稳转细胞株。
10.一种携带BCR-ABL1基因激酶区双位点突变的细胞株，其特征在于，采用如权利要求7至9任一项所述的构建携带BCR-ABL1基因激酶区双位点突变的细胞株的方法得到。
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