CN113025657A - Adeno-associated virus for bimodal imaging and application thereof - Google Patents
Adeno-associated virus for bimodal imaging and application thereof Download PDFInfo
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Abstract
The invention discloses an adeno-associated virus for bimodal imaging and application thereof. The CAG promoter, the AQP1 gene, the 2A connecting sequence, the EGFP gene, the WPRE post-transcriptional regulatory element and the polyA sequence are sequentially inserted into the ITR of the AAV vector plasmid. After the prepared recombinant adeno-associated virus infects living nerve cells of animals, the expression of AQP1 and EGFP protein can be mediated in the nerve cells, so that the expression region has green fluorescence signals, and simultaneously, diffusion weighted MRI signals can be changed, thereby having the characteristic of bimodal imaging. The expression level of the exogenous gene transduced by the viral vector disclosed by the invention can be observed in situ in living tissues through noninvasive magnetic resonance imaging, can also be observed in isolated tissues through fluorescence imaging, and can be used for magnetic resonance imaging radiography, neuron marking, neural loop tracing and the like.
Description
Technical Field
The invention belongs to the technical field of virus vectors, and particularly relates to an adeno-associated virus carrying a fusion gene of type 1 aquaporin and green fluorescent protein and application thereof.
Background
The detection of gene expression in living animals by non-invasive imaging methods is critical for the fields of gene transduction, gene therapy, cell transplantation, cell therapy, etc., and in addition, methods for detecting gene expression in vivo will contribute to basic biological studies. In gene transduction or cell transplantation, a reporter gene is usually used to label the expression of a target gene or gene of interest. Currently, the most commonly used reporter genes are those based on fluorescent proteins and chemiluminescent proteins. Since light is difficult to penetrate deep tissues, optical imaging is difficult to obtain the gene expression result of opaque deep tissues at the living body level, and therefore, in the existing research, most of the gene expression is shown by the in vitro optical imaging, such as using green fluorescent protein EGFP gene as a reporter gene and using fluorescence imaging to observe the gene expression result. The isolated optical image can achieve good spatial resolution and sensitivity, but the result cannot show the level of gene expression in a living body, and a long-term experiment for carrying out multiple observations on the same experimental object cannot be realized.
Magnetic Resonance Imaging (MRI) is an imaging method combining many advantages of non-invasive, non-ionizing radiation, high-penetrability, and suitability for soft tissue, and has been widely used in clinical diagnosis and scientific research. And higher spatial and temporal resolution can be achieved for human and, at present, mammalian model animal (large mouse) MRI, which is commonly used. Therefore, there has been a great deal of research focused on constructing or defining reporter genes based on magnetic resonance imaging. At present, reporter genes based on magnetic resonance imaging contrast are mainly divided into two main categories. One class is based on metalloproteins and metal ion transporters, which enhance relaxation, primarily by enriching for paramagnetic metal ions, resulting in a change in the T1 or T2 weighted signal. The other is based on chemical exchange saturation transfer, which produces MRI contrast by exchangeable protons and the principle of chemical exchange saturation transfer. Among the limitations of the first class of reporter genes are the bioavailability of the metal ions, and the toxicity of the metal ions. The second category of reporter genes has the limitation of low detection sensitivity and requires high expression (2.5. mu.M) to produce a visible contrast effect.
In recent years, Aquaporin (AQP) genes are reported to be used as magnetic resonance imaging reporter genes, and over-expression of AQP1 protein can generate DWI-MRI signal change and does not cause obvious damage to cells or tissues. Diffusion-weighted imaging (DWI) is a widely used and mature MRI technique with applications in basic biophysical studies and diagnosis of disease. DWI generally produces diffusion weighted contrast by applying a pair of gradient magnetic fields to dephase spins in diffusing water molecules. Aquaporins are a group of highly conserved transmembrane transporters, enriched in the cell membrane in the form of tetramers, expressed in many types of cells and highly permselective for water. According to literature reports, the overexpression of the type 1 aquaporin (AQP1) can promote the transmembrane diffusion of water, thereby enhancing the contrast of DWI MRI, presenting a dark signal on DWI-MRI images and a high signal on an apparent diffusion coefficient ADC map. As an MRI reporter gene, the AQP1 has high sensitivity (500nM) and is independent of metal ions, thereby avoiding the defects of the two types of reporter genes.
At present, no report of using the AQP1 gene carried by a viral vector directly for MRI imaging contrast in living animals is available. The virus as a gene carrier can mediate the expression of the exogenous gene in various types of cells, can limit the expression of the exogenous gene in specific types of cells through molecular elements, and can control the expression of the exogenous gene in a specific period of time (conditional expression) through medicaments. Among many types of viral vectors, recombinant adeno-associated virus (rAAV) has the advantages of low toxicity, low immunogenicity, wide host infection range, and stable and durable expression of foreign genes, and thus has been widely used in neural circuit tracing and gene therapy. According to the results of the early-stage exploration in the laboratory, compared with the commonly used neurotropic virus vectors, namely herpes simplex virus HSV and vesicular stomatitis virus VSV, the rAAV vector can be expressed in the animal body for a longer time, so that the expression level is higher. And the AAV packaging cycle is short, and the purified virus can be obtained more quickly for in vivo experiments.
In conclusion, using rAAV carrying a foreign gene fused to AQP1 would make it possible to monitor the expression level of the foreign gene at the in vivo level by diffusion-weighted MRI. The rAAV can be possibly applied to the fields of magnetic resonance imaging radiography, neuron marking, neural loop tracing, gene therapy monitoring, cell therapy monitoring and the like.
Disclosure of Invention
The invention aims to provide a recombinant adenovirus vector capable of monitoring gene expression by using magnetic resonance imaging and fluorescence imaging bimodal imaging, wherein the expression level of an exogenous gene transduced by the vector can be observed in situ in living tissues through noninvasive magnetic resonance imaging and can also be observed in vitro tissues through fluorescence imaging. The vector can be used in the fields of magnetic resonance imaging radiography, neuron marking, neural loop tracing, gene therapy monitoring, cell therapy monitoring and the like.
In order to achieve the purpose, the invention adopts the following technical scheme:
the recombinant adenovirus plasmid is characterized in that a CAG promoter (shown in SEQ ID NO. 1), an AQP1 gene (shown in SEQ ID NO. 2), a 2A connecting sequence (shown in SEQ ID NO. 3), an EGFP gene (shown in SEQ ID NO. 4), a WPRE post-transcription regulatory element (shown in SEQ ID NO. 5) and a polyA sequence (shown in SEQ ID NO. 6) are sequentially inserted into the ITR of an AAV vector plasmid to construct a recombinant adeno-associated virus core plasmid pAAV2-CAG-AQP 1-2A-EGFP-WPRE-pA.
The recombinant adeno-associated virus is prepared by taking pAAV2-CAG-AQP1-2A-EGFP-WPRE-pA as a core plasmid, and the specific method comprises the following steps: co-transfecting HEK293T cells with helper plasmids, serotype plasmids and core plasmids required by packaging rAAV, collecting cell precipitates after centrifugation, and purifying recombinant adeno-associated virus rAAV2-CAG-AQP 1-2A-EGFP-WPRE-pA.
The application of the recombinant adeno-associated virus rAAV2-CAG-AQP1-2A-EGFP-WPRE-pA comprises the following steps: the virus can mediate neurons to express AQP1 protein and EGFP protein, further AQP1 protein influences DWI-MRI signals, EGFP generates fluorescence signals, and therefore the recombinant adeno-associated virus can be used as a bimodal contrast carrier for magnetic resonance imaging and fluorescence imaging and used for preparing magnetic resonance imaging contrast agents or nerve markers and tracers.
Compared with the prior art, the invention has the following advantages:
1. the recombinant adeno-associated virus provided by the invention has wide application, and relates to a plurality of fields of magnetic resonance imaging radiography, neuron marking, neural loop tracing, gene therapy monitoring, cell therapy monitoring and the like. The recombinant adeno-associated virus can be used as a tumor tracer to infect tumor cells, and can be used for identifying experimental animal tumor models and tumor metastasis by magnetic resonance imaging and fluorescence imaging. It can also be used as cell tracer to infect cells in cell therapy, and can be used for detecting transplantation, metastasis and proliferation of cells by using magnetic resonance imaging and fluorescence imaging. Besides being used alone, other functional genes, such as genes for gene therapy, can be inserted into the genome of the recombinant adeno-associated virus. After the coupling expression of the functional gene with AQP1 and EGFP is realized, the expression level of the functional gene in the tissue can be detected at the living body level by using magnetic resonance imaging, and the expression condition of the functional gene can be indicated at the ex vivo level by fluorescence imaging.
2. The rAAV is used as a gene vector of the AQP1 and the EGFP, has the advantages of stable and durable exogenous gene expression, wide host range, low toxicity, low immunogenicity and the like, is widely applied to scientific research of various species, and is approved to be used as a human gene therapy medicament. The safety, stability and high efficiency of the method are verified in many aspects.
3. According to the invention, AQP1 is used as the MRI reporter gene, the DWI-MRI detection sensitivity is high (500nM), and the metal ion is not depended on, so that the defect of the MRI reporter gene based on metalloprotein and chemical exchange saturation transfer is avoided, and the AQP1 is a novel MRI reporter gene with wide application potential.
4. The recombinant virus rAAV-CAG-AQP1-2A-EGFP-WPRE-pA uses a high-efficiency CAG promoter, so that the high expression quantity of AQP1 and EGFP protein is ensured, and the high abundance of AQP1 protein ensures the MRI contrast effect.
5. In order to improve the stability of the target gene after transcription, WPRE (human polyporus frondosus) posttranscriptional regulatory elements and polyA sequences are added at the 3' end of the target gene.
Drawings
FIG. 1 is a schematic diagram of rAAV genome carrying AQP1 gene and EGFP gene expression cassette.
FIG. 2 is a DWI-MRI image of pAAV-CAG-AQP1-2A-EGFP-WPRE-pA plasmid transfected into 293 cells.
FIG. 3 shows the MRI imaging results of the brains of mice injected with rAAV2-CAG-AQP1-2A-EGFP-WPRE-pA (right side) and control virus rAAV2-Ef1 alpha-EGFP-WPRE-pA (left side). A is a schematic diagram of a coronal plane of a brain of a virus injection mouse, B is a DWI-MRI image of a brain horizontal plane of the mouse, and C is a DWI-MRI signal intensity value statistical result of two virus infection areas.
FIG. 4 shows the AQP1 protein immunohistochemistry and fluorescence imaging results of mouse brains injected with rAAV2-CAG-AQP1-2A-EGFP-WPRE-pA (right side) and control virus rAAV2-Ef1 alpha-EGFP-WPRE-pA (left side). A is the result of green fluorescent protein imaging, B is the result of AQP1 immunohistochemistry, C is the result of DAPI cell nucleus counterstaining, and D is the result of red, green and blue combined channel imaging.
Detailed Description
The technical solutions of the present invention, if not specifically mentioned, are conventional in the art, and the reagents and materials, if not specifically mentioned, are commercially available. The specific embodiments illustrated herein are merely illustrative of the invention and are not intended to be limiting.
Example 1: construction of plasmid pAAV-CAG-AQP1-2A-EGFP-WPRE-pA carrying AQP1 and EGFP genes and preparation of recombinant adeno-associated virus rAAV-CAG-AQP1-2A-EGFP-WPRE-pA
In order to improve the expression level of a target gene, a eukaryotic strong promoter CAG promoter is selected. To express AQP1 gene fused with green fluorescent protein (EGFP) reporter gene, we selected the 2A self-splicing polypeptide coding sequence as the junction sequence of AQP1 gene and EGFP gene. In order to improve the stability of the target gene after transcription, WPRE (human polyporus frondosus) posttranscriptional regulatory elements and polyA sequences are added at the 3' end of the target gene. Inserting a CAG promoter (shown in SEQ ID NO. 1), an AQP1 gene (shown in SEQ ID NO. 2), a 2A connecting sequence (shown in SEQ ID NO. 3), an EGFP gene (shown in SEQ ID NO. 4), a WPRE post-transcriptional regulatory element (shown in SEQ ID NO. 5) and a polyA sequence (shown in SEQ ID NO. 6) in the middle of an AAV terminal repetitive sequence (ITR) in a core plasmid pAAV-MCS for packaging rAAV by using a conventional molecular cloning method; the plasmid pAAV-CAG-AQP1-2A-EGFP-WPRE-pA containing the needed rAAV genome is obtained through construction. pAAV-CAG-AQP1-2A-EGFP-WPRE-pA genome schematic (FIG. 1).
The packaging and purification steps of the recombinant adeno-associated virus rAAV are as follows:
in order to improve the efficiency of rAAV infection of living bodies and facilitate the preparation of rAAV, a commonly used type 2 rAAV is selected. HEK293T cells were first plated into 10cm cell dishes and cultured to 80% density, and three plasmids required for packaging rAAV (helper plasmid pAAV-helper, serotype plasmid pAAV-RC2, core plasmid pAAV-CAG-AQP1-2A-EGFP-WPRE-pA) were co-transfected into HEK293T cells at molar ratio (1:1: 1). After transfection for 72h, the adherent cells producing the virus were scraped off by cell scraping and centrifuged, and the centrifuged cell pellet was collected. Then, cell pellet lysate is treated with nuclease, and rAAV in the virus supernatant is concentrated with PEG/NaCl pellets. And (3) separating and purifying the lysate supernatant containing the rAAV by iodixanol density gradient ultracentrifugation, subpackaging the purified rAAV, and freezing and storing in a refrigerator at the temperature of-80 ℃. The titer of rAAV virus was determined by real-time fluorescent quantitative PCR using a CFX Connect fluorescent quantitative PCR instrument from BioRad.
Example 2: MRI imaging observation of pAAV-CAG-AQP1-2A-EGFP-WPRE-pA plasmid after transfection of BHK cells
To verify the MRI contrast effect of pAAV-CAG-AQP1-2A-EGFP-WPRE-pA plasmid mediated expression of AQP1, BHK cells were transfected with pAAV-CAG-AQP1-2A-EGFP-WPRE-pA plasmid (AQP1-EGFP) using Lipfectamine 2000, and at the same time, pAAV-CAG-AQP1-WPRE-pA plasmid (AQP1) expressing only AQP1 protein and pAAV-CAG-EGFP-WPRE-pA plasmid (AQP1-EGFP) expressing only EGFP protein were transfected as controls. FIG. 2 shows the DWI-MRI imaging results of cell pellets after transfection of BHK cells with different plasmids. Cells in which pAAV-CAG-AQP1-2A-EGFP-WPRE-pA plasmid (AQP1-EGFP) was transfected exhibited the darkest DWI signal intensity. The result shows that compared with the single expression of the AQP1 protein, the expression of the AQP1 protein and the EGFP protein simultaneously can generate stronger DWI-MRI contrast effect.
Example 3: MRI imaging observation of mouse brain injected with rAAV2-CAG-AQP1-2A-EGFP-WPRE-pA and control virus rAAV2-Ef1 alpha-EGFP-WPRE-pA
rAAV2-CAG-AQP1-2A-EGFP-WPRE-pA virus is injected into the right dorsal striatal brain region (CPu, coordinate: AP-0.5 mm; ML-2 mm; DV-3.3mm) of mouse brain through brain stereotaxic injection, and the titer is 6 x 1011vg/ml, injection volume 2. mu.L. Control virus rAAV2-Ef1 alpha-EGFP-WPRE-pA was injected into the left dorsal striatal brain region (coordinates: AP-0.5 mm; ML 2 mm; DV-3.3mm) at a titer of 2.4X 1012vg/ml, injection volume 0.5. mu.L. A schematic of the virus injection is shown in FIG. 3A.
After 3 weeks, the animal brains were subjected to in vivo MRI observations using a 7.0T magnetic resonance imager, wherein the diffusion-weighted imaging sequence used was a STEAM-based SE-DWI sequence with the parameters: TR is 3000ms, TE is 24ms, the average number of times is 2, and the total sequence duration is 51min12 s. The diffusion gradient duration is 7ms, the diffusion gradient interval time is 100ms, and the b value is 1000s mm-2. The FOV was 2.5X 2.2cm, the spatial resolution 0.195X 0.171mm and the layer thickness 0.8 mm. The MRI results (fig. 3B) indicated that the virus successfully infected neurons and expressed AQP1 protein sufficient to alter the DWI-MRI signal in the right CPU, whereas the left CPU injected with the control virus did not present a DWI dark signal. FIG. 3C shows a statistical comparison of DWI-MRI signal intensity values for both striations, with the right striation having significantly lower DWI-MRI signal intensity than the left. This result indicates that the AQP1 protein enhances diffusion of water molecules, reduces DWI-MRI signals, and produces MRI contrast that can be observed at the in vivo level.
Example 4: AQP1 protein immunohistochemistry and fluorescence imaging of mouse brain injected with rAAV2-CAG-AQP1-2A-EGFP-WPRE-pA and control virus rAAV2-Ef1 alpha-EGFP-WPRE-pA
rAAV2-CAG-AQP1-2A-EGFP-WPRE-pA virus geneStereotactically injecting the supernatant into the right dorsal striatal brain region (CPu, coordinate: AP-0.5 mm; ML-2 mm; DV-3.3mm) of mouse brain with titer of 6 × 1011vg/ml, injection volume 2. mu.L. Control virus rAAV2-Ef1 alpha-EGFP-WPRE-pA was injected into the left dorsal striatal brain region (coordinates: AP-0.5 mm; ML 2 mm; DV-3.3mm) at a titer of 2.4X 1012vg/ml, injection volume 0.5. mu.L. A schematic of the virus injection is shown in FIG. 3A. After 3 weeks, live MRI observations were made of the animal brain using a 7.0T magnetic resonance imager. After the MRI experiment, mice were anesthetized, and after cardiac perfusion, mouse brains were removed and cryo-sectioned. Mouse brain sections were then immunohistochemically stained with AQP1 antibody from a mouse to show highly expressed brain regions of AQP 1. The results are shown in fig. 4, where green is the fluorescent signal generated by the EGFP protein, red is the signal of AQP1 immunohistochemistry, the left striatum has only a green fluorescent signal, and the right striatum has not only a green fluorescent signal but also a red fluorescent signal. The result shows that the rAAV2-CAG-AQP1-2A-EGFP-WPRE-pA can mediate the expression of AQP1 protein and EGFP protein expressed by neurons, further, the AQP1 protein influences DWI-MRI signals, and the EGFP generates fluorescence signals. Therefore, the rAAV2-CAG-AQP1-2A-EGFP-WPRE-pA can be used as a bimodal contrast carrier for magnetic resonance imaging and fluorescence imaging.
Sequence listing
<110> institute of precision measurement, science and technology innovation, of the Chinese academy of sciences
<120> adeno-associated virus for bimodal imaging and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1460
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg 60
tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg 120
gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt 180
acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg 240
accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg 300
gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc cccaccccca 360
attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg gggggggggg 420
gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt 480
gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg 540
cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg ggagtcgctg cgacgctgcc 600
ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc tgactgaccg 660
cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct 720
tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag gggctccggg 780
agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg tgcgtgggga 840
gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc 900
tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg gggcggtgcc ccgcggtgcg 960
gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg gggtgagcag 1020
ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc cgagttgctg 1080
agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg ctcgccgtgc 1140
cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct cgggccgggg 1200
agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga ggcgcggcga 1260
gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt cctttgtccc 1320
aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc gggcgcgggg 1380
cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt gcgtcgccgc 1440
gccgccgtcc ccttctccct 1460
<210> 2
<211> 807
<212> DNA
<213> Artificial Sequence (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> Artificial Sequence (Artificial Sequence)
<400> 3
gctactaact ttagcctgct gaagcaggcc ggggacgtgg aagagaaccc tggccct 57
<210> 4
<211> 717
<212> DNA
<213> Artificial Sequence (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> Artificial Sequence (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> Artificial Sequence (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. The recombinant adenovirus plasmid is characterized in that a CAG promoter, an AQP1 gene, a 2A connecting sequence, an EGFP gene, a WPRE post-transcriptional regulatory element and a polyA sequence are sequentially inserted in the middle of ITR of an AAV vector plasmid.
2. A recombinant adeno-associated virus, characterized in that, the recombinant adenovirus plasmid of claim 1 is used as a core plasmid to prepare the recombinant adeno-associated virus.
3. Use of the recombinant adenovirus plasmid of claim 1 or the recombinant adeno-associated virus of claim 2 in the preparation of a contrast agent for magnetic resonance imaging.
4. Use of the recombinant adenovirus plasmid of claim 1 or the recombinant adeno-associated virus of claim 2 in the preparation of neural markers and tracers.
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| CN113667671A (en) * | 2021-08-20 | 2021-11-19 | 深圳市恩辑生物科技有限公司 | Mini promoter pRTN1 and application thereof |
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| CN113981005A (en) * | 2021-11-04 | 2022-01-28 | 中国科学院精密测量科学与技术创新研究院 | In-vivo tracing nerve connection adeno-associated virus and application thereof |
| CN114231565A (en) * | 2021-11-04 | 2022-03-25 | 中国科学院精密测量科学与技术创新研究院 | rAAV capable of being used for in vivo detection of cell type specific nerve connection and application thereof |
| CN114107387A (en) * | 2021-11-05 | 2022-03-01 | 中国科学院精密测量科学与技术创新研究院 | Adeno-associated virus for whole-brain astrocyte living imaging and application thereof |
| CN114848850A (en) * | 2022-04-28 | 2022-08-05 | 武汉大学 | Application of RS1 gene in preparation of XLRS therapeutic agent and therapeutic agent |
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