CN111471716B - 一种基于重组腺相关病毒8型的microRNA-212-3P海绵的构建及其应用 - Google Patents

一种基于重组腺相关病毒8型的microRNA-212-3P海绵的构建及其应用 Download PDF

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CN111471716B
CN111471716B CN202010432475.3A CN202010432475A CN111471716B CN 111471716 B CN111471716 B CN 111471716B CN 202010432475 A CN202010432475 A CN 202010432475A CN 111471716 B CN111471716 B CN 111471716B
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杨文卓
刘瑞麟
孙波
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Shanghai Tongji Hospital
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Abstract

本发明涉及一种基于重组腺相关病毒8型的microRNA‑212‑3P海绵的构建及其应用。本发明明确了下调肝脏miR‑212‑3P的表达可降低肝脏脂质积累，预防高脂饮食所致的非酒精性脂肪性肝病；构建了一种microRNA‑212‑3P海绵，基于rAAV8载体递送该microRNA‑212‑3P海绵，这种基因疗法被证实其对肝脏脂质积累、血清脂质含量的降低作用显著，取得良好的实验性NAFLD防治效果。本发明为非酒精性脂肪性肝病的基因治疗提供了新的工具。

Description

一种基于重组腺相关病毒8型的microRNA-212-3P海绵的构建 及其应用
技术领域
本发明涉及生物医药技术领域，具体地说，涉及一种基于重组腺相关病毒8型的microRNA-212-3P海绵的构建及其应用。
背景技术
非酒精性脂肪性肝病(Nonalcoholic fatty liver disease,NAFLD)通常始于单纯脂肪变性，其特征是甘油三酸酯(TG)积聚在肝细胞脂质滴内，进而可发展为非酒精性脂肪性肝炎，肝硬化，甚至是肝细胞癌。有效防治NAFLD对改善人们生活质量及疾病预后有重要意义。目前临床上尚无针对NAFLD的特效防治药物，生活方式干预仍是NAFLD首选的防治手段，包括健康饮食及运动。临床研究显示运动能有效防止NAFLD的发生与发展。然而临床上却存在相当部分病人因生活习惯或疾病不能坚持或耐受运动，导致NAFLD控制不良而进展至肝衰竭甚至肝癌。寻找运动治疗NAFLD的作用靶点，有助于发掘新的治疗NAFLD的方法和策略(如研发调控相关靶点的药物或工具)，在NAFLD病人，特别是不能耐受运动治疗的病人中可模拟运动达到治疗目的，将对改善患者预后具有良好的临床价值和积极的社会意义。
微小RNA(MicroRNA，miRNA，miR)在NAFLD发生中的作用正日益受到重视。已有研究表明微小RNA参与NAFLD，但是否参与介导运动对NAFLD的保护尚待阐明。我们前期的研究(Xiao J,Bei Y,Liu J,et al.miR-212downregulation contributes to the protectiveeffect of exercise against non-alcoholic fatty liver via targeting FGF-21.JCell Mol Med.2016Feb；20(2):204-16)显示：运动改善小鼠NAFLD伴随miR-212下调；细胞水平过表达miR-212可导致肝细胞脂质沉积，抑制miR-212具有反向作用，机制研究发现该过程由其靶基因FGF-21介导。
而由于前期研究仅局限于体外细胞层面发现miR-212与肝细胞脂质沉积的关系，且miR-212与肝脏疾病的具体关系目前并不明朗，例如文献(孙飞.血清miR-132和miR-212检测在肝硬化诊断中的应用价值.《临床医药文献电子杂志》.2016,3(28):5689-5690)公开了临床肝硬化患者血清miR-212表达明显低于健康对照，差异有统计学意义(P＜0.05)，因此，我们还不知晓miR-212在NAFLD治疗中的意义。再者，如果miR-212可作为NAFLD的治疗靶点，那么开发高效的药物则对于实现其在临床的应用来说非常重要。
miRNA海绵是一种miRNA靶基因的竞争性抑制剂，是将若干个miRNA的反义序列串联在一起，连接到合适的载体中表达，其转录物“吸附”相应的miRNA，与miRNA靶基因形成竞争，导致靶基因去抑制化。
目前未见以miR-212-3P为靶点设计的能高效治疗NAFLD的miRNA海绵产品。
发明内容
本发明的目的是针对现有技术中的不足，提供一种基于重组腺相关病毒8型的microRNA-212-3P海绵及其制备方法和应用。
第一方面，本发明提供了一种miRNA-212-3P海绵表达载体，构建方法为：在基本载体pHBAAV-U6-MCS-CMV-EGFP的多克隆位点连入序列如SEQ ID NO:3的第442-569bp所示的序列。
作为一个优选例，所述miRNA-212-3P海绵表达载体的序列如SEQ ID NO:3所示。
第二方面，本发明提供了所述miRNA-212-3P海绵表达载体在制备预防或治疗非酒精性脂肪性肝病的药物中的应用。
第三方面，本发明提供了所述miRNA-212-3P海绵表达载体的制备方法。
第四方面，本发明提供了一种腺相关病毒，制备所述腺相关病毒采用的穿梭质粒是如上所述的miRNA-212-3P海绵表达载体。
作为一个优选例，制备所述腺相关病毒采用的编码rep和cap蛋白编码基因的pAAV-RC为8型AAV。
更优选地，所述腺相关病毒的制备方法包含以下步骤：将如上所述的miRNA-212-3P海绵表达载体，与pAAV-RC质粒和pHelper质粒共转染AAV-293细胞。
第五方面，本发明提供了所述腺相关病毒在制备预防或治疗非酒精性脂肪性肝病的药物中的应用。
第六方面，本发明提供了所述腺相关病毒的制备方法。
本发明优点在于：
1、本发明明确了下调肝脏miR-212-3P的表达，降低了肝脏脂质积累，可以预防高脂饮食所致的NAFLD，为后期新药开发和临床治疗提供了明确的靶点和实验基础。
2、本发明构建了miRNA-212-3P海绵表达载体，由于在基本载体选择、miRNA反义结合位点的序列和数量、间隔序列等方面设计恰当，该miRNA-212-3P海绵表达载体对于miRNA-212-3P的敲降效果显著高于研究过程中构建的其他海绵表达载体，动物实验也表明其对肝脏脂质积累、血清脂质含量的降低作用非常突出，显著优于其他海绵表达载体，取得了预料不到的技术效果，因此在临床药物开发中具有重要价值。
3、本发明在腺病毒包装中使用pAAV-RC作为递送载体，其是8型AAV载体，对肝脏亲和力较好。
附图说明
附图1：microRNA表达变化的韦恩图。
附图2：流式细胞术：调控miR-212-3P表达对脂质生成的影响。*P＜0.05，**P＜0.01，***P＜0.001，VS FFA+NC；#P＜0.05，##P＜0.01，###P＜0.001，VS BSA+NC。
附图3：尼罗红染色：调控miR-212-3P表达对脂质生成的影响。*P＜0.05，**P＜0.01，***P＜0.001，VS FFA+NC；#P＜0.05，##P＜0.01，###P＜0.001，VS BSA+NC。
附图4：各组小鼠肝脏病理切片油红O染色。
附图5：各种小鼠血清及肝脏脂质变化。*P＜0.05，**P＜0.01，***P＜0.001，VSSD；#P＜0.05，##P＜0.01，###P＜0.001，VS HFD。
附图6：A，三组肝脏变化(肉眼观)；B，敲减效率统计；C，脂滴减少统计；D，油红O变化；E，ALT和AST变化；F，三组肝脏TG、血清TG、血清NEFA变化。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
实施例1运动通过miR-212-3P调控非酒精性脂肪性肝病中的肝脏脂质积累1、方法
将40只雄性C57/BL6小鼠随机分组，分为正常饮食组、正常饮食&运动组、高脂饮食组、高脂饮食&运动组(每组各10只)。正常饮食组和高脂饮食组分别饲喂标准饲料和高脂饲料，16周；正常饮食&运动组和高脂饮食&运动组在饲喂对应饲料的同时，予以60min/d,10m/min的跑步锻炼，共16周。四组小鼠于16周末腹腔麻醉后摘眼球法采血后处死动物，剪下完整肝组织标本，称重后取部分右叶肝脏以4％中性甲醛固定，用于病理检测；将余下肝脏于液氮中速冻，-80℃冰箱内保存。血清于-20℃冰箱内保存，用于血清学肝功能检测。
人肝癌细胞(HepG2)分别转染miR-212-3P mimics和miR-212-3P inhibitor，并向培养基中分别添加长链脂肪酸(FFA)和牛血清蛋白(BSA)，具体分组为：
Figure BDA0002501074400000041
*NC:negative control2、结果
(1)同正常饮食组小鼠相比，高脂饮食组小鼠肝脏miR-212-3P显著上升；同高脂饮食组小鼠相比，高脂饮食&运动组小鼠肝脏miR-212-3P下调最为显著(图1)。
(2)在FFA/BSA(即高脂/低脂)培养环境下，HepG2细胞转染miR-212-3P mimics，与对照组相比，上调miR-212-3P表达，流式及尼罗红染色显示细胞内脂质积累明显增多；而HepG2细胞转染miR-212-3P inhibitor，与对照组相比，下调miR-212-3P表达，流式及尼罗红染色显示胞内脂质积累明显减少(图2、图3)。
(3)证实miR-212-3P在运动治疗NAFLD过程中发挥了脂质调控作用。
实施例2rAAV8-miR-212-3P-Sponge可以有效预防高脂饮食所致的非酒精性脂肪性肝病中的脂质积累
1、方法
1.1腺相关病毒的制备
1.1.1rAAV8-miR-212-3P-Sponge
miR-212-3p序列：TAACAGTCTCCAGTCACGGCCA(SEQ ID NO:1)
其反向互补序列：TGGCCGTGACTGGAGACTGTTA(SEQ ID NO:2)
使用pHBAAV-U6-MCS-CMV-EGFP作为基本载体，酶切，回收载体，连入目的片段，转化感受态细胞DH5α，测序鉴定，构建得到载体pHBAAV-U6-miR-212-3P-Sponge-CMV-EGFP(序列如SEQ ID NO:3所示)，连入载体的目的片段序列如SEQ ID NO:3的第442-569bp所示。
然后将制备好的质粒pHBAAV-U6-miR-212-3P-Sponge-CMV-EGFP与pAAV-RC(序列如SEQ ID NO:4所示)和pHelper(序列如SEQ ID NO:5所示)，共转染AAV-293细胞，转染后6h更换为完全培养基，72h后，收集富含腺相关病毒颗粒的AAV-293细胞，反复冻融裂解细胞后，收集含病毒颗粒的上清液，利用全能核酸酶处理和柱纯化方法，得到高滴度的纯化腺相关病毒颗粒。1.1.2rAAV8-miR-212-3P Control
使用pHBAAV-U6-MCS-CMV-EGFP基本载体质粒作为对照。
1.1.3过表达载体
以pHBAAV-U6-MCS-CMV-EGFP为基本载体，根据mmu-miR-212-3P的成熟体序列设计引物，安排引物合成。将单链的引物退火成双链oligo序列，连接入双酶切线性化的载体。转化子送往测序公司进行测序验证。测序验证正确的克隆，进行高纯度质粒抽提。获得过表达载体
pHBAAV-U6-miR-212-3P-CMV-EGFP(序列如SEQ ID NO:6所示)。
1.2给药及检测
将24只雄性C57/BL6小鼠随机分为4组，分别为正常饮食+rAAV8-miR-212-3PControl组(SD组)、正常饮食+rAAV-miR-212-3P Over expression组(SD&OE组)、高脂饮食+rAAV8-miR-212-3P Control组(HFD组)、高脂饮食+rAAV8-miR-212-3P Sponge组(HFD&Sponge组)，每组6只。每组小鼠尾静脉注射对应腺相关病毒100μl(1*10^12vg/ml)，饲喂相应饲料16周。于16周末腹腔麻醉后摘眼球法采血后处死动物，剪下完整肝组织标本，称重后取部分右叶肝脏以4％中性甲醛固定，用于病理检测；将余下肝脏于液氮中速冻，-80℃冰箱内保存。血清于-20℃冰箱内保存，用于血清学肝功能检测。
2、结果
(1)同正常饮食对照组相比，上调肝脏miR-212-3P表达，肝内脂质积累增多，血清游离脂肪酸显著升高；而在高脂饮食组中，下调miR-212-3P表达，肝内脂质含量明显下降，血清游离脂肪酸含量显著降低。结果证实下调肝脏miR-212-3P的表达可以有效预防NAFLD的发生发展(图4、图5)。
(2)基于rAAV8载体递送miRNA-212-3P-Sponge的基因疗法可以显著改善饮食所致的非酒精性脂肪性肝病，降低肝内脂质积累：根据imageJ定量脂滴数量，相比于HFD组，HFD&Sponge组肝脏脂滴减少89.3％；相比于HFD组，HFD&Sponge组肝脏TG下降66.6％，血清TG下降37.4％，血清NEFA下降54.7％。
实施例3rAAV8-miR-212-3P-Sponge的筛选
本研究设计了多种miR-212-3P海绵，现选取部分结果作为示例，如下：
1、方法
1.1腺相关病毒的制备
1.1.1rAAV8-miR-212-3P-Sponge
同实施例2。
1.1.2rAAV8-miR-212-3P-Sponge对照
制备方法同rAAV8-miR-212-3P-Sponge，不同之处仅在于用SEQ ID NO:7替换连入pHBAAV-U6-MCS-CMV-EGFP载体的目的片段。
1.2给药及检测
将18只6-8周C57/BL6小鼠随机分为3组，分别为高脂饮食+基本载体组(Contrl)、高脂饮食+rAAV8-miR-212-3P Sponge对照组(Sponge Contrl)、高脂饮食+rAAV8-miR-212-3PSponge组(Sponge)。按照实施例2的方法给药和检测。
2、结果
图6中A显示了三组给药后的肝脏变化；图6中B表明rAAV8-miR-212-3P Sponge和rAAV8-miR-212-3P Sponge对照均能降低miR-212-3P水平，其中rAAV8-miR-212-3P Sponge的敲减效率要高于rAAV8-miR-212-3P Sponge对照，差异具有统计学意义(P<0.05)；图6中C表明rAAV8-miR-212-3P Sponge和rAAV8-miR-212-3P Sponge对照均能减少肝脏中脂滴形成，其中Sponge组脂滴含量显著低于Sponge Contrl组，差异具有统计学意义(P<0.05)；图6中D为油红O染色照片，可见各组脂滴的变化；图6中E表明rAAV8-miR-212-3P Sponge和rAAV8-miR-212-3P Sponge对照均无肝毒性；图6中F显示了三组肝脏TG、血清TG、血清NEFA的变化，可见相比于Contrl组，Sponge组和Sponge Contrl组的肝脏TG、血清TG、血清NEFA均有所下降，其中Sponge组下降最为明显，与Contrl组相比，肝脏TG差异有统计学意义(P<0.01)，血清NEFA差异有统计学意义(P<0.001)，与Sponge Contrl组相比，肝脏TG差异有统计学意义(P<0.05)，血清NEFA差异有统计学意义(P<0.01)。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 上海市同济医院
<120> 一种基于重组腺相关病毒8型的microRNA-212-3P海绵的构建及其应用
<130> /
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> 人工序列
<400> 1
taacagtctc cagtcacggc ca 22
<210> 2
<211> 22
<212> DNA
<213> 人工序列
<400> 2
tggccgtgac tggagactgt ta 22
<210> 3
<211> 5811
<212> DNA
<213> 人工序列
<400> 3
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggcggcc gcacgcgtac 60
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 120
gatccgacgc cgccatctct aggcccgcgc cggccccctc gcacagactt gtgggagaag 180
ctcggctact cccctgcccc ggttaatttg catataatat ttcctagtaa ctatagaggc 240
ttaatgtgcg ataaaagaca gataatctgt tctttttaat actagctaca ttttacatga 300
taggcttgga tttctataag agatacaaat actaaattat tattttaaaa aacagcacaa 360
aaggaaactc accctaactg taaagtaatt gtgtgttttg agactataaa tatcccttgg 420
agaaaagcct tgtttgaaac aacagaattc tggccgtgac tggagactgt tatatactgg 480
ccgtgactgg agactgttaa catctggccg tgactggaga ctgttatctt catggccgtg 540
actggagact gttatttttt ggatccacac cgcggccgct aacaacatct agggaatact 600
tgaggcactt ccccaaactc cggccgcttt tttctgcagg ggcggcctcg tgatacgcct 660
atttttatag gttaatgtcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc 720
caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg 780
gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca 840
tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc 900
ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt 960
attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata 1020
gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt 1080
ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca 1140
aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 1200
tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg 1260
atccagcctc cgtaccggtt cgaacaggta agcgccccta aaatcccatg gtgagcaagg 1320
gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg 1380
gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc 1440
tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc 1500
tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct 1560
tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg 1620
gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg 1680
agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca 1740
actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga 1800
acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc 1860
agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc 1920
agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 1980
tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagctcgaa tcaagcttat 2040
cgataaaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat 2100
gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct 2160
tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag 2220
gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 2280
cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc 2340
ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct 2400
cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg 2460
ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg 2520
gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 2580
cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga 2640
taccgtcgat cgagaaatcg atcgcccggg tggcatccct gtgacccctc cccagtgcct 2700
ctcctggccc tggaagttgc cactccagtg cccaccagcc ttgtcctaat aaaattaagt 2760
tgcatcattt tgtctgacta ggtgtccttc tataatatta tggggtggag gggggtggta 2820
tggagcaagg ggcaagttgg gaagacaacc tgtagggcct gcggggtcta ttgggaacca 2880
agctggagtg cagtggcaca atcttggctc actgcaatct ccgcctcctg ggttcaagcg 2940
attctcctgc ctcagcctcc cgagttgttg ggattccagg catgcatgac caggctcagc 3000
taatttttgt ttttttggta gagacggggt ttcaccatat tggccaggct ggtctccaac 3060
tcctaatctc aggtgatcta cccaccttgg cctcccaaat tgctgggatt acaggcgtga 3120
accactgctc ccttccctgt cctttccgga cacgtgcgga ccgagcggcc gcaacgccag 3180
caacgcggcc tttttacggt tcctggcctt ttgctgggcg cctgatgcgg tattttctcc 3240
ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg 3300
tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc 3360
cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg 3420
ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg 3480
gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg 3540
atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt 3600
ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt 3660
gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt 3720
taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc 3780
cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg 3840
tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca 3900
gaggttttca ccgtcatcac cgaaacgcgc gacgcggaac ccctatttgt ttatttttct 3960
aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat 4020
attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg 4080
cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg 4140
aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc 4200
ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat 4260
gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact 4320
attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca 4380
tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact 4440
tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg 4500
atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg 4560
agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg 4620
aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg 4680
caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag 4740
ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc 4800
gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga 4860
tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat 4920
atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc 4980
tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag 5040
accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct 5100
gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac 5160
caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc 5220
tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg 5280
ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt 5340
tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt 5400
gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc 5460
tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca 5520
gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata 5580
gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg 5640
ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct 5700
gttgcggtcg ttgcgccgga aaaatgccaa ggaccggaaa acgaccggaa aacgagtgta 5760
caagaaagga cgcaataggg gactaagaca cctattggca taatggcgga a 5811
<210> 4
<211> 7334
<212> DNA
<213> 人工序列
<400> 4
ctaatgcggt tcgcgcgcgc tctagaacta gtggatcgag cgcgcggaag ttcctattct 60
ctagaaagta taggaacttc gaattgacta aagccaaata gaaaattatt cagttcctgg 120
cttaagtttt taaaagtgat attatttatt tggttgtaac caaccaaaag aatgtaaata 180
atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc 240
ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat 300
tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag 360
cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggctct tttctttgtg 420
caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg 480
aaatccatgg ttttgggacg tttcctgagt cagattcgcg aaaaactgat tcagagaatt 540
taccgcggga tcgagccgac tttgccaaac tggttcgcgg tcacaaagac cagaaatggc 600
gccggaggcg ggaacaaggt ggtggatgag tgctacatcc ccaattactt gctccccaaa 660
acccagcctg agctccagtg ggcgtggact aatatggaac agtatttaag cgcctgtttg 720
aatctcacgg agcgtaaacg gttggtggcg cagcatctga cgcacgtgtc gcagacgcag 780
gagcagaaca aagagaatca gaatcccaat tctgatgcgc cggtgatcag atcaaaaact 840
tcagccaggt acatggagct ggtcgggtgg ctcgtggaca aggggattac ctcggagaag 900
cagtggatcc aggaggacca ggcctcatac atctccttca atgcggcctc caactcgcgg 960
tcccaaatca aggctgcctt ggacaatgcg ggaaagatta tgagcctgac taaaaccgcc 1020
cccgactacc tggtgggcca gcagcccgtg gaggacattt ccagcaatcg gatttataaa 1080
attttggaac taaacgggta cgatccccaa tatgcggctt ccgtctttct gggatgggcc 1140
acgaaaaagt tcggcaagag gaacaccatc tggctgtttg ggcctgcaac taccgggaag 1200
accaacatcg cggaggccat agcccacact gtgcccttct acgggtgcgt aaactggacc 1260
aatgagaact ttcccttcaa cgactgtgtc gacaagatgg tgatctggtg ggaggagggg 1320
aagatgaccg ccaaggtcgt ggagtcggcc aaagccattc tcggaggaag caaggtgcgc 1380
gtggaccaga aatgcaagtc ctcggcccag atagacccga ctcccgtgat cgtcacctcc 1440
aacaccaaca tgtgcgccgt gattgacggg aactcaacga ccttcgaaca ccagcagccg 1500
ttgcaagacc ggatgttcaa atttgaactc acccgccgtc tggatcatga ctttgggaag 1560
gtcaccaagc aggaagtcaa agactttttc cggtgggcaa aggatcacgt ggttgaggtg 1620
gagcatgaat tctacgtcaa aaagggtgga gccaagaaaa gacccgcccc cagtgacgca 1680
gatataagtg agcccaaacg ggtgcgcgag tcagttgcgc agccatcgac gtcagacgcg 1740
gaagcttcga tcaactacgc agacaggtac caaaacaaat gttctcgtca cgtgggcatg 1800
aatctgatgc tgtttccctg cagacaatgc gagagaatga atcagaattc aaatatctgc 1860
ttcactcacg gacagaaaga ctgtttagag tgctttcccg tgtcagaatc tcaacccgtt 1920
tctgtcgtca aaaaggcgta tcagaaactg tgctacattc atcatatcat gggaaaggtg 1980
ccagacgctt gcactgcctg cgatctggtc aatgtggatt tggatgactg catctttgaa 2040
caataaatga tttaaatcag gtatggctgc cgatggttat cttccagatt ggctcgagga 2100
caacctctct gagggcattc gcgagtggtg ggcgctgaaa cctggagccc cgaagcccaa 2160
agccaaccag caaaagcagg acgacggccg gggtctggtg cttcctggct acaagtacct 2220
cggacccttc aacggactcg acaaggggga gcccgtcaac gcggcggacg cagcggccct 2280
cgagcacgac aaggcctacg accagcagct gcaggcgggt gacaatccgt acctgcggta 2340
taaccacgcc gacgccgagt ttcaggagcg tctgcaagaa gatacgtctt ttgggggcaa 2400
cctcgggcga gcagtcttcc aggccaagaa gcgggttctc gaacctctcg gtctggttga 2460
ggaaggcgct aagacggctc ctggaaagaa gagaccggta gagccatcac cccagcgttc 2520
tccagactcc tctacgggca tcggcaagaa aggccaacag cccgccagaa aaagactcaa 2580
ttttggtcag actggcgact cagagtcagt tccagaccct caacctctcg gagaacctcc 2640
agcagcgccc tctggtgtgg gacctaatac aatggctgca ggcggtggcg caccaatggc 2700
agacaataac gaaggcgccg acggagtggg tagttcctcg ggaaattggc attgcgattc 2760
cacatggctg ggcgacagag tcatcaccac cagcacccga acctgggccc tgcccaccta 2820
caacaaccac ctctacaagc aaatctccaa cgggacatcg ggaggagcca ccaacgacaa 2880
cacctacttc ggctacagca ccccctgggg gtattttgac tttaacagat tccactgcca 2940
cttttcacca cgtgactggc agcgactcat caacaacaac tggggattcc ggcccaagag 3000
actcagcttc aagctcttca acatccaggt caaggaggtc acgcagaatg aaggcaccaa 3060
gaccatcgcc aataacctca ccagcaccat ccaggtgttt acggactcgg agtaccagct 3120
gccgtacgtt ctcggctctg cccaccaggg ctgcctgcct ccgttcccgg cggacgtgtt 3180
catgattccc cagtacggct acctaacact caacaacggt agtcaggccg tgggacgctc 3240
ctccttctac tgcctggaat actttccttc gcagatgctg agaaccggca acaacttcca 3300
gtttacttac accttcgagg acgtgccttt ccacagcagc tacgcccaca gccagagctt 3360
ggaccggctg atgaatcctc tgattgacca gtacctgtac tacttgtctc ggactcaaac 3420
aacaggaggc acggcaaata cgcagactct gggcttcagc caaggtgggc ctaatacaat 3480
ggccaatcag gcaaagaact ggctgccagg accctgttac cgccaacaac gcgtctcaac 3540
gacaaccggg caaaacaaca atagcaactt tgcctggact gctgggacca aataccatct 3600
gaatggaaga aattcattgg ctaatcctgg catcgctatg gcaacacaca aagacgacga 3660
ggagcgtttt tttcccagta acgggatcct gatttttggc aaacaaaatg ctgccagaga 3720
caatgcggat tacagcgatg tcatgctcac cagcgaggaa gaaatcaaaa ccactaaccc 3780
tgtggctaca gaggaatacg gtatcgtggc agataacttg cagcagcaaa acacggctcc 3840
tcaaattgga actgtcaaca gccagggggc cttacccggt atggtctggc agaaccggga 3900
cgtgtacctg cagggtccca tctgggccaa gattcctcac acggacggca acttccaccc 3960
gtctccgctg atgggcggct ttggcctgaa acatcctccg cctcagatcc tgatcaagaa 4020
cacgcctgta cctgcggatc ctccgaccac cttcaaccag tcaaagctga actctttcat 4080
cacgcaatac agcaccggac aggtcagcgt ggaaattgaa tgggagctgc agaaggaaaa 4140
cagcaagcgc tggaaccccg agatccagta cacctccaac tactacaaat ctacaagtgt 4200
ggactttgct gttaatacag aaggcgtgta ctctgaaccc cgccccattg gcacccgtta 4260
cctcacccgt aatctgtaat tgcctgttaa tcaataaacc ggttgattcg tttcagttga 4320
actttggtct ctgcgaaggg cgaattcgtt taaacctgca ggactagagg tcctgtatta 4380
gaggtcacgt gagtgttttg cgacattttg cgacaccatg tggtcacgct gggtatttaa 4440
gcccgagtga gcacgcaggg tctccatttt gaagcgggag gtttgaacgc gcagccgcca 4500
agccgaattc tgcagatatc catcacactg gcggccgctc gactagagcg gccgccaccg 4560
cggtggagct ccagcttttg ttcactggcc gtcgttttac attgcgttgc gctcactgcc 4620
cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 4680
gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 4740
ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 4800
agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 4860
ccgtaaaaag gccgcgttgc tggcgttaaa ttgtaagcgt taatattttg ttaaaattcg 4920
cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc 4980
cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga 5040
gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg 5100
atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag 5160
cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga 5220
acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg 5280
tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg 5340
cgtcacattt ccccgaaaag tgccacccgc ggaaccccta tttgtttatt tttctaaata 5400
cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 5460
aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 5520
ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 5580
cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 5640
agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 5700
gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 5760
cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 5820
gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 5880
ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 5940
gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 6000
gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 6060
cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 6120
ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 6180
gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 6240
gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 6300
gagataggtg cctcactgat taagcattgg taatgacagt ctggttcaaa tgagtatata 6360
tgaaatctaa ctaaattttg aagtaaaaat taaattttcc tagatccact tctaggaaaa 6420
actattagag tactggtttt agggaattgc actcaaaagc aaggtgactc gcagtctggg 6480
gcatcttttc tagtttccta gaagttgaga tccttttttt ctgcgcgtaa tctgctgctt 6540
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 6600
tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 6660
gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 6720
gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 6780
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 6840
acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 6900
agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 6960
cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 7020
tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 7080
gagcctatgg aaacccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg 7140
cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt 7200
tgggtaacgc cagggttttc ccagtcacga cgtttttaca ctttatgctt ccggctcgta 7260
tgttgcacct tttgtgagcg gataacaaca ccttaaagtg tcaggaaaca gctatgacca 7320
ccttaaagtg tgta 7334
<210> 5
<211> 11635
<212> DNA
<213> 人工序列
<400> 5
ggtacccaac tccatgctta acagtcccca ggtacagccc accctgcgtc gcaaccagga 60
acagctctac agcttcctgg agcgccactc gccctacttc cgcagccaca gtgcgcagat 120
taggagcgcc acttcttttt gtcacttgaa aaacatgtaa aaataatgta ctaggagaca 180
ctttcaataa aggcaaatgt ttttatttgt acactctcgg gtgattattt accccccacc 240
cttgccgtct gcgccgttta aaaatcaaag gggttctgcc gcgcatcgct atgcgccact 300
ggcagggaca cgttgcgata ctggtgttta gtgctccact taaactcagg cacaaccatc 360
cgcggcagct cggtgaagtt ttcactccac aggctgcgca ccatcaccaa cgcgtttagc 420
aggtcgggcg ccgatatctt gaagtcgcag ttggggcctc cgccctgcgc gcgcgagttg 480
cgatacacag ggttgcagca ctggaacact atcagcgccg ggtggtgcac gctggccagc 540
acgctcttgt cggagatcag atccgcgtcc aggtcctccg cgttgctcag ggcgaacgga 600
gtcaactttg gtagctgcct tcccaaaaag ggtgcatgcc caggctttga gttgcactcg 660
caccgtagtg gcatcagaag gtgaccgtgc ccggtctggg cgttaggata cagcgcctgc 720
atgaaagcct tgatctgctt aaaagccacc tgagcctttg cgccttcaga gaagaacatg 780
ccgcaagact tgccggaaaa ctgattggcc ggacaggccg cgtcatgcac gcagcacctt 840
gcgtcggtgt tggagatctg caccacattt cggccccacc ggttcttcac gatcttggcc 900
ttgctagact gctccttcag cgcgcgctgc ccgttttcgc tcgtcacatc catttcaatc 960
acgtgctcct tatttatcat aatgctcccg tgtagacact taagctcgcc ttcgatctca 1020
gcgcagcggt gcagccacaa cgcgcagccc gtgggctcgt ggtgcttgta ggttacctct 1080
gcaaacgact gcaggtacgc ctgcaggaat cgccccatca tcgtcacaaa ggtcttgttg 1140
ctggtgaagg tcagctgcaa cccgcggtgc tcctcgttta gccaggtctt gcatacggcc 1200
gccagagctt ccacttggtc aggcagtagc ttgaagtttg cctttagatc gttatccacg 1260
tggtacttgt ccatcaacgc gcgcgcagcc tccatgccct tctcccacgc agacacgatc 1320
ggcaggctca gcgggtttat caccgtgctt tcactttccg cttcactgga ctcttccttt 1380
tcctcttgcg tccgcatacc ccgcgccact gggtcgtctt cattcagccg ccgcaccgtg 1440
cgcttacctc ccttgccgtg cttgattagc accggtgggt tgctgaaacc caccatttgt 1500
agcgccacat cttctctttc ttcctcgctg tccacgatca cctctgggga tggcgggcgc 1560
tcgggcttgg gagaggggcg cttctttttc tttttggacg caatggccaa atccgccgtc 1620
gaggtcgatg gccgcgggct gggtgtgcgc ggcaccagcg catcttgtga cgagtcttct 1680
tcgtcctcgg actcgagacg ccgcctcagc cgcttttttg ggggcgcgcg gggaggcggc 1740
ggcgacggcg acggggacga cacgtcctcc atggttggtg gacgtcgcgc cgcaccgcgt 1800
ccgcgctcgg gggtggtttc gcgctgctcc tcttcccgac tggccatttc cttctcctat 1860
aggcagaaaa agatcatgga gtcagtcgag aaggaggaca gcctaaccgc cccctttgag 1920
ttcgccacca ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca 1980
cccccgcttg aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac 2040
gacgaggatc gctcagtacc aacagaggat aaaaagcaag accaggacga cgcagaggca 2100
aacgaggaac aagtcgggcg gggggaccaa aggcatggcg actacctaga tgtgggagac 2160
gacgtgctgt tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag 2220
cgcagcgatg tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctgttc 2280
tcaccgcgcg taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc 2340
aacttctacc ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa 2400
aactgcaaga tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc 2460
ttgcggcagg gcgctgtcat acctgatatc gcctcgctcg acgaagtgcc aaaaatcttt 2520
gagggtcttg gacgcgacga gaaacgcgcg gcaaacgctc tgcaacaaga aaacagcgaa 2580
aatgaaagtc actgtggagt gctggtggaa cttgagggtg acaacgcgcg cctagccgtg 2640
ctgaaacgca gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag 2700
gttatgagca cagtcatgag cgagctgatc gtgcgccgtg cacgacccct ggagagggat 2760
gcaaacttgc aagaacaaac cgaggagggc ctacccgcag ttggcgatga gcagctggcg 2820
cgctggcttg agacgcgcga gcctgccgac ttggaggagc gacgcaagct aatgatggcc 2880
gcagtgcttg ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg 2940
cagcgcaagc tagaggaaac gttgcactac acctttcgcc agggctacgt gcgccaggcc 3000
tgcaaaattt ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa 3060
aaccgcctcg ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac 3120
gtccgcgact gcgtttactt atttctgtgc tacacctggc aaacggccat gggcgtgtgg 3180
cagcaatgcc tggaggagcg caacctaaag gagctgcaga agctgctaaa gcaaaacttg 3240
aaggacctat ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacattatc 3300
ttccccgaac gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc 3360
atgttgcaaa actttaggaa ctttatccta gagcgttcag gaattctgcc cgccacctgc 3420
tgtgcgcttc ctagcgactt tgtgcccatt aagtaccgtg aatgccctcc gccgctttgg 3480
ggtcactgct accttctgca gctagccaac taccttgcct accactccga catcatggaa 3540
gacgtgagcg gtgacggcct actggagtgt cactgtcgct gcaacctatg caccccgcac 3600
cgctccctgg tctgcaattc gcaactgctt agcgaaagtc aaattatcgg tacctttgag 3660
ctgcagggtc cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg 3720
ctgtggacgt cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt 3780
aggttctacg aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc 3840
cagggccaca tccttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta 3900
cgaaagggac ggggggttta cctggacccc cagtccggcg aggagctcaa cccaatcccc 3960
ccgccgccgc agccctatca gcagccgcgg gcccttgctt cccaggatgg cacccaaaaa 4020
gaagctgcag ctgccgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc 4080
agaggaggtt ttggacgagg aggaggagat gatggaagac tgggacagcc tagacgaagc 4140
ttccgaggcc gaagaggtgt cagacgaaac accgtcaccc tcggtcgcat tcccctcgcc 4200
ggcgccccag aaattggcaa ccgttcccag catcgctaca acctccgctc ctcaggcgcc 4260
gccggcactg cctgttcgcc gacccaaccg tagatgggac accactggaa ccagggccgg 4320
taagtctaag cagccgccgc cgttagccca agagcaacaa cagcgccaag gctaccgctc 4380
gtggcgcggg cacaagaacg ccatagttgc ttgcttgcaa gactgtgggg gcaacatctc 4440
cttcgcccgc cgctttcttc tctaccatca cggcgtggcc ttcccccgta acatcctgca 4500
ttactaccgt catctctaca gcccctactg caccggcggc agcggcagcg gcagcaacag 4560
cagcggtcac acagaagcaa aggcgaccgg atagcaagac tctgacaaag cccaagaaat 4620
ccacagcggc ggcagcagca ggaggaggag cgctgcgtct ggcgcccaac gaacccgtat 4680
cgacccgcga gcttagaaat aggatttttc ccactctgta tgctatattt caacaaagca 4740
ggggccaaga acaagagctg aaaataaaaa acaggtctct gcgctccctc acccgcagct 4800
gcctgtatca caaaagcgaa gatcagcttc ggcgcacgct ggaagacgcg gaggctctct 4860
tcagcaaata ctgcgcgctg actcttaagg actagtttcg cgccctttct caaatttaag 4920
cgcgaaaact acgtcatctc cagcggccac acccggcgcc agcacctgtc gtcagcgcca 4980
ttatgagcaa ggaaattccc acgccctaca tgtggagtta ccagccacaa atgggacttg 5040
cggctggagc tgcccaagac tactcaaccc gaataaacta catgagcgcg ggaccccaca 5100
tgatatcccg ggtcaacgga atccgcgccc accgaaaccg aattctcctc gaacaggcgg 5160
ctattaccac cacacctcgt aataacctta atccccgtag ttggcccgct gccctggtgt 5220
accaggaaag tcccgctccc accactgtgg tacttcccag agacgcccag gccgaagttc 5280
agatgactaa ctcaggggcg cagcttgcgg gcggctttcg tcacagggtg cggtcgcccg 5340
ggcgttttag ggcggagtaa cttgcatgta ttgggaattg tagttttttt aaaatgggaa 5400
gtgacgtatc gtgggaaaac ggaagtgaag atttgaggaa gttgtgggtt ttttggcttt 5460
cgtttctggg cgtaggttcg cgtgcggttt tctgggtgtt ttttgtggac tttaaccgtt 5520
acgtcatttt ttagtcctat atatactcgc tctgtacttg gcccttttta cactgtgact 5580
gattgagctg gtgccgtgtc gagtggtgtt ttttaatagg tttttttact ggtaaggctg 5640
actgttatgg ctgccgctgt ggaagcgctg tatgttgttc tggagcggga gggtgctatt 5700
ttgcctaggc aggagggttt ttcaggtgtt tatgtgtttt tctctcctat taattttgtt 5760
atacctccta tgggggctgt aatgttgtct ctacgcctgc gggtatgtat tcccccgggc 5820
tatttcggtc gctttttagc actgaccgat gttaaccaac ctgatgtgtt taccgagtct 5880
tacattatga ctccggacat gaccgaggaa ctgtcggtgg tgctttttaa tcacggtgac 5940
cagttttttt acggtcacgc cggcatggcc gtagtccgtc ttatgcttat aagggttgtt 6000
tttcctgttg taagacaggc ttctaatgtt taaatgtttt tttttttgtt attttatttt 6060
gtgtttaatg caggaacccg cagacatgtt tgagagaaaa atggtgtctt tttctgtggt 6120
ggttccggaa cttacctgcc tttatctgca tgagcatgac tacgatgtgc ttgctttttt 6180
gcgcgaggct ttgcctgatt ttttgagcag caccttgcat tttatatcgc cgcccatgca 6240
acaagcttac ataggggcta cgctggttag catagctccg agtatgcgtg tcataatcag 6300
tgtgggttct tttgtcatgg ttcctggcgg ggaagtggcc gcgctggtcc gtgcagacct 6360
gcacgattat gttcagctgg ccctgcgaag ggacctacgg gatcgcggta tttttgttaa 6420
tgttccgctt ttgaatctta tacaggtctg tgaggaacct gaatttttgc aatcatgatt 6480
cgctgcttga ggctgaaggt ggagggcgct ctggagcaga tttttacaat ggccggactt 6540
aatattcggg atttgcttag agacatattg ataaggtggc gagatgaaaa ttatttgggc 6600
atggttgaag gtgctggaat gtttatagag gagattcacc ctgaagggtt tagcctttac 6660
gtccacttgg acgtgagggc agtttgcctt ttggaagcca ttgtgcaaca tcttacaaat 6720
gccattatct gttctttggc tgtagagttt gaccacgcca ccggagggga gcgcgttcac 6780
ttaatagatc ttcattttga ggttttggat aatcttttgg aataaaaaaa aaaaaacatg 6840
gttcttccag ctcttcccgc tcctcccgtg tgtgactcgc agaacgaatg tgtaggttgg 6900
ctgggtgtgg cttattctgc ggtggtggat gttatcaggg cagcggcgca tgaaggagtt 6960
tacatagaac ccgaagccag ggggcgcctg gatgctttga gagagtggat atactacaac 7020
tactacacag agcgagctaa gcgacgagac cggagacgca gatctgtttg tcacgcccgc 7080
acctggtttt gcttcaggaa atatgactac gtccggcgtt ccatttggca tgacactacg 7140
accaacacga tctcggttgt ctcggcgcac tccgtacagt agggatcgcc tacctccttt 7200
tgagacagag acccgcgcta ccatactgga ggatcatccg ctgctgcccg aatgtaacac 7260
tttgacaatg cacaacgtga gttacgtgcg aggtcttccc tgcagtgtgg gatttacgct 7320
gattcaggaa tgggttgttc cctgggatat ggttctgacg cgggaggagc ttgtaatcct 7380
gaggaagtgt atgcacgtgt gcctgtgttg tgccaacatt gatatcatga cgagcatgat 7440
gatccatggt tacgagtcct gggctctcca ctgtcattgt tccagtcccg gttccctgca 7500
gtgcatagcc ggcgggcagg ttttggccag ctggtttagg atggtggtgg atggcgccat 7560
gtttaatcag aggtttatat ggtaccggga ggtggtgaat tacaacatgc caaaagaggt 7620
aatgtttatg tccagcgtgt ttatgagggg tcgccactta atctacctgc gcttgtggta 7680
tgatggccac gtgggttctg tggtccccgc catgagcttt ggatacagcg ccttgcactg 7740
tgggattttg aacaatattg tggtgctgtg ctgcagttac tgtgctgatt taagtgagat 7800
cagggtgcgc tgctgtgccc ggaggacaag gcgtctcatg ctgcgggcgg tgcgaatcat 7860
cgctgaggag accactgcca tgttgtattc ctgcaggacg gagcggcggc ggcagcagtt 7920
tattcgcgcg ctgctgcagc accaccgccc tatcctgatg cacgattatg actctacccc 7980
catgtaggcg tggacttccc cttcgccgcc cgttgagcaa ccgcaagttg gacagcagcc 8040
tgtggctcag cagctggaca gcgacatgaa cttaagcgag ctgcccgggg agtttattaa 8100
tatcactgat gagcgtttgg ctcgacagga aaccgtgtgg aatataacac ctaagaatat 8160
gtctgttacc catgatatga tgctttttaa ggccagccgg ggagaaagga ctgtgtactc 8220
tgtgtgttgg gagggaggtg gcaggttgaa tactagggtt ctgtgagttt gattaaggta 8280
cggtgatcaa tataagctat gtggtggtgg ggctatacta ctgaatgaaa aatgacttga 8340
aattttctgc aattgaaaaa taaacacgtt gaaacataac atgcaacagg ttcacgattc 8400
tttattcctg ggcaatgtag gagaaggtgt aagagttggt agcaaaagtt tcagtggtgt 8460
attttccact ttcccaggac catgtaaaag acatagagta agtgcttacc tcgctagttt 8520
ctgtggattc actagaatcg atgtaggatg ttgcccctcc tgacgcggta ggagaagggg 8580
agggtgccct gcatgtctgc cgctgctctt gctcttgccg ctgctgagga ggggggcgca 8640
tctgccgcag caccggatgc atctgggaaa agcaaaaaag gggctcgtcc ctgtttccgg 8700
aggaatttgc aagcggggtc ttgcatgacg gggaggcaaa cccccgttcg ccgcagtccg 8760
gccggcccga gactcgaacc gggggtcctg cgactcaacc cttggaaaat aaccctccgg 8820
ctacagggag cgagccactt aatgctttcg ctttccagcc taaccgctta cgccgcgcgc 8880
ggccagtggc caaaaaagct agcgcagcag ccgccgcgcc tggaaggaag ccaaaaggag 8940
cgctcccccg ttgtctgacg tcgcacacct gggttcgaca cgcgggcggt aaccgcatgg 9000
atcacggcgg acggccggat ccggggttcg aaccccggtc gtccgccatg atacccttgc 9060
gaatttatcc accagaccac ggaagagtgc ccgcttacag gctctccttt tgcacggtct 9120
agagcgtcaa cgactgcgca cgcctcaccg gccagagcgt cccgaccatg gagcactttt 9180
tgccgctgcg caacatctgg aaccgcgtcc gcgactttcc gcgcgcctcc accaccgccg 9240
ccggcatcac ctggatgtcc aggtacatct acggattacg tcgacgttta aaccatatga 9300
tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 9360
aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 9420
tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 9480
tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 9540
cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 9600
agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 9660
tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 9720
aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 9780
ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 9840
cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 9900
accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 9960
ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 10020
ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 10080
gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 10140
aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 10200
gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 10260
gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 10320
cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 10380
gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 10440
gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 10500
ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 10560
tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 10620
ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 10680
cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 10740
accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 10800
cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 10860
tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 10920
cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 10980
acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 11040
atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 11100
tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 11160
aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt 11220
aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag 11280
aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga 11340
acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg 11400
aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc 11460
ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg 11520
aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc 11580
gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgatg gatcc 11635
<210> 6
<211> 5747
<212> DNA
<213> 人工序列
<400> 6
aacgccagca acgcggcctt tttacggttc ctggcctttt gctggcggcc gcacgcgtac 60
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 120
gatccgacgc cgccatctct aggcccgcgc cggccccctc gcacagactt gtgggagaag 180
ctcggctact cccctgcccc ggttaatttg catataatat ttcctagtaa ctatagaggc 240
ttaatgtgcg ataaaagaca gataatctgt tctttttaat actagctaca ttttacatga 300
taggcttgga tttctataag agatacaaat actaaattat tattttaaaa aacagcacaa 360
aaggaaactc accctaactg taaagtaatt gtgtgttttg agactataaa tatcccttgg 420
agaaaagcct tgtttgaaac aaattcgtaa cagtctccag tcacggccat tcaagagatg 480
gccgtgtgga gactgttatt ttttgccgcg gccgctaaca acatctaggg aatacttgag 540
gcacttcccc aaactccggc cgcttttttc tgcaggggcg gcctcgtgat acgcctattt 600
ttataggtta atgtcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac 660
gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact 720
ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa 780
gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg 840
cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta 900
gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg 960
tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 1020
caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 1080
ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag 1140
atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc 1200
agcctccgta ccggttcgaa caggtaagcg cccctaaaat cccatggtga gcaagggcga 1260
ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 1320
caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa 1380
gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac 1440
ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa 1500
gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa 1560
ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 1620
gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 1680
caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt 1740
caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 1800
cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 1860
cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 1920
cgccgccggg atcactctcg gcatggacga gctgtacaag ctcgaatcaa gcttatcgat 1980
aaaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg 2040
ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc 2100
gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt 2160
tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca 2220
ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc 2280
ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc 2340
tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc 2400
tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc 2460
tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc 2520
ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc 2580
gtcgatcgag aaatcgatcg cccgggtggc atccctgtga cccctcccca gtgcctctcc 2640
tggccctgga agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca 2700
tcattttgtc tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga 2760
gcaaggggca agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct 2820
ggagtgcagt ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc 2880
tcctgcctca gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat 2940
ttttgttttt ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct 3000
aatctcaggt gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca 3060
ctgctccctt ccctgtcctt tccggacacg tgcggaccga gcggccgcaa cgccagcaac 3120
gcggcctttt tacggttcct ggccttttgc tgggcgcctg atgcggtatt ttctccttac 3180
gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 3240
ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 3300
gccttagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 3360
ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 3420
ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 3480
acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 3540
actggaacaa cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg 3600
atttcggtct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 3660
aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 3720
tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 3780
ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 3840
ttttcaccgt catcaccgaa acgcgcgacg cggaacccct atttgtttat ttttctaaat 3900
acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 3960
aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 4020
attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 4080
tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 4140
gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 4200
cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 4260
tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 4320
agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 4380
tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 4440
tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 4500
tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 4560
acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 4620
accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 4680
tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 4740
cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 4800
tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 4860
actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 4920
tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 4980
cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 5040
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 5100
tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt 5160
gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 5220
gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 5280
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 5340
acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 5400
agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 5460
cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 5520
tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 5580
gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctgttg 5640
cggtcgttgc gccggaaaaa tgccaaggac cggaaaacga ccggaaaacg agtgtacaag 5700
aaaggacgca ataggggact aagacaccta ttggcataat ggcggaa 5747
<210> 7
<211> 129
<212> DNA
<213> 人工序列
<400> 7
acagaattct ggccgtgact ggagactgtt atatactggc cgtgactgga gactgttaac 60
ttactggccg tgactggaga ctgttatctt catggccgtg actggagact gttatttttt 120
ggatccaca 129

Claims (9)

1.一种miRNA-212-3P海绵表达载体，其特征在于，构建方法为：在基本载体pHBAAV-U6-MCS-CMV-EGFP的多克隆位点连入序列如SEQ ID NO:3的第442-569bp所示的序列。
2.根据权利要求1所述的miRNA-212-3P海绵表达载体，其特征在于，所述miRNA-212-3P海绵表达载体的序列如SEQ ID NO:3所示。
3.权利要求1或2所述的miRNA-212-3P海绵表达载体在制备预防或治疗非酒精性脂肪性肝病的药物中的应用。
4.权利要求1或2所述的miRNA-212-3P海绵表达载体的制备方法，其特征在于，在基本载体pHBAAV-U6-MCS-CMV-EGFP的多克隆位点连入序列如SEQ ID NO:3的第442-569bp所示的序列，所述miRNA-212-3P海绵表达载体的序列如SEQ ID NO:3所示。
5.一种腺相关病毒，其特征在于，制备所述腺相关病毒采用的穿梭质粒是权利要求1或2所述的miRNA-212-3P海绵表达载体。
6.根据权利要求5所述的腺相关病毒，其特征在于，制备所述腺相关病毒采用的编码rep和cap蛋白编码基因的pAAV-RC为8型AAV。
7.根据权利要求6所述的腺相关病毒，其特征在于，所述腺相关病毒的制备方法包含以下步骤：将权利要求1或2的miRNA-212-3P海绵表达载体，与pAAV-RC质粒和pHelper质粒共转染AAV-293细胞。
8.权利要求5-7任一所述的腺相关病毒在制备预防或治疗非酒精性脂肪性肝病的药物中的应用。
9.权利要求5-7任一所述的腺相关病毒的制备方法，其特征在于，将权利要求1或2的miRNA-212-3P海绵表达载体，与pAAV-RC质粒和pHelper质粒共转染AAV-293细胞。
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