CN113005146A - 一种重组质粒及其构建方法、重组影像系统与应用 - Google Patents
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Abstract
本发明公开一种重组质粒及其构建方法、重组影像系统与应用。本发明针对MSI1/AGO2的蛋白‑蛋白交互作用，就生物光学‑萤光共振能量转移技术与萤光素酶分离互补系统在定性与定量上的可靠度、以及萤光素酶分离互补系统的优化进行探讨。经过测试不同长度与弹性/电荷分布的连接肽(linker peptide)，成功建构出一组较萤火虫萤光素酶(fireflyluciferase,FLuc)亮度高上千倍的桡脚类萤光素酶(Gaussia luciferase,GLuc)的重组影像系统；同时，萤火虫萤光素酶将作为定量分析时的标准化讯号。本发明将主要应用于临床前的细胞实验与动物实验，能够即时并且非侵入性地测量并定量脑肿瘤的治疗效果。
Description
技术领域
本发明涉及生物医药技术领域，尤其涉及一种重组质粒及其构建方法、重组影像系统与应用。
背景技术
恶性肿瘤的可视化在迈入21世纪的今日，无论在动物模式、药物前期开发、药代动力学、乃至于序贯治疗计画决断等方面，已经占据了生物医学方面科研面项的极大声量。分子影像学乃是一门针对分子机制可视化、具现化以及可量化等方面的学问。运用各种不同的分子设计与成像平台来达到非侵入式的分析。从20世纪60年代，绿色萤光蛋白为日本学者下村脩自海洋生物分离并定义以来，运用光学原理的各种影像技术应运而生。随着美籍华裔科学家钱永健对于绿色萤光蛋白的各种改良与研发，生物萤光影像在现今科研已经占有一席不可抹灭的地位。时至今日，各种运用萤光、生物冷光乃至于核子医学等多模态分子影像技术进行的科研项目不胜枚举。针对蛋白-蛋白交互作用的分子影像学研究已经颇具历史。在早期蛋白质组学领域，利用酵母菌双杂合系统(Yeast-2-hybrid system)已经相当成熟，然而其大多应用于真核生物，在哺乳类细胞内的效果并不显著。在生物化学的范畴内，免疫共沉淀实验被广为应用于验证蛋白-蛋白交互作用的主要工具，然而此技术完全无法应用于细胞，更遑论活体内的应用。随着萤光蛋白与生物光学的广泛研究，利用两个供体与受体生色团(chromophore)重叠的发散与激发光频谱形成的能量传递，可以做为特定分子足够靠近的证据，如此技术被称之为荧光共振能量转移(FRET)。而这项技术也在共聚焦显微成像技术成熟的今日，被大量地使用在动态观察分子交互作用的研究上。相较于前述几项技术，FRET在细胞生理学上的应用可见一斑，由于其具备量化与追踪的功能，使得分子信息得以可视化，可谓一大进步；然而，此技术由于仰赖雷射激活，而使得活体内的应用备受限制。一项取代激发光的生物光学-萤光共振能量转移技术(BRET)应运而生。此技术使用萤光素酶作为激发光源，由于萤光素酶发光仅需能量与受质，因此不受激发光源穿透组织的能量衰减影响，可以有效地将交互作用实时地藉由光学讯号表达出来。近来，另一类型的蛋白-蛋白交互作用影像系统被相继开发面世，萤光素酶分离互补系统(split luciferasecomplementation system)作为代表性的设计而为人熟知。概念上，首先将萤光素酶分为两段，将两个可能互结合的受测蛋白质分别标志上其中一段萤光素酶。倘若两个受测蛋白质因为蛋白-蛋白交互作用而足够靠近，两个萤光素酶片段或其结构将有可能重新结合而重新激活萤光素酶活性，发出光讯号。为了增加萤光素酶再激活的效率，许多修正版本的设计也如雨后春笋般出现，如使用Ozawa等人发表来自集胞藻属的内含肽(intein)序列加于萤光素酶的一端增加重合的机率，并且有效地非侵入地侦测动物体内的光讯号。
根据Inouye等人的研究说明桡脚类海洋生物分离之萤光素酶(Gaussialuciferase)蛋白质结构中，有两处明显具功能的催化域(catalytic domain)，其存在左右了此萤光素酶与其受质腔肠素(coelenterazine)交互作用而放出光亮的能力。相较于常见的萤火虫萤光素酶，Gaussia萤光素酶不需要耗能(ATP)，就可以与腔肠素作用并发出近千倍的亮度。不谋而合地，Renilla萤光素酶也是与腔肠素作用，虽然放出的光讯号较Gaussia较低，但一个关键因素在于Gaussia萤光素酶属于外泌蛋白，于胞浆制造后会被运输至胞外，而使量化效果出现误差。相对地，Renilla萤光素酶则存留于胞内，使放出的光讯号真实反映出发生之事件数(如蛋白-蛋白交互作用)。Paulmurugan等人曾利用Renilla萤光素酶分离互补技术(Split Renillia Luciferase Complementation)建立蛋白-蛋白交互作用成像系统，并且进一步的在小动物体内验证这项发展。前述研究中，Paulmurugan与Gambhir等人根据Renilla的催化域位置进行多个不同的蛋白截断试验，并且将截短Renilla萤光素酶与受测蛋白质MyoD间使用常见的中性连接肽(GGGGS)2进行串接；不过连接肽的存在是否影响萤光素酶的互补重合，仍待研究。Chen等人的回顾文章中整理了常用的几种连接肽，具较佳弹性的甘氨酸富集与丝氨酸交替之组合，如前述的(GGGGS)n，经常被使用于各种融合荧光蛋白等，避免影响生色团的物理共振现象。此外，也有较硬的连接肽如(EAAAK)n或(XP)n等仍待探讨。另一方面，Gaussia萤光素酶(约20千道尔顿)是一个相较于Renilla萤光素酶(36千道尔顿)或萤火虫萤光素酶(约61千道尔顿)更有效率且更小的萤光素酶。这意味着针对Gaussia萤光素酶的造影平台开发，是极具潜力且有效的方法。早前，Remy等人于2006年的一份研究中同样针对明显较多光讯号量的Gaussia萤光素酶作为蛋白结合作用的验证工具。Neveu等人在2012年的一份研究，应用了前述Remy开发的技术，将病毒感染过程可视化作为高通量筛选平台。
发明内容
本发明为运用分子影像之药物筛选平台，运用于不同的疾病模式进行规划与设计。动物模式的建立对于药物的前期开发有着举足轻重的角色。在迈入临床试验之前，无论是安全性测试、最大耐受剂量测试、毒理病理测试以及药代动力学等缺一不可。然而，动物个体间的差异往往造成实验结果的不确定性；同时，大量实验动物的需求也与世界趋势相悖。因此，将分子影像技术应用于动物模式开发，有效缩小实验动物数量上的需求，并且将个体间差异极小化，可望对科研领域的新药前期开发时程有效缩短，并可将此技术延伸至针对序贯治疗的安排做有效反馈。
本发明旨在创建一个针对MSI1/AGO2蛋白-蛋白交互作用解构化的高通量药筛平台，非但用于细胞层级，更能扩大至动物模式。其也是一种可定性、定量、高通量且稳定的影像平台，针对多型性胶质母细胞瘤生长速率、恶性度、耐药性以至于药物开发提供强而有力的信息。
具体地，本发明的技术方案如下：
一种重组质粒的构建方法，其中，包括步骤：
使用聚合酶链式反应将AGO2植入限制性内切酶切位HindIII与BamHI后进行扩增，得到HindIII-AGO2-BamHI片段；
将HindIII-AGO2-BamHI片段引入pcDNA载体的多克隆位点，得到pcDNA-AGO2-C1；
使用聚合酶链式反应将桡脚类萤光素酶GLuc的C端蛋白进行扩增，加入限制性内切酶切位BamHI与XbaI，并加入(GGGGS)2连接肽，得到(GGGGS)2-GLuc(C)片段；
将(GGGGS)2-GLuc(C)片段使用克隆技术载入pcDNA-AGO2-C1中，得到pcDNA-AGO2-GLuc(C)重组质粒。
一种重组质粒，其采用本发明如上所述的方法构建得到。
一种重组质粒的构建方法，其中，包括步骤：
以pCMV-GLuc1质粒为载体，使用聚合酶链式反应将桡脚类萤光素酶GLuc的N端加上(GGGGS)2连接肽与限制性内切酶切位BamHI与XbaI后，克隆制成pCMV-GLuc(N)；
将MSI1使用PCR扩增技术放大并纯化后加上限制性内切酶切位HindIII与BamHI，经克隆技术将MSI1载入pCMV-GLuc(N)中，得到pCMV-MSI1-GLuc(N)。
一种重组质粒，其采用本发明如上所述的方法构建得到。
一种融合蛋白，其由如上所述两种重组质粒融合得到。
一种重组影像系统，其包括本发明如上所述的两种重组质粒。
本发明如上所述的融合蛋白在作为或制备具有示踪性质产品中的应用。
本发明如上所述的融合蛋白在用于制备脑肿瘤治疗药物中的应用。
有益效果：本发明基于MSI1/AGO2的蛋白-蛋白交互作用，生物光学-萤光共振能量转移技术与萤光素酶分离互补系统，成功建构出一组较萤火虫萤光素酶(fireflyluciferase,FLuc)亮度高上千倍的桡脚类萤光素酶(Gaussia luciferase,GLuc)的重组影像系统；同时，萤火虫萤光素酶将作为定量分析时的标准化讯号。本发明将主要应用于临床前的细胞实验与动物实验，能够即时并且非侵入性地测量并定量脑肿瘤的治疗效果。
附图说明
图1为pcDNA-AGO2-C1以及pCMV-GLuc(N)的结构示意图。
图2为pcDNA-AGO2-GLuc(C)以及pCMV-MSI1-GLuc(N)的结构示意图。
图3为MSI1-Gluc(N)与AGO2-CGluc(C)的融合蛋白产物示意图。
图4为本发明重组影像系统的工作原理示意图。
图5为克隆制造的多个不同连接肽的重组质粒。
图6为不同片段冷光素酶的正常表现量示意图。
图7为正常与压力环境下的冷光量变化示意图。
图8为经不同压力诱发之相对冷光量测试结果示意图。
图9为于同一皮下植有脑肿瘤的小鼠使用本实施例影像系统，于给予治疗(Temozolomide)前后之影像示意图。
具体实施方式
本发明提供一种重组质粒及其构建方法、重组影像系统与应用，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
Argonaute-2(AGO2)蛋白质是RNA诱导沉默复合体的主要成员，并与非编译核糖核酸的调控有关。Musashi1(MSI1)蛋白质是一个已知的干细胞生物标记并被报导经由核糖核酸标靶而抑制基因转译。先前研究发现恶性胶质细胞瘤检体与药物抗性及MSI1表现有高度关联性，研究证实:1)MSI1高度表现会明显增加神经胶质恶性脑瘤之抗药性；2)更进一步观察到MSI1有往细胞质集中的现象发生；3)MSI1在抗癌药物刺激下，会明显造成细胞核内MSI1朝细胞质转移，并由AGO2与MSI1协同增强多形性胶质母细胞瘤(GBM)的抗药性与复发的分子作用机制。
基于此，本发明针对MSI1/AGO2的蛋白-蛋白交互作用，就生物光学-萤光共振能量转移技术与萤光素酶分离互补系统在定性与定量上的可靠度、以及萤光素酶分离互补系统的优化进行探讨。经过测试不同长度与弹性/电荷分布的连接肽(linker peptide)，成功建构出一组较萤火虫萤光素酶(firefly luciferase,FLuc)亮度高上千倍的桡脚类萤光素酶(Gaussia luciferase,GLuc)的重组影像系统；同时，萤火虫萤光素酶将作为定量分析时的标准化讯号。本发明将主要应用于临床前的细胞实验与动物实验，能够即时并且非侵入性地测量并定量脑肿瘤的治疗效果。本影像系统具有以下优势：
一、本重组影像系统即时将脑肿瘤细胞对压力反应可视化；
二、本重组影像系统由两个单独的质粒构成，具有高的稳定度；
三、冷光讯号为常用萤火虫冷光酶的千倍以上；
四、冷光讯号于3-5分钟即褪去，可用于持续的影像观测。
具体地，本发明提供的两种重组质粒，其结构如图1-2所示，其构建方法具体如下：
1、pcDNA-AGO2-GLuc(C)重组质粒
使用聚合酶链式反应将AGO2移除终止密码子并且植入限制性内切酶切位HindIII与BamHI后进行扩增，得到HindIII-AGO2-BamHI片段。然后以pcDNA质粒为载体，将HindIII-AGO2-BamHI片段引入pcDNA载体的多克隆位点，建构第一中间物pcDNA-AGO2-C1，其具有序列表中SEQ ID No.1所示的核苷酸序列；
以pCMV-GLuc1质粒为载体，使用聚合酶链式反应将桡脚类萤光素酶(GLuc)的C端蛋白进行扩增(107-185aa及终止密码子)，加入限制性内切酶切位BamHI与XbaI，并加入(GGGGS)2连接肽。将(GGGGS)2-GLuc(C)片段使用克隆技术载入前述pcDNA-AGO2-C1中间物，制成产物pcDNA-AGO2-GLuc(C)，其具有序列表中SEQ ID No.2所示的核苷酸序列。
2、pCMV-MSI1-GLuc(N)重组质粒
以pCMV-GLuc1质粒为载体，使用聚合酶链式反应将桡脚类萤光素酶(GLuc)的N端(1-106aa)加上(GGGGS)2连接肽与限制性内切酶切位BamHI与XbaI后，克隆制成第二中间物pCMV-GLuc(N)，其具有序列表中SEQ ID No.3所示的核苷酸序列；
将MSI1使用PCR扩增技术放大并纯化后加上限制性内切酶切位HindIII与BamHI，经克隆技术将MSI1安装于第二中间物pCMV-GLuc(N)之中，构成pCMV-MSI1-GLuc(N)，其具有序列表中SEQ ID No.4所示的核苷酸序列。
本发明提供的融合蛋白见图3所示，其由如上所述两种重组质粒融合得到。
图4为本发明重组影像系统的工作原理示意图，如图所示，将GLuc之C端与N端于第106号氨基酸处断开，分别利用分子克隆将C侧片段(106氨基酸长度)与N侧片段(78氨基酸长度)融合于AGO2与MSI1蛋白的C侧，透过连接肽(GGGGS)2来减低主蛋白质可能造成的荧光素酶功能影响。MSI1在癌症细胞遭受压力，尤其是抗癌药物治疗后，会进行胞内运输，自细胞核移动至胞浆，同时与位处胞浆的AGO2发生蛋白蛋白交互作用。本发明利用这个特点，借由克隆于MSI1与AGO2上的两个荧光素酶片段重合，进而产生生物冷光讯号。借由影像撷取系统如盘式冷光仪(活体外)或是IVIS(活体)等造影并进行量化处理。
下面通过具体的实施例对本发明作进一步地说明。
根据图5，本实施例测试不同连接肽，以试图比较不同长度与弹性的连接肽是否影响冷光重合的效果。主要以两种类型(GGGGS)的弹性型与(PGPGP)的较硬型做比较。同时也比较单一连接肽或双倍连接肽(GGGGS-GGGGS；记为(GGGGS)2)的成效。克隆方式与前述质粒构筑相同，主要于引物设计上放入不同长度的连接肽基因序列来达成。研究发现：在缺乏连接肽的情况下，冷光讯号微乎其微。采用上述两种形式的连接肽进行测试，借由蛋白蛋白交互作用后的光学效果以及交互作用前的基础值来厘清最适合之设计。
应用与测试方式具体如下：
1、材料：
纯化并且经分光光度计量化之纯净质粒(浓度1ug/ul；260/280比介于1.8-1.9)；
脑肿瘤细胞株DBTRG-05MG(CRL-2020,ATCC,美国)；
六孔细胞培养盘(NUNC,Thermo Fisher,美国)；
细胞培养液(高糖DMEM+10％胎牛血清+1％综合抗生素)；
细胞转染试剂(JetPrime,Polyplus,法国)；
盘式冷光仪(Victor3 Multilabel Plate Reader,PerkinElmer,美国)；
活体光学影像仪(In Vivo Imaging System Spectrum CT,PerkinElmer,美国)；
双报导光学套件(Dual Reporter Assay System,Promega,美国)。
2、操作步骤如下：
第0天(准备)：
脑肿瘤DBTRG-05MG细胞经至少3次继代培养后，于6孔细胞盘之每孔种植1x105细胞。放入细胞培养箱培养隔夜(至少16小时)。
质粒抽提、定量、以及质保。
第1天(转染)：
将脑肿瘤细胞取出，更换为1毫升新鲜培养基后放回培养箱备用。
依照转染试剂说明，将每孔合计2微克的质粒与200微升转染缓冲液混合，震荡10秒后简单离心备用。
如单一质粒转染，则为2微克；如为两质粒组合转染，则维持比例1:1，即两质粒各加入1微克/孔；如为三质粒组合(包含定量用控制组萤火虫荧光酶)，则质量比例为0.6:1.2:1.2。
将4微升转染试剂加入前述质粒混合缓冲液后，震荡并简单离心后放置室温反应约10分钟。
将所有混合液使用滴入方式加入指定之孔内，开始转染。
第2天(恢复)：
加入转染试剂24小时后更换为新鲜培养基，将盘放回箱内持续培养。
第3天(量化分析)：
依照说明使用双报导光学套件，使用盘式冷光仪之自动注入系统进行冷光受质D-luciferin与Coelenterazine的注入与终止反应来进行光学讯号撷取。
如使用活体影像系统，则于手动加入冷光受质后整盘放入机器，使用LivingImage 4.0软件进行自动冷光影像撷取。量化数值设定阈值为20％进行ROI圈选与计算。
图6为不同片段冷光素酶的背景值，由于冷光素酶经过截切并截短，其蛋白功能可能部分存在。经过测试，决大部分的截短冷光素酶至多不出现超过10％的正常表现量。相对冷光量以GLuc冷光读值除以FLuc冷光读值；所有细胞皆为FLuc之稳定克隆细胞。
图7为正常与压力环境下的冷光量变化，结果显示两质粒皆使用(GGGGS)2连接肽的状况下(即bGS)，能够提供最有效的报导功能，连结肽重组效果为最佳。
图8为经不同压力诱发之相对冷光量测试，显示多数压力途径皆可被此影像系统感知。
图9为动物活体测试，于同一皮下植有脑肿瘤的小鼠使用此影像系统，于给予治疗(Temozolomide)前后之影像。显示本重组影像系统可有效观测治疗状态。
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。
序列表
<110> 香港理工大学深圳研究院
<120> 一种重组质粒及其构建方法、重组影像系统与应用
<160> 4
<210> 1
<211> 8952
<212> DNA
<213> 人工序列（rengongxulie）
<400> 1
acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat 60
tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga 120
ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt 180
ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca 240
ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg 300
tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 360
tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa 420
tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct tatgtactcg 480
ggagccggcc ccgcacttgc acctcctgcg ccgccgcccc ccatccaagg atatgccttc 540
aagcctccac ctagacccga ctttgggacc tccgggagaa caatcaaatt acaggccaat 600
ttcttcgaaa tggacatccc caaaattgac atctatcatt atgaattgga tatcaagcca 660
gagaagtgcc cgaggagagt taacagggaa atcgtggaac acatggtcca gcactttaaa 720
acacagatct ttggggatcg gaagcccgtg tttgacggca ggaagaatct atacacagcc 780
atgccccttc cgattgggag ggacaaggtg gagctggagg tcacgctgcc aggagaaggc 840
aaggatcgca tcttcaaggt gtccatcaag tgggtgtcct gcgtgagctt gcaggcgtta 900
cacgatgcac tttcagggcg gctgcccagc gtcccttttg agacgatcca ggccctggac 960
gtggtcatga ggcacttgcc atccatgagg tacacccccg tgggccgctc cttcttcacc 1020
gcgtccgaag gctgctctaa ccctcttggc gggggccgag aagtgtggtt tggcttccat 1080
cagtccgtcc ggccttctct ctggaaaatg atgctgaata ttgatgtgtc agcaacagcg 1140
ttttacaagg cacagccagt aatcgagttt gtttgtgaag ttttggattt taaaagtatt 1200
gaagaacaac aaaaacctct gacagattcc caaagggtaa agtttaccaa agaaattaaa 1260
ggtctaaagg tggagataac gcactgtggg cagatgaaga ggaagtaccg tgtctgcaat 1320
gtgacccggc ggcccgccag tcaccaaaca ttcccgctgc agcaggagag cgggcagacg 1380
gtggagtgca cggtggccca gtatttcaag gacaggcaca agttggttct gcgctacccc 1440
cacctcccat gtttacaagt cggacaggag cagaaacaca cctaccttcc cctggaggtc 1500
tgtaacattg tggcaggaca aagatgtatt aaaaaattaa cggacaatca gacctcaacc 1560
atgatcagag cgactgctag gtcggcgccc gatcggcaag aagagattag caaattgatg 1620
cgaagtgcaa gtttcaacac agatccatac gtccgtgaat ttggaatcat ggtcaaagat 1680
gagatgacag acgtgactgg gcgggtgctg cagccgccct ccatcctcta cgggggcagg 1740
aataaagcta ttgcgacccc tgtccagggc gtctgggaca tgcggaacaa gcagttccac 1800
acgggcatcg agatcaaggt gtgggccatt gcgtgcttcg ccccccagcg ccagtgcacg 1860
gaagtccatc tgaagtcctt cacagagcag ctcagaaaga tctcgagaga cgccggcatg 1920
cccatccagg gccagccgtg cttctgcaaa tacgcgcagg gggcggacag cgtggagccc 1980
atgttccggc acctgaagaa cacgtatgcg ggcctgcagc tggtggtggt catcctgccc 2040
ggcaagacgc ccgtgtacgc cgaggtcaag cgcgtgggag acacggtgct ggggatggcc 2100
acgcagtgcg tgcagatgaa gaacgtgcag aggaccacgc cacagaccct gtccaacctc 2160
tgcctgaaga tcaacgtcaa gctgggaggc gtgaacaaca tcctgctgcc ccagggcagg 2220
ccgccggtgt tccagcagcc cgtcatcttt ctgggagcag acgtcactca cccccccgcc 2280
ggggatggga agaagccctc cattgccgcc gtggtgggca gcatggacgc ccaccccaat 2340
cgctactgcg ccaccgtgcg cgtgcagcag caccggcagg agatcataca agacctggcc 2400
gccatggtcc gcgagctcct catccagttc tacaagtcca cgcgcttcaa gcccacccgc 2460
atcatcttct accgcgacgg tgtctctgaa ggccagttcc agcaggttct ccaccacgag 2520
ttgctggcca tccgtgaggc ctgtatcaag ctagaaaaag actaccagcc cgggatcacc 2580
ttcatcgtgg tgcagaagag gcaccacacc cggctcttct gcactgacaa gaacgagcgg 2640
gttgggaaaa gtggaaacat tccagcaggc acgactgtgg acacgaaaat cacccacccc 2700
accgagttcg acttctacct gtgtagtcac gctggcatcc aggggacaag caggccttcg 2760
cactatcacg tcctctggga cgacaatcgt ttctcctctg atgagctgca gatcctaacc 2820
taccagctgt gtcacaccta cgtgcgctgc acacgctccg tgtccatccc agcgccagca 2880
tactacgctc acctggtggc cttccgggcc aggtaccacc tggtggataa ggaacatgac 2940
agtgctgaag gaagccatac ctctgggcag agtaacgggc gagaccacca agcactggcc 3000
aaggcggtcc aggttcacca agacactctg cgcaccatgt actttgctgg tggaggcggt 3060
tcaggtggag gcggatcctc gagatggtga gcaagggcga ggagctgttc accggggtgg 3120
tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtccgcggcg 3180
agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca 3240
agctgcccgt gccctggccc accctcgtga ccaccttcgg ctacggcgtg gcctgcttca 3300
gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct 3360
acgtccagga gcgcaccatc tctttcaagg acgacggtac ctacaagacc cgcgccgagg 3420
tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 3480
aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac aacgtctata 3540
tcacggccga caagcagaag aacggcatca aggctaactt caagatccgc cacaacgttg 3600
aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 3660
ccgtgctgct gcccgacaac cactacctga gccatcagtc cgccctgagc aaagacccca 3720
acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg attacacatg 3780
gcatggacga gctgtacaag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 3840
tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 3900
aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 3960
gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 4020
aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 4080
ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 4140
gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 4200
tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 4260
gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 4320
attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 4380
cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 4440
ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 4500
aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 4560
agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 4620
ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 4680
catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 4740
aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 4800
agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 4860
aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 4920
gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa 4980
gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag 5040
cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta 5100
caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct 5160
tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt 5220
cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc 5280
catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc 5340
gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca 5400
tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct 5460
cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga 5520
tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag 5580
cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg 5640
gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg 5700
atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt 5760
ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg 5820
atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac 5880
cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag 5940
ggcaaaggaa tagcacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt 6000
gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 6060
gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag 6120
caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 6180
gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt 6240
ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 6300
caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 6360
cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 6420
gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 6480
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 6540
ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 6600
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 6660
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 6720
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 6780
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 6840
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 6900
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 6960
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 7020
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 7080
cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 7140
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtttttttgt 7200
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 7260
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 7320
atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 7380
aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 7440
ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 7500
tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 7560
ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 7620
tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 7680
aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 7740
gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 7800
tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 7860
cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 7920
tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 7980
ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 8040
cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 8100
actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 8160
ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 8220
aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 8280
ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 8340
atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 8400
tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc 8460
tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag 8520
tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag 8580
aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt 8640
tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 8800
ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 8860
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 8920
actttccatt gacgtcaatg ggtggagtat tt 8952
<210> 2
<211> 8670
<212> DNA
<213> 人工序列（rengongxulie）
<400> 2
acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat 60
tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga 120
ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt 180
ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca 240
ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg 300
tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 360
tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa 720
tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct tatgtactcg 780
ggagccggcc ccgcacttgc acctcctgcg ccgccgcccc ccatccaagg atatgccttc 840
aagcctccac ctagacccga ctttgggacc tccgggagaa caatcaaatt acaggccaat 900
ttcttcgaaa tggacatccc caaaattgac atctatcatt atgaattgga tatcaagcca 960
gagaagtgcc cgaggagagt taacagggaa atcgtggaac acatggtcca gcactttaaa 1020
acacagatct ttggggatcg gaagcccgtg tttgacggca ggaagaatct atacacagcc 1080
atgccccttc cgattgggag ggacaaggtg gagctggagg tcacgctgcc aggagaaggc 1140
aaggatcgca tcttcaaggt gtccatcaag tgggtgtcct gcgtgagctt gcaggcgtta 1200
cacgatgcac tttcagggcg gctgcccagc gtcccttttg agacgatcca ggccctggac 1260
gtggtcatga ggcacttgcc atccatgagg tacacccccg tgggccgctc cttcttcacc 1320
gcgtccgaag gctgctctaa ccctcttggc gggggccgag aagtgtggtt tggcttccat 1380
cagtccgtcc ggccttctct ctggaaaatg atgctgaata ttgatgtgtc agcaacagcg 1440
ttttacaagg cacagccagt aatcgagttt gtttgtgaag ttttggattt taaaagtatt 1500
gaagaacaac aaaaacctct gacagattcc caaagggtaa agtttaccaa agaaattaaa 1560
ggtctaaagg tggagataac gcactgtggg cagatgaaga ggaagtaccg tgtctgcaat 1620
gtgacccggc ggcccgccag tcaccaaaca ttcccgctgc agcaggagag cgggcagacg 1680
gtggagtgca cggtggccca gtatttcaag gacaggcaca agttggttct gcgctacccc 1740
cacctcccat gtttacaagt cggacaggag cagaaacaca cctaccttcc cctggaggtc 1800
tgtaacattg tggcaggaca aagatgtatt aaaaaattaa cggacaatca gacctcaacc 1860
atgatcagag cgactgctag gtcggcgccc gatcggcaag aagagattag caaattgatg 1920
cgaagtgcaa gtttcaacac agatccatac gtccgtgaat ttggaatcat ggtcaaagat 1980
gagatgacag acgtgactgg gcgggtgctg cagccgccct ccatcctcta cgggggcagg 2040
aataaagcta ttgcgacccc tgtccagggc gtctgggaca tgcggaacaa gcagttccac 2100
acgggcatcg agatcaaggt gtgggccatt gcgtgcttcg ccccccagcg ccagtgcacg 2160
gaagtccatc tgaagtcctt cacagagcag ctcagaaaga tctcgagaga cgccggcatg 2220
cccatccagg gccagccgtg cttctgcaaa tacgcgcagg gggcggacag cgtggagccc 2280
atgttccggc acctgaagaa cacgtatgcg ggcctgcagc tggtggtggt catcctgccc 2340
ggcaagacgc ccgtgtacgc cgaggtcaag cgcgtgggag acacggtgct ggggatggcc 2400
acgcagtgcg tgcagatgaa gaacgtgcag aggaccacgc cacagaccct gtccaacctc 2460
tgcctgaaga tcaacgtcaa gctgggaggc gtgaacaaca tcctgctgcc ccagggcagg 2520
ccgccggtgt tccagcagcc cgtcatcttt ctgggagcag acgtcactca cccccccgcc 2580
ggggatggga agaagccctc cattgccgcc gtggtgggca gcatggacgc ccaccccaat 2640
cgctactgcg ccaccgtgcg cgtgcagcag caccggcagg agatcataca agacctggcc 2700
gccatggtcc gcgagctcct catccagttc tacaagtcca cgcgcttcaa gcccacccgc 2760
atcatcttct accgcgacgg tgtctctgaa ggccagttcc agcaggttct ccaccacgag 2820
ttgctggcca tccgtgaggc ctgtatcaag ctagaaaaag actaccagcc cgggatcacc 2880
ttcatcgtgg tgcagaagag gcaccacacc cggctcttct gcactgacaa gaacgagcgg 2940
gttgggaaaa gtggaaacat tccagcaggc acgactgtgg acacgaaaat cacccacccc 3000
accgagttcg acttctacct gtgtagtcac gctggcatcc aggggacaag caggccttcg 3060
cactatcacg tcctctggga cgacaatcgt ttctcctctg atgagctgca gatcctaacc 3120
taccagctgt gtcacaccta cgtgcgctgc acacgctccg tgtccatccc agcgccagca 3180
tactacgctc acctggtggc cttccgggcc aggtaccacc tggtggataa ggaacatgac 3240
agtgctgaag gaagccatac ctctgggcag agtaacgggc gagaccacca agcactggcc 3300
aaggcggtcc aggttcacca agacactctg cgcaccatgt actttgctgg tggaggcggt 3360
tcaggtggag gcggatccgg cataggcgag gcgatcgtcg acattcctga gattcctggg 3420
ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 3480
acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 3540
ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 3600
ggggccggtg gtgactaatc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg 3660
ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 3720
ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 3780
aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 3840
gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 3900
agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt 3960
gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 4020
gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 4080
gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 4140
tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 4200
ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 4260
atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 4320
aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag 4380
ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 4440
agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 4500
tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 4560
ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 4620
aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag 4680
gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc 4740
acgtgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt 4800
tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct 4860
tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca 4920
aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg 4980
acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca 5040
cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca 5100
tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc 5160
aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg 5220
tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg 5280
atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt 5340
tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg 5400
aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt 5460
tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat 5520
cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg 5580
ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat 5640
ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg 5700
atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg 5760
caaaggaata gcacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg 5820
gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc 5880
tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca 5940
atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 6000
ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg 6060
cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 6120
acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 6180
cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 6240
attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 6300
cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 6360
caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 6420
caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 6480
ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 6540
cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 6600
ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 6660
tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 6720
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 6780
ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 6840
ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 6900
gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 6960
aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ttttttgttt 7020
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 7080
cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 7140
caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7200
gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7260
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7320
cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7380
caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7440
gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7500
gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7560
cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7620
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7680
gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7740
ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7800
gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7860
cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7920
tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7980
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 8040
atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 8100
ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 8160
gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8220
acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc 8280
tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta 8340
gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa 8400
tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg 8460
acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 8520
atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 8580
cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 8640
tttccattga cgtcaatggg tggagtattt 8670
<210> 3
<211> 5801
<212> DNA
<213> 人工序列（rengongxulie）
<400> 3
gacggatcgg gagatctccc gatcccctat ggtcgactct 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 720
attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 780
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 840
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 900
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 960
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 1020
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 1080
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 1140
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 1200
gagctcggat ccatgggagt caaagttctg tttgccctga tctgcatcgc tgtggccgag 1260
gccaagccca ccgagaacaa cgaagacttc aacatcgtgg ccgtggccag caacttcgcg 1320
accacggatc tcgatgctga ccgcgggaag ttgcccggca agaagctgcc gctggaggtg 1380
ctcaaagaga tggaagccaa tgcccggaaa gctggctgca ccaggggctg tctgatctgc 1440
ctgtcccaca tcaagtgcac gcccaagatg aagaagttca tcccaggacg ctgccacacc 1500
tacgaaggcg acaaagagtc cgcacagggc tgatctagaa ataattctta ctgtcatgcc 1560
aagtaagatg cttttctgtg ctgcaatagc aggcatgctg gggatgcggt gggctctatg 1620
gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc 1680
ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 1740
gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 1800
ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc tttacggcac 1860
ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag 1920
acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 1980
actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgggg 2040
atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc 2100
tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc aggcagaagt 2160
atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 2220
gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 2280
actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 2340
ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag 2400
tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata 2460
tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 2520
ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 2580
caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 2640
gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg 2700
cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 2760
gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 2820
caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 2880
cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 2940
actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 3000
gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc 3060
gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 3120
ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 3180
cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 3240
atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 3300
gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt 3360
tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg 3420
gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt 3480
ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 3540
catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 3600
tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 3660
tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 3720
aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 3780
ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 3840
gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 3900
tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 3960
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 4020
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 4080
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 4140
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4200
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 4260
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4320
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 4380
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 4440
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 4500
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 4560
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 4620
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 4680
aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 4740
ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 4800
acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 4860
ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 4920
gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 4980
taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 5040
tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 5100
gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 5160
cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 5220
aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 5280
cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 5340
tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 5400
gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 5460
tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 5520
gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 5580
ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 5640
cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 5700
agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 5760
gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c 5801
<210> 4
<211> 6899
<212> DNA
<213> 人工序列（rengongxulie）
<400> 4
gacggatcgg gagatctccc gatcccctat ggtcgactct 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 720
attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 780
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 840
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 900
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 960
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 1020
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 1080
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 1140
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttatggag 1200
actgacgcgc cccagcccgg cctcgcctcc ccggactcgc cgcacgaccc ctgcaagatg 1260
ttcatcgggg gactcagttg gcagactacg caggaagggc tgcgcgaata cttcggccag 1320
ttcggggagg tgaaggagtg tctggtgatg cgggaccccc tgaccaagag atccaggggt 1380
ttcggcttcg tcactttcat ggaccaggcg ggggtggata aagtgctggc gcaatcgcgg 1440
cacgagctcg actccaaaac aattgaccct aaggtggcct tccctcggcg agcacagccc 1500
aagatggtga ctcgaacgaa gaagatcttt gtgggggggc tgtcggtgaa caccacggtg 1560
gaggacgtga agcaatattt tgagcagttt gggaaggtgg acgacgccat gctgatgttt 1620
gacaaaacca ccaaccggca ccgagggttc gggtttgtca cgtttgagag tgaggacatc 1680
gtggagaaag tgtgtgaaat tcattttcat gaaatcaaca acaaaatggt ggaatgtaag 1740
aaagctcagc caaaggaggt gatgtcgcca acgggctcag cccgggggag gtctcgagtc 1800
atgccctacg gaatggacgc cttcatgctg ggcatcggca tgctgggtta cccaggtttc 1860
caagccacaa cctacgccag ccggagttat acaggcctcg cccctggcta cacctaccag 1920
ttccccgaat tccgtgtaga gcggacccct ctcccgagcg ccccagtcct ccccgagctt 1980
acagccattc ctctcactgc ctacggacca atggcggcgg cagcggcggc agcggctgtg 2040
gttcgaggga caggctctca cccctggacg atggctcccc ctccaggttc gactcccagc 2100
cgcacagggg gcttcctggg gaccaccagc cccggcccca tggccgagct ctacggggcg 2160
gccaaccagg actcgggggt cagcagttac atcagcgccg ccagccctgc ccccagcacc 2220
ggcttcggcc acagtcttgg gggccctttg attgccacag ccttcaccaa tgggtaccac 2280
ggtggaggcg gttcaggtgg aggcggatcc atgggagtca aagttctgtt tgccctgatc 2340
tgcatcgctg tggccgaggc caagcccacc gagaacaacg aagacttcaa catcgtggcc 2400
gtggccagca acttcgcgac cacggatctc gatgctgacc gcgggaagtt gcccggcaag 2460
aagctgccgc tggaggtgct caaagagatg gaagccaatg cccggaaagc tggctgcacc 2520
aggggctgtc tgatctgcct gtcccacatc aagtgcacgc ccaagatgaa gaagttcatc 2580
ccaggacgct gccacaccta cgaaggcgac aaagagtccg cacagggctg atctagaaat 2640
aattcttact gtcatgccaa gtaagatgct tttctgtgct gcaatagcag gcatgctggg 2700
gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 2760
ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 2820
accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 2880
gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg gcatcccttt agggttccga 2940
tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 3000
gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 3060
agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 3120
ttataaggga ttttggggat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 3180
tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 3240
ccccaggcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga 3300
aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 3360
accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat 3420
tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc 3480
tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag 3540
ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt 3600
cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta 3660
ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg 3720
tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa 3780
ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct 3840
gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg 3900
caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca 3960
atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat 4020
cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac 4080
gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc 4140
gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa 4200
aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag 4260
gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc 4320
ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt 4380
cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca 4440
acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa 4500
tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct 4560
tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 4620
caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 4680
tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat 4740
ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 4800
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 4860
cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 4920
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 4980
ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 5040
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 5100
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 5160
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 5220
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 5280
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat 5340
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 5400
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5460
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5520
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5580
gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5640
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 5700
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5760
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 5820
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 5880
atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 5940
tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 6000
gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 6060
ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 6120
caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 6180
cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 6240
cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 6300
cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 6360
agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 6420
tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 6480
agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 6540
atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 6600
ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 6660
cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 6720
caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 6780
attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 6840
agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc 6899
Claims (8)
1.一种重组质粒的构建方法，其特征在于，包括步骤：
使用聚合酶链式反应将AGO2植入限制性内切酶切位HindIII与BamHI后进行扩增，得到HindIII-AGO2-BamHI片段；
将HindIII-AGO2-BamHI片段引入pcDNA载体的多克隆位点，得到pcDNA-AGO2-C1；
使用聚合酶链式反应将桡脚类萤光素酶GLuc的C端蛋白进行扩增，加入限制性内切酶切位BamHI与XbaI，并加入(GGGGS)2连接肽，得到(GGGGS)2-GLuc(C)片段；
将(GGGGS)2-GLuc(C)片段使用克隆技术载入pcDNA-AGO2-C1中，得到pcDNA-AGO2-GLuc(C)重组质粒。
2.一种重组质粒，其特征在于，采用权利要求1所述的方法构建得到。
3.一种重组质粒的构建方法，其特征在于，包括步骤：
以pCMV-GLuc1质粒为载体，使用聚合酶链式反应将桡脚类萤光素酶GLuc的N端加上(GGGGS)2连接肽与限制性内切酶切位BamHI与XbaI后，克隆制成pCMV-GLuc(N)；
将MSI1使用PCR扩增技术放大并纯化后加上限制性内切酶切位HindIII与BamHI，经克隆技术将MSI1载入pCMV-GLuc(N)中，得到pCMV-MSI1-GLuc(N)。
4.一种重组质粒，其特征在于，采用权利要求3所述的方法构建得到。
5.一种融合蛋白，其特征在于，由权利要求2所述的重组质粒和权利要求4所述的重组质粒融合得到。
6.一种重组影像系统，其特征在于，包括权利要求2所述的重组质粒和权利要求4所述的重组质粒。
7.权利要求5所述的融合蛋白在作为或制备具有示踪性质产品中的应用。
8.权利要求5所述的融合蛋白在用于制备脑肿瘤治疗药物中的应用。
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