CN108690839B - Tp53基因敲除的动物模型的构建方法及其短肽 - Google Patents
Tp53基因敲除的动物模型的构建方法及其短肽 Download PDFInfo
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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Tp53基因进行准确、高效的打靶，获得Tp53基因敲除大鼠。本发明还提供了一种敲除Tp53基因的方法以及一种Tp53基因敲除的动物模型的构建方法，由此得到的大鼠可用于肿瘤相关的研究，包括肿瘤分子生物学研究、肿瘤疾病研究和致癌性检验等，具有广泛的应用前景。
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Tp53基因的方法、构建Tp53基因敲除的动物模型的方法。
背景技术
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。目前在肿瘤生物学研究中最常使用的是移植性肿瘤动物模型和诱导性肿瘤动物模型。但移植性肿瘤模型制备过程要求很高，需要无菌操作，且肿瘤细胞形态、染色体含量等对模型效果影响较大；而诱导性模型由于需要使用化学物质或病毒进行诱发，制瘤周期、类型、诱导时间及恶性程度和个体差异方面都有很大差异。
相比之下，自发性肿瘤模型具有独特的优点：首先是自发性肿瘤通常与人类所患的肿瘤更为相似，有利于将动物实验结果推用到人；其次是这类肿瘤发生的条件比较自然，有可能通过细致观察和统计分析而发现原来没有发现的环境的或其它的致癌因素，可以着重观察遗传因素在肿瘤发生上的作用。目前常见的自发性肿瘤模型比较少，主要是利用近交系动物在一定年龄内可发生一定比率的某种自发性癌症的特点，培育了一些小鼠自发肿瘤模型。但这种制备方法周期长，大多数模型只发生单一肿瘤，且不容易同时获得大批病程相似的自发瘤动物，限制了自发性肿瘤模型在研究中的应用。
TP53基因是(tumor protein p53)是目前发现的与肿瘤相关性最高的一种抑癌基因，且常与其他的基因协同作用，是多种肿瘤的分子标志物。TP53基因位于人类染色体17p13上，编码393个氨基酸的P53蛋白。P53蛋白自1979年发现以来，一直都是生物学领域中研究热度最高的蛋白。P53蛋白包括3个主要的结构域：N末端反式激活位点中间的DNA结合区域和c末端寡聚位点；是细胞生长、增殖和损伤修复的重要调节因子，在细胞处于应激状态时可被诱导表达，当细胞受到各种理化或生物因素的影响致使DNA受损时，P53蛋白使细胞停止于G1/S期，让细胞修复损伤而重新进入细胞周期，如损伤未能修复则促进细胞衰老或凋亡。现有研究表明TP53基因突变或缺失现象与50％以上的人类肿瘤有关联，携带有TP53突变型基因的人类个体表现出较高的癌变倾向，这种遗传性的癌症易感性综合征被称为李弗劳梅尼综合症(Li-Fraumeni Syndrome)。此外，还有研究表明动脉粥样硬化的发生可能与Tp53基因表达较低有关。
就Tp53基因相关的动物模型而言，目前已有多种Trp53基因缺失和多种点突变小鼠，且广泛应用于肿瘤研究及寻找癌症治疗方法当中。已有研究表明，这些小鼠模型通常都能正常发育，与未发生Trp53基因突变或缺失的野生型小鼠相比，这些小鼠模型在任何时间段内发生各种肿瘤的速度都要快得多。其中，Trp53–/–小鼠平均出现肿瘤的年龄是4.5个月，到了10月龄时所有的Trp53–/–小鼠都会死于癌症。
大鼠作为啮齿类模式动物，具有比小鼠更大的体型和与人类更类似的生理学特征，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。与小鼠相比，大鼠更适合于心血管疾病、神经系统疾病、代谢系统疾病等的研究。尤其是在肿瘤研究方面，大鼠癌症易感模型的体形大，生理结构更接近人类，预期大鼠肿瘤模型具有更长的荷瘤生存期，肿瘤生长的体积可以更大，弥补小鼠模型先天的不足。但是，由于胚胎干细胞提取和培养方法在大鼠上还不成熟，长期以来利用传统方法制备大鼠基因敲除模型比较困难，在肿瘤研究方面，大鼠癌症易感模型较少。
目前已有部分Tp53基因敲除大鼠，如Chang Tong等人通过利用ES细胞同源重组方法制备了Tp53基因敲除大鼠(DA背景)，该大鼠2-5号外显子缺失；对该方法得到的Tp53基因敲除大鼠进一步的研究表明，Tp53-/-DA大鼠存活不超过6个月，易发展为血管肉瘤和淋巴瘤；Tp53+/-DA大鼠可存活约12个月，并表现出多种肉瘤(a broader varity ofsarcomas)，其中约20％的雌性大鼠表现出乳腺癌。但是由于大鼠ES细胞体外易分化，建系较难，导致利用该方法制备除DA品系以外其它品系的基因敲除大鼠模型无法复制和应用。
Ruben van Boxtel等人通过ENU介导的随机诱导大鼠6号外显子突变(C273X)，得到了在Wistar背景的Tp53缺陷大鼠，该突变的纯合子主要表达肉瘤，存活约4个月，杂合子在约43周时主要发展为骨肉瘤(Am J Pathol.2011Oct；179(4):1616–1622)。但是，ENU诱导方法随机性强，基因突变结果不稳定，存在难以重复且在长期交配繁殖中极易丢失等问题。
Sarah A.Hansen等人通过回交的方式得到Tp53基因敲除大鼠(F344背景)，发现该大鼠雌性纯合子有一部分因为神经缺陷胚胎致死(Sarah A.Hansenet al，Dis ModelMech.2016Oct 1；9(10):1139–1146)。但是，利用回交的方法制备基因敲除大鼠的周期长，且模型背景不纯，实验结果不稳定，重复性差。
综上所述，上述三种制备方法均有一定的缺陷。不仅如此，具有同样基因突变的不同品系动物，其对实验结果将产生不同的结果，需要针对不同品系动物的遗传背景和特点，建立相应基因突变模型，因此，上述方法均无法适用于SD大鼠模型的制备，而SD大鼠作为最早使用的实验动物，因生长快、繁殖性能好，广泛用于糖尿病模型、肾衰模型、抗体制备模型、且心脏病模型以及安全性评价等，对相关研究具有重要意义。
鉴于此，目前亟待提出一种SD背景的Tp53基因敲除大鼠模型的构建方法。
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p53基因敲除大鼠模型制备过程中存在的成本高、制备周期长、背景不纯的问题，进而提供一种制备成本低、打靶载体构建简单、制备周期短、背景单一的构建敲除Tp53基因的动物模型方法。
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Tp53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；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Tp53基因的方法，采用所述短肽对Tp53基因进行打靶。
本发明提供的Tp53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Tp53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Tp53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Tp53基因进行准确、高效的打靶，快速获得Tp53基因敲除大鼠；
(2)本发明所述的Tp53基因敲除的动物模型的构建方法，实现了对SD大鼠的Tp53基因敲除模型构建，便于癌症及相关药物开发的基础研究和临床前研究，通过本方法建立的癌症易感大鼠可广泛用于肿瘤相关的研究，包括肿瘤分子生物学研究、肿瘤疾病研究和致癌性检验等；
(3)本发明所述的Tp53基因敲除的动物模型的构建方法，制备和筛选流程简单，且得到的动物模型背景单一。
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Tp53基因敲除大鼠打靶后目的基因序列测序分析结果。
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；
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1 Tp53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：
LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGRPALE；
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：
LTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALE。
其设计方法如下：
以SD大鼠为例，首先，根据Tp53基因的序列特征，选择第2外显子上的序列作为TALEN作用的靶位点，所述第2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’-ATGGAGGATTCACAGTCGGATATGAGCATCGAGCTCCCTCTGAGTCAGGAGACATTTTCATGCTTATGGAAACT-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2-18位核苷酸，为NCBI登录号为NC_005109.4的第7603-7619位核苷酸序列，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19-36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37-53位核苷酸，为NCBI登录号为NC_005109.4的第7638-7654位核苷酸序列，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 Tp53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：
CTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTCACGCCTGAGCAGGTAGTGGCTATTGCATCCAACATCGGGGGCAGACCCGCACTGGAG；
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：
CTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTCACGCCTGAGCAGGTAGTGGCTATTGCATCCAACGGAGGGGGCAGACCCGCACTGGAG。
实施例3 包含Tp53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：
AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGGCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGTAGATTTGAGAACTTTGGGATATTCACAGCAGCAGCAGGAAAAGATCAAGCCCAAAGTGAGGTCGACAGTCGCGCAGCATCACGAAGCGCTGGTGGGTCATGGGTTTACACATGCCCACATCGTAGCCTTGTCGCAGCACCCTGCAGCCCTTGGCACGGTCGCCGTCAAGTACCAGGACATGATTGCGGCGTTGCCGGAAGCCACACATGAGGCGATCGTCGGTGTGGGGAAACAGTGGAGCGGAGCCCGAGCGCTTGAGGCCCTGTTGACGGTCGCGGGAGAGCTGAGAGGGCCTCCCCTTCAGCTGGACACGGGCCAGTTGCTGAAGATCGCGAAGCGGGGAGGAGTCACGGCGGTCGAGGCGGTGCACGCGTGGCGCAATGCGCTCACGGGAGCACCCCTCAACCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTCACGCCTGAGCAGGTAGTGGCTATTGCATCCAACATCGGGGGCAGACCCGCACTGGAGTCAATCGTGGCCCAGCTTTCGAGGCCGGACCCCGCGCTGGCCGCACTCACTAATGATCATCTTGTAGCGCTGGCCTGCCTCGGCGGACGACCCGCCTTGGATGCGGTGAAGAAGGGGCTCCCGCACGCGCCTGCATTGATTAAGCGGACCAACAGAAGGATTCCCGAGAGGACATCACATCGAGTGGCAGGTTCCCAACTCGTGAAGAGTGAACTTGAGGAGAAAAAGTCGGAGCTGCGGCACAAATTGAAATACGTACCGCATGAATACATCGAACTTATCGAAATTGCTAGGAACTCGACTCAAGACAGAATCCTTGAGATGAAGGTAATGGAGTTCTTTATGAAGGTTTATGGATACCGAGGGAAGCATCTCGGTGGATCACGAAAACCCGACGGAGCAATCTATACGGTGGGGAGCCCGATTGATTACGGAGTGATCGTCGACACGAAAGCCTACAGCGGTGGGTACAATCTTCCCATCGGGCAGGCAGATGAGATGgAgCGTTATGTCGAAGAAAATCAGACCAGGgACAAACACcTCAATCCAAATGAGTGGTGGAAAGTGTATCCTTCATCAGTGACCGAGTTTAAGTTTTTGTTTGTCTCTGGGCATTTCAAAGGCAACTATAAGGCCCAGCTCACACGGTTGAATCACATTACGAACTGCAATGGTGCGGTTTTGTCCGTAGAGGAACTGCTCATTGGTGGAGAAATGATCAAAGCGGGAACTCTGACACTGGAAGAAGTCAGACGCAAGTTTAACAATGGCGAGATCAATTTCCGCTCATAAAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGCGCTGCGATGATACCGCGAGAACCACGCTCACCGGCTCCGGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATCGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATATTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTCAGTGTTACAACCAATTAACCAATTCTGAACATTATCGCGAGCCCATTTATACCTGAATATGGCTCATAACACCCCTTGCTCATGACCAAAATCCCTTAACGTGAGTTACGCGCGCGTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGCCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGGCGAGAGTAGGGAACTGCCAGGCATCAAACTAAGCAGAAGGCCCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTCTGTGTTGTAAAACGACGGCCAGTCTTAAGCTCGGGCCCCCTGGGCGGTTCTGATAACGAGTAATCGTTAATCCGCAAATAACGTAAAAACCCGCTTCGGCGGGTTTTTTTATGGGGGGAGTTTAGGGAAAGAGCATTTGTCAGAATATTTAAGGGCGCCTGTCACTTTGCTTGATATATGAGAATTATTTAACCTTATAAATGAGAAAAAAGCAACGCACTTTAAATAAGATACGTTGCTTTTTCGATTGATGAACACCTATAATTAAACTATTCATCTATTATTTATGATTTTTTGTATATACAATATTTCTAGTTTGTTAAAGAGAATTAAGAAAATAAATCTCGAAAATAATAAAGGGAAAATCAGTTTTTGATATCAAAATTATACATGTCAACGATAATACAAAATATAATACAAACTATAAGATGTTATCAGTATTTATTATCATTTAGAATAAATTTTGTGTCGCCCTTAATTGTGAGCGGATAACAATTACGAGCTTCATGCACAGTGGCGTTGACATTGATTATTGACTAGTCCAAACAATTCTGCAGGAATCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGATTTGATACCGCGGGCCCTAG；
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：
AGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGGCCACCATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGTAGATTTGAGAACTTTGGGATATTCACAGCAGCAGCAGGAAAAGATCAAGCCCAAAGTGAGGTCGACAGTCGCGCAGCATCACGAAGCGCTGGTGGGTCATGGGTTTACACATGCCCACATCGTAGCCTTGTCGCAGCACCCTGCAGCCCTTGGCACGGTCGCCGTCAAGTACCAGGACATGATTGCGGCGTTGCCGGAAGCCACACATGAGGCGATCGTCGGTGTGGGGAAACAGTGGAGCGGAGCCCGAGCGCTTGAGGCCCTGTTGACGGTCGCGGGAGAGCTGAGAGGGCCTCCCCTTCAGCTGGACACGGGCCAGTTGCTGAAGATCGCGAAGCGGGGAGGAGTCACGGCGGTCGAGGCGGTGCACGCGTGGCGCAATGCGCTCACGGGAGCACCCCTCAACCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCCACGACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACATCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACAACGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTGACCCCTGAGCAGGTGGTGGCCATCGCCAGCAACGGCGGCGGCAAGCAGGCCCTGGAGACCGTGCAGAGGCTGCTGCCTGTGCTGTGCCAGGCCCACGGCCTCACGCCTGAGCAGGTAGTGGCTATTGCATCCAACGGAGGGGGCAGACCCGCACTGGAGTCAATCGTGGCCCAGCTTTCGAGGCCGGACCCCGCGCTGGCCGCACTCACTAATGATCATCTTGTAGCGCTGGCCTGCCTCGGCGGACGACCCGCCTTGGATGCGGTGAAGAAGGGGCTCCCGCACGCGCCTGCATTGATTAAGCGGACCAACAGAAGGATTCCCGAGAGGACATCACATCGAGTGGCAGGTTCCCAACTCGTGAAGAGTGAACTTGAGGAGAAAAAGTCGGAGCTGCGGCACAAATTGAAATACGTACCGCATGAATACATCGAACTTATCGAAATTGCTAGGAACTCGACTCAAGACAGAATCCTTGAGATGAAGGTAATGGAGTTCTTTATGAAGGTTTATGGATACCGAGGGAAGCATCTCGGTGGATCACGAAAACCCGACGGAGCAATCTATACGGTGGGGAGCCCGATTGATTACGGAGTGATCGTCGACACGAAAGCCTACAGCGGTGGGTACAATCTTCCCATCGGGCAGGCAGATGAGATGCAACGTTATGTCAAAGAAAATCAGACCAGGAACAAACACATCAATCCAAATGAGTGGTGGAAAGTGTATCCTTCATCAGTGACCGAGTTTAAGTTTTTGTTTGTCTCTGGGCATTTCAAAGGCAACTATAAGGCCCAGCTCACACGGTTGAATCGCAAGACGAACTGCAATGGTGCGGTTTTGTCCGTAGAGGAACTGCTCATTGGTGGAGAAATGATCAAAGCGGGAACTCTGACACTGGAAGAAGTCAGACGCAAGTTTAACAATGGCGAGATCAATTTCCGCTCATAAAAAATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAGCACGTGATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAGCACGTGCTACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGCGCTGCGATGATACCGCGAGAACCACGCTCACCGGCTCCGGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATCGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATATTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTCAGTGTTACAACCAATTAACCAATTCTGAACATTATCGCGAGCCCATTTATACCTGAATATGGCTCATAACACCCCTTGCTCATGACCAAAATCCCTTAACGTGAGTTACGCGCGCGTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGCCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGGCGAGAGTAGGGAACTGCCAGGCATCAAACTAAGCAGAAGGCCCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTCTGTGTTGTAAAACGACGGCCAGTCTTAAGCTCGGGCCCCCTGGGCGGTTCTGATAACGAGTAATCGTTAATCCGCAAATAACGTAAAAACCCGCTTCGGCGGGTTTTTTTATGGGGGGAGTTTAGGGAAAGAGCATTTGTCAGAATATTTAAGGGCGCCTGTCACTTTGCTTGATATATGAGAATTATTTAACCTTATAAATGAGAAAAAAGCAACGCACTTTAAATAAGATACGTTGCTTTTTCGATTGATGAACACCTATAATTAAACTATTCATCTATTATTTATGATTTTTTGTATATACAATATTTCTAGTTTGTTAAAGAGAATTAAGAAAATAAATCTCGAAAATAATAAAGGGAAAATCAGTTTTTGATATCAAAATTATACATGTCAACGATAATACAAAATATAATACAAACTATAAGATGTTATCAGTATTTATTATCATTTAGAATAAATTTTGTGTCGCCCTTAATTGTGAGCGGATAACAATTACGAGCTTCATGCACAGTGGCGTTGACATTGATTATTGACTAGTCCAAACAATTCTGCAGGAATCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGATTTGATACCGCGGGCCCTAG。
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 Tp53基因的敲除
本实施例的Tp53基因敲除的方法，采用实施例1中所述的短肽对Tp53基因进行打靶。
作为上述方法的具体应用，本实施例提供Tp53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Tp53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Tp53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 Tp53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Tp53基因靶序列是否被成功敲除。
取实施例4中构建得到的SD大鼠2只，分别编号1、2，对其进行DNA测序，其测序分析结果如图3所示，由测序结果可知，两只被测SD大鼠在Tp53基因靶位点发生了DNA序列的片段缺失，造成了移码突变，进而导致大鼠体内的的Tp53基因功能丧失。
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；编号为1的Tp53基因被敲除的SD大鼠的靶位点序列，具有如SEQ ID NO：9所示的序列结构；编号为2的Tp53基因被敲除的SD大鼠的靶位点序列，具有如SEQ ID NO：10所示的序列结构，三者的对比见表1。
表1Tp53基因敲除大鼠靶序列测定结果
注：Δ12为删除12bp，In2为插入2bp，Δ1为删除1bp，[]中为具体删除的核苷酸序列，<>中为插入的核苷酸序列。
由上述对比可知，实施例4的方法制备得到的Tp53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，均缺失和/或插入若干个碱基。具体而言，编号为1的Tp53基因被敲除的SD大鼠缺失12个碱基，并随机插入2个碱基；编号为2的Tp53基因被敲除的SD大鼠缺失1个碱基。
因此利用本发明公开的方法可以用于啮齿类Tp53基因组编辑和遗传改造，制备啮齿动物突变体。
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> Tp53基因敲除的动物模型的构建方法及其短肽
<130> PB710028N
<160> 10
<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 His Asp 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
50 55 60
Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His
65 70 75 80
Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Gly 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 His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
Val Ala Ile Ala Ser Asn Gly 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 Asn Gly Gly Lys Gln Ala Leu Glu
210 215 220
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 Ile 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 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
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Ile 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 Asn 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 Asn Ile Gly Gly Lys Gln Ala Leu Glu
485 490 495
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 Ile Gly Gly Arg Pro Ala
515 520 525
Leu Glu
530
<210> 2
<211> 564
<212> PRT
<213> 人工合成
<400> 2
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Ile 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 Asn Ile Gly
35 40 45
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
65 70 75 80
Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val
85 90 95
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
100 105 110
Ser Asn Ile 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 Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg
145 150 155 160
Leu Leu Pro Val Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val
165 170 175
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 His Asp Gly Gly Lys Gln Ala Leu Glu
210 215 220
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 Ile 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 His Asp 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
355 360 365
Leu Cys Gln Ala His Gly Leu Thr Pro Glu Gln Val Val Ala Ile Ala
370 375 380
Ser Asn Ile 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 His Asp 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 Ile 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 Asn Asn Gly Gly Lys Gln Ala Leu Glu
485 490 495
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 Gly Gly Gly Lys Gln Ala
515 520 525
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala His Gly
530 535 540
Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Arg
545 550 555 560
Pro Ala Leu Glu
<210> 3
<211> 1590
<212> DNA
<213> 人工合成
<400> 3
ctgacccctg agcaggtggt ggccatcgcc agccacgacg gcggcaagca ggccctggag 60
accgtgcaga ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg 120
gtggccatcg ccagccacga cggcggcaag caggccctgg agaccgtgca gaggctgctg 180
cctgtgctgt gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagccac 240
gacggcggca agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc 300
cacggcctga cccctgagca ggtggtggcc atcgccagca acggcggcgg caagcaggcc 360
ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg cccacggcct gacccctgag 420
caggtggtgg ccatcgccag ccacgacggc ggcaagcagg ccctggagac cgtgcagagg 480
ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg agcaggtggt ggccatcgcc 540
agcaacggcg gcggcaagca ggccctggag accgtgcaga ggctgctgcc tgtgctgtgc 600
caggcccacg gcctgacccc tgagcaggtg gtggccatcg ccagcaacaa cggcggcaag 660
caggccctgg agaccgtgca gaggctgctg cctgtgctgt gccaggccca cggcctgacc 720
cctgagcagg tggtggccat cgccagcaac atcggcggca 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 ccagcaacat cggcggcaag caggccctgg agaccgtgca gaggctgctg 1200
cctgtgctgt gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagcaac 1260
aacggcggca agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc 1320
cacggcctga cccctgagca ggtggtggcc atcgccagca acaacggcgg caagcaggcc 1380
ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg cccacggcct gacccctgag 1440
caggtggtgg ccatcgccag caacatcggc ggcaagcagg ccctggagac cgtgcagagg 1500
ctgctgcctg tgctgtgcca ggcccacggc ctcacgcctg agcaggtagt ggctattgca 1560
tccaacatcg ggggcagacc cgcactggag 1590
<210> 4
<211> 1692
<212> DNA
<213> 人工合成
<400> 4
ctgacccctg agcaggtggt ggccatcgcc agcaacatcg gcggcaagca ggccctggag 60
accgtgcaga ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg 120
gtggccatcg ccagcaacat 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 caacggcggc ggcaagcagg ccctggagac cgtgcagagg 480
ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg agcaggtggt ggccatcgcc 540
agccacgacg gcggcaagca ggccctggag accgtgcaga ggctgctgcc tgtgctgtgc 600
caggcccacg gcctgacccc tgagcaggtg gtggccatcg ccagccacga cggcggcaag 660
caggccctgg agaccgtgca gaggctgctg cctgtgctgt gccaggccca cggcctgacc 720
cctgagcagg tggtggccat cgccagcaac atcggcggca agcaggccct ggagaccgtg 780
cagaggctgc tgcctgtgct gtgccaggcc cacggcctga cccctgagca ggtggtggcc 840
atcgccagcc acgacggcgg 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 ccagcaacat cggcggcaag caggccctgg agaccgtgca gaggctgctg 1200
cctgtgctgt gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagccac 1260
gacggcggca agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc 1320
cacggcctga cccctgagca ggtggtggcc atcgccagca acatcggcgg caagcaggcc 1380
ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg cccacggcct gacccctgag 1440
caggtggtgg ccatcgccag caacaacggc ggcaagcagg ccctggagac cgtgcagagg 1500
ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg agcaggtggt ggccatcgcc 1560
agcaacggcg gcggcaagca ggccctggag accgtgcaga ggctgctgcc tgtgctgtgc 1620
caggcccacg gcctcacgcc tgagcaggta gtggctattg catccaacgg agggggcaga 1680
cccgcactgg ag 1692
<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 ccagccacga cggcggcaag caggccctgg 660
agaccgtgca gaggctgctg cctgtgctgt gccaggccca cggcctgacc cctgagcagg 720
tggtggccat cgccagccac gacggcggca agcaggccct ggagaccgtg cagaggctgc 780
tgcctgtgct gtgccaggcc cacggcctga cccctgagca ggtggtggcc atcgccagcc 840
acgacggcgg caagcaggcc ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg 900
cccacggcct gacccctgag caggtggtgg ccatcgccag caacggcggc ggcaagcagg 960
ccctggagac cgtgcagagg ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg 1020
agcaggtggt ggccatcgcc agccacgacg gcggcaagca ggccctggag accgtgcaga 1080
ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg gtggccatcg 1140
ccagcaacgg cggcggcaag caggccctgg agaccgtgca gaggctgctg cctgtgctgt 1200
gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagcaac aacggcggca 1260
agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc cacggcctga 1320
cccctgagca ggtggtggcc atcgccagca acatcggcgg 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 cgccagcaac atcggcggca agcaggccct ggagaccgtg cagaggctgc 1800
tgcctgtgct gtgccaggcc cacggcctga cccctgagca ggtggtggcc atcgccagca 1860
acaacggcgg caagcaggcc ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg 1920
cccacggcct gacccctgag caggtggtgg ccatcgccag caacaacggc ggcaagcagg 1980
ccctggagac cgtgcagagg ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg 2040
agcaggtggt ggccatcgcc agcaacatcg 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 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> 9316
<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 ccagcaacat cggcggcaag caggccctgg 660
agaccgtgca gaggctgctg cctgtgctgt gccaggccca cggcctgacc cctgagcagg 720
tggtggccat cgccagcaac atcggcggca 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 agcaacggcg gcggcaagca ggccctggag accgtgcaga 1080
ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg gtggccatcg 1140
ccagccacga cggcggcaag caggccctgg agaccgtgca gaggctgctg cctgtgctgt 1200
gccaggccca cggcctgacc cctgagcagg tggtggccat cgccagccac gacggcggca 1260
agcaggccct ggagaccgtg cagaggctgc tgcctgtgct gtgccaggcc cacggcctga 1320
cccctgagca ggtggtggcc atcgccagca acatcggcgg caagcaggcc ctggagaccg 1380
tgcagaggct gctgcctgtg ctgtgccagg cccacggcct gacccctgag caggtggtgg 1440
ccatcgccag ccacgacggc 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 cgccagcaac atcggcggca agcaggccct ggagaccgtg cagaggctgc 1800
tgcctgtgct gtgccaggcc cacggcctga cccctgagca ggtggtggcc atcgccagcc 1860
acgacggcgg caagcaggcc ctggagaccg tgcagaggct gctgcctgtg ctgtgccagg 1920
cccacggcct gacccctgag caggtggtgg ccatcgccag caacatcggc ggcaagcagg 1980
ccctggagac cgtgcagagg ctgctgcctg tgctgtgcca ggcccacggc ctgacccctg 2040
agcaggtggt ggccatcgcc agcaacaacg gcggcaagca ggccctggag accgtgcaga 2100
ggctgctgcc tgtgctgtgc caggcccacg gcctgacccc tgagcaggtg gtggccatcg 2160
ccagcaacgg cggcggcaag caggccctgg agaccgtgca gaggctgctg cctgtgctgt 2220
gccaggccca cggcctcacg cctgagcagg tagtggctat tgcatccaac ggagggggca 2280
gacccgcact ggagtcaatc gtggcccagc tttcgaggcc ggaccccgcg ctggccgcac 2340
tcactaatga tcatcttgta gcgctggcct gcctcggcgg acgacccgcc ttggatgcgg 2400
tgaagaaggg gctcccgcac gcgcctgcat tgattaagcg gaccaacaga aggattcccg 2460
agaggacatc acatcgagtg gcaggttccc aactcgtgaa gagtgaactt gaggagaaaa 2520
agtcggagct gcggcacaaa ttgaaatacg taccgcatga atacatcgaa cttatcgaaa 2580
ttgctaggaa ctcgactcaa gacagaatcc ttgagatgaa ggtaatggag ttctttatga 2640
aggtttatgg ataccgaggg aagcatctcg gtggatcacg aaaacccgac ggagcaatct 2700
atacggtggg gagcccgatt gattacggag tgatcgtcga cacgaaagcc tacagcggtg 2760
ggtacaatct tcccatcggg caggcagatg agatgcaacg ttatgtcaaa gaaaatcaga 2820
ccaggaacaa acacatcaat ccaaatgagt ggtggaaagt gtatccttca tcagtgaccg 2880
agtttaagtt tttgtttgtc tctgggcatt tcaaaggcaa ctataaggcc cagctcacac 2940
ggttgaatcg caagacgaac tgcaatggtg cggttttgtc cgtagaggaa ctgctcattg 3000
gtggagaaat gatcaaagcg ggaactctga cactggaaga agtcagacgc aagtttaaca 3060
atggcgagat caatttccgc tcataaaaaa tcagcctcga ctgtgccttc tagttgccag 3120
ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact 3180
gtcctttcct aataaaatga ggaaattgca tcacaacact caaccctatc tcggtctatt 3240
cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt 3300
aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc 3360
cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag 3420
gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta 3480
gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc 3540
cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc 3600
ctctgcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg 3660
caaaaagctc ccgggagctt gtatatccat tttcggatct gatcagcacg tgatgaaaaa 3720
gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc 3780
cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg 3840
gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt 3900
ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt 3960
cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct 4020
gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc 4080
tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca 4140
atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca 4200
aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct 4260
ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa 4320
tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg 4380
ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga 4440
gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg 4500
ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt 4560
cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac 4620
tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga 4680
agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagca 4740
cgtgctacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt 4800
tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc 4860
ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 4920
tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 4980
tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc 5040
attaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 5100
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 5160
ccagcgctgc gatgataccg cgagaaccac gctcaccggc tccggattta tcagcaataa 5220
accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 5280
agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 5340
acgttgttgc catcgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 5400
tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 5460
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 5520
tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 5580
ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 5640
gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 5700
tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 5760
ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 5820
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 5880
cacggaaatg ttgaatactc atattcttcc tttttcaata ttattgaagc atttatcagg 5940
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 6000
tcagtgttac aaccaattaa ccaattctga acattatcgc gagcccattt atacctgaat 6060
atggctcata acaccccttg ctcatgacca aaatccctta acgtgagtta cgcgcgcgtc 6120
gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 6180
tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 6240
gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 6300
accaaatact gttcttctag tgtagccgta gttagcccac cacttcaaga actctgtagc 6360
accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 6420
gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 6480
ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 6540
atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 6600
gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 6660
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 6720
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 6780
gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 6840
tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 6900
cgagcgcagc gagtcagtga gcgaggaagc ggaaggcgag agtagggaac tgccaggcat 6960
caaactaagc agaaggcccc tgacggatgg cctttttgcg tttctacaaa ctctttctgt 7020
gttgtaaaac gacggccagt cttaagctcg ggccccctgg gcggttctga taacgagtaa 7080
tcgttaatcc gcaaataacg taaaaacccg cttcggcggg tttttttatg gggggagttt 7140
agggaaagag catttgtcag aatatttaag ggcgcctgtc actttgcttg atatatgaga 7200
attatttaac cttataaatg agaaaaaagc aacgcacttt aaataagata cgttgctttt 7260
tcgattgatg aacacctata attaaactat tcatctatta tttatgattt tttgtatata 7320
caatatttct agtttgttaa agagaattaa gaaaataaat ctcgaaaata ataaagggaa 7380
aatcagtttt tgatatcaaa attatacatg tcaacgataa tacaaaatat aatacaaact 7440
ataagatgtt atcagtattt attatcattt agaataaatt ttgtgtcgcc cttaattgtg 7500
agcggataac aattacgagc ttcatgcaca gtggcgttga cattgattat tgactagtcc 7560
aaacaattct gcaggaatct agttattaat agtaatcaat tacggggtca ttagttcata 7620
gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 7680
ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 7740
ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 7800
atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 7860
cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 7920
tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca 7980
tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag 8040
cgatgggggc gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc 8100
ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt 8160
ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg 8220
cggggagtcg ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg 8280
cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc 8340
tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg 8400
tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc 8460
gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga 8520
gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc 8580
cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg 8640
tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct 8700
gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg 8760
gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg 8820
ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc 8880
cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag 8940
ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg 9000
caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg 9060
ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg 9120
tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg 9180
tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca 9240
gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattgatttg 9300
ataccgcggg ccctag 9316
<210> 7
<211> 74
<212> DNA
<213> 人工合成
<400> 7
atggaggatt cacagtcgga tatgagcatc gagctccctc tgagtcagga gacattttca 60
tgcttatgga aact 74
<210> 8
<211> 54
<212> DNA
<213> 人工合成
<400> 8
tccctctgag tcaggagaca ttttcatgct tatggaaact gtgagtggat ctta 54
<210> 9
<211> 56
<212> DNA
<213> 人工合成
<400> 9
tccctctgag tcaggaaaga cattttcatg cttatggaaa ctgtgagtgg atctta 56
<210> 10
<211> 54
<212> DNA
<213> 人工合成
<400> 10
tccctctgag tcaggagaca ttttcatgct tatggaaact gtgagtggat ctta 54
Claims (10)
1.一对蛋白，其特征在于，所述蛋白包括第一蛋白与第二蛋白；所述第一蛋白的氨基酸序列如SEQ ID NO：1所示；所述第二蛋白的氨基酸序列如SEQ ID NO：2所示。
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中所述的第一蛋白与所述第二蛋白。
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的序列如SEQ ID NO：3所示；所述第二转录激活子样效应因子的序列如SEQID NO：4所示。
4.一种包含权利要求2或3中所述的一对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载体为能编码FokI核酸酶的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载体的序列如SEQ ID NO：5所示；所述第二DNA载体的序列如SEQID NO：6所示。
7.一种敲除啮齿类动物Tp53基因的方法，其特征在于，采用权利要求1所述的一对蛋白或权利要求2所述的一对转录激活子样效应因子对Tp53基因进行打靶，所述啮齿类动物为SD大鼠。
8.一种Tp53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；
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。
9.根据权利要求8所述的Tp53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Tp53基因的靶位点，构建如权利要求2或3所述的一对转录激活子样效应因子，并将其插入载体，即得。
10.根据权利要求8所述的构建方法，其特征在于，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。
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