CN113493805A - 人类血清素神经元tph2报告基因细胞系的构建方法及应用 - Google Patents

人类血清素神经元tph2报告基因细胞系的构建方法及应用 Download PDF

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CN113493805A
CN113493805A CN202110764505.5A CN202110764505A CN113493805A CN 113493805 A CN113493805 A CN 113493805A CN 202110764505 A CN202110764505 A CN 202110764505A CN 113493805 A CN113493805 A CN 113493805A
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陆建峰
许婷
曹立宁
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Shanghai East Hospital Tongji University Affiliated East Hospital
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Abstract

本发明属于生物技术领域，具体涉及一种稳定的TPH2报告基因人多能干细胞系的构建方法与应用。本发明还包括一种表达血清素神经元TPH2报告基因的载体系统，所述的载体系统包括但不限于：靶向人类血清素神经元TPH2基因终止密码子附近的单链向导RNA的向导载体，和携带人类血清素神经元TPH2基因终止密码子附近同源臂的供体载体。本发明还包括转入上述载体系统的TPH2报告基因人多能干细胞系的构建方法，本发明可应用于鉴定干细胞分化来源的人类血清素神经元，以及在活细胞状态下对血清素神经元的形态和功能进行研究。

Description

人类血清素神经元TPH2报告基因细胞系的构建方法及应用
技术领域
本发明属于生物技术领域，涉及一种血清素神经元TPH2报告基因人多能干细胞系的构建方法与应用。
背景技术
在中枢神经系统中，TPH2基因在血清素(五羟色胺)神经元中特异表达，其编码的色氨酸羟化酶二是血清素合成的关键限速酶，在血清素的合成过程中负责催化色氨酸转变成五羟色氨酸，最后通过氨基酸脱羧反应生成五羟色胺。
脑内血清素神经元具有调控情绪和呼吸等功能，与重度抑郁症以及婴儿猝死综合等疾病息息相关，然而血清素神经元调控情绪以及呼吸的具体机制目前尚不明确。虽然体外诱导人多能干细胞可以分化得到血清素神经元，但是最终分化体系中非血清素神经元仍然占据较高比例，活细胞状态下研究血清素神经元的形态和功能也面临巨大挑战。
因此，建立荧光标记的血清素神经元的报告细胞系将有利于人类血清素神经元的相关研究，为重度抑郁症等疾病的药物筛选提供广阔的应用平台。
发明内容
本发明旨在建立TPH2基因的报告细胞系，来指针分化体系中的人类血清素神经元，解决分化体系中多种细胞混合条件下对血清素神经元进行研究的难题。
一方面，本发明提供了一种表达血清素神经元TPH2报告基因的载体系统，所述的载体系统包括但不限于：
靶向人类血清素神经元TPH2基因终止密码子附近的单链向导RNA的向导载体，和
携带人类血清素神经元TPH2基因终止密码子附近同源臂的供体载体。
本发明中，单链向导RNA(sgRNA)覆盖了TPH2基因终止密码子，报告基因插入后可破坏原始单链向导RNA识别序列，从而防止被Cas9蛋白二次切割。
TPH2基因终止密码子附近的单链向导RNA是指TPH2基因终止密码子上下游100bp以内TPH2基因终止密码子附近同源臂是指距离终止密码子上下游各1500bp左右。
在本发明的一个实施例中，TPH2基因终止密码子附近同源臂如SEQ ID NO 21或者SEQ ID NO 22所示。
较好的，靶向人类血清素神经元TPH2基因终止密码子附近的单链向导RNA的向导载体选自：PX330-sgT、PX458-sgT或者PX459-sgT。
在本发明的一个实施例中，PX330-sgT的序列如SEQ ID NO 19所示。
另一方面，本发明提供了一种稳定的血清素神经元TPH2报告基因人多能干细胞系的构建方法，包括以下步骤：
(1)构建携带靶向TPH2基因终止密码子附近的单链向导RNA的向导载体PX330-sgT；
获得靶向人类TPH2基因终止密码子附近的sgRNA，合成两条寡聚核苷酸链，退火并磷酸化后插入到PX330的BbSI位点，重组载体转化大肠杆菌，挑取单克隆菌进行测序验证重组成功的质粒PX330-sgT；
(2)构建报告基因供体载体的工具载体pUC19-T2A-EGFP-CMV-PuroR；
从AAVS1-TRE3G-EGFP中扩增出puromycin(嘌呤霉素)的抗性基因，克隆到FLAG-HA-pcDNA3.1-表达载体的CMV启动子后，得到pcDNA3.1-CMV-PuroR-polyA载体，从此载体中扩增CMV-PuroR-polyA序列，同时从PX458中扩增T2A-EGFP，将扩增的两个片段重组到pUC19载体的多克隆位点，得到工具载体pUC19-T2A-EGFP-CMV-PuroR；
(3)构建TPH2报告基因的供体载体TPH2-T2A-EGFP；
从人的基因组中扩增TPH2终止密码子前后1.5kb的左右同源臂，THL和THR，在工具载体的左右两侧的多克隆位点分别插入THL和THR，得到TPH2报告基因供体载体TPH2-T2A-EGFP；
(4)TPH2报告基因人多能干细胞系的构建；
将向导载体PX330-sgT和供体载体TPH2-T2A-EGFP电转到人多能干细胞H9，经puromycin筛选，获得抗性克隆后，提取细胞基因组进行PCR扩增鉴定插入片段，并测序，最终获得稳定的EGFP标记的报告血清素神经元TPH2基因的人多能干细胞系。
较好的，所述步骤1)中构建向导载体的工具载体包括但不限于：PX330、PX458或者PX459中的一种或者几种。
较好的，所述步骤2)中用于构建打靶载体工具载体的报告基因元件包括但不限于tdTomato,YFP,RFP或者EGFP中的一种或者几种。
较好的，所述步骤2)中用于构建打靶载体工具载体的抗性筛选元件包括但不限于PuroR,NeoR。
较好的，所述步骤3)是从工具载体pUC19-T2A-EGFP-CMV-PuroR出发，插入TPH2基因的左右同源臂。
较好的，所述步骤4)中电转的人多能干细胞系包括但不限于人胚胎干细胞系或者诱导多能干细胞系(例如H1,RUES1,RUES2以及人体细胞重编程来源的多能干细胞)。
在本发明的优选实施例中，人类血清素神经元TPH2报告基因稳定细胞系的构建方法如下：
1.1构建携带靶向TPH2基因终止密码子附近的单一向导RNA(single guide RNA)的向导载体(例如，PX459-sgT或者PX330-sgT)；
1.2构建携带TPH2基因终止密码子附近同源臂的供体载体(TPH2-T2A-EGFP)；
1.3将PX459-sgT和TPH2-T2A-EGFP载体电转到人多能干细胞系中，经puromycin抗性筛选得到稳定的EGFP标记TPH2基因的人多能干细胞系。
上述人类血清素神经元TPH2报告基因稳定细胞系的构建方法中，所述步骤1.1中用于构建携带sgRNA的打靶载体包括PX330/PX458/PX459，优选PX330。靶向TPH2终止密码子附近的寡链核苷酸退火形成双链后,通过酶切连接插入到PX330载体的BbSI位点，重组载体经大肠杆菌扩增获得无内毒素的PX330-sgT质粒，序列如表1SEQ ID NO 19所示。
所述步骤1.2中供体载体(TPH2-T2A-EGFP)的报告基因可选用mCherry,RFP,GFP,tdTomato等,本发明选用EGFP。
供体载体的构建步骤分为两步，第一步构建报告基因的工具载体，第二步在工具载体中插入TPH2终止密码子前后各近1.5kb的同源臂。
较好的，报告基因的工具载体构建步骤如下：
1.从AAVS1-TRE3G-EGFP(addgene#52343)扩增出PuroR，通过同源重组克隆到FLAG-HA-pcDNA3.1-(addgene#52535)的NheI和AflII酶切位点之间，同时去除NheI和AflII的酶切位点，得到pcDNA3.1-CMV-PuroR-polyA,序列如表2所示。
2.从pcDNA3.1-CMV-PuroR-polyA中扩增CMV-PuroR-polyA-AflII-NheI，通过引物加入AfLII和NheI两个酶切位点。
3.从PX458(addgene#48138)中扩增T2A-EGFP序列。
4.将2,3步骤得到的两个片段同源重组到pUC19(addgene#50005)的BamHI和KpnI两个酶切位点之间，得到pUC19-T2A-EGFP-CMV-PuroR工具载体，序列如表三所示。
5.将人基因组中扩增的TPH2终止密码子前后1.5kb左右的同源臂同源重组组装到工具载体pUC19-T2A-EGFP-CMV-PuroR的多克隆位点，重组载体经大肠杆菌扩增得到无内毒素的TPH2-T2A-EGFP质粒，序列如表四所示。
再一方面，本发明提供了使用上述血清素神经元TPH2报告基因人多能干细胞系的构建方法获得的血清素神经元TPH2报告基因人多能干细胞系。
较好的，所述的血清素神经元TPH2报告基因人多能干细胞系源自H9。
再一方面，本发明还提供了上述血清素神经元TPH2报告基因多能干细胞系的应用，包括所述的血清素神经元TPH2报告基因人多能干细胞系在制备血清素神经元的定向分化药物、清素神经元的形态和功能研究方面、血清素神经元的定向分化药物筛选等方面的应用。
在本发明的另一个优选实施例中，人类血清素神经元TPH2报告基因多能干细胞系的鉴定方法如下：
根据已发表的实验方案(PMID:26655496)体外诱导TPH2基因报告人多能干细胞系分化得到血清素神经元，荧光显微镜下可见绿色荧光表达，可直观观察分化效率；细胞免疫荧光检测红色荧光标记血清素与绿色EGFP荧光细胞共定位；记录绿色荧光标记的血清素神经元的电生理，提高了血清素神经元电生理检测效率和准确性。
本发明公开了一种人类血清素神经元TPH2报告基因人多能干细胞系的构建方法以及应用。绿色荧光指示多能干细胞分化来源的人类血清素神经元，促进了活细胞状态下人类血清素神经元的形态和功能的研究，为重度抑郁症等疾病药物筛选提供精确且便利的平台。
附图说明
图1为报告基因工具载体图示。
图2为TPH2报告基因载体图示。
图3为TPH2报告基因人多能干细胞系的构建图示。
图4为细胞系鉴定PCR扩增图。
图5为血清素神经元与绿色荧光共定位图。
图6为绿色荧光标记的血清素神经元在电压刺激下的钠钾电流。
图7为绿色荧光标记的血清素神经元在电流刺激下诱发的动作电位和自发动作电位。
具体实施方式
实施例1：构建含有报告基因和独立启动子启动的抗性筛选基因的工具载体
(1)从AAVS1-TRE3G-EGFP(addgene#52343)质粒中扩增puromycin的抗性基因PuroR并纯化；
正向引物：5’-gggagacccaagctgATGACCGAGTACAAGCCCACGG-3’(SEQ ID NO 1)
反向引物：5’-gatcagcggtttaaaTCAGGCACCGGGCTTGCG-3’(SEQ ID NO 2)
引物5’端划线小写字母标注的序列为同源臂序列。
(2)FLAG-HA-pcDNA3.1-(addgene#52535)载体NheI，AflII(NEB)双酶切并纯化回收。
(3)使用同源重组试剂盒(近岸蛋白)将(1)中的PuroR序列重组到(2)中的酶切纯化的FLAG-HA-pcDNA3.1-载体的NheI和AflII之间，去除原载体上NheI和AflII的酶切位点，得到pcDNA3.1-CMV-PuroR-polyA。
(4)从(3)中的载体pcDNA3.1-CMV-PuroR-polyA中扩增CMV-PuroR-polyA序列，并纯化；
正向引物：5’-catgctggggaGGCCAGATATACGCGTTGAC-3’(SEQ ID NO 3)
反向引物：5’-agtgaattcgagctcggtaccgcgCTTAAGCCATAGAGCCCACCGCATC-3’(SEQID NO 4)
引物5’端划线小写字母标注的为同源臂序列，反向引物上携带AflII-NheI酶切位点序列。
(5)从PX458(addgene#48138)中扩增T2A-EGFP,并纯化；
正向引物：5’-caggtcgactctagaggatccGAGGGCAGAGGAAGTCTGCTA-3’(SEQ ID NO5)
反向引物：5’-tatctggccTCCCCAGCATGCCTGCTATTC-3’(SEQ ID NO 6)
引物5’端划线小写字母标注的序列为同源臂序列
(6)pUC19(addgene#50005)BamHI,KpnI酶切纯化回收；
(7)使用同源重组试剂盒(近岸蛋白)将(3)和(5)中扩增的CMV-PuroR-polyA和T2A-EGFP两个片段连接到(6)中的pUC19载体的BamHI和KpnI之间，得到pUC19-T2A-EGFP-CMV-PuroR质粒。
实施例2：在实施例1中构建的工具载体中插入TPH2基因的左右同源臂，构建TPH2报告基因供体载体
(1)从H9中提取基因组DNA，并以此作为模板扩增TPH2终止密码子前后的左右同源臂片段并纯化。
左同源臂扩增引物：
正向引物：5’-cttgcatgcctgcagTCTCTCTGCCTCTCTCTCCC-3’(SEQ ID NO 7)
反向引物：5’-acttcctctgccctcAATCCCCAGATATTGGTTCATTTT-3’(SEQ ID NO 8)
右同源臂扩增引物：
正向引物：5’-ctatggcttaagcgcTGATGCCTGGAACTATGTTGT-3’(SEQ ID NO 9)
反向引物：5’-agtgaattcgagctcGATTTCTGACCCTTTGTGGGC-3’(SEQ ID NO 10)
(2)pUC19-T2A-EGFP-CMV-PuroR使用KpnI、NheI、SalI、BamHI酶切纯化。
(3)分别将左右同源臂重组到pUC19-T2A-EGFP-CMV-PuroR的SalI、BamHI和KpnINheI之间，转化DH5α感受态挑取单克隆菌落，测序验证重组成功的供体载体TPH2-T2A-EGFP。
实施例3：构建靶向TPH2终止密码子附近的打靶载体PX330-sgT
(1)使用sgRNA设计网站(网址：https://zlab.bio/guide-design-resources)获得一系列候选sgRNA序列，选择覆盖TPH2终止密码子且切割效率高、脱靶效率低的sgRNA,具体序列如下：
sgT-F:5’-caccGATATCTGGGGATTTGATGCC-3’(SEQ ID NO 11)
sgT-R:5’-aaacGGCATCAAATCCCCAGATATC-3’(SEQ ID NO 12)
小写字母序列是BbSI位点酶切后互补的粘性末端，划线序列为终止密码子
(2)寡链退火并磷酸化形成双链SgT。
(3)将露出粘性末端的双链sgT通过酶切连接插入到PX330(addgene#42230)的BbSI酶切位点，转化感受态挑取单克隆菌落，测序鉴定得到PX330-sgT载体。
实施例4：TPH2-EGFP人多能干细胞系的构建
(1)使用TryPLE将人多能干细胞消化成单细胞
(2)1*106细胞重悬到400ul HEPES电转缓冲液，加入5μg PX330-sgT和10μg TPH2-T2A-EGFP，并转移到宽度4mm的电击杯中，放入到伯乐电穿孔仪(Biorad GenePulserXcell)
(3)电转参数设置为：指数波，电压250V，电容500μF，执行电转程序
(4)按照8*103/cm2密度,将细胞种到提前铺了X射线处理的小鼠成纤维细胞的六孔板
(5)电转24小时后，经0.4μg/ml嘌呤霉素药筛一周
(6)电转后10-17天挑取单克隆细胞，进行扩增
(7)提取细胞基因组DNA，进行PCR扩增鉴定成功打靶的TPH2-EGFP报告血清素神经元的细胞系。
(8)鉴定原理示意图如图3所示，若5TP和3TP有扩增产物，而TN在无扩增产物则左右同源臂之间的T2A-EGFP-CMV-PuroR序列完整插入到TPH2基因的终止密码子5’端，且是纯合子；若5TP、3TP以及TN都有扩增产物，则只在一条染色体上插入，即为杂合子；若5TP、3TP无扩增产物，而TN有扩增产物则未插入；若5TP和3TP只有一条有扩增产物则为部分插入。TPH2报告基因细胞系为杂合子，鉴定如图4所示。
(9)鉴定序列如下：
5TP序列扩增序列长度为1623bp；
5TP-F：5’-AGACCTGGACTAAAGCCCCA-3’(SEQ ID NO 13)
5TP-R：5’-TCGACGTCACCGCATGTTAG-3’(SEQ ID NO 14)
3TP序列扩增序列长度为1848bp；
3TP-F：5’-CGGTGCCTGATTTAAACCGC-3’(SEQ ID NO 15)
3TP-R：5’-CTCCCTGATGTGTCTTTTAGCTCT-3’(SEQ ID NO 16)
TN序列扩增序列长度为507bp；
TN-F：5’-CCCTCGTACCAATGAGGGTT-3’(SEQ ID NO 17)
TN-R：5’-AGATGCAGTTTGGTTAAGGACA-3’(SEQ ID NO 18)。
实施例5：TPH2报告基因人多能干细胞系向血清素神经元定向诱导分化
将TPH2报告基因人多能干细胞按3-5*104/cm2种到Matrigel(Corning,货号354277)包被的六孔板，使用TeSRTM-E8TM(Stem Cell technology,货号05990)培养。细胞接种2-4天后，密度达到80％左右换神经诱导培养基(DMEM/F12(Gibco,货号11330-032):neurobasal(Gibco,货号21103049)(1:1),1×N2(Gibco,货号17502048),1×B27(Gibco,货号12587010),1×Non-Essential amino acids(Gibco,货号11140050),1×GlutaMAX(Gibco,货号35050061),2μM SB431542(TargetMol,货号T1726),2μM DMH1(TargetMol,货号T1942),1.4μM CHIR99021(TargetMol,货号T2310))培养一周。第二周1:3传到新的六孔板，在第一周培液的基础上添加0.5μg/ml SHH C25II(R&D Systems,货号464-SH-025)。第二周末1:3传代到六孔板，在第二周的基础上添加10ng/ml FGF4(PeproTech,货号100-31)培养,第三周末使用tryPLE(Gibco,货号12604021)消化成单细胞，每块玻片(提前铺PO(sigma-Ardrich,货号P3655)-laminin(Thermo fisher,货号23017015))种1万个血清素前体细胞,传代第二天换成神经分化培养基(neurobasal，1×N2，1×B27，1×NEAA，0.2mMvitamin C(sigma-Ardrich,货号A4403)，2.5μM DAPT(TargetMol,货号T6202)，10ng/mlGDNF(PeproTech,货号450-10)，10ng/ml BDNF(PeproTech,货号450-02)，1ng/ml TGFβ3(PeproTech,货号100-36E)，10ng/ml IGF1(PeproTech,货号100-11))。
神经元分化培养基培养两周后，细胞免疫荧光检测血清素以及绿色荧光蛋白的表达，如图5，绿色荧光(TPH2-EGFP)与红色荧光(5-HT)共定位。神经分化培养基培养四周检测表达绿色荧光蛋白的血清素神经元的电生理活动，如图6。全细胞膜片钳记录定向分化带绿色荧光的血清素神经元Na+/K+电流，Na+/K+通道在-40mV至+40mV下触发了向内的Na+电流和向外的K+电流。如图7，全细胞膜片钳记录定向分化带绿色荧光的血清素神经元的动作电位，从-14pA到+14pA注入电流触发的动作电位。
表1到表6的序列如下：
表1
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagagataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtagaaagtaataatttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaagtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg atatctgggg atttgatgccgttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagtcggtgctttt ttgttttaga gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgccaattctgca gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgaccgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac gtcaatgggtggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctattgacgtcaatg acggtaaatg gcccgcctgg cattgtgccc agtacatgac cttatgggac tttcctacttggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactctccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatgggggcggggg gggggggggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagccaatcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct ataaaaagcgaagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgccgcccgcccc ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgggctgtaatta gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaattacctggagcac ctgcctgaaa tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacgacggagacta caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcggaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaactctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaacaccgaccggca cagcatcaag aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggccacccggctg aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagagatcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtggaagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaagtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatctatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgacaacagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccatcaacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaatctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcctgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacacctacgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaagaacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctgagcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggcagcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgacggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgaggaactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatcccccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaaggacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggggaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagtggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaacgagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtgaaatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacctgctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgcttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgctgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgctgaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgacgacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatcaacggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacagaaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc ccaggtgtccggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc cgccattaag aagggcatcctgcagacagt gaaggtggtg gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgatcgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatcgaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcagaacgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaaccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaacaaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaagaagatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg acaatctgaccaaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacccggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgacaagctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttccagttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc cgtcgtgggaaccgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacgtgcggaagat gatcgccaag agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaacatcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgagacaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctgagcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcctgcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttcgacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactgaagagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactttctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttcgagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccctgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgaggataatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagcgagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcaccgggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccctgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggacgccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcgacaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag taagaattcc tagagctcgctgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttgaccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtaggtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcaggcatgctgggg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgctcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcgagcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacaccgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttacgcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc ttcctttctcgccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctttacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagacggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaacactcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaaatgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcactctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcgccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgtgtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttataggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacccctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgcttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgcggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttgggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaagaacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgggcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaaaagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactgcggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatgggggatcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacaccacgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttcccggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggctggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactggggccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaaatagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcatatatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataatctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaaggatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagcggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcagataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgcctacatacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggttggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagcccagcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttcccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagcttccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgatttttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctggccttttgctg gccttttgct cacatgt (SEQ ID NO 19)
表2：pcDNA3.1-CMV-PuroR-polyA(SEQ ID NO 20)gacggatcgg gagatctcccgatcccctat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctgcttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccgacaattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacgcgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatatatggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccattgacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggagtatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacgtcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggcagtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtggatagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcaccaaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgtacggtgggag gtctatataa gcagagctct ctggctaact agagaaccca ctgcttactg gcttatcgaaattaatacga ctcactatag ggagacccaa gctgatgacc gagtacaagc ccacggtgcg cctcgccacccgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccacaccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggctcgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtcgaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgcagcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcggcgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgagcgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggcttcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgcctgatttaaa ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccccgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcgcattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattgggaagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctggggctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagcgtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgttcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggcacctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggtttttcgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaaccctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagctgatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccaggctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtccccaggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgcccctaactccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaattttttttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttggaggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca agagacaggatgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggctattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgcaggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcgcggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaagggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaaagtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccaccaagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctggacgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcgaggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctggattcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattgctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgcagcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa atgaccgaccaagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcggaatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccaccccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaagcatttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg tctgtataccgtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctcacaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaactcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatgaatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcgctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacagaatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggccgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcagaggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctcctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctcatagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccccccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgacttatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagttcttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagccagttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggtttttttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggggtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttcacctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctgacagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgcctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgataccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgcagaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtagttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgtttggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaaaagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggttatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtactcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggataataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctcaaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatcttttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataagggcgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattgtctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccccgaaaagtgc cacctgacgt c
表3：pUC19-T2A-EGFP-CMV-PuroR(SEQ ID NO 21)gagataccta cagcgtgagctatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaacaggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccacctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacgcggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctgattctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgcagcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccgattcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgtgagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaattgtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcaggtcgactctag aggatccgag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggcccagtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaacggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttcatctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtgcttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtccaggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcgacaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaagctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtgaacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacacccccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaagaccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatggacgagctgt acaaggaatt ctaactagag ctcgctgatc agcctcgact gtgccttcta gttgccagccatctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaataaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcaggacagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggaggcc agatatacgc gttgacattgattattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgcgttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataatgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggtaaactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggtaaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacgtattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttgactcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca aaatcaacgggactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggaggtctatataag cagagctctc tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgactcactatagg gagacccaag ctgatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgtccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccggaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggcaaggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggcggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatggaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccgaccaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggtgcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcaccgccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgatttaaaccgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttccttgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgagtaggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagcaggcatgctg gggatgcggt gggctctatg gcttaagcgc gctagcggta ccgagctcga attcactggccgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccccctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctgaatggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtgcactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgacgcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgcatgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctatttttataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcggaacccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaatgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttttgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcagttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccgaagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgccgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcacagaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataacactgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatgggggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgacaccacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagcttcccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttccggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactggggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaacgaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactcatatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgataatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatcaaaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctaccagcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcgcagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgcctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgggttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacagcccagcttgg agcgaacgac ctacaccgaa ct
表4:左同源臂序列(THL)(SEQ ID NO 22)tctctctgcc tctctctccc aactaatttcccccttaaat taagaaaaag aattaagagg agtatttctc aatatatagt tcacaaccac ttatattggagtcaacttgg atgcctttaa aatgaagatt tctaggtttc atccagacct gccaaatgaa attgagaccctggatatctg cattttcata agctcctcag tgattcttat gcacattgca tttttagaac catcaatctagggtctataa ataggcagca catggcagga aatgaccact aacttttaac atcactgaat ttatcttgagaatatagtca ttcattgttc cctgctgttc ttagtgctag tcatgtagtg agctgaacgg agctaatgatggttcaaaga gtaattacgc cagctttcct gtttttctaa tacctgctct tggcataact ctgactattaatattagctc cttttttcct caagtggcaa tatttatctg ctgtatattg ctctgctgta cttggtggaaggtaaatgaa tgctttttaa gtatcctcaa atatacaggc ccactccaga ctctgttgat acacagtacctggcctcaaa atatctccca atgaatagac aaatagttta gtcagagccc caattacctc tccatgcagataattattac ttctgccatt taagtgaaat tttagtacat tgcttatagc aataaatttt ggagcatatttatgctgtcc ctgcttctat tcatagcata gatttaaaat cccatttctg tgggggaata gctctgaactgtccttcagt atgaatttgg ctggtaataa attaaataat accacaatct cattttctcc cacagtacctagcatactta aaaaagcaaa acaaacaaac aaccaaacaa aatcctagtt tccaatttac cctgcacacaggagagttcc atatttcatg acagactagt aactgagcag ctctgtagga ttaccgagct atcaaatgtgttgtaaaaat gtgatgtcat ggagcttcgg aagtctcatt acagagttta acaggttttg tggtatattttgcagcacgc cctttctgac aaggcatgtg tgaaagcctt tgacccaaag acaacttgct tacaggaatgccttatcacc accttccagg aagcctactt tgtttcagaa agttttgaag aagccaaaga aaagatgaggtaaacttttt tttcctccta gctagagaaa ataacttttt atttttctgt ctctattcct tccttttatctatccctcgt accaatgagg gttgatcaca tctctttcta cttctgttta ttctgcaggg actttgcaaagtcaattacc cgtcccttct cagtatactt caatccctac acacagagta ttgaaattct gaaagacaccagaagtattg aaaatgtggt gcaggacctt cgcagcgact tgaatacagt gtgtgatgct ttaaacaaaatgaaccaata tctggggatt
表5：右同源臂序列(THR)(SEQ ID NO 23)tgatgcctgg aactatgttg ttgccagcatgatctttttg gggcttagca gcagttcagt caatgtcata taacgcaaat aaccttctgt gtcatggcttggctaataag catgcaattc catatatcta taccatcttg taactcactg tgttagtata taaagcaccataagaaatcc aatggcagat aaccactcat tgtatgaaat aacgtattat gtttaaacat cttaaaaagatttgacattc ctgcttagtg tccttaacca aactgcatct agttaaaatt tgtaacaaat agccctcttatgagtctcat ttatgccctt ttctttttca gatctaagcc tttcctctgt gttcattaga taaaatgaaaaaaagcagtg aagctgtttc cattttcaat agtatcagtg ttttcacgca ttatttgaga taaacccagaattgtaggaa acttcccatc acaataacaa aggttcaata ttctatttca aaaattgttg aggtaacacagcagttggaa tgatttttag gttgagtatt tacacaatgc aagaaaacac ctttttacaa atggaattatgtaggttgcg ttgaccttgt agaacctgag ttatgacaag cttcctgaag tattttggaa gatagtacttccggaaagga cattaggaaa gactaaacag tggacaatca atcttgggac tatgaatttt atgctggaataaagtaaatt atcatgttca ggtgtttaaa caattttttt ctggtcagag gagaataaaa tcacagagctataaagatca tctttgattt cagagatgtg gaaattgagt cacagagaag ttgtgatttg cctgaagcagtacagtgagt tgtacactaa cattcacagt ccattgcact ttccttttgt tttgctgata aattcgtcccagtgcttgca tgtgtttctg tgtcatccga gatgggaaac ttcatcttgg tgtgtgcatg tgttaatatgtcattggaga taggaaataa tgcctatcca aactaatgtt tatcagtaaa tagaacttgg tttgtggtcaagggttgcca actgtatata tgttgtttcc cctcacagct gcaaatcagg ccaaatctat ggaaggttctccactttcct aaagtttggg caactttggg taaacagtag tgatgcagca cacagttttg gtggtctttgcagtaaaaga tgaaattaag tggttaagtg aacaaataga aaactgctga gagtgaatga atttctttggattccctcac tgaacataac tactgttctt ctgattctgc atttggtagt aaggaacact ggactggggaaggaaggggc aagaaaaact agagaaaaaa aatgacaaaa taacctttag caatttaaaa tatttacttgagcaatttcc aaagatgttc ttttagtaag aaattacata taattttaaa ctaattcata ctcttatgtggagcccacaa agggtcagaa atc
表6：TPH2-T2A-EGFP(SEQ ID NO 24)gagataccta cagcgtgagc tatgagaaagcgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgcacgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttgagcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggcctttttacggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggataaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcagtgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatgcagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctcactcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggataacaatttca cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagt ctctctgcctctctctccca actaatttcc cccttaaatt aagaaaaaga attaagagga gtatttctca atatatagttcacaaccact tatattggag tcaacttgga tgcctttaaa atgaagattt ctaggtttca tccagacctgccaaatgaaa ttgagaccct ggatatctgc attttcataa gctcctcagt gattcttatg cacattgcatttttagaacc atcaatctag ggtctataaa taggcagcac atggcaggaa atgaccacta acttttaacatcactgaatt tatcttgaga atatagtcat tcattgttcc ctgctgttct tagtgctagt catgtagtgagctgaacgga gctaatgatg gttcaaagag taattacgcc agctttcctg tttttctaat acctgctcttggcataactc tgactattaa tattagctcc ttttttcctc aagtggcaat atttatctgc tgtatattgctctgctgtac ttggtggaag gtaaatgaat gctttttaag tatcctcaaa tatacaggcc cactccagactctgttgata cacagtacct ggcctcaaaa tatctcccaa tgaatagaca aatagtttag tcagagccccaattacctct ccatgcagat aattattact tctgccattt aagtgaaatt ttagtacatt gcttatagcaataaattttg gagcatattt atgctgtccc tgcttctatt catagcatag atttaaaatc ccatttctgtgggggaatag ctctgaactg tccttcagta tgaatttggc tggtaataaa ttaaataata ccacaatctcattttctccc acagtaccta gcatacttaa aaaagcaaaa caaacaaaca accaaacaaa atcctagtttccaatttacc ctgcacacag gagagttcca tatttcatga cagactagta actgagcagc tctgtaggattaccgagcta tcaaatgtgt tgtaaaaatg tgatgtcatg gagcttcgga agtctcatta cagagtttaacaggttttgt ggtatatttt gcagcacgcc ctttctgaca aggcatgtgt gaaagccttt gacccaaagacaacttgctt acaggaatgc cttatcacca ccttccagga agcctacttt gtttcagaaa gttttgaagaagccaaagaa aagatgaggt aaactttttt ttcctcctag ctagagaaaa taacttttta tttttctgtctctattcctt ccttttatct atccctcgta ccaatgaggg ttgatcacat ctctttctac ttctgtttattctgcaggga ctttgcaaag tcaattaccc gtcccttctc agtatacttc aatccctaca cacagagtattgaaattctg aaagacacca gaagtattga aaatgtggtg caggaccttc gcagcgactt gaatacagtgtgtgatgctt taaacaaaat gaaccaatat ctggggattg agggcagagg aagtctgcta acatgcggtgacgtcgagga gaatcctggc ccagtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggtcgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctacggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgaccaccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtccgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgcgccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggaggacggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaagcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgccgaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagcacccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgccgccgggatc actctcggca tggacgagct gtacaaggaa ttctaactag agctcgctga tcagcctcgactgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgccactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctattctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggaggccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttcatagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgacccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtcaatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgccccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggactttcctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatcaatgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagtttgttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggcggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttactggcttatcga aattaatacg actcactata gggagaccca agctgatgac cgagtacaag cccacggtgcgcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgccacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacgcgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgccggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccggctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctggccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtggaggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacgagcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgcaagcccggtg cctgatttaa accgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtttgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgaggaaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaagggggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttaagc gctgatgcctggaactatgt tgttgccagc atgatctttt tggggcttag cagcagttca gtcaatgtca tataacgcaaataaccttct gtgtcatggc ttggctaata agcatgcaat tccatatatc tataccatct tgtaactcactgtgttagta tataaagcac cataagaaat ccaatggcag ataaccactc attgtatgaa ataacgtattatgtttaaac atcttaaaaa gatttgacat tcctgcttag tgtccttaac caaactgcat ctagttaaaatttgtaacaa atagccctct tatgagtctc atttatgccc ttttcttttt cagatctaag cctttcctctgtgttcatta gataaaatga aaaaaagcag tgaagctgtt tccattttca atagtatcag tgttttcacgcattatttga gataaaccca gaattgtagg aaacttccca tcacaataac aaaggttcaa tattctatttcaaaaattgt tgaggtaaca cagcagttgg aatgattttt aggttgagta tttacacaat gcaagaaaacacctttttac aaatggaatt atgtaggttg cgttgacctt gtagaacctg agttatgaca agcttcctgaagtattttgg aagatagtac ttccggaaag gacattagga aagactaaac agtggacaat caatcttgggactatgaatt ttatgctgga ataaagtaaa ttatcatgtt caggtgttta aacaattttt ttctggtcagaggagaataa aatcacagag ctataaagat catctttgat ttcagagatg tggaaattga gtcacagagaagttgtgatt tgcctgaagc agtacagtga gttgtacact aacattcaca gtccattgca ctttccttttgttttgctga taaattcgtc ccagtgcttg catgtgtttc tgtgtcatcc gagatgggaa acttcatcttggtgtgtgca tgtgttaata tgtcattgga gataggaaat aatgcctatc caaactaatg tttatcagtaaatagaactt ggtttgtggt caagggttgc caactgtata tatgttgttt cccctcacag ctgcaaatcaggccaaatct atggaaggtt ctccactttc ctaaagtttg ggcaactttg ggtaaacagt agtgatgcagcacacagttt tggtggtctt tgcagtaaaa gatgaaatta agtggttaag tgaacaaata gaaaactgctgagagtgaat gaatttcttt ggattccctc actgaacata actactgttc ttctgattct gcatttggtagtaaggaaca ctggactggg gaaggaaggg gcaagaaaaa ctagagaaaa aaaatgacaa aataacctttagcaatttaa aatatttact tgagcaattt ccaaagatgt tcttttagta agaaattaca tataattttaaactaattca tactcttatg tggagcccac aaagggtcag aaatcgagct cgaattcact ggccgtcgttttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcgccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcgaatggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctcagtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccctgacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtcagaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggttaatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaacccctatttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttcaataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggcattttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtgcacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacgttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaagagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagcatcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggccaacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcatgtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacgatgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggcaacaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggctggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccagatggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatagacagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatatactttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctcatgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatcttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtggtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagataccaaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacatacctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggactcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagcttggagcgaac gacctacacc gaact
对比例1：单链向导RNA靶向序列未覆盖终止密码子导致Cas9蛋白二次切割
(1)使用sgRNA设计网站(网址：https://zlab.bio/guide-design-resources)获得一系列候选sgRNA序列，选择距离终止密码子上游6bp且切割效率高、脱靶效率低的sgRNA，具体序列如下：
sgT-F1:caccGACAAAATGAACCAATATCTG(SEQ ID NO 25)
sgT-R1:aaacCAGATATTGGTTCATTTTGTC(SEQ ID NO 26)
划线序列是BbSI位点酶切后互补的粘性末端。
(2)根据实施例3所述的方法获得PX330-sgT1载体。
(3)根据实施例4所述的方法获得TPH2-EGFP报告血清素神经元的细胞系，并进行鉴定。测序结果显示虽然在TPH2终止密码子上游成功插入了报告基因，但是由于sgRNA又重新识别靶向序列，引导Cas9蛋白二次切割，导致TPH2基因在该位点发生碱基缺失。本发明专利将sgRNA序列覆盖TPH2基因终止密码子，插入报告基因后原始sgRNA识别序列被破坏，从而防止二次切割。
对比例2：TPH2-EGFP人多能干细胞系的构建方法的优化
将质粒导入到细胞种常见的方法包括脂质体转染，PEI转和电穿孔等，我们尝试并比较了脂质体转染，PEI转和电穿孔方法的转染效率，发现电穿孔的效率更高。并对电穿孔的过程进一步优化，具体实施方法如下：
(1)根据实施例4所述的步骤(1)-(4)获得电转的细胞，按照2*103/cm2、8*103/cm2、3.2*104/cm2的密度种到铺了X射线处理过的小鼠成纤维细胞的六孔板。
(2)根据实施例4所述的步骤(6)进行药筛，电转后10-17天，2*103/cm2最终存活的克隆少，3.2*104/cm2接种密度最终克隆密度大，不方便挑取单克隆，8*103/cm2接种密度最为合适。
对比例3：TPH2-EGFP人多能干细胞系的构建方法的优化
(1)根据实施例4所述的步骤(1)-(4)获得电转后的细胞，分别在电转24小时后使用0.2μg/ml、0.3μg/ml、0.4μg/ml、0.5μg/ml嘌呤霉素药筛一周。
(2)根据实施例4所述的步骤(6)-(9)挑取克隆进行验证，最终0.4μg/ml嘌呤霉素处理组的阳性克隆的概率最高。
对比例4：无荧光标记的血清素神经元的电生理检测
无荧光标记的分化来源的血清素神经元电生理检测，通常需要检测多个细胞，并给检测的细胞注入荧光染料，最终通过细胞免疫荧光检测血清素神经元标志物，两者共定位才能确定检测的神经元即为血清素神经元。本发明专利通过TPH2-EGFP绿色荧光指示血清素神经元，从而减少电生理检测过程的工作量，提高检测效率。
综上所述，本发明的TPH2报告基因人多能干细胞系体外定向诱导分化来源的血清素神经元特异性表达绿色荧光蛋白，可以合成血清素神经递质并具有成熟的电生理功能。绿色荧光标记的血清素神经元有利于在混合的分化体系中指示出血清素神经元，从而有利于对人类血清素元形态和功能的研究，在重度抑郁症等疾病的药物筛选方面具有广阔的应用前景和巨大的市场价值。
申请人声明，本发明通过实施例详细说明了TPH2报告基因人多能干细胞系的构建方法以及应用，但本发明不局限于实施例所述的实验方法。对本发明的任何改进，包括对本发明内所使用的细胞系以及质粒的替换、具体实施方案等，均属于本发明的保护范围和公开范围之内。
上述相关说明以及对实施例的描述是为便于该技术领域的普通技术人员能理解和应用本发明。熟悉本领域技术的人员显然可以容易地对这些内容做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述相关说明以及对实施例的描述，本领域的技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 上海市东方医院
同济大学
<120> 人类血清素神经元TPH2报告基因细胞系的构建方法及应用
<160> 26
<170> SIPOSequenceListing 1.0
<210> 1
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gggagaccca agctgatgac cgagtacaag cccacgg 37
<210> 2
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gatcagcggt ttaaatcagg caccgggctt gcg 33
<210> 3
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
catgctgggg aggccagata tacgcgttga c 31
<210> 4
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
agtgaattcg agctcggtac cgcgcttaag ccatagagcc caccgcatc 49
<210> 5
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
caggtcgact ctagaggatc cgagggcaga ggaagtctgc ta 42
<210> 6
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tatctggcct ccccagcatg cctgctattc 30
<210> 7
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cttgcatgcc tgcagtctct ctgcctctct ctccc 35
<210> 8
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
acttcctctg ccctcaatcc ccagatattg gttcatttt 39
<210> 9
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ctatggctta agcgctgatg cctggaacta tgttgt 36
<210> 10
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
agtgaattcg agctcgattt ctgacccttt gtgggc 36
<210> 11
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
caccggatat ctggggattt gatgcc 26
<210> 12
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aaacggcatc aaatccccag atatc 25
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
agacctggac taaagcccca 20
<210> 14
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tcgacgtcac cgcatgttag 20
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
cggtgcctga tttaaaccgc 20
<210> 16
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
ctccctgatg tgtcttttag ctct 24
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ccctcgtacc aatgagggtt 20
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
agatgcagtt tggttaagga ca 22
<210> 19
<211> 8487
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg atatctgggg atttgatgcc gttttagagc tagaaatagc aagttaaaat 300
aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgttttaga 360
gctagaaata gcaagttaaa ataaggctag tccgttttta gcgcgtgcgc caattctgca 420
gacaaatggc tctagaggta cccgttacat aacttacggt aaatggcccg cctggctgac 480
cgcccaacga cccccgccca ttgacgtcaa tagtaacgcc aatagggact ttccattgac 540
gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata 600
tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattgtgccc 660
agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta 720
ttaccatggt cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc 780
cacccccaat tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg 840
gggggggggg gcggggcgag gggcggggcg gggcgaggcg gagaggtgcg gcggcagcca 900
atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg cggcggccct 960
ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc cccgtgcccc 1020
gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg 1080
tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt 1140
ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa 1200
tcactttttt tcaggttgga ccggtgccac catggactat aaggaccacg acggagacta 1260
caaggatcat gatattgatt acaaagacga tgacgataag atggccccaa agaagaagcg 1320
gaaggtcggt atccacggag tcccagcagc cgacaagaag tacagcatcg gcctggacat 1380
cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa 1440
attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct 1500
gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag 1560
atacaccaga cggaagaacc ggatctgcta tctgcaagag atcttcagca acgagatggc 1620
caaggtggac gacagcttct tccacagact ggaagagtcc ttcctggtgg aagaggataa 1680
gaagcacgag cggcacccca tcttcggcaa catcgtggac gaggtggcct accacgagaa 1740
gtaccccacc atctaccacc tgagaaagaa actggtggac agcaccgaca aggccgacct 1800
gcggctgatc tatctggccc tggcccacat gatcaagttc cggggccact tcctgatcga 1860
gggcgacctg aaccccgaca acagcgacgt ggacaagctg ttcatccagc tggtgcagac 1920
ctacaaccag ctgttcgagg aaaaccccat caacgccagc ggcgtggacg ccaaggccat 1980
cctgtctgcc agactgagca agagcagacg gctggaaaat ctgatcgccc agctgcccgg 2040
cgagaagaag aatggcctgt tcggaaacct gattgccctg agcctgggcc tgacccccaa 2100
cttcaagagc aacttcgacc tggccgagga tgccaaactg cagctgagca aggacaccta 2160
cgacgacgac ctggacaacc tgctggccca gatcggcgac cagtacgccg acctgtttct 2220
ggccgccaag aacctgtccg acgccatcct gctgagcgac atcctgagag tgaacaccga 2280
gatcaccaag gcccccctga gcgcctctat gatcaagaga tacgacgagc accaccagga 2340
cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct gagaagtaca aagagatttt 2400
cttcgaccag agcaagaacg gctacgccgg ctacattgac ggcggagcca gccaggaaga 2460
gttctacaag ttcatcaagc ccatcctgga aaagatggac ggcaccgagg aactgctcgt 2520
gaagctgaac agagaggacc tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc 2580
ccaccagatc cacctgggag agctgcacgc cattctgcgg cggcaggaag atttttaccc 2640
attcctgaag gacaaccggg aaaagatcga gaagatcctg accttccgca tcccctacta 2700
cgtgggccct ctggccaggg gaaacagcag attcgcctgg atgaccagaa agagcgagga 2760
aaccatcacc ccctggaact tcgaggaagt ggtggacaag ggcgcttccg cccagagctt 2820
catcgagcgg atgaccaact tcgataagaa cctgcccaac gagaaggtgc tgcccaagca 2880
cagcctgctg tacgagtact tcaccgtgta taacgagctg accaaagtga aatacgtgac 2940
cgagggaatg agaaagcccg ccttcctgag cggcgagcag aaaaaggcca tcgtggacct 3000
gctgttcaag accaaccgga aagtgaccgt gaagcagctg aaagaggact acttcaagaa 3060
aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa gatcggttca acgcctccct 3120
gggcacatac cacgatctgc tgaaaattat caaggacaag gacttcctgg acaatgagga 3180
aaacgaggac attctggaag atatcgtgct gaccctgaca ctgtttgagg acagagagat 3240
gatcgaggaa cggctgaaaa cctatgccca cctgttcgac gacaaagtga tgaagcagct 3300
gaagcggcgg agatacaccg gctggggcag gctgagccgg aagctgatca acggcatccg 3360
ggacaagcag tccggcaaga caatcctgga tttcctgaag tccgacggct tcgccaacag 3420
aaacttcatg cagctgatcc acgacgacag cctgaccttt aaagaggaca tccagaaagc 3480
ccaggtgtcc ggccagggcg atagcctgca cgagcacatt gccaatctgg ccggcagccc 3540
cgccattaag aagggcatcc tgcagacagt gaaggtggtg gacgagctcg tgaaagtgat 3600
gggccggcac aagcccgaga acatcgtgat cgaaatggcc agagagaacc agaccaccca 3660
gaagggacag aagaacagcc gcgagagaat gaagcggatc gaagagggca tcaaagagct 3720
gggcagccag atcctgaaag aacaccccgt ggaaaacacc cagctgcaga acgagaagct 3780
gtacctgtac tacctgcaga atgggcggga tatgtacgtg gaccaggaac tggacatcaa 3840
ccggctgtcc gactacgatg tggaccatat cgtgcctcag agctttctga aggacgactc 3900
catcgacaac aaggtgctga ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc 3960
ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg cagctgctga acgccaagct 4020
gattacccag agaaagttcg acaatctgac caaggccgag agaggcggcc tgagcgaact 4080
ggataaggcc ggcttcatca agagacagct ggtggaaacc cggcagatca caaagcacgt 4140
ggcacagatc ctggactccc ggatgaacac taagtacgac gagaatgaca agctgatccg 4200
ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc gatttccgga aggatttcca 4260
gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc cacgacgcct acctgaacgc 4320
cgtcgtggga accgccctga tcaaaaagta ccctaagctg gaaagcgagt tcgtgtacgg 4380
cgactacaag gtgtacgacg tgcggaagat gatcgccaag agcgagcagg aaatcggcaa 4440
ggctaccgcc aagtacttct tctacagcaa catcatgaac tttttcaaga ccgagattac 4500
cctggccaac ggcgagatcc ggaagcggcc tctgatcgag acaaacggcg aaaccgggga 4560
gatcgtgtgg gataagggcc gggattttgc caccgtgcgg aaagtgctga gcatgcccca 4620
agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc ttcagcaaag agtctatcct 4680
gcccaagagg aacagcgata agctgatcgc cagaaagaag gactgggacc ctaagaagta 4740
cggcggcttc gacagcccca ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa 4800
gggcaagtcc aagaaactga agagtgtgaa agagctgctg gggatcacca tcatggaaag 4860
aagcagcttc gagaagaatc ccatcgactt tctggaagcc aagggctaca aagaagtgaa 4920
aaaggacctg atcatcaagc tgcctaagta ctccctgttc gagctggaaa acggccggaa 4980
gagaatgctg gcctctgccg gcgaactgca gaagggaaac gaactggccc tgccctccaa 5040
atatgtgaac ttcctgtacc tggccagcca ctatgagaag ctgaagggct cccccgagga 5100
taatgagcag aaacagctgt ttgtggaaca gcacaagcac tacctggacg agatcatcga 5160
gcagatcagc gagttctcca agagagtgat cctggccgac gctaatctgg acaaagtgct 5220
gtccgcctac aacaagcacc gggataagcc catcagagag caggccgaga atatcatcca 5280
cctgtttacc ctgaccaatc tgggagcccc tgccgccttc aagtactttg acaccaccat 5340
cgaccggaag aggtacacca gcaccaaaga ggtgctggac gccaccctga tccaccagag 5400
catcaccggc ctgtacgaga cacggatcga cctgtctcag ctgggaggcg acaaaaggcc 5460
ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag taagaattcc tagagctcgc 5520
tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg 5580
ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt 5640
gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc 5700
aagggggagg attgggaaga gaatagcagg catgctgggg agcggccgca ggaaccccta 5760
gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca 5820
aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc 5880
tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac 5940
cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg 6000
tggtggttac gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg 6060
ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 6120
ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 6180
tgggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 6240
tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta 6300
tctcgggcta ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa 6360
atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt 6420
tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc 6480
cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac 6540
aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac 6600
gcgcgagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa 6660
tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 6720
tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 6780
ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 6840
ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 6900
aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 6960
gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 7020
ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 7080
gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 7140
cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 7200
cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 7260
acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 7320
caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 7380
taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 7440
ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 7500
aatctggagc cggtgagcgt ggaagccgcg gtatcattgc agcactgggg ccagatggta 7560
agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 7620
atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 7680
tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 7740
tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 7800
gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 7860
taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 7920
aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 7980
ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 8040
catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 8100
ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 8160
ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 8220
agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 8280
taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 8340
atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 8400
cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 8460
ccttttgctg gccttttgct cacatgt 8487
<210> 20
<211> 5921
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctgatgacc 900
gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc 960
ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac 1020
atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc 1080
aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc 1140
gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg 1200
ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg 1260
tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc 1320
gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt cctggagacc 1380
tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc 1440
gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc ctgatttaaa 1500
ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc 1560
cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga 1620
aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga 1680
cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat 1740
ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg cgccctgtag 1800
cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag 1860
cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 1920
tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 1980
cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata 2040
gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 2100
aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc 2160
gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattaatt 2220
ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt 2280
atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 2340
gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 2400
actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 2460
ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag 2520
tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata 2580
tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 2640
ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 2700
caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 2760
gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg 2820
cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 2880
gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 2940
caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 3000
cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 3060
actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 3120
gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc 3180
gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 3240
ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 3300
cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 3360
atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 3420
gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt 3480
tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg 3540
gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt 3600
ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 3660
catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 3720
tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 3780
tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 3840
aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 3900
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 3960
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 4020
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 4080
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 4140
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 4200
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 4260
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4320
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 4380
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4440
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 4500
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 4560
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 4620
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 4680
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 4740
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 4800
aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 4860
ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 4920
acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 4980
ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 5040
gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 5100
taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 5160
tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 5220
gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 5280
cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 5340
aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 5400
cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 5460
tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 5520
gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 5580
tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 5640
gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 5700
ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 5760
cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 5820
agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 5880
gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c 5921
<210> 21
<211> 5232
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 60
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 120
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 180
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 240
acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 300
ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac 360
gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc 420
tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa 480
agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc 540
tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca 600
cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagg tcgactctag 660
aggatccgag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc 720
agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg 780
cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg 840
caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct 900
cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca 960
gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt 1020
caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt 1080
gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa 1140
gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg 1200
catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga 1260
ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta 1320
cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct 1380
gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaaggaatt 1440
ctaactagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 1500
tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 1560
taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 1620
gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc tggggaggcc 1680
agatatacgc gttgacattg attattgact agttattaat agtaatcaat tacggggtca 1740
ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct 1800
ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta 1860
acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac 1920
ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt 1980
aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag 2040
tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat 2100
gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat 2160
gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc 2220
ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc 2280
tggctaacta gagaacccac tgcttactgg cttatcgaaa ttaatacgac tcactatagg 2340
gagacccaag ctgatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt 2400
ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac 2460
cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg 2520
cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg 2580
gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc 2640
cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca 2700
ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg 2760
caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt 2820
gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt 2880
caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa 2940
gcccggtgcc tgatttaaac cgctgatcag cctcgactgt gccttctagt tgccagccat 3000
ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc 3060
tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg 3120
ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg 3180
gggatgcggt gggctctatg gcttaagcgc gctagcggta ccgagctcga attcactggc 3240
cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc 3300
agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 3360
ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc tccttacgca 3420
tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct ctgatgccgc 3480
atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 3540
gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 3600
gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 3660
ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 3720
tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 3780
gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 3840
acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 3900
cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 3960
catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 4020
tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 4080
cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 4140
accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 4200
cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 4260
ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 4320
accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 4380
ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 4440
attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 4500
ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 4560
tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 4620
tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 4680
gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 4740
tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 4800
ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 4860
ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 4920
agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 4980
cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt 5040
caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 5100
tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 5160
ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 5220
ctacaccgaa ct 5232
<210> 22
<211> 1520
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
tctctctgcc tctctctccc aactaatttc ccccttaaat taagaaaaag aattaagagg 60
agtatttctc aatatatagt tcacaaccac ttatattgga gtcaacttgg atgcctttaa 120
aatgaagatt tctaggtttc atccagacct gccaaatgaa attgagaccc tggatatctg 180
cattttcata agctcctcag tgattcttat gcacattgca tttttagaac catcaatcta 240
gggtctataa ataggcagca catggcagga aatgaccact aacttttaac atcactgaat 300
ttatcttgag aatatagtca ttcattgttc cctgctgttc ttagtgctag tcatgtagtg 360
agctgaacgg agctaatgat ggttcaaaga gtaattacgc cagctttcct gtttttctaa 420
tacctgctct tggcataact ctgactatta atattagctc cttttttcct caagtggcaa 480
tatttatctg ctgtatattg ctctgctgta cttggtggaa ggtaaatgaa tgctttttaa 540
gtatcctcaa atatacaggc ccactccaga ctctgttgat acacagtacc tggcctcaaa 600
atatctccca atgaatagac aaatagttta gtcagagccc caattacctc tccatgcaga 660
taattattac ttctgccatt taagtgaaat tttagtacat tgcttatagc aataaatttt 720
ggagcatatt tatgctgtcc ctgcttctat tcatagcata gatttaaaat cccatttctg 780
tgggggaata gctctgaact gtccttcagt atgaatttgg ctggtaataa attaaataat 840
accacaatct cattttctcc cacagtacct agcatactta aaaaagcaaa acaaacaaac 900
aaccaaacaa aatcctagtt tccaatttac cctgcacaca ggagagttcc atatttcatg 960
acagactagt aactgagcag ctctgtagga ttaccgagct atcaaatgtg ttgtaaaaat 1020
gtgatgtcat ggagcttcgg aagtctcatt acagagttta acaggttttg tggtatattt 1080
tgcagcacgc cctttctgac aaggcatgtg tgaaagcctt tgacccaaag acaacttgct 1140
tacaggaatg ccttatcacc accttccagg aagcctactt tgtttcagaa agttttgaag 1200
aagccaaaga aaagatgagg taaacttttt tttcctccta gctagagaaa ataacttttt 1260
atttttctgt ctctattcct tccttttatc tatccctcgt accaatgagg gttgatcaca 1320
tctctttcta cttctgttta ttctgcaggg actttgcaaa gtcaattacc cgtcccttct 1380
cagtatactt caatccctac acacagagta ttgaaattct gaaagacacc agaagtattg 1440
aaaatgtggt gcaggacctt cgcagcgact tgaatacagt gtgtgatgct ttaaacaaaa 1500
tgaaccaata tctggggatt 1520
<210> 23
<211> 1523
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
tgatgcctgg aactatgttg ttgccagcat gatctttttg gggcttagca gcagttcagt 60
caatgtcata taacgcaaat aaccttctgt gtcatggctt ggctaataag catgcaattc 120
catatatcta taccatcttg taactcactg tgttagtata taaagcacca taagaaatcc 180
aatggcagat aaccactcat tgtatgaaat aacgtattat gtttaaacat cttaaaaaga 240
tttgacattc ctgcttagtg tccttaacca aactgcatct agttaaaatt tgtaacaaat 300
agccctctta tgagtctcat ttatgccctt ttctttttca gatctaagcc tttcctctgt 360
gttcattaga taaaatgaaa aaaagcagtg aagctgtttc cattttcaat agtatcagtg 420
ttttcacgca ttatttgaga taaacccaga attgtaggaa acttcccatc acaataacaa 480
aggttcaata ttctatttca aaaattgttg aggtaacaca gcagttggaa tgatttttag 540
gttgagtatt tacacaatgc aagaaaacac ctttttacaa atggaattat gtaggttgcg 600
ttgaccttgt agaacctgag ttatgacaag cttcctgaag tattttggaa gatagtactt 660
ccggaaagga cattaggaaa gactaaacag tggacaatca atcttgggac tatgaatttt 720
atgctggaat aaagtaaatt atcatgttca ggtgtttaaa caattttttt ctggtcagag 780
gagaataaaa tcacagagct ataaagatca tctttgattt cagagatgtg gaaattgagt 840
cacagagaag ttgtgatttg cctgaagcag tacagtgagt tgtacactaa cattcacagt 900
ccattgcact ttccttttgt tttgctgata aattcgtccc agtgcttgca tgtgtttctg 960
tgtcatccga gatgggaaac ttcatcttgg tgtgtgcatg tgttaatatg tcattggaga 1020
taggaaataa tgcctatcca aactaatgtt tatcagtaaa tagaacttgg tttgtggtca 1080
agggttgcca actgtatata tgttgtttcc cctcacagct gcaaatcagg ccaaatctat 1140
ggaaggttct ccactttcct aaagtttggg caactttggg taaacagtag tgatgcagca 1200
cacagttttg gtggtctttg cagtaaaaga tgaaattaag tggttaagtg aacaaataga 1260
aaactgctga gagtgaatga atttctttgg attccctcac tgaacataac tactgttctt 1320
ctgattctgc atttggtagt aaggaacact ggactgggga aggaaggggc aagaaaaact 1380
agagaaaaaa aatgacaaaa taacctttag caatttaaaa tatttacttg agcaatttcc 1440
aaagatgttc ttttagtaag aaattacata taattttaaa ctaattcata ctcttatgtg 1500
gagcccacaa agggtcagaa atc 1523
<210> 24
<211> 8245
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 60
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 120
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 180
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 240
acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga 300
ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac 360
gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc 420
tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa 480
agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc 540
tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca 600
cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagt ctctctgcct 660
ctctctccca actaatttcc cccttaaatt aagaaaaaga attaagagga gtatttctca 720
atatatagtt cacaaccact tatattggag tcaacttgga tgcctttaaa atgaagattt 780
ctaggtttca tccagacctg ccaaatgaaa ttgagaccct ggatatctgc attttcataa 840
gctcctcagt gattcttatg cacattgcat ttttagaacc atcaatctag ggtctataaa 900
taggcagcac atggcaggaa atgaccacta acttttaaca tcactgaatt tatcttgaga 960
atatagtcat tcattgttcc ctgctgttct tagtgctagt catgtagtga gctgaacgga 1020
gctaatgatg gttcaaagag taattacgcc agctttcctg tttttctaat acctgctctt 1080
ggcataactc tgactattaa tattagctcc ttttttcctc aagtggcaat atttatctgc 1140
tgtatattgc tctgctgtac ttggtggaag gtaaatgaat gctttttaag tatcctcaaa 1200
tatacaggcc cactccagac tctgttgata cacagtacct ggcctcaaaa tatctcccaa 1260
tgaatagaca aatagtttag tcagagcccc aattacctct ccatgcagat aattattact 1320
tctgccattt aagtgaaatt ttagtacatt gcttatagca ataaattttg gagcatattt 1380
atgctgtccc tgcttctatt catagcatag atttaaaatc ccatttctgt gggggaatag 1440
ctctgaactg tccttcagta tgaatttggc tggtaataaa ttaaataata ccacaatctc 1500
attttctccc acagtaccta gcatacttaa aaaagcaaaa caaacaaaca accaaacaaa 1560
atcctagttt ccaatttacc ctgcacacag gagagttcca tatttcatga cagactagta 1620
actgagcagc tctgtaggat taccgagcta tcaaatgtgt tgtaaaaatg tgatgtcatg 1680
gagcttcgga agtctcatta cagagtttaa caggttttgt ggtatatttt gcagcacgcc 1740
ctttctgaca aggcatgtgt gaaagccttt gacccaaaga caacttgctt acaggaatgc 1800
cttatcacca ccttccagga agcctacttt gtttcagaaa gttttgaaga agccaaagaa 1860
aagatgaggt aaactttttt ttcctcctag ctagagaaaa taacttttta tttttctgtc 1920
tctattcctt ccttttatct atccctcgta ccaatgaggg ttgatcacat ctctttctac 1980
ttctgtttat tctgcaggga ctttgcaaag tcaattaccc gtcccttctc agtatacttc 2040
aatccctaca cacagagtat tgaaattctg aaagacacca gaagtattga aaatgtggtg 2100
caggaccttc gcagcgactt gaatacagtg tgtgatgctt taaacaaaat gaaccaatat 2160
ctggggattg agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc 2220
ccagtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 2280
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 2340
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 2400
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 2460
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 2520
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 2580
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 2640
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 2700
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 2760
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 2820
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 2880
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaaggaa 2940
ttctaactag agctcgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg 3000
tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct 3060
aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg 3120
gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat gctggggagg 3180
ccagatatac gcgttgacat tgattattga ctagttatta atagtaatca attacggggt 3240
cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc 3300
ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag 3360
taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc 3420
acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg 3480
gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc 3540
agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca 3600
atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca 3660
atgggagttt gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg 3720
ccccattgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc 3780
tctggctaac tagagaaccc actgcttact ggcttatcga aattaatacg actcactata 3840
gggagaccca agctgatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac 3900
gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac 3960
accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg 4020
cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc 4080
tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg 4140
gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg 4200
caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag 4260
ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg 4320
gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc 4380
ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc 4440
aagcccggtg cctgatttaa accgctgatc agcctcgact gtgccttcta gttgccagcc 4500
atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 4560
cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 4620
ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc 4680
tggggatgcg gtgggctcta tggcttaagc gctgatgcct ggaactatgt tgttgccagc 4740
atgatctttt tggggcttag cagcagttca gtcaatgtca tataacgcaa ataaccttct 4800
gtgtcatggc ttggctaata agcatgcaat tccatatatc tataccatct tgtaactcac 4860
tgtgttagta tataaagcac cataagaaat ccaatggcag ataaccactc attgtatgaa 4920
ataacgtatt atgtttaaac atcttaaaaa gatttgacat tcctgcttag tgtccttaac 4980
caaactgcat ctagttaaaa tttgtaacaa atagccctct tatgagtctc atttatgccc 5040
ttttcttttt cagatctaag cctttcctct gtgttcatta gataaaatga aaaaaagcag 5100
tgaagctgtt tccattttca atagtatcag tgttttcacg cattatttga gataaaccca 5160
gaattgtagg aaacttccca tcacaataac aaaggttcaa tattctattt caaaaattgt 5220
tgaggtaaca cagcagttgg aatgattttt aggttgagta tttacacaat gcaagaaaac 5280
acctttttac aaatggaatt atgtaggttg cgttgacctt gtagaacctg agttatgaca 5340
agcttcctga agtattttgg aagatagtac ttccggaaag gacattagga aagactaaac 5400
agtggacaat caatcttggg actatgaatt ttatgctgga ataaagtaaa ttatcatgtt 5460
caggtgttta aacaattttt ttctggtcag aggagaataa aatcacagag ctataaagat 5520
catctttgat ttcagagatg tggaaattga gtcacagaga agttgtgatt tgcctgaagc 5580
agtacagtga gttgtacact aacattcaca gtccattgca ctttcctttt gttttgctga 5640
taaattcgtc ccagtgcttg catgtgtttc tgtgtcatcc gagatgggaa acttcatctt 5700
ggtgtgtgca tgtgttaata tgtcattgga gataggaaat aatgcctatc caaactaatg 5760
tttatcagta aatagaactt ggtttgtggt caagggttgc caactgtata tatgttgttt 5820
cccctcacag ctgcaaatca ggccaaatct atggaaggtt ctccactttc ctaaagtttg 5880
ggcaactttg ggtaaacagt agtgatgcag cacacagttt tggtggtctt tgcagtaaaa 5940
gatgaaatta agtggttaag tgaacaaata gaaaactgct gagagtgaat gaatttcttt 6000
ggattccctc actgaacata actactgttc ttctgattct gcatttggta gtaaggaaca 6060
ctggactggg gaaggaaggg gcaagaaaaa ctagagaaaa aaaatgacaa aataaccttt 6120
agcaatttaa aatatttact tgagcaattt ccaaagatgt tcttttagta agaaattaca 6180
tataatttta aactaattca tactcttatg tggagcccac aaagggtcag aaatcgagct 6240
cgaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 6300
ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 6360
ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt 6420
ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc agtacaatct 6480
gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct 6540
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 6600
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga 6660
tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca 6720
cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 6780
tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 6840
gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 6900
ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 6960
cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 7020
ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 7080
cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 7140
tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 7200
tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 7260
tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 7320
ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 7380
tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 7440
cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 7500
gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 7560
ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 7620
acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 7680
cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 7740
atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 7800
tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 7860
tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 7920
aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 7980
aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt 8040
taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 8100
taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 8160
agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 8220
tggagcgaac gacctacacc gaact 8245
<210> 25
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
caccgacaaa atgaaccaat atctg 25
<210> 26
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
aaaccagata ttggttcatt ttgtc 25

Claims (11)

1.一种表达血清素神经元TPH2报告基因的载体系统，其特征在于：所述的载体系统包括但不限于：
靶向人类血清素神经元TPH2基因终止密码子附近的单链向导RNA的向导载体，和
携带人类血清素神经元TPH2基因终止密码子附近同源臂的供体载体。
2.如权利要求1所述的载体系统，其特征在于：
靶向人类血清素神经元TPH2基因终止密码子附近的单链向导RNA的向导载体选自：PX330-sgT、PX458-sgT或者PX459-sgT。
3.一种稳定的血清素神经元TPH2报告基因人多能干细胞系的构建方法，其特征在于：包括以下步骤：
(1)构建携带靶向TPH2基因终止密码子附近的单链向导RNA的向导载体PX330-sgT；
获得靶向人类TPH2基因终止密码子附近的sgRNA，合成两条寡聚核苷酸链，退火并磷酸化后插入到PX330的BbSI位点，重组载体转化大肠杆菌，挑取单克隆菌进行测序验证重组成功的质粒PX330-sgT；
(2)构建报告基因供体载体的工具载体pUC19-T2A-EGFP-CMV-PuroR；
从AAVS1-TRE3G-EGFP中扩增出puromycin的抗性基因，克隆到FLAG-HA-pcDNA3.1-表达载体的CMV启动子后，得到pcDNA3.1-CMV-PuroR-polyA载体，从此载体中扩增CMV-PuroR-polyA序列，同时从PX458中扩增T2A-EGFP，将扩增的两个片段重组到pUC19载体的多克隆位点，得到工具载体pUC19-T2A-EGFP-CMV-PuroR；
(3)构建TPH2报告基因的供体载体TPH2-T2A-EGFP；
从人的基因组中扩增TPH2终止密码子前后分别1.5kb的左右同源臂，THL和THR，在工具载体的左右两侧的多克隆位点分别插入THL和THR，得到TPH2报告基因供体载体TPH2-T2A-EGFP；
(4)TPH2报告基因人多能干细胞系的构建；
将向导载体PX330-sgT和供体载体TPH2-T2A-EGFP电转到人多能干细胞H9(WA09,WiCell)，经puromycin筛选，获得抗性克隆后，提取细胞基因组进行PCR扩增鉴定插入片段，并测序，最终获得稳定的EGFP标记的报告血清素神经元TPH2基因的人多能干细胞系。
4.根据权利要求3所述的血清素神经元TPH2报告基因人多能干细胞系的构建方法，其特征在于：
所述步骤1)中构建向导载体的工具载体包括但不限于：PX330、PX458或者PX459中的一种或者几种。
5.根据权利要求3所述的血清素神经元TPH2报告基因人多能干细胞系的构建方法，其特征在于：
所述步骤2)中用于构建打靶载体工具载体的报告基因元件包括但不限于tdTomato,YFP,RFP或者EGFP中的一种或者几种。
6.根据权利要求3所述的血清素神经元TPH2报告基因人多能干细胞系的构建方法，其特征在于：
所述步骤2)中用于构建打靶载体工具载体的抗性筛选元件包括但不限于PuroR、NeoR。
7.根据权利要求3所述的血清素神经元TPH2报告基因人多能干细胞系的构建方法，其特征在于：
所述步骤3)是从工具载体pUC19-T2A-EGFP-CMV-PuroR出发，插入TPH2基因的左右同源臂。
8.根据权利要求3-7任意一项所述的血清素神经元TPH2报告基因人多能干细胞系的构建方法，其特征在于：
所述步骤4)中电转的人多能干细胞系包括但不限于人胚胎干细胞系或者诱导多能干细胞系。
9.使用权利要求3-7中任意一种的血清素神经元TPH2报告基因人多能干细胞系的构建方法获得的血清素神经元TPH2报告基因人多能干细胞系。
10.根据权利要求9所述的血清素神经元TPH2报告基因人多能干细胞系，其特征在于，所述的细胞源自H9。
11.权利要求9或者10所述的血清素神经元TPH2报告基因多能干细胞系的应用，其特征在于，所述的血清素神经元TPH2报告基因人多能干细胞系在制备血清素神经元的定向分化药物、血清素神经元的形态和功能研究方面、血清素神经元的定向分化药物筛选方面的应用。
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