CN108690840A - Apoe基因敲除的动物模型的构建方法及其短肽 - Google Patents
Apoe基因敲除的动物模型的构建方法及其短肽 Download PDFInfo
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- CN108690840A CN108690840A CN201710225752.1A CN201710225752A CN108690840A CN 108690840 A CN108690840 A CN 108690840A CN 201710225752 A CN201710225752 A CN 201710225752A CN 108690840 A CN108690840 A CN 108690840A
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- dna vector
- apoe
- small peptide
- transcriptional activation
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Classifications
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Abstract
本发明提供一对短肽、一对转录激活子样效应因子、一对DNA载体，本发明所述的短肽能够对大鼠Apoe基因进行准确、高效的打靶，快速获得Apoe基因敲除大鼠。本发明还提供了一种敲除Apoe基因的方法以及一种Apoe基因敲除的动物模型的构建方法，本发明的敲除Apoe基因的方法敲除基因结果更为可靠，并且可实现在大鼠成年初期即表现出高血脂特征。
Description
技术领域
本发明属于生物技术领域，具体涉及一对短肽、一对转录激活子样效应因子及敲除Apoe基因的方法、构建Apoe基因敲除的动物模型的的方法。
背景技术
ApoE蛋白是一种载脂蛋白，存在于极低密度脂蛋白、乳糜微粒、中间密度脂蛋白和高密度脂蛋白中，主要在肝脏和脑组织中合成，参与脂蛋白的转化与代谢过程，是脂类代谢中的重要配体。已有研究表明ApoE基因缺陷或基因变异与心血管疾病、阿尔兹海默症、年龄相关性黄斑变性等疾病有关，是高胆固醇血症和动脉粥样硬化的主要基因。
全世界范围内针对人类不同疾病进行治病机理研究和药物开发从未停止，但由于受到人类自身伦理和技术实现的限制，很多研究不能在人类自体上直接进行，而需要在与人类相近的动物体上进行实验。因此，为进一步研究ApoE基因，进而为人类疾病治疗提供理论依据，早在1992年，Andrew和Zhang等人分别使用胚胎干细胞基因敲除技术成功培育了Apoe-/-小鼠(Plump,A.S.et al.Cell 71:343-353,1992，Zhang,S.H.et al.Science 258:468-471,1992.)，这些小鼠即使在低脂肪、低胆固醇饮食中也表现出严重的高胆固醇血症；通过饲喂含胆固醇的高脂饮食，Apoe-/-小鼠可出现明显的自发性动脉粥样硬化(atherosclerosis，AS)斑块。此后，人们还制备了多种Apoe基因或多基因敲除及修饰小鼠，并在Apoe基因功能研究和作为疾病模型得到广泛应用。
但是，上述模型多采用有肥胖倾向的C57BL/6小鼠为背景进行改造，研究表明，针对C57BL/6小鼠使用高脂饲料4周即可形成肥胖模型(刘芳等，中国药理学通报，2013Mar 29(3)：360-5)，因此，C57BL/6小鼠本身具有一定偏好性，制备C57BL/6小鼠的敲除Apoe基因模型的方法很难应用于其它品系动物模型的制备。
大鼠作为啮齿类模式动物，已经成为人疾病相关基础研究和药物开发不可或缺的动物模型。近年来通过基因工程技术改造大鼠特定基因从而得到特定基因型和表型的模式大鼠已经成为研究热点。与小鼠模型相比，大鼠在解剖、生理方面与人相似，生长代谢快，更为重要的是，大鼠脑体积和体型更大，而动脉粥样硬化等疾病研究需涉及解剖心脑血管操作，大鼠更容易进行手术操作，尤其是在血管腔内的操作；大鼠可采血量大，也可以动态监测大鼠动脉粥样硬化过程中血液学指标的变化过程。虽然，一直以来大鼠都是药理学研究的首选动物，并且被广泛应用于心血管疾病、神经性疾病的研究领域，但是由于胚胎干细胞提取和培养方法在大鼠上还未成熟，利用传统胚胎干细胞打靶方法制备大鼠基因敲除模型比较困难。
针对上述问题，中国期刊《实验动物与比较医学》(Aug.2015，35(4):277-282)中公开的《应用Talen法制备Apoe基因敲除大鼠模型》提出了解决方案，通过Talen法得到了敲除Apoe基因的大鼠模型。但是，上述方法仍存在以下问题：(1)仅在第二外显子缺失了1个碱基，虽然也造成了移码突变，且经检测无Apoe蛋白表达，但仍存在由排在缺失碱基之后的起始密码子恢复Apoe蛋白表达的可能(因密码子排列顺序不唯一的特性，编码序列中任何一个编码甲硫氨酸的密码子都可能成为起始密码子，启动后续DNA的转录和蛋白翻译)；(2)采用上述方法构建的大鼠模型仅在大月龄的大鼠(6-7个月龄)中表现出血脂中胆固醇水平高的特征，并不能确定是否与Apoe基因敲除或基因序列缺失程度有关，而SD大鼠在2个月就达到性成熟，该模型也未能在SD大鼠发育成熟早期表现出高血脂特征，导致相关研究的不完善。需要建立更好的模型进行进一步验证Apoe基因敲除与高血脂的相关性。
发明内容
本发明所要解决的第一个技术问题是现有技术中对于Apoe基因敲除大鼠模型的制备中，仍存在恢复Apoe蛋白表达的可能、且无法在大鼠成年初期使其表现出高血脂特征的问题，进而提供一种敲除基因结果更为可靠、并且可在大鼠成年初期即使其表现出高血脂特征的Apoe基因敲除的大鼠模型的构建方法。
本发明所要解决的第二个技术问题是提供一对短肽、一对可编码所述短肽的转录激活子样效应因子、一种包含所述转录激活子样效应因子的DNA载体。
本发明所要解决的第三个技术问题是提供一种敲除Apoe基因的方法。
为此，本发明提供的一对短肽，所述短肽包括第一短肽与第二短肽；所述第一短肽具有如SEQ ID NO：1所示的序列结构；所述第二短肽具有如SEQ ID NO：2所示的序列结构。
本发明提供的一对转录激活子样效应因子，所述转录激活子样效应因子包括第一转录激活子样效应因子与第二转录激活子样效应因子；所述第一转录激活子样效应因子与所述第二转录激活子样效应因子分别转录翻译得到所述的第一短肽与所述第二短肽。
优选地，所述第一转录激活子样效应因子具有如SEQ ID NO：3所示的序列结构；所述第二转录激活子样效应因子具有如SEQ ID NO：4所示的序列结构。
本发明提供的包含所述转录激活子样效应因子的DNA载体；所述DNA载体为可编码FokI核酸的DNA载体。
优选地，所述DNA载体包括第一DNA载体与第二DNA载体。所述第一DNA载体具有如SEQ ID NO：5所示的序列结构；所述第二DNA载体具有如SEQ ID NO：6所示的序列结构。
本发明还提供了一种敲除Apoe基因的方法，采用所述的短肽对Apoe基因进行打靶。
本发明提供的Apoe基因敲除的动物模型的构建方法，包括如下步骤：取原核期受精卵,将所述DNA载体的体外转录产物导入所述原核期受精卵的细胞质或细胞核中，再将其移植至受体的输卵管，经生育、繁殖，即得。
优选地，所述DNA载体通过如下方法制备得到：首先，确定敲除Apoe基因的靶位点，构建所述转录激活子样效应因子，并将其插入载体，即得。
进一步优选地，所述DNA载体的体外转录产物通过如下方法制备得到：先使用体外转录试剂盒将所述DNA载体转录成mRNA，再使用加尾试剂盒在所述mRNA的3’末端加尾,即得所述DNA载体的体外转录产物。
进一步优选地，所述的Apoe基因敲除的动物模型的构建方法，所述原核期受精卵为啮齿类动物的原核期受精卵；所述啮齿类动物为SD大鼠、Wistar大鼠、LEA品系大鼠、Fischer品系大鼠、F344大鼠、F6大鼠和黑刺鼠中的一种。
本发明的上述技术方案，相比现有技术具有以下优点：
(1)本发明提供了一对短肽，利用这对短肽构建获得的一对转录激活子样效应因子(TALE)能够特异性地识别大鼠Apoe基因上的两段相邻核苷酸序列；利用这对转录激活子样效应因子构建获得的一对转录激活子样效应因子核酸酶(TALENs)，能够对大鼠Apoe基因进行准确、高效的打靶，快速获得Apoe基因敲除大鼠；
(2)采用本发明的方法，可以准确、高效、快速的对啮齿动物的Apoe基因组进行编辑和遗传改造，进而制备啮齿动物突变体；
(3)采用本发明的方法，对大鼠的Apoe基因进行敲除，可实现在第3外显子敲除13个碱基，大大提高了敲除基因结果的可靠性；另外，经验证表明，可实现在大鼠月龄达到2个月后的大鼠成年初期，即表现出高血脂特征，使大鼠可更早地应用于相关研究中。
附图说明
为了使本发明的内容更容易被清楚的理解，下面结合附图，对本发明作进一步详细的说明，其中，
图1：实施例3中所述第一DNA载体的构建示意图；
图2：实施例3中所述第二DNA载体的构建示意图；
图3：实施例5中Apoe基因敲除大鼠打靶后目的基因序列测序分析结果；
图4：实施例5中Apoe基因敲除大鼠与野生型SD大鼠的Western Bot分析结果图；
图5：Apoe基因敲除大鼠与野生型SD大鼠基础血脂水平测试结果。
具体实施方式
本发明下述各实施例中，使用的设备和材料是从以下所指出的几家公司获得：
SD大鼠购自北京维通利华实验动物技术有限公司；
Ambion体外转录试剂盒购自Thermo Fisher，货号：AM1344；
Ambion加尾试剂盒购自Thermo Fisher，货号：AM1350；
血脂CHO测定试剂盒购自POINTE Scientific，货号：C7009-25；
需要说明的是，以上仅给出其中一种型号的产品予以阐述本发明的效果，市售各型号的产品之间效果并无差异。
实施例1Apoe基因特异TALEN序列的设计和识别模块的构建
本实施例中的一对短肽，所述短肽包括第一短肽与第二短肽；所述第一短肽具有如SEQ ID NO：1所示的序列结构，所述SEQ ID NO：1的序列结构具体为：
LTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGRPALE；
所述第二短肽具有如SEQ ID NO：2所示的序列结构，所述SEQ ID NO：2的序列结构具体为：
LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGRPALE。
其设计方法如下：
以SD大鼠为例，首先，根据Apoe基因的序列特征，选择第3外显子上的序列作为TALEN作用的靶位点，所述第3外显子具有如SEQ ID NO：7所示的核苷酸序列，所述SEQ IDNO：7的序列结构具体为：
5’-GATGCCTGGCCGAGGGAGAGCTGGAGGTGACAGATCAGCTCCCAGGGCAAAGCGACCAACCCTGGGAGCAGGCCCTGAACCGCTTCTGGGATTACCTGCGCTGGGTGCAGACGCTTTCTGACCAGGTCCAGGAAGAGCTGCAGAGCTCCCAAGTCACACAGGAACTGAC-3’
将所述SEQ ID NO：7的序列结构中T(Nn)A的核苷酸序列作为TALEN作用靶序列，其中N为A，G，T和C中的任一碱基，n为13至21之间的任一数字；因此，得到所述靶序列具有如SEQ ID NO：8所示的序列结构，所述SEQ ID NO：8的序列结构具体为：
所述靶序列包括5’端的L序列、间隔序列以及3’端的R序列。其中，所述L序列为所述靶序列的2-17位核苷酸，即在所述靶序列中标记为Ⅰ的序列；所述间隔序列为所述靶序列的18-34位核苷酸，即在所述靶序列中标记为Ⅱ的序列；所述R序列为所述靶序列的35-50位核苷酸，即在所述靶序列中标记为Ⅲ的序列。
为识别所述靶序列，构建所述第一短肽以识别所述L序列；构建所述第二短肽以识别所述R序列。
实施例2 Apoe基因敲除转录激活子样效应因子
为编码实施例1中所述的第一短肽、第二短肽，本实施例构建一对转录激活子样效应因子，所述转录激活子样效应因子包括第一转录激活子样效应因子与第二转录激活子样效应因子；所述第一转录激活子样效应因子与所述第二转录激活子样效应因子分别转录翻译得到实施例1中所述的第一短肽与所述第二短肽。
所述第一转录激活子样效应因子具有如SEQ ID NO：3所示的序列结构，所述SEQID NO：3的序列结构具体为：
CTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTCACGCCTGAGCAGGTAGTGGCTATTGCATCCAACAACGGGGGCAGACCCGCACTGGAG；
所述第二转录激活子样效应因子具有如SEQ ID NO：4所示的序列结构，所述SEQID NO：4的序列结构具体为：
CTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTCACGCCTGAGCAGGTAGTGGCTATTGCATCCAACATCGGGGGCAGACCCGCACTGGAG。
实施例3包含Apoe基因敲除转录激活子样效应因子的DNA载体
本实施例的DNA载体包含实施例2中所述的转录激活子样效应因子；作为本实施例的具体实现方式，所述DNA载体为可编码FokI核酸的DNA载体。
具体而言，所述DNA载体为DNA质粒，所述DNA载体包括第一DNA载体与第二DNA载体。
如图1所示为所述第一DNA载体构建示意图，所述第一DNA载体可识别实施例1中所述的L序列，具有如SEQ ID NO：5所示的序列结构，所述SEQ ID NO：5的序列结构具体为：
AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGGCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGTAGATTTGAGAACTTTGGGATATTCACAGCAGCAGCAGGAAAAGATCAAGCCCAAAGTGAGGTCGACAGTCGCGCAGCATCACGAAGCGCTGGTGGGTCATGGGTTTACACATGCCCACATCGTAGCCTTGTCGCAGCACCCTGCAGCCCTTGGCACGGTCGCCGTCAAGTACCAGGACATGATTGCGGCGTTGCCGGAAGCCACACATGAGGCGATCGTCGGTGTGGGGAAACAGTGGAGCGGAGCCCGAGCGCTTGAGGCCCTGTTGACGGTCGCGGGAGAGCTGAGAGGGCCTCCCCTTCAGCTGGACACGGGCCAGTTGCTGAAGATCGCGAAGCGGGGAGGAGTCACGGCGGTCGAGGCGGTGCACGCGTGGCGCAATGCGCTCACGGGAGCACCCCTCAACCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTCACGCCTGAGCAGGTAGTGGCTATTGCATCCAACAACGGGGGCAGACCCGCACTGGAGTCAATCGTGGCCCAGCTTTCGAGGCCGGACCCCGCGCTGGCCGCACTCACTAATGATCATCTTGTAGCGCTGGCCTGCCTCGGCGGACGACCCGCCTTGGATGCGGTGAAGAAGGGGCTCCCGCACGCGCCTGCATTGATTAAGCGGACCAACAGAAGGATTCCCGAGAGGACATCACATCGAGTGGCAGGTTCCCAACTCGTGAAGAGTGAACTTGAGGAGAAAAAGTCGGAGCTGCGGCACAAATTGAAATACGTACCGCATGAATACATCGAACTTATCGAAATTGCTAGGAACTCGACTCAAGACAGAATCCTTGAGATGAAGGTAATGGAGTTCTTTATGAAGGTTTATGGATACCGAGGGAAGCATCTCGGTGGATCACGAAAACCCGACGGAGCAATCTATACGGTGGGGAGCCCGATTGATTACGGAGTGATCGTCGACACGAAAGCCTACAGCGGTGGGTACAATCTTCCCATCGGGCAGGCAGATGAGATGgAgCGTTATGTCGAAGAAAATCAGACCAGGgACAAACACcTCAATCCAAATGAGTGGTGGAAAGTGTATCCTTCATCAGTGACCGAGTTTAAGTTTTTGTTTGTCTCTGGGCATTTCAAAGGCAACTATAAGGCCCAGCTCACACGGTTGAATCACATTACGAACTGCAATGGTGCGGTTTTGTCCGTAGAGGAACTGCTCATTGGTGGAGAAATGATCAAAGCGGGAACTCTGACACTGGAAGAAGTCAGACGCAAGTTTAACAATGGCGAGATCAATTTCCGCTCATAAAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGCGCTGCGATGATACCGCGAGAACCACGCTCACCGGCTCCGGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATCGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATATTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTCAGTGTTACAACCAATTAACCAATTCTGAACATTATCGCGAGCCCATTTATACCTGAATATGGCTCATAACACCCCTTGCTCATGACCAAAATCCCTTAACGTGAGTTACGCGCGCGTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGCCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGGCGAGAGTAGGGAACTGCCAGGCATCAAACTAAGCAGAAGGCCCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTCTGTGTTGTAAAACGACGGCCAGTCTTAAGCTCGGGCCCCCTGGGCGGTTCTGATAACGAGTAATCGTTAATCCGCAAATAACGTAAAAACCCGCTTCGGCGGGTTTTTTTATGGGGGGAGTTTAGGGAAAGAGCATTTGTCAGAATATTTAAGGGCGCCTGTCACTTTGCTTGATATATGAGAATTATTTAACCTTATAAATGAGAAAAAAGCAACGCACTTTAAATAAGATACGTTGCTTTTTCGATTGATGAACACCTATAATTAAACTATTCATCTATTATTTATGATTTTTTGTATATACAATATTTCTAGTTTGTTAAAGAGAATTAAGAAAATAAATCTCGAAAATAATAAAGGGAAAATCAGTTTTTGATATCAAAATTATACATGTCAACGATAATACAAAATATAATACAAACTATAAGATGTTATCAGTATTTATTATCATTTAGAATAAATTTTGTGTCGCCCTTAATTGTGAGCGGATAACAATTACGAGCTTCATGCACAGTGGCGTTGACATTGATTATTGACTAGTCCAAACAATTCTGCAGGAATCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGATTTGATACCGCGGGCCCTAG；
如图2所示为所述第二DNA载体构建示意图，所述第二DNA载体可识别实施例1中所述的R序列，具有如SEQ ID NO：6所示的序列结构，所述SEQ ID NO：6的序列结构具体为：
AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGGCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGTAGATTTGAGAACTTTGGGATATTCACAGCAGCAGCAGGAAAAGATCAAGCCCAAAGTGAGGTCGACAGTCGCGCAGCATCACGAAGCGCTGGTGGGTCATGGGTTTACACATGCCCACATCGTAGCCTTGTCGCAGCACCCTGCAGCCCTTGGCACGGTCGCCGTCAAGTACCAGGACATGATTGCGGCGTTGCCGGAAGCCACACATGAGGCGATCGTCGGTGTGGGGAAACAGTGGAGCGGAGCCCGAGCGCTTGAGGCCCTGTTGACGGTCGCGGGAGAGCTGAGAGGGCCTCCCCTTCAGCTGGACACGGGCCAGTTGCTGAAGATCGCGAAGCGGGGAGGAGTCACGGCGGTCGAGGCGGTGCACGCGTGGCGCAATGCGCTCACGGGAGCACCCCTCAACCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTCACGCCTGAGCAGGTAGTGGCTATTGCATCCAACATCGGGGGCAGACCCGCACTGGAGTCAATCGTGGCCCAGCTTTCGAGGCCGGACCCCGCGCTGGCCGCACTCACTAATGATCATCTTGTAGCGCTGGCCTGCCTCGGCGGACGACCCGCCTTGGATGCGGTGAAGAAGGGGCTCCCGCACGCGCCTGCATTGATTAAGCGGACCAACAGAAGGATTCCCGAGAGGACATCACATCGAGTGGCAGGTTCCCAACTCGTGAAGAGTGAACTTGAGGAGAAAAAGTCGGAGCTGCGGCACAAATTGAAATACGTACCGCATGAATACATCGAACTTATCGAAATTGCTAGGAACTCGACTCAAGACAGAATCCTTGAGATGAAGGTAATGGAGTTCTTTATGAAGGTTTATGGATACCGAGGGAAGCATCTCGGTGGATCACGAAAACCCGACGGAGCAATCTATACGGTGGGGAGCCCGATTGATTACGGAGTGATCGTCGACACGAAAGCCTACAGCGGTGGGTACAATCTTCCCATCGGGCAGGCAGATGAGATGCAACGTTATGTCAAAGAAAATCAGACCAGGAACAAACACATCAATCCAAATGAGTGGTGGAAAGTGTATCCTTCATCAGTGACCGAGTTTAAGTTTTTGTTTGTCTCTGGGCATTTCAAAGGCAACTATAAGGCCCAGCTCACACGGTTGAATCGCAAGACGAACTGCAATGGTGCGGTTTTGTCCGTAGAGGAACTGCTCATTGGTGGAGAAATGATCAAAGCGGGAACTCTGACACTGGAAGAAGTCAGACGCAAGTTTAACAATGGCGAGATCAATTTCCGCTCATAAAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGCGCTGCGATGATACCGCGAGAACCACGCTCACCGGCTCCGGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATCGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATATTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTCAGTGTTACAACCAATTAACCAATTCTGAACATTATCGCGAGCCCATTTATACCTGAATATGGCTCATAACACCCCTTGCTCATGACCAAAATCCCTTAACGTGAGTTACGCGCGCGTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGCCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGGCGAGAGTAGGGAACTGCCAGGCATCAAACTAAGCAGAAGGCCCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTCTGTGTTGTAAAACGACGGCCAGTCTTAAGCTCGGGCCCCCTGGGCGGTTCTGATAACGAGTAATCGTTAATCCGCAAATAACGTAAAAACCCGCTTCGGCGGGTTTTTTTATGGGGGGAGTTTAGGGAAAGAGCATTTGTCAGAATATTTAAGGGCGCCTGTCACTTTGCTTGATATATGAGAATTATTTAACCTTATAAATGAGAAAAAAGCAACGCACTTTAAATAAGATACGTTGCTTTTTCGATTGATGAACACCTATAATTAAACTATTCATCTATTATTTATGATTTTTTGTATATACAATATTTCTAGTTTGTTAAAGAGAATTAAGAAAATAAATCTCGAAAATAATAAAGGGAAAATCAGTTTTTGATATCAAAATTATACATGTCAACGATAATACAAAATATAATACAAACTATAAGATGTTATCAGTATTTATTATCATTTAGAATAAATTTTGTGTCGCCCTTAATTGTGAGCGGATAACAATTACGAGCTTCATGCACAGTGGCGTTGACATTGATTATTGACTAGTCCAAACAATTCTGCAGGAATCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGATTTGATACCGCGGGCCCTAG。
需要说明的是，本领域技术人员可根据实际情况更换载体类型，只要实现得到的DNA载体包含实施例2中所述的转录激活子样效应因子即可，其制备方法可采用现有技术中将外源DNA插入DNA载体中的一般操作即可。
实施例4 Apoe基因的敲除
本实施例的Apoe基因敲除的方法，采用实施例1中所述的短肽对Apoe基因进行打靶。
作为上述方法的具体应用，本实施例提供Apoe基因敲除的动物模型的构建方法，包括如下步骤：取原核期受精卵,将实施例3中所述DNA载体的体外转录产物导入所述原核期受精卵的细胞质或细胞核中，再将其移植至受体的输卵管，即可。
作为本实施例的优选实施方式，所述原核期受精卵为SD大鼠。即Apoe基因敲除的SD大鼠模型的构建方法。具体包括如下步骤：
取SD大鼠的原核期受精卵，利用显微注射仪将实施例3中制备得到的所述DNA载体的体外转录产物注射至SD大鼠的原核期受精卵的细胞质或细胞核中(本领域技术人员可参照《小鼠胚胎操作实验手册(第三版)》中的方法进行受精卵的显微注射)；然后，将注射后的所述受精卵转移至培养液中培养，然后移植至受体母鼠的输卵管中，所述受体母鼠生产后即得Apoe基因敲除的SD大鼠。
其中，所述DNA载体的体外转录产物通过如下方法制备得到：先使用体外转录试剂盒将所述DNA载体转录成mRNA，再使用加尾试剂盒在所述mRNA的3’末端加尾,即得所述DNA载体的体外转录产物。
需要说明的是，所述培养液的组分并不唯一，本领域技术人员可根据实际情况选择、配制相应的培养液，只要能够实现注射后的受精卵的短暂培养即可，并不影响本发明技术效果的实现。例如，可采用KSOM培养液。
实施例5 Apoe基因敲除SD大鼠的鉴定
本实施例对本发明的技术方案进行效果验证，为此，本实施例对实施例4中构建得到的SD大鼠模型进行以下实验：
实验一、基因型鉴定
利用DNA测序技术检验实施例4中构建得到的SD大鼠体细胞中的Apoe基因靶序列是否被成功敲除。
其测序分析结果如图3所示，由测序结果可知，被测SD大鼠在Apoe基因靶位点发生了突变，突变包括13个碱基的缺失。由于DNA序列的碱基缺失造成编码区的移码突变，进而导致被测SD大鼠体内的Apoe基因功能的丧失。
实施例4中构建得到的Apoe基因被敲除的SD大鼠的靶位点序列，具有如SEQ IDNO：9所示的序列结构；野生型SD大鼠靶位点序列，具有如SEQ ID NO：8所示的序列结构，两者的对比见表1。
表1 Apoe基因敲除大鼠与野生型大鼠靶序列对比
由上述对比可知，实施例4中构建得到的Apoe基因被敲除的SD大鼠的靶位点序列，相较于野生型SD大鼠靶位点序列，缺失13个碱基，缺失的碱基为上表中标记为Ⅳ的序列。
实验二、表达情况分析
取实施例4中构建得到的SD大鼠，即F0代成功敲除13bp的阳性Apoe基因的SD大鼠，将其与野生型SD大鼠进行交配，由此获得F1代SD大鼠，通过实验一中的方法进行基因型鉴定，选取Apoe基因敲除阳性杂合鼠，再进行雌雄互相交配，由此获得F2代SD大鼠，然后通过实验一中的方法基因型鉴定，筛选Apoe基因敲除阳性纯合大鼠(Apoe-/-)用于后续实验。
取1只所述Apoe基因敲除阳性纯合大鼠(Apoe-/-)，另取1只野生型SD大鼠作为对照，将2只大鼠处死后取脾和肝组织，提取组织总蛋白，选用β-actin为内参，应用WesternBlot方法分别检测组织中Apoe蛋白的表达水平。
其检测分析结果如图4所示，由检测结果可知，所述野生型SD大鼠中可见Apoe的表达，所述Apoe基因敲除阳性纯合大鼠(Apoe-/-)的脾脏和肝中未见Apoe蛋白表达。由此表明，本发明的方法制备的模型大鼠实现了Apoe基因的敲除。
实验三、表达情况分析
取实施例4中构建得到的Apoe基因敲除的SD大鼠(以下简称Apoe-/-大鼠)，并取野生型SD大鼠作为对照，在两组SD大鼠2个月龄时进行心脏采血，收集Apoe-/-大鼠及野生型SD大鼠的血液，将两组血液在25℃下，以3000r/min的转速离心20min，取上清液(即为血清样品)，采用血脂CHO测定试剂盒进行血脂检测。
其检测结果如图5所示，由检测结果可知，Apoe-/-大鼠的基础血脂水平明显高于野生型大鼠。由此表明，本发明的方法制备的模型大鼠在成年初期即表现出高血脂特征。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
SEQUENCE LISTING
<110> 北京百奥赛图基因生物技术有限公司
<120> Apoe基因敲除的动物模型的构建方法及其短肽
<130> PB710029N
<160> 9
<170> PatentIn version 3.3
<210> 1
<211> 530
<212> PRT
<213> 人工合成
<400> 1
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
1 5 10 15
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly
35 40 45
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
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Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
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Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
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Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
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Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
115 120 125
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
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Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
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Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
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Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
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Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
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Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala
245 250 255
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
340 345 350
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
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Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
385 390 395 400
Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
405 410 415
Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
420 425 430
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
435 440 445
Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
450 455 460
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
465 470 475 480
Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu
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Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
500 505 510
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Arg Pro Ala
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Leu Glu
530
<210> 2
<211> 530
<212> PRT
<213> 人工合成
<400> 2
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys
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Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
20 25 30
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly
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Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn
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Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
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Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
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Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
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Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
130 135 140
Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
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Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
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Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val
180 185 190
Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
195 200 205
Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu
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Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr
225 230 235 240
Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala
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Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
260 265 270
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys
275 280 285
Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala
290 295 300
His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly
305 310 315 320
Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys
325 330 335
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
340 345 350
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
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Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
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Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu
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Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala
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Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
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Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
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Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val
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Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu
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Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu
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<210> 3
<211> 1590
<212> DNA
<213> 人工合成
<400> 3
ctgacccctg agcaggtggt ggccatcgcc agcaacggcg gcggcaagca ggccctggag 60
accgtgcaga ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg 120
gtggccatcg ccagccacga cggcggcaag caggccctgg agaccgtgca gaggctgctg 180
cctgtgctgt gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagcaac 240
ggcggcggca agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc 300
cacggcctga cccctgagca ggtggtggcc atcgccagca acaacggcgg caagcaggcc 360
ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg cccacggcct gacccctgag 420
caggtggtgg ccatcgccag caacaacggc ggcaagcagg ccctggagac cgtgcagagg 480
ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg agcaggtggt ggccatcgcc 540
agcaacaacg gcggcaagca ggccctggag accgtgcaga ggctgctgcc tgtgctgtgc 600
caggcccacg gcctgacccc tgagcaggtg gtggccatcg ccagcaacat cggcggcaag 660
caggccctgg agaccgtgca gaggctgctg cctgtgctgt gccaggccca cggcctgacc 720
cctgagcagg tggtggccat cgccagcaac ggcggcggca agcaggccct ggagaccgtg 780
cagaggctgc tgcctgtgct gtgccaggcc cacggcctga cccctgagca ggtggtggcc 840
atcgccagca acggcggcgg caagcaggcc ctggagaccg tgcagaggct gctgcctgtg 900
ctgtgccagg cccacggcct gacccctgag caggtggtgg ccatcgccag caacatcggc 960
ggcaagcagg ccctggagac cgtgcagagg ctgctgcctg tgctgtgcca ggcccacggc 1020
ctgacccctg agcaggtggt ggccatcgcc agccacgacg gcggcaagca ggccctggag 1080
accgtgcaga ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg 1140
gtggccatcg ccagccacga cggcggcaag caggccctgg agaccgtgca gaggctgctg 1200
cctgtgctgt gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagcaac 1260
ggcggcggca agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc 1320
cacggcctga cccctgagca ggtggtggcc atcgccagca acaacggcgg caagcaggcc 1380
ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg cccacggcct gacccctgag 1440
caggtggtgg ccatcgccag ccacgacggc ggcaagcagg ccctggagac cgtgcagagg 1500
ctgctgcctg tgctgtgcca ggcccacggc ctcacgcctg agcaggtagt ggctattgca 1560
tccaacaacg ggggcagacc cgcactggag 1590
<210> 4
<211> 1590
<212> DNA
<213> 人工合成
<400> 4
ctgacccctg agcaggtggt ggccatcgcc agccacgacg gcggcaagca ggccctggag 60
accgtgcaga ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg 120
gtggccatcg ccagcaacgg cggcggcaag caggccctgg agaccgtgca gaggctgctg 180
cctgtgctgt gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagcaac 240
aacggcggca agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc 300
cacggcctga cccctgagca ggtggtggcc atcgccagca acatcggcgg caagcaggcc 360
ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg cccacggcct gacccctgag 420
caggtggtgg ccatcgccag ccacgacggc ggcaagcagg ccctggagac cgtgcagagg 480
ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg agcaggtggt ggccatcgcc 540
agccacgacg gcggcaagca ggccctggag accgtgcaga ggctgctgcc tgtgctgtgc 600
caggcccacg gcctgacccc tgagcaggtg gtggccatcg ccagcaacat cggcggcaag 660
caggccctgg agaccgtgca gaggctgctg cctgtgctgt gccaggccca cggcctgacc 720
cctgagcagg tggtggccat cgccagcaac aacggcggca agcaggccct ggagaccgtg 780
cagaggctgc tgcctgtgct gtgccaggcc cacggcctga cccctgagca ggtggtggcc 840
atcgccagca acaacggcgg caagcaggcc ctggagaccg tgcagaggct gctgcctgtg 900
ctgtgccagg cccacggcct gacccctgag caggtggtgg ccatcgccag caacggcggc 960
ggcaagcagg ccctggagac cgtgcagagg ctgctgcctg tgctgtgcca ggcccacggc 1020
ctgacccctg agcaggtggt ggccatcgcc agccacgacg gcggcaagca ggccctggag 1080
accgtgcaga ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg 1140
gtggccatcg ccagccacga cggcggcaag caggccctgg agaccgtgca gaggctgctg 1200
cctgtgctgt gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagcaac 1260
atcggcggca agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc 1320
cacggcctga cccctgagca ggtggtggcc atcgccagca acaacggcgg caagcaggcc 1380
ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg cccacggcct gacccctgag 1440
caggtggtgg ccatcgccag caacaacggc ggcaagcagg ccctggagac cgtgcagagg 1500
ctgctgcctg tgctgtgcca ggcccacggc ctcacgcctg agcaggtagt ggctattgca 1560
tccaacatcg ggggcagacc cgcactggag 1590
<210> 5
<211> 9214
<212> DNA
<213> 人工合成
<400> 5
agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca 60
ctataggggc caccatggac tataaggacc acgacggaga ctacaaggat catgatattg 120
attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg 180
gagtcccagc agccgtagat ttgagaactt tgggatattc acagcagcag caggaaaaga 240
tcaagcccaa agtgaggtcg acagtcgcgc agcatcacga agcgctggtg ggtcatgggt 300
ttacacatgc ccacatcgta gccttgtcgc agcaccctgc agcccttggc acggtcgccg 360
tcaagtacca ggacatgatt gcggcgttgc cggaagccac acatgaggcg atcgtcggtg 420
tggggaaaca gtggagcgga gcccgagcgc ttgaggccct gttgacggtc gcgggagagc 480
tgagagggcc tccccttcag ctggacacgg gccagttgct gaagatcgcg aagcggggag 540
gagtcacggc ggtcgaggcg gtgcacgcgt ggcgcaatgc gctcacggga gcacccctca 600
acctgacccc tgagcaggtg gtggccatcg ccagcaacgg cggcggcaag caggccctgg 660
agaccgtgca gaggctgctg cctgtgctgt gccaggccca cggcctgacc cctgagcagg 720
tggtggccat cgccagccac gacggcggca agcaggccct ggagaccgtg cagaggctgc 780
tgcctgtgct gtgccaggcc cacggcctga cccctgagca ggtggtggcc atcgccagca 840
acggcggcgg caagcaggcc ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg 900
cccacggcct gacccctgag caggtggtgg ccatcgccag caacaacggc ggcaagcagg 960
ccctggagac cgtgcagagg ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg 1020
agcaggtggt ggccatcgcc agcaacaacg gcggcaagca ggccctggag accgtgcaga 1080
ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg gtggccatcg 1140
ccagcaacaa cggcggcaag caggccctgg agaccgtgca gaggctgctg cctgtgctgt 1200
gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagcaac atcggcggca 1260
agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc cacggcctga 1320
cccctgagca ggtggtggcc atcgccagca acggcggcgg caagcaggcc ctggagaccg 1380
tgcagaggct gctgcctgtg ctgtgccagg cccacggcct gacccctgag caggtggtgg 1440
ccatcgccag caacggcggc ggcaagcagg ccctggagac cgtgcagagg ctgctgcctg 1500
tgctgtgcca ggcccacggc ctgacccctg agcaggtggt ggccatcgcc agcaacatcg 1560
gcggcaagca ggccctggag accgtgcaga ggctgctgcc tgtgctgtgc caggcccacg 1620
gcctgacccc tgagcaggtg gtggccatcg ccagccacga cggcggcaag caggccctgg 1680
agaccgtgca gaggctgctg cctgtgctgt gccaggccca cggcctgacc cctgagcagg 1740
tggtggccat cgccagccac gacggcggca agcaggccct ggagaccgtg cagaggctgc 1800
tgcctgtgct gtgccaggcc cacggcctga cccctgagca ggtggtggcc atcgccagca 1860
acggcggcgg caagcaggcc ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg 1920
cccacggcct gacccctgag caggtggtgg ccatcgccag caacaacggc ggcaagcagg 1980
ccctggagac cgtgcagagg ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg 2040
agcaggtggt ggccatcgcc agccacgacg gcggcaagca ggccctggag accgtgcaga 2100
ggctgctgcc tgtgctgtgc caggcccacg gcctcacgcc tgagcaggta gtggctattg 2160
catccaacaa cgggggcaga cccgcactgg agtcaatcgt ggcccagctt tcgaggccgg 2220
accccgcgct ggccgcactc actaatgatc atcttgtagc gctggcctgc ctcggcggac 2280
gacccgcctt ggatgcggtg aagaaggggc tcccgcacgc gcctgcattg attaagcgga 2340
ccaacagaag gattcccgag aggacatcac atcgagtggc aggttcccaa ctcgtgaaga 2400
gtgaacttga ggagaaaaag tcggagctgc ggcacaaatt gaaatacgta ccgcatgaat 2460
acatcgaact tatcgaaatt gctaggaact cgactcaaga cagaatcctt gagatgaagg 2520
taatggagtt ctttatgaag gtttatggat accgagggaa gcatctcggt ggatcacgaa 2580
aacccgacgg agcaatctat acggtgggga gcccgattga ttacggagtg atcgtcgaca 2640
cgaaagccta cagcggtggg tacaatcttc ccatcgggca ggcagatgag atggagcgtt 2700
atgtcgaaga aaatcagacc agggacaaac acctcaatcc aaatgagtgg tggaaagtgt 2760
atccttcatc agtgaccgag tttaagtttt tgtttgtctc tgggcatttc aaaggcaact 2820
ataaggccca gctcacacgg ttgaatcaca ttacgaactg caatggtgcg gttttgtccg 2880
tagaggaact gctcattggt ggagaaatga tcaaagcggg aactctgaca ctggaagaag 2940
tcagacgcaa gtttaacaat ggcgagatca atttccgctc ataaaaaatc agcctcgact 3000
gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3060
gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc acaacactca 3120
accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 3180
taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca 3240
gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 3300
caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 3360
aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc 3420
cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 3480
tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt 3540
tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga 3600
tcagcacgtg atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga 3660
aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt 3720
cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt 3780
ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt 3840
gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg 3900
tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga 3960
ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg 4020
accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc 4080
ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc 4140
tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc 4200
ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg 4260
gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc 4320
gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc 4380
aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag 4440
cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt 4500
ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg 4560
gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc 4620
gagggcaaag gaatagcacg tgctacgaga tttcgattcc accgccgcct tctatgaaag 4680
gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct 4740
catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata 4800
aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 4860
tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag 4920
cttggcgtaa tcatggtcat taccaatgct taatcagtga ggcacctatc tcagcgatct 4980
gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 5040
agggcttacc atctggcccc agcgctgcga tgataccgcg agaaccacgc tcaccggctc 5100
cggatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 5160
ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 5220
cagttaatag tttgcgcaac gttgttgcca tcgctacagg catcgtggtg tcacgctcgt 5280
cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 5340
ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 5400
tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 5460
catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 5520
gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 5580
gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 5640
tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 5700
catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 5760
aaaagggaat aagggcgaca cggaaatgtt gaatactcat attcttcctt tttcaatatt 5820
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 5880
aaaataaaca aataggggtc agtgttacaa ccaattaacc aattctgaac attatcgcga 5940
gcccatttat acctgaatat ggctcataac accccttgct catgaccaaa atcccttaac 6000
gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 6060
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 6120
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 6180
cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt tagcccacca 6240
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 6300
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 6360
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 6420
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 6480
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 6540
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 6600
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 6660
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 6720
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 6780
tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aaggcgagag 6840
tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc tttttgcgtt 6900
tctacaaact ctttctgtgt tgtaaaacga cggccagtct taagctcggg ccccctgggc 6960
ggttctgata acgagtaatc gttaatccgc aaataacgta aaaacccgct tcggcgggtt 7020
tttttatggg gggagtttag ggaaagagca tttgtcagaa tatttaaggg cgcctgtcac 7080
tttgcttgat atatgagaat tatttaacct tataaatgag aaaaaagcaa cgcactttaa 7140
ataagatacg ttgctttttc gattgatgaa cacctataat taaactattc atctattatt 7200
tatgattttt tgtatataca atatttctag tttgttaaag agaattaaga aaataaatct 7260
cgaaaataat aaagggaaaa tcagtttttg atatcaaaat tatacatgtc aacgataata 7320
caaaatataa tacaaactat aagatgttat cagtatttat tatcatttag aataaatttt 7380
gtgtcgccct taattgtgag cggataacaa ttacgagctt catgcacagt ggcgttgaca 7440
ttgattattg actagtccaa acaattctgc aggaatctag ttattaatag taatcaatta 7500
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 7560
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 7620
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 7680
ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 7740
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 7800
cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt 7860
tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt 7920
tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg 7980
cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga 8040
gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa 8100
agcgaagcgc gcggcgggcg gggagtcgct gcgacgctgc cttcgccccg tgccccgctc 8160
cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 8220
cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 8280
ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 8340
gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 8400
gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 8460
gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 8520
caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 8580
cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 8640
tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 8700
ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 8760
cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 8820
atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 8880
atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 8940
caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 9000
tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 9060
ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 9120
cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 9180
tggcaaagaa ttgatttgat accgcgggcc ctag 9214
<210> 6
<211> 9214
<212> DNA
<213> 人工合成
<400> 6
agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca 60
ctataggggc caccatggac tataaggacc acgacggaga ctacaaggat catgatattg 120
attacaaaga cgatgacgat aagatggccc caaagaagaa gcggaaggtc ggtatccacg 180
gagtcccagc agccgtagat ttgagaactt tgggatattc acagcagcag caggaaaaga 240
tcaagcccaa agtgaggtcg acagtcgcgc agcatcacga agcgctggtg ggtcatgggt 300
ttacacatgc ccacatcgta gccttgtcgc agcaccctgc agcccttggc acggtcgccg 360
tcaagtacca ggacatgatt gcggcgttgc cggaagccac acatgaggcg atcgtcggtg 420
tggggaaaca gtggagcgga gcccgagcgc ttgaggccct gttgacggtc gcgggagagc 480
tgagagggcc tccccttcag ctggacacgg gccagttgct gaagatcgcg aagcggggag 540
gagtcacggc ggtcgaggcg gtgcacgcgt ggcgcaatgc gctcacggga gcacccctca 600
acctgacccc tgagcaggtg gtggccatcg ccagccacga cggcggcaag caggccctgg 660
agaccgtgca gaggctgctg cctgtgctgt gccaggccca cggcctgacc cctgagcagg 720
tggtggccat cgccagcaac ggcggcggca agcaggccct ggagaccgtg cagaggctgc 780
tgcctgtgct gtgccaggcc cacggcctga cccctgagca ggtggtggcc atcgccagca 840
acaacggcgg caagcaggcc ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg 900
cccacggcct gacccctgag caggtggtgg ccatcgccag caacatcggc ggcaagcagg 960
ccctggagac cgtgcagagg ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg 1020
agcaggtggt ggccatcgcc agccacgacg gcggcaagca ggccctggag accgtgcaga 1080
ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg gtggccatcg 1140
ccagccacga cggcggcaag caggccctgg agaccgtgca gaggctgctg cctgtgctgt 1200
gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagcaac atcggcggca 1260
agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc cacggcctga 1320
cccctgagca ggtggtggcc atcgccagca acaacggcgg caagcaggcc ctggagaccg 1380
tgcagaggct gctgcctgtg ctgtgccagg cccacggcct gacccctgag caggtggtgg 1440
ccatcgccag caacaacggc ggcaagcagg ccctggagac cgtgcagagg ctgctgcctg 1500
tgctgtgcca ggcccacggc ctgacccctg agcaggtggt ggccatcgcc agcaacggcg 1560
gcggcaagca ggccctggag accgtgcaga ggctgctgcc tgtgctgtgc caggcccacg 1620
gcctgacccc tgagcaggtg gtggccatcg ccagccacga cggcggcaag caggccctgg 1680
agaccgtgca gaggctgctg cctgtgctgt gccaggccca cggcctgacc cctgagcagg 1740
tggtggccat cgccagccac gacggcggca agcaggccct ggagaccgtg cagaggctgc 1800
tgcctgtgct gtgccaggcc cacggcctga cccctgagca ggtggtggcc atcgccagca 1860
acatcggcgg caagcaggcc ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg 1920
cccacggcct gacccctgag caggtggtgg ccatcgccag caacaacggc ggcaagcagg 1980
ccctggagac cgtgcagagg ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg 2040
agcaggtggt ggccatcgcc agcaacaacg gcggcaagca ggccctggag accgtgcaga 2100
ggctgctgcc tgtgctgtgc caggcccacg gcctcacgcc tgagcaggta gtggctattg 2160
catccaacat cgggggcaga cccgcactgg agtcaatcgt ggcccagctt tcgaggccgg 2220
accccgcgct ggccgcactc actaatgatc atcttgtagc gctggcctgc ctcggcggac 2280
gacccgcctt ggatgcggtg aagaaggggc tcccgcacgc gcctgcattg attaagcgga 2340
ccaacagaag gattcccgag aggacatcac atcgagtggc aggttcccaa ctcgtgaaga 2400
gtgaacttga ggagaaaaag tcggagctgc ggcacaaatt gaaatacgta ccgcatgaat 2460
acatcgaact tatcgaaatt gctaggaact cgactcaaga cagaatcctt gagatgaagg 2520
taatggagtt ctttatgaag gtttatggat accgagggaa gcatctcggt ggatcacgaa 2580
aacccgacgg agcaatctat acggtgggga gcccgattga ttacggagtg atcgtcgaca 2640
cgaaagccta cagcggtggg tacaatcttc ccatcgggca ggcagatgag atgcaacgtt 2700
atgtcaaaga aaatcagacc aggaacaaac acatcaatcc aaatgagtgg tggaaagtgt 2760
atccttcatc agtgaccgag tttaagtttt tgtttgtctc tgggcatttc aaaggcaact 2820
ataaggccca gctcacacgg ttgaatcgca agacgaactg caatggtgcg gttttgtccg 2880
tagaggaact gctcattggt ggagaaatga tcaaagcggg aactctgaca ctggaagaag 2940
tcagacgcaa gtttaacaat ggcgagatca atttccgctc ataaaaaatc agcctcgact 3000
gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3060
gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc acaacactca 3120
accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 3180
taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca 3240
gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 3300
caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 3360
aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc 3420
cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 3480
tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt 3540
tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga 3600
tcagcacgtg atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga 3660
aaagttcgac agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt 3720
cagcttcgat gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt 3780
ctacaaagat cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt 3840
gcttgacatt ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg 3900
tgtcacgttg caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga 3960
ggccatggat gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg 4020
accgcaagga atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc 4080
ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc 4140
tctcgatgag ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc 4200
ggatttcggc tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg 4260
gagcgaggcg atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc 4320
gtggttggct tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc 4380
aggatcgccg cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag 4440
cttggttgac ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt 4500
ccgatccgga gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg 4560
gaccgatggc tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc 4620
gagggcaaag gaatagcacg tgctacgaga tttcgattcc accgccgcct tctatgaaag 4680
gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct 4740
catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata 4800
aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg 4860
tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag 4920
cttggcgtaa tcatggtcat taccaatgct taatcagtga ggcacctatc tcagcgatct 4980
gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg 5040
agggcttacc atctggcccc agcgctgcga tgataccgcg agaaccacgc tcaccggctc 5100
cggatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa 5160
ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc 5220
cagttaatag tttgcgcaac gttgttgcca tcgctacagg catcgtggtg tcacgctcgt 5280
cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 5340
ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 5400
tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 5460
catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt 5520
gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata 5580
gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 5640
tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 5700
catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa 5760
aaaagggaat aagggcgaca cggaaatgtt gaatactcat attcttcctt tttcaatatt 5820
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 5880
aaaataaaca aataggggtc agtgttacaa ccaattaacc aattctgaac attatcgcga 5940
gcccatttat acctgaatat ggctcataac accccttgct catgaccaaa atcccttaac 6000
gtgagttacg cgcgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 6060
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 6120
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 6180
cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt tagcccacca 6240
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 6300
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 6360
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 6420
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 6480
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 6540
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 6600
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 6660
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 6720
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 6780
tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aaggcgagag 6840
tagggaactg ccaggcatca aactaagcag aaggcccctg acggatggcc tttttgcgtt 6900
tctacaaact ctttctgtgt tgtaaaacga cggccagtct taagctcggg ccccctgggc 6960
ggttctgata acgagtaatc gttaatccgc aaataacgta aaaacccgct tcggcgggtt 7020
tttttatggg gggagtttag ggaaagagca tttgtcagaa tatttaaggg cgcctgtcac 7080
tttgcttgat atatgagaat tatttaacct tataaatgag aaaaaagcaa cgcactttaa 7140
ataagatacg ttgctttttc gattgatgaa cacctataat taaactattc atctattatt 7200
tatgattttt tgtatataca atatttctag tttgttaaag agaattaaga aaataaatct 7260
cgaaaataat aaagggaaaa tcagtttttg atatcaaaat tatacatgtc aacgataata 7320
caaaatataa tacaaactat aagatgttat cagtatttat tatcatttag aataaatttt 7380
gtgtcgccct taattgtgag cggataacaa ttacgagctt catgcacagt ggcgttgaca 7440
ttgattattg actagtccaa acaattctgc aggaatctag ttattaatag taatcaatta 7500
cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 7560
gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 7620
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 7680
ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 7740
atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 7800
cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt 7860
tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt 7920
tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg 7980
cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga 8040
gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa 8100
agcgaagcgc gcggcgggcg gggagtcgct gcgacgctgc cttcgccccg tgccccgctc 8160
cgccgccgcc tcgcgccgcc cgccccggct ctgactgacc gcgttactcc cacaggtgag 8220
cgggcgggac ggcccttctc ctccgggctg taattagcgc ttggtttaat gacggcttgt 8280
ttcttttctg tggctgcgtg aaagccttga ggggctccgg gagggccctt tgtgcggggg 8340
gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc 8400
gctgcccggc ggctgtgagc gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt 8460
gcgcgagggg agcgcggccg ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa 8520
caaaggctgc gtgcggggtg tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt 8580
cgggctgcaa ccccccctgc acccccctcc ccgagttgct gagcacggcc cggcttcggg 8640
tgcggggctc cgtacggggc gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca 8700
ggtgggggtg ccgggcgggg cggggccgcc tcgggccggg gagggctcgg gggaggggcg 8760
cggcggcccc cggagcgccg gcggctgtcg aggcgcggcg agccgcagcc attgcctttt 8820
atggtaatcg tgcgagaggg cgcagggact tcctttgtcc caaatctgtg cggagccgaa 8880
atctgggagg cgccgccgca ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg 8940
caggaaggaa atgggcgggg agggccttcg tgcgtcgccg cgccgccgtc cccttctccc 9000
tctccagcct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 9060
ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 9120
cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 9180
tggcaaagaa ttgatttgat accgcgggcc ctag 9214
<210> 7
<211> 169
<212> DNA
<213> 人工合成
<400> 7
gatgcctggc cgagggagag ctggaggtga cagatcagct cccagggcaa agcgaccaac 60
cctgggagca ggccctgaac cgcttctggg attacctgcg ctgggtgcag acgctttctg 120
accaggtcca ggaagagctg cagagctccc aagtcacaca ggaactgac 169
<210> 8
<211> 51
<212> DNA
<213> 人工合成
<400> 8
ttctgggatt acctgcgctg ggtgcagacg ctttctgacc aggtccagga a 51
<210> 9
<211> 38
<212> DNA
<213> 人工合成
<400> 9
ttctgggatt acctgcgctt tctgaccagg tccaggaa 38
Claims (10)
1.一对短肽，其特征在于，所述短肽包括第一短肽与第二短肽；所述第一短肽具有如SEQ ID NO：1所示的序列结构；所述第二短肽具有如SEQ ID NO：2所示的序列结构。
2.一对转录激活子样效应因子，其特征在于，所述转录激活子样效应因子包括第一转录激活子样效应因子与第二转录激活子样效应因子；所述第一转录激活子样效应因子与所述第二转录激活子样效应因子分别转录翻译得到权利要求1所述的第一短肽与所述第二短肽。
3.根据权利要求2中所述的一对转录激活子样效应因子，其特征在于，所述第一转录激活子样效应因子具有如SEQ ID NO：3所示的序列结构；所述第二转录激活子样效应因子具有如SEQ ID NO：4所示的序列结构。
4.一种包含权利要求2或3中所述的转录激活子样效应因子的DNA载体。
5.根据权利要求4中所述的DNA载体，其特征在于，所述DNA载体为可编码FokI核酸的DNA载体。
6.根据权利要求4或5中所述的DNA载体，其特征在于，所述DNA载体包括第一DNA载体与第二DNA载体；所述第一DNA载体具有如SEQ ID NO：5所示的序列结构；所述第二DNA载体具有如SEQ ID NO：6所示的序列结构。
7.一种敲除Apoe基因的方法，其特征在于，采用权利要求1或2所述的短肽对Apoe基因进行打靶。
8.一种Apoe基因敲除的动物模型的构建方法，其特征在于，包括如下步骤：取原核期受精卵,将权利要求4-6中任意一项所述DNA载体的体外转录产物导入所述原核期受精卵的细胞质或细胞核中，再将其移植至受体的输卵管，经生育、繁殖，即得。
9.根据权利要求8所述的Apoe基因敲除的动物模型的构建方法，其特征在于，所述DNA载体通过如下方法制备得到：首先，确定敲除Apoe基因的靶位点，构建如权利要求2或3所述的转录激活子样效应因子，并将其插入载体，即得；优选地，所述DNA载体的体外转录产物通过如下方法制备得到：先使用体外转录试剂盒将所述DNA载体转录成mRNA，再使用加尾试剂盒在所述mRNA的3’末端加尾,即得所述DNA载体的体外转录产物。
10.根据权利要求8或9所述的Apoe基因敲除的动物模型的构建方法，其特征在于，所述原核期受精卵为啮齿类动物的原核期受精卵；所述啮齿类动物为SD大鼠、Wistar大鼠、LEA品系大鼠、Fischer品系大鼠、F344大鼠、F6大鼠和黑刺鼠中的一种。
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