CN114107387A - 一种可用于全脑星形胶质细胞活体成像的腺相关病毒及其应用 - Google Patents

一种可用于全脑星形胶质细胞活体成像的腺相关病毒及其应用 Download PDF

Info

Publication number
CN114107387A
CN114107387A CN202111310451.1A CN202111310451A CN114107387A CN 114107387 A CN114107387 A CN 114107387A CN 202111310451 A CN202111310451 A CN 202111310451A CN 114107387 A CN114107387 A CN 114107387A
Authority
CN
China
Prior art keywords
egfp
aqp1
astrocytes
associated virus
gfap
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111310451.1A
Other languages
English (en)
Inventor
王杰
柳壮
李梅
徐富强
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Precision Measurement Science and Technology Innovation of CAS
Original Assignee
Institute of Precision Measurement Science and Technology Innovation of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Precision Measurement Science and Technology Innovation of CAS filed Critical Institute of Precision Measurement Science and Technology Innovation of CAS
Priority to CN202111310451.1A priority Critical patent/CN114107387A/zh
Publication of CN114107387A publication Critical patent/CN114107387A/zh
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0097—Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1896—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes not provided for elsewhere, e.g. cells, viruses, ghosts, red blood cells, virus capsides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/34—Vector systems having a special element relevant for transcription being a transcription initiation element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/36—Vector systems having a special element relevant for transcription being a transcription termination element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Radiology & Medical Imaging (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明公开了一种可用于全脑星形胶质细胞活体成像的腺相关病毒及其应用，该rAAV载体通过启动子GFAP驱动水通道蛋白AQP1与绿色荧光蛋白EGFP在星形胶质细胞中特异表达，AQP1蛋白将改变扩散加权MRI信号水平，EGFP将产生荧光信号，因而该重组腺相关病毒可作为星形胶质细胞磁共振成像与荧光成像的双模态造影载体，并且在活体水平实现了对整个大脑的星形胶质细胞检测，可用于制备星形胶质细胞活体水平的标记物或示踪剂。

Description

一种可用于全脑星形胶质细胞活体成像的腺相关病毒及其 应用
技术领域
本发明属于病毒载体技术领域，具体涉及一种携带GFAP启动子和1型水通道蛋白的腺相关病毒及其应用。
背景技术
蛋白质造影剂的基因编码和细胞生产能力为细胞功能的体内研究创造了前所未有的机会，而它们的基因工程亲和性使其物理、化学和生物学特性的原子水平设计成为可能。在这方面，很少有技术能像绿色荧光蛋白(GFP)那样产生如此巨大的影响。GFP可以在细胞内进行基因编码，与细胞内部的生命周期和分子信号建立密切联系。然而，由于光在生物组织中的强散射，荧光成像的穿透深度有限，超过了一毫米这限制了它在透明动物、小型模型生物和外科手术进入区域的使用。在完整生物体背景下研究细胞功能的需求、基于细胞的诊断和治疗制剂的开发以及复杂生物材料的工程技术的推动下，对深入研究细胞功能的需求巨大。与荧光成像不同，磁共振成像(MRI)可以穿透完整组织的深处，从而在临床医学中得到广泛应用。在过去的20年里，大量的努力致力于开发这些非侵入性成像模式的报告基因--用于MRI的“GFP”。磁共振成像(MRI)是观察生物体内特定细胞和分子功能的一项强有力的技术。然而，与光学显微镜相比，荧光蛋白报告可以显示从基因表达和化学信号到生物力学的数百个细胞功能，迄今为止，磁共振成像可用的此类报告相对较少。开发核磁共振可检测的生物分子的努力主要集中在含有或运输顺磁性金属的蛋白质上，用于T1和T2弛豫增强，或用于化学交换饱和转移的大量可交换质子。最新生物分子MRI造影剂之一是使用水通道蛋白(Aquaporin,AQP)作为扩散加权成像(DWI)的报告基因。DWI是一种广泛应用于解剖成像的技术，在该技术中，扩散加权通常通过应用一对脉冲磁场梯度来实现，这一对脉冲磁场梯度根据核自旋在特定时间段内扩散的程度来使核自旋相去相。因此，扩散较自由的水分子具有较严重的脱相质子自旋，在DWI中呈现较暗。
星形胶质细胞是特化的神经胶质细胞，其数量超过神经元五倍。它们连续地覆盖整个中枢神经系统(CNS)，与多种类型的细胞相互作用，包括神经元、其他胶质细胞和血管，并在健康的中枢神经系统中发挥许多基本的复杂功能。星形胶质细胞存在于大脑的所有区域；事实上，它们构成了近一半的脑细胞。它们在滋养神经元和调节细胞外空间的离子和神经递质浓度方面发挥着重要作用。星形胶质细胞通过调节其代谢活动，控制神经递质水平、调节细胞外钾离子，从而影响神经细胞放电的阈值，并释放促进突触形成和修剪的分子。大脑不同部位的星形胶质细胞不一样，例如，在海马体中，星形胶质细胞更靠近兴奋性神经突触，并且对神经递质谷氨酸的反应比对纹状体的反应更强。因为它表明星形胶质细胞可能是脑区特异性的，甚至是神经回路特异性的，对星形胶质细胞的功能作用及结构的深入研究是解答此类问题的根本前提。
整合以上两种神经网络研究的优势方法，申请人开发了一种新型磁共振技术，通过可见而又能标记全脑星形胶质细胞结构的分子探针来实现活体长时程解析胶质细胞的网络的目的。
发明内容
本发明的目的在于构建一种同时可以用荧光成像和磁共振成像的双模态载体工具检测活体星形胶质细胞的变化的病毒载体。该载体工具可通过荧光成像离体观察信号，转导的AQP1蛋白在星形胶质细胞中表达，该病毒载体靶向星形胶质细胞，即可以通过无创的磁共振成像检测活体组织中的星形胶质细胞原位信号。
为了实现上述目的，本发明采用以下技术方案：
重组腺病毒质粒构建，该质粒是在AAV载体质粒的ITR中间依次插入GFAP启动子(SEQ ID NO.1所示)、AQP1基因(SEQ ID NO.2所示)、2A连接序列(SEQ ID NO.3所示)、EGFP基因(SEQ ID NO.4所示)、WPRE转录后调控元件(SEQ ID NO.5所示)以及polyA序列(SEQ IDNO.6所示)，构建重组腺相关病毒核心质粒pAAV-GFAP-AQP1-2A-EGFP-WPRE-pA。
重组腺相关病毒制备，方法如下：将包装rAAV所需的辅助质粒pAAV-helper、血清型质粒AAV、核心质粒pAAV-GFAP-AQP1-2A-EGFP-WPRE-pA共同转染HEK293细胞，收集离心后的细胞沉淀纯化重组腺相关病rAAV-GFAP-AQP1-2A-EGFP-WPRE-pA。
重组腺相关病毒rAAV-GFAP-AQP1-2A-EGFP-WPRE-pA的应用：该病毒可介导星形胶质细胞表达AQP1蛋白和EGFP蛋白，进而AQP1蛋白将影响DWI-MRI信号，EGFP将产生荧光信号，因而该重组腺相关病毒可作为星形胶质细胞磁共振成像与荧光成像的双模态造影载体，用于制备星形胶质细胞活体水平的标记物或示踪剂。
与现有监控星形胶质细胞的技术相比，本发明具有以下优点：
1.本发明中重组病毒rAAV-GFAP-AQP1-2A-EGFP-WPRE-pA使用高效率的GFAP启动子，保障了AQP1与EGFP蛋白的定位表达效果，而高丰度的AQP1蛋白保证了MRI的造影效果。
2.本发明rAAV载体在基因治疗中广泛应用，具有宿主范围广泛，毒性低，对宿主的免疫原性相比其它病毒载体比较低，且在宿主中能持续稳定表达等优点。
3.本发明中提出的重组腺相关病毒应用主要是活体全脑星形胶质细胞的检测，除了监测作用，病毒载体上还能再插入其它功能基因，用于神经疾病的治疗。功能基因在载体上表达，可以磁共振成像检测活体水平功能基因的表达水平，也可以通过荧光成像离体检测功能基因表达。
附图说明
图1为携带AQP1基因及EGFP基因的表达框的示意图。
图2为rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA病毒(图2A)和对照病毒(图2B)感染全脑星形胶质细胞3周后检测DWI核磁信号的结果。
图3为rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA病毒感染皮层区域的DWI-MRI信号三次扫描的强度值统计结果。
图4为感染rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA后矢状面脑片GFAP免疫组化的结果。A为DAPI细胞核复染的成像结果，B为绿色荧光蛋白成像的结果，C为GFAP免疫组化的成像结果，D为红色、绿色和蓝色的组合通道成像结果。
具体实施方式
本发明所述技术方案，如未特别说明，均为本领域的常规技术，所述试剂与材料，如未特别说明，均来源于商业渠道。此处展示的具体实施例仅用于解释本发明，而不用于限定本发明。
实施例1：携带AQP1与EGFP基因的质粒pAAV-GFAP-AQP1-2A-EGFP-WPRE-pA的构建与重组腺相关病毒rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA的制备
为了靶向星形胶质细胞，我们选取了人源的GFAP启动子。为了使AQP1基因与绿色荧光蛋白(EGFP)报告基因表达，我们选取了2A自剪切多肽编码序列作为AQP1基因与EGFP基因的连接序列。为了提高目的基因转录后的稳定性，我们在目的基因的3’端增加了WPRE转录后调控元件以及polyA序列。我们利用常规分子克隆方法在包装rAAV的核心质粒pAAV-MCS中的AAV的末端重复序列(ITR)中间插入GFAP启动子(SEQ ID NO.1所示)、AQP1基因(SEQID NO.2所示)、2A连接序列(SEQ ID NO.3所示)、EGFP基因(SEQ ID NO.4所示)、WPRE转录后调控元件(SEQ ID NO.5所示)以及polyA序列(SEQ ID NO.6所示)，携带AQP1与EGFP基因的表达框如图1所示，由此构建重组腺相关病毒核心质粒pAAV-GFAP-AQP1-2A-EGFP-WPRE-pA质粒。
重组腺相关病毒rAAV的包装和纯化步骤如下：
为了提高rAAV感染活体的效率和便于rAAV的制备，我们选取跨血脑屏障常用的AAV-PHP.eB。首先将HEK293细胞铺到10cm细胞皿中培养至80％密度，将包装rAAV所需的三个质粒(辅助质粒pAAV-helper、血清型质粒AAV-PHP.eB、核心质粒pAAV-GFAP-AQP1-2A-EGFP-WPRE-pA)以摩尔比(1:1:1)共同转染HEK293细胞。转染72h后，用细胞刮将产毒的贴壁细胞刮下后离心，收集离心后的细胞沉淀。然后，将细胞沉淀裂解液用核酸酶处理，病毒上清液中的rAAV用PEG/NaCl沉淀浓缩。将含有rAAV的裂解液上清用碘克沙醇密度梯度超速离心分离纯化，最后将纯化得到的rAAV分装后，在-80度冰箱冻存。使用BioRad公司的CFXConnect荧光定量PCR仪进行实时荧光定量PCR测定rAAV病毒的滴度。
实施例2：注射rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA及对照病毒rAAV-PHP.eB-GFAP-EGFP-WPRE-pA的小鼠大脑的MRI成像观察
将rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA病毒和对照病毒rAAV-PHP.eB-GFAP-EGFP-WPRE-pA分别通过尾静脉注射到小鼠体内，其滴度为1×1012vg/ml，注射体积为100μL。
0、2、3周时间点，使用7.0T磁共振成像仪对动物大脑进行活体MRI观察，其中用到的扩散加权成像的序列为基于STEAM的SE-DWI序列，参数为：TR＝3000ms，TE＝24ms，平均次数为2，序列总时长为51min12s。扩散梯度持续时间为7ms，扩散梯度间隔时间为100ms，b值为1000s·mm-2。FOV为2.5×2.2cm，空间分辨率为0.195×0.171mm，层厚为0.8mm。3周后MRI结果(图2)表明rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA病毒成功感染星形胶质细胞，并在大部分脑区表达出足够改变DWI-MRI信号的AQP1蛋白，而注射了对照病毒的动物未呈现DWI信号的改变。图3显示了一侧皮层的DWI-MRI信号强度值的统计比较结果，3周的ADC值明显高于起始的ADC值。此结果表明AQP1蛋白增强了水分子的扩散，降低了DWI-MRI信号，产生了可在活体水平观察的MRI对比度。
实施3：注射rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA病毒的小鼠大脑的GFAP蛋白免疫组化及荧光成像
将rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA病毒通过尾静脉注射到小鼠体内。3周的MRI实验后，麻醉小鼠，进行心脏灌流后取出鼠脑并做冰冻切片。随后使用羊来源的GFAP抗体对小鼠大脑切片进行免疫组化染色，以显示病毒高表达的脑区。结果见图4，其中绿色为EGFP蛋白产生的荧光信号，红色为GFAP免疫组化的信号。结果表明，rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA可介导星形胶质细胞表达AQP1蛋白和EGFP蛋白，进而AQP1蛋白将影响DWI-MRI信号，EGFP将产生荧光信号。因此，rAAV-PHP.eB-GFAP-AQP1-2A-EGFP-WPRE-pA可作为磁共振成像与荧光成像的全脑星形胶质细胞造影载体。
序列表
<110> 中国科学院精密测量科学与技术创新研究院
<120> 一种可用于全脑星形胶质细胞活体成像的腺相关病毒及其应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2207
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
cccacctccc tctctgtgct gggactcaca gagggagacc tcaggaggca gtctgtccat 60
cacatgtcca aatgcagagc ataccctggg ctgggcgcag tggcgcacaa ctgtaattcc 120
agcactttgg gaggctgatg tggaaggatc acttgagccc agaagttcta gaccagcctg 180
ggcaacatgg caagacccta tctctacaaa aaaagttaaa aaatcagcca cgtgtggtga 240
cacacacctg tagtcccagc tattcaggag gctgaggtga ggggatcact taaggctggg 300
aggttgaggc tgcagtgagt cgtggttgcg ccactgcact ccagcctggg caacagtgag 360
accctgtctc aaaagacaaa aaaaaaaaaa aaaaaaaaaa gaacatatcc tggtgtggag 420
taggggacgc tgctctgaca gaggctcggg ggcctgagct ggctctgtga gctggggagg 480
aggcagacag ccaggccttg tctgcaagca gacctggcag cattgggctg gccgcccccc 540
agggcctcct cttcatgccc agtgaatgac tcaccttggc acagacacaa tgttcggggt 600
gggcacagtg cctgcttccc gccgcacccc agcccccctc aaatgccttc cgagaagccc 660
attgagcagg gggcttgcat tgcaccccag cctgacagcc tggcatcttg ggataaaagc 720
agcacagccc cctaggggct gcccttgctg tgtggcgcca ccggcggtgg agaacaaggc 780
tctattcagc ctgtgcccag gaaaggggat caggggatgc ccaggcatgg acagtgggtg 840
gcaggggggg agaggagggc tgtctgcttc ccagaagtcc aaggacacaa atgggtgagg 900
ggactgggca gggttctgac cctgtgggac cagagtggag ggcgtagatg gacctgaagt 960
ctccagggac aacagggccc aggtctcagg ctcctagttg ggcccagtgg ctccagcgtt 1020
tccaaaccca tccatcccca gaggttcttc ccatctctcc aggctgatgt gtgggaactc 1080
gaggaaataa atctccagtg ggagacggag gggtggccag ggaaacgggg cgctgcagga 1140
ataaagacga gccagcacag ccagctcatg tgtaacggct ttgtggagct gtcaaggcct 1200
ggtctctggg agagaggcac agggaggcca gacaaggaag gggtgacctg gagggacaga 1260
tccaggggct aaagtcctga taaggcaaga gagtgccggc cccctcttgc cctatcagga 1320
cctccactgc cacatagagg ccatgattga cccttagaca aagggctggt gtccaatccc 1380
agcccccagc cccagaactc cagggaatga atgggcagag agcaggaatg tgggacatct 1440
gtgttcaagg gaaggactcc aggagtctgc tgggaatgag gcctagtagg aaatgaggtg 1500
gcccttgagg gtacagaaca ggttcattct tcgccaaatt cccagcacct tgcaggcact 1560
tacagctgag tgagataatg cctgggttat gaaatcaaaa agttggaaag caggtcagag 1620
gtcatctggt acagcccttc cttccctttt tttttttttt tttttgtgag acaaggtctc 1680
tctctgttgc ccaggctgga gtggcgcaaa cacagctcac tgcagcctca acctactggg 1740
ctcaagcaat cctccagcct cagcctccca aagtgctggg attacaagca tgagccaccc 1800
cactcagccc tttccttcct ttttaattga tgcataataa ttgtaagtat tcatcatggt 1860
ccaaccaacc ctttcttgac ccaccttcct agagagaggg tcctcttgct tcagcggtca 1920
gggccccaga cccatggtct ggctccaggt accacctgcc tcatgcagga gttggcgtgc 1980
ccaggaagct ctgcctctgg gcacagtgac ctcagtgggg tgaggggagc tctccccata 2040
gctgggctgc ggcccaaccc caccccctca ggctatgcca gggggtgttg ccaggggcac 2100
ccgggcatcg ccagtctagc ccactccttc ataaagccct cgcatcccag gagcgagcag 2160
agccagagca ggttggagag gagacgcatc acctccgctg ctcgccg 2207
<210> 2
<211> 807
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggccagcg agttcaagaa gaagctgttc tggagggccg tggtggctga gttcctggcc 60
accaccctgt tcgtgttcat cagcatcggc agcgccctgg gcttcaagta ccctgtgggc 120
aacaaccaga cagccgtgca agacaacgtg aaggtgtccc tggccttcgg cctgagcatc 180
gccaccctgg cccagtcggt gggacacatc tccggcgccc acctgaaccc tgccgtgacc 240
ctgggcctgc tgctgagctg tcagatcagc atctttagag ccctgatgta catcatcgcc 300
cagtgtgtgg gtgccatcgt ggccaccgcc atcctgagcg gcatcaccag cagcctgacc 360
ggcaacagcc tgggcagaaa cgacctggcc gacggcgtga acagcggcca gggcctgggc 420
atcgagatca tcggcacact gcagctggtg ctgtgcgtgc tggctacaac cgacaggagg 480
agaagagacc tgggaggatc cgcccctctg gctattggcc tgagcgtggc cctgggacat 540
ctgctggcta ttgattatac aggatgtggc attaatcctg ctagatcttt cggttctgct 600
gtgatcacac ataatttctc taatcactgg atcttttggg tgggcccatt catcggcgga 660
gccctggccg tgctgatcta tgattttatt ctggctccaa gatctagtga tctgactgat 720
agagtgaagg tgtggacatc cggacaggtg gaagagtatg accttgatgc tgatgatatt 780
aatagtaggg tagagatgaa gcctaaa 807
<210> 3
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gctactaact ttagcctgct gaagcaggcc ggggacgtgg aagagaaccc tggccct 57
<210> 4
<211> 717
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gtgagcaagg tcgaggagct gttcaccggc gtggtgccca tcctggtgga gctggacggc 60
gacgtgaacg gccacaagtt cagcgtgagc ggcgagggcg agggcgacgc cacctacggc 120
aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctg 180
gtgaccaccc tgacctacgg cgtgcagtgc ttcagcaggt accccgacca catgaagcag 240
cacgacttct tcaagagcgc catgcccgag ggctacgtgc aggagaggac catcttcttc 300
aaggacgacg gcaactacaa gaccagggcc gaggtgaagt tcgagggcga caccctggtg 360
aacaggatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct gggccacaag 420
ctggagtaca actacaacag ccacaacgtg tacatcatgg ccgacaagca gaagaacggc 480
atcaaggtga acttcaagat caggcacaac atcgaggacg gcagcgtgca gctggccgac 540
cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600
ctgagcaccc agagcgccct gagcaaggac cccaacgaga agagggacca catggtgctg 660
ctggagttcg tgaccgccgc cggcatcacc ctgggcatgg acgagctgta caagtaa 717
<210> 5
<211> 589
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60
ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120
atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180
tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240
ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300
attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360
ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420
gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480
aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540
cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589
<210> 6
<211> 477
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60
gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 120
ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 180
acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg 240
ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg 300
ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg 360
ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct 420
tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtcctt 477

Claims (4)

1.重组腺病毒质粒，其特征在于，在AAV载体质粒的ITR中间依次插入GFAP启动子、AQP1基因、2A连接序列、EGFP基因、WPRE转录后调控序列和polyA终止密码子序列。
2.重组腺相关病毒，其特征在于，以权利要求1所述重组腺病毒质粒为核心质粒制备重组腺相关病毒。
3.权利要求1所述的重组腺病毒质粒或权利要求2所述的重组腺相关病毒在制备星形胶质细胞磁共振成像造影剂中的应用。
4.权利要求1所述的重组腺病毒质粒或权利要求2所述的重组腺相关病毒在制备星形胶质细胞活体水平的标记物及示踪剂中的应用。
CN202111310451.1A 2021-11-05 2021-11-05 一种可用于全脑星形胶质细胞活体成像的腺相关病毒及其应用 Pending CN114107387A (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111310451.1A CN114107387A (zh) 2021-11-05 2021-11-05 一种可用于全脑星形胶质细胞活体成像的腺相关病毒及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111310451.1A CN114107387A (zh) 2021-11-05 2021-11-05 一种可用于全脑星形胶质细胞活体成像的腺相关病毒及其应用

Publications (1)

Publication Number Publication Date
CN114107387A true CN114107387A (zh) 2022-03-01

Family

ID=80381437

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111310451.1A Pending CN114107387A (zh) 2021-11-05 2021-11-05 一种可用于全脑星形胶质细胞活体成像的腺相关病毒及其应用

Country Status (1)

Country Link
CN (1) CN114107387A (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023168400A3 (en) * 2022-03-03 2023-11-23 Research Institute At Nationwide Children's Hospital Materials and methods for the treatment of eif2b5 mutations and diseases resulting therefrom

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010071454A1 (en) * 2008-12-17 2010-06-24 Auckland Uniservices Limited Adeno-associated viral vectors and uses thereof
CN113025657A (zh) * 2021-03-05 2021-06-25 中国科学院精密测量科学与技术创新研究院 一种可用于双模态成像的腺相关病毒及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010071454A1 (en) * 2008-12-17 2010-06-24 Auckland Uniservices Limited Adeno-associated viral vectors and uses thereof
CN113025657A (zh) * 2021-03-05 2021-06-25 中国科学院精密测量科学与技术创新研究院 一种可用于双模态成像的腺相关病毒及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GRY FLUGE VINDEDAL: "Molecular and functional aspects of astrocytes at the brain-blood interface" *
JARRED M. GRIFFIN等: "Astrocyte-selective AAV gene therapy through the endogenous GFAP promoter results in robust transduction in the rat spinal cord following injury" *
MEI LI等: "In vivo imaging of astrocytes in the whole brain with engineered AAVs and diffusion-weighted magnetic resonance imaging" *
黄吉炜: "胶质瘤特异性4optHBS-Msi1/GFAP启动子的构建评价及其在基因治疗中的初步应用" *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023168400A3 (en) * 2022-03-03 2023-11-23 Research Institute At Nationwide Children's Hospital Materials and methods for the treatment of eif2b5 mutations and diseases resulting therefrom

Similar Documents

Publication Publication Date Title
Okada et al. In vivo imaging of engrafted neural stem cells: its application in evaluating the optimal timing of transplantation for spinal cord injury
CN113025657A (zh) 一种可用于双模态成像的腺相关病毒及其应用
Sasaki et al. Identified olfactory ensheathing cells transplanted into the transected dorsal funiculus bridge the lesion and form myelin
CN101815522B (zh) 诊断、预防或治疗与表达IL‑8或GRO‑α的细胞有关的疾病的组合物
Syková et al. Magnetic resonance tracking of transplanted stem cells in rat brain and spinal cord
EP3403675B1 (en) Adeno-associated virus virion for use in treatment of epilepsy
Le Blon et al. Intracerebral transplantation of interleukin 13-producing mesenchymal stem cells limits microgliosis, oligodendrocyte loss and demyelination in the cuprizone mouse model
CN114231565A (zh) 一种可用于活体检测细胞类型特异神经连接的rAAV及其应用
Cushnie et al. Using rAAV2-retro in rhesus macaques: promise and caveats for circuit manipulation
CN114107387A (zh) 一种可用于全脑星形胶质细胞活体成像的腺相关病毒及其应用
Wang et al. Spinal cord injury target-immunotherapy with TNF-α autoregulated and feedback-controlled human umbilical cord mesenchymal stem cell derived exosomes remodelled by CRISPR/Cas9 plasmid
Szulc et al. Bright Ferritin—a Reporter Gene Platform for On-Demand, Longitudinal Cell Tracking on MRI
Le et al. Characterization of an orthotopic rat model of glioblastoma using multiparametric magnetic resonance imaging and bioluminescence imaging
Neyrinck et al. The human somatostatin receptor type 2 as an imaging and suicide reporter gene for pluripotent stem cell-derived therapy of myocardial infarction
Sandu et al. Molecular imaging for stem cell transplantation in neuroregenerative medicine
CN110025768B (zh) 一种眼部疾病动物模型的构建方法及其应用
Cicchetti et al. Dual‐modality in vivo monitoring of subventricular zone stem cell migration and metabolism
CN113981005A (zh) 一种活体示踪神经连接的腺相关病毒及其应用
WO2020107566A1 (zh) 一种小分子蛋白质及其应用
US20160324989A1 (en) Methods for enhancing the delivery of active agents
Schomann et al. Lentiviral transduction and subsequent loading with nanoparticles do not affect cell viability and proliferation in hair‐follicle‐bulge‐derived stem cells in vitro
JP3410738B2 (ja) ミクログリアからなる医薬用キャリアー
Manganas et al. Stem cell therapy for central nervous system demyelinating disease
Islam et al. Transplantation of human embryonic stem cells alleviates motor dysfunction in AAV2-Htt171-82Q transfected rat model of Huntington’s disease
CN113652402A (zh) 一种诱导胶质细胞转分化为功能性神经元的方法及其应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination