CN112680443A - Promoter pCalm1 and application thereof - Google Patents
Promoter pCalm1 and application thereof Download PDFInfo
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- CN112680443A CN112680443A CN202011435366.3A CN202011435366A CN112680443A CN 112680443 A CN112680443 A CN 112680443A CN 202011435366 A CN202011435366 A CN 202011435366A CN 112680443 A CN112680443 A CN 112680443A
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Abstract
The invention discloses a promoter pCalm1 and application thereof, wherein the nucleotide sequence of the promoter pCalm1 is shown as SEQ ID NO: 1 is shown. The promoter pCalm1 can be applied to recombinant adeno-associated virus, so that exogenous genes can be efficiently expressed in a neocortex, and the promoter pCalm1 has wide application prospect in the field of neural loop tracing and gene therapy.
Description
Technical Field
The invention belongs to the technical field of nerve engineering, and particularly relates to a promoter pCalm1 and application thereof.
Background
The neocortex is a part of the mammalian central nervous system and is involved in a variety of important functions including sensation, perception, voluntary movement, learning, cognition, and the like. The analysis of the neural circuits connecting the neocortex to the respective nuclei can help us to better understand the higher functions of the brain. The research on neocortex can help us to understand the pathogenesis of cortical-related neurological diseases and further search a treatment method thereof. However, there is no viral tool available to efficiently label neocortex, which has hampered neocortex studies.
Adeno-associated virus is a single-stranded DNA virus without an envelope. The recombinant adeno-associated virus (rAAV) is a recombinant virus which is reconstructed on the basis of wild AAV. The recombinant adeno-associated virus has become a virus tool widely used in the field of neuroscience due to the advantages of long-term stable expression of mediated genes in mammals, low immunogenicity, wide host range and the like, and is also a gene therapy vector tool most widely applied in clinical experiments of central nervous system diseases at present.
In the process of applying rAAV to neuroscience research and clinical treatment, the key point of gene delivery is how to improve the space-time specificity and the expression level of gene expression. Strategies to regulate foreign gene expression directly affect the efficiency and safety of gene delivery. For neuroscience research, to achieve labeling of specific neurons in a particular neural circuit, different brain regions or nuclei are labeled. For gene therapy of nervous system diseases, the manipulation of neurons in the whole brain or in different subtypes is required to intervene in different nervous system diseases. One important approach is to select rAAV viruses of different serotypes. Among the presently available reports, there are AAV serotypes suitable for use in the nervous system: type 1, type 2, type 5, type 6, type 8, type 9, DJ, PHP.B, PHP.eB, PHP.S, Retro, rh10, etc. Different serotypes of viruses have different infection efficiencies and spreading capacities for different brain regions, and therefore, different rAAV serotypes can be selected as gene delivery vehicles according to the subject or target of treatment. However, due to the complexity of the central nervous system, these serotypes are far from meeting the current requirements for gene delivery specificity. Another approach is to use tissue-specific promoters in the rAAV genome. Promoters are cis-acting elements essential in the regulation of gene expression. Under the control of promoters with expression specificity, exogenous genes are generally expressed only in certain specific cell types or brain regions. In the rAAV, the specific promoter is used for driving gene expression, so that the gene expression efficiency can be improved, and the immunoreaction generated in the rAAV infection process is reduced.
Disclosure of Invention
In order to solve the problems of the background art, the invention aims to provide a promoter pCalm1 for efficiently expressing neocortex and application thereof. The promoter pCalm1 can be applied to recombinant adeno-associated virus, so that exogenous genes can be efficiently expressed in a neocortex, and the promoter pCalm1 has wide application prospect in the field of neural loop tracing and gene therapy.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows: in one aspect, the invention provides a promoter pCalm1, wherein the nucleotide sequence of the promoter pCalm1 is as shown in SEQ ID NO: 1 is shown.
Further, the promoter pCalm1 is a partial sequence of calmodulin 1(Calm1) gene, which is selected from a partial sequence near the transcription start site of Calm1 gene, and the first exon and second exon partial sequences, which are spliced into a sequence of 2327bp in total.
Further, the promoter pCalm1 was expressed efficiently in neocortex.
In another aspect, the present invention provides a recombinant adeno-associated virus vector comprising the promoter described above.
Further, the recombinant adeno-associated viral vector includes Paav-pCalm 1-fluorescent protein, Paav-pCalm 1-functional protein or Paav-pCalm 1-therapeutic protein.
Further, the fluorescent protein includes eYFP, tdTomato;
preferably, the functional protein comprises a optogenetically-related protein or a chemically genetically-related protein.
The Paav-pCalm 1-fluorescent protein (fluorescent protein including eYFP, tdTomato) can realize the nerve loop multicolor tracing. Paav-pCalm 1-functional proteins (including optogenetically related proteins or chemogenetically related proteins) can make recombinant adeno-associated viruses that activate or inhibit specific neurons. The Paav-pCalm 1-therapeutic protein can be used in gene therapy.
Further, the recombinant adeno-associated virus vector Paav-pCalm1-eYFP adopts Paav-hSyn-eYFP as a skeleton vector, hSyn is replaced in the vector to be pCalm1 promoter, and the recombinant adeno-associated virus vector Paav-pCalm1-eYFP is obtained.
Further, the preparation method of the recombinant adeno-associated virus vector Paav-pCalm1-eYFP comprises the following steps: cloning the promoter pCalm1 into a PUC-57 vector to obtain a PUC57-pCalm1 vector; treating Paav-hSyn-eYFP vector by using restriction enzymes MluI-HF and BamHI-HF, and treating PUC57-pCalm1 vector by using restriction enzymes MluI-HF and BamHI-HF; and (3) recovering, purifying and connecting the two enzyme digestion products to obtain the recombinant adeno-associated virus vector Paav-pCalm 1-eYFP.
In another aspect, the present invention provides a recombinant adeno-associated virus comprising any one of the recombinant adeno-associated virus vectors described above.
Further, the recombinant adeno-associated virus comprises AAV 8-retroro-pCalm 1-fluorescent protein, AAV 8-retroro-pCalm 1-functional protein or AAV 8-retroro-pCalm 1-therapeutic protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the functional protein comprises a optogenetically-related protein or a chemically genetically-related protein.
The recombinant adeno-associated virus AAV 8-retroro-pCalm 1-eYFP and AAV 8-retroro-pCalm 1-tdTomato can effectively reverse transsynaptic marking striatum-cortex neural circuits and express eYFP or tdTomato with high fluorescence intensity.
In still another aspect, the present invention provides a kit comprising the promoter pCalm1 described above, the recombinant adeno-associated vector described above, or the recombinant adeno-associated virus described above.
In still another aspect, the invention provides an application of the promoter pCalm1, the recombinant adeno-associated vector, the recombinant adeno-associated virus or the kit in the preparation of a neural circuit tracing and/or nerve cell manipulation and/or gene therapy reagent.
In yet another aspect, the present invention provides a neural network tracer comprising a Paav-pCalm 1-fluorescent protein and/or a recombinant adeno-associated virus comprising a Paav-pCalm 1-fluorescent protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the recombinant adeno-associated virus comprises AAV8-retro-pCalm1-eYFP, AAV8-retro-pCalm 1-tdTomato.
In a further aspect, the invention provides a use of the neural network tracer described above in striatum-cortex loop tracing.
Compared with the prior art, the invention has the following beneficial effects:
(1) the promoter pCalm1 has high-efficiency expression efficiency in neocortex, can realize high-efficiency expression of exogenous genes in neocortex, improves the expression specificity after virus infection, and has wide application prospect in the fields of neural loop tracing and gene therapy;
(2) the promoter pCalm1 of the invention has no cytotoxicity in bacteria and mammalian cells;
(3) the recombinant adeno-associated virus vector adopts pCalm1 as a promoter, and has high-efficiency expression efficiency in neocortex;
(4) the recombinant adeno-associated virus can efficiently and reversely mark the neural circuit from striatum to cortex in the brain, and has high marking efficiency and strong infection capacity.
Drawings
FIG. 1 is a flow chart of the construction of the Paav-pCalm1-eYFP vector of the present invention;
FIG. 2 is a graph showing the expression efficiency of AAV8-retro-pCalm1-eYFP in striatum and cortex.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
EXAMPLE 1 construction of pUC57-pCalm1 vector
The pCalm1 promoter is a partial sequence of calmodulin 1(Calm1) gene, which selects a partial sequence near the transcription start site of Calm1 gene and partial sequences of the first exon and the second exon, and splices them into a total sequence of 2327 bp. The nucleotide sequence of the promoter pCalm1 is shown as SEQ ID NO: 1 is shown.
The promoter pCalm1 is artificially synthesized and then cloned in a PUC-57 vector to obtain a PUC57-pCalm1 vector.
Example 2 construction of recombinant adeno-associated Virus vector Paav-pCalm1-eYFP
In this example, because YFP is used as a reporter gene of a promoter, when constructing a recombinant vector, a recombinant adeno-associated vector Paav-hSyn-eYFP containing YFP gene is selected as a vector backbone to connect with a pCalm1 promoter, and the following steps are performed:
as shown in FIG. 1, Paav-hSyn-eYFP vector is treated with restriction enzymes MluI-HF and BamHI-HF, and PUC57-pCalm1 vector is treated with restriction enzymes MluI-HF and BamHI-HF, and then digested at 37 ℃ for 3h, wherein the digestion systems are shown in tables 1 and 2, and after recovering the digestion products, ligation is performed at 16 ℃ for 30min by using a ligation premix (2 × ligation premix, TAKARA), and the system is shown in Table 3, thus obtaining the successfully ligated Paav-hSyn-eYFP vector.
TABLE 1 Paav-hSyn-Eyfp vector restriction enzyme System
| Reagent | Dosage of |
| Enzyme digestion Buffer solution 10 XCutSmart Buffer | 5μL |
| MluI-HF | 1μL |
| BamHI-HF | 1μL |
| Paav-hSyn-Eyfp | 1μg |
| ddH2O | Make up to 50 μ L |
TABLE 2 PUC57-pCalm1 vector restriction enzyme system
| Reagent | Dosage of |
| Enzyme digestion Buffer solution 10 XCutSmart Buffer | 5μL |
| MluI-HF | 1μL |
| BamHI-HF | 1μL |
| PUC57-pCalm1 | 1μg |
| ddH2O | Make up to 50mL |
TABLE 3 connection System
| Reagent | Dosage of |
| Connecting the premix 2 × ligation premix | 5μL |
| Paav-eYFP connecting skeleton | 0.5μL |
| pCalm1 ligation fragment | 4.5μL |
Example 3 preparation of recombinant adeno-associated Virus AAV8retro-pCalm1-eYFP
This example uses a three plasmid co-transfection method to prepare the virus. Before virus preparation, plasmids required by packaging virus are mentioned, including a recombinant adeno-associated virus vector Paav-pCalm1-eYFP, a packaging plasmid AAV8-retro-RCB and a helper plasmid. The method comprises the following specific steps:
preparing cells: 293T cells were plated on plates containing whole medium (10% fetal bovine serum, 1% double antibody) and cultured in an incubator.
Preparation of transfection reagent: 5.25mL of ultrapure water, 75. mu.g of packaged plasmid, 75. mu.g of recombinant plasmid, 75. mu.g of helper plasmid and 800. mu.L of calcium chloride solution were aspirated and gently mixed. To the above reagent, an equal volume of 2 × HBS was added, vortexed, and allowed to stand for 30 min.
Transfection of cells: mu.L of chloroquine was added to each dish of cells, and the cells were incubated after addition of transfection reagent.
And (3) virus collection: after 72h, the transfected cells were collected in a centrifuge tube and centrifuged at 3000rpm for 30 min. The supernatant was discarded and cell lysate was added, followed by lysis of cells by repeated freeze-thaw. A cell disruption solution containing the virus was obtained.
And (3) purifying the virus: the cell disruption solution was centrifuged at 11500rpm for 30min, the supernatant was discarded and 200. mu.L of HBS was added thereto, followed by mixing, 200. mu.L of chloroform was added thereto, followed by centrifugation at 12000rpm for 5min, the supernatant was taken, 100. mu.L of 2.5mM NaCl and 100. mu.L of 40% PEG8000 were added thereto, followed by mixing by vortexing, and the mixture was cooled overnight in a refrigerator at 4 ℃. The overnight sample was centrifuged at 12000rpm for 30min, the supernatant was discarded, 30. mu.L of HBS and 0.5. mu.L of nuclease were added, and the mixture was allowed to stand for 30 min. Then 30. mu.L of chloroform was added and centrifuged at 12000rpm for 5 min. After purification, the product was stored in a freezer at-80 ℃.
And (3) determining the titer: the titer of the purified virus was measured by using AAV titer measurement dye-based fluorescent quantitation kit (TAKARA Co.) and found to be 1.0X 1013VG/mL。
Example 4 recombinant adeno-associated Virus labeling mouse striatum-cortical neural circuits
The purified recombinant adeno-associated virus is injected into the right striatum of a mouse (AP: + 0.62; R: + 1.75; D: 3.5) by stereotaxic localization. The recombinant adeno-associated viral capsid is AAV8-retro, has the ability to infect retroviruses, and is capable of infecting neurons of the cortex upstream of the striatum into which it is injected. Three weeks later, mice were perfused to harvest brains, stained by immunohistochemical sectioning, and brain slice results were observed. The experimental results are shown in fig. 2, eYFP has high expression efficiency in mouse neocortex.
In conclusion, the invention is verified on mice, and the promoter pCalm1 is proved to have high expression level in neocortex.
The above description is only a specific embodiment of the present invention, and not all embodiments, and any equivalent modifications of the technical solutions of the present invention, which are made by those skilled in the art through reading the present specification, are covered by the claims of the present invention.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute of Chinese academy of sciences
<120> promoter pCalm1 and application thereof
<130> CP120010890C
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 2327
<212> DNA
<213> Artificial sequence
<400> 1
cttccccaag gccaaccacc gtgtcctcaa cacagtcctc tcgcctctac agctaaggtt 60
ggattccgcc tccccgcggc tgtcctcagc ccctagatct cctccaggtc ctcccctgtg 120
ggggacccag gattggtggc tctgcaacct cgaagccacc aggagtggtg ggagggggga 180
cacacaggtg aagtggggct gagtgacgga cacgcgcctg gaagcccggg acggcccccc 240
agatttcgct acgcaggcat tccagctcta gaggactctt gcagcaacag accagcaccc 300
tcgctcaggt ctccgactcc ccagtgaccc ctgcccgcgt aatgggcccg ccgcgccggg 360
cgcgcctggg gggcggagcc ttgcggtgag gtccggctgc cgcagcgccg ccctgcgcga 420
ggcggtactg ccgcggcgga gggataccgc gcaccgtata tatatcgcgg cacacaggct 480
cgcgctacgg cagtggtgct gggagtctcg tgtccgtggt gccgttactc gaagtcgggc 540
ggcggctgag gctcagcgca caacgcaggt agcgcgttag cagcagcaga agcggaggca 600
cctcggcggt cacagcccct gcgctggtgc agccaccctc gcctgctccg ttcttccttc 660
cttcgctcgc accatggtag gttgggatcg gcagtggcag acgtagcagc tgcccgagct 720
ttctgaacag atttgtagtt gagtcttggg gtctccccct tttcctggtt gaggttaccc 780
cctaccccat tttgccaggt gttacgcagc agctgcagtg aaaggggatg tattttcggg 840
tttgcatctc cgctctggct gccagagaac ctcctctgag ttttgggcgc ctctggggct 900
ggactctaca gtgggctgtg cgcgggctgg agccgctgcc acagctgcac cggggccttc 960
cgcatccacc ccctccgcca gccccgcacc cactgcatcc aacgcgccgc acccgggctg 1020
cctgcagcgc agcgcctcgg ccggggccag cggggcgggg gagccgggcg gggcaggccg 1080
cgacgtcccg gtagtcagtc cccgaccgcg gagaatgaat gggtcgcggc gagccgggag 1140
acgtacaggg cttggaccgc ggcctcgggg tgtcctcagc caccttcaga ccccctgacc 1200
agcccagtcg ggagtccccc ctcgcttgcg gtcccctcct tcctcccggg gggcgcggcg 1260
cgggcgtgag ctgggaagga aggagccggg gaagggtggg gttgggggca ggaaggcgaa 1320
ggctgggggg cggagagggc ggaagcggcc gccctgcgcc cgggcggggc cccgcagggt 1380
ggccgcgcgc gtggctgggc cgtgcgacct cggtccccct cccggtgccg cgcgagacgg 1440
gcgcgatcgc tcgtgtccgc ggggaggccc cttcccgcgg gaggtgctcg gctcgcgcta 1500
attggggcgg cagggcgggg gaggagagag ttggcgcgct cctcggtttc cattagagac 1560
gcaaagtttc tgctggggag gaggcggcgg caggctcagt gcctggggga gcaggagcgg 1620
tgggaggtgc tggagatagg cagcaccacg gtcctggggc ccctttgggt cttcacccta 1680
gtgcggcccc tcgcccggca tgccccttgt ctctcgctgc tccctgggcg gcagggagat 1740
gcccatctgt acgtttccgc tttctctgcc gcctctggat tgccagatgc gaatatgtgg 1800
ttggccgtgt gcgtgtgccg tagccccctt ctccctcgcc aggcagagct gacatcacca 1860
gcagcacctt cagccctggc aaggtggcta ggtcgttctc ggcaggttct ggaacgcctt 1920
tgacccaaag ccagaatgac ttggggttga ggtgggggag tcgttgtccc gaaggtagat 1980
gcacttgaca ggcctctagt cagagaattt tcccgaggag gtggaactcc tctccttggt 2040
gagctggttt ctcctacgag gtgtgacagt gcttcctgtc gtgcaagact gacatggcga 2100
gcattacata aatcgtgtct ccatttgcct taaaagtagc ttccagtaat ggatagaagg 2160
gaagacccac ggtaccccag tactaatgaa ttcagcacac ttttaaaata ctgattagga 2220
agaaaagccc cacaatttta aagtataatt aggaaagcta ggttattatc aaccaaactt 2280
gtctagatta aattatgatc ttttctttct ctgtgtaggt gggatcc 2327
Claims (10)
1. A promoter pCalm1, wherein the nucleotide sequence of the promoter pCalm1 is as shown in SEQ ID NO: 1 is shown.
2. The promoter pCalm1 of claim 1, wherein the promoter pCalm1 is highly expressed in neocortex.
3. A recombinant adeno-associated virus vector comprising the promoter according to claim 1 or 2.
4. The recombinant adeno-associated viral vector according to claim 3 wherein the recombinant adeno-associated viral vector comprises a Paav-pCalm 1-fluorescent protein, a Paav-pCalm 1-functional protein or a Paav-pCalm 1-therapeutic protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the functional protein comprises a optogenetically-related protein or a chemically genetically-related protein.
5. A recombinant adeno-associated virus comprising the recombinant adeno-associated virus vector according to claim 3 or 4.
6. The recombinant adeno-associated virus according to claim 5 wherein the recombinant adeno-associated virus comprises AAV8-retro-pCalm 1-fluorescent protein, AAV8-retro-pCalm 1-functional protein or AAV8-retro-pCalm 1-therapeutic protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the functional protein comprises a optogenetically-related protein or a chemically genetically-related protein.
7. A kit comprising the promoter pCalm1 of any one of claims 1-2, the recombinant adeno-associated vector of any one of claims 3-4, or the recombinant adeno-associated virus of any one of claims 5-6.
8. Use of the promoter pCalm1 of any one of claims 1 to 2, the recombinant adeno-associated vector of any one of claims 3 to 4, the recombinant adeno-associated virus of any one of claims 5 to 6, or the kit of claim 7 for the preparation of a neural circuit tracing and/or manipulating nerve cells and/or gene therapy reagents.
9. A neural network tracer comprising Paav-pCalm 1-fluorescent protein and/or a recombinant adeno-associated virus comprising Paav-pCalm 1-fluorescent protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the recombinant adeno-associated virus comprises AAV8-retro-pCalm1-eYFP, AAV8-retro-pCalm 1-tdTomato.
10. Use of a neural network tracer according to claim 9 in striatal-cortical loop tracing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022166413A1 (en) * | 2021-02-02 | 2022-08-11 | 中国科学院深圳先进技术研究院 | Promoter pcalm2 and application thereof |
| WO2023283749A1 (en) * | 2021-07-13 | 2023-01-19 | 深圳市恩辑生物科技有限公司 | Mini-promoter pcalm1 and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130224836A1 (en) * | 2010-10-27 | 2013-08-29 | Jichi Medical University | Adeno-Associated Virus Virion for Gene Transfer to Nervous System Cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20130224836A1 (en) * | 2010-10-27 | 2013-08-29 | Jichi Medical University | Adeno-Associated Virus Virion for Gene Transfer to Nervous System Cells |
Non-Patent Citations (5)
| Title |
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| BONGMIN BAE等: "Elimination of Calm1 long 3’-UTR mRNA isoform by CRISPR–Cas9 gene editing impairs dorsal root ganglion development and hippocampal neuron activation in mice", 《RNA (NEW YORK, N.Y.)》 * |
| GENBANK: "Mus musculus strain C57BL/6J chromosome 12, GRCm39", 《GENBANK》 * |
| TOUTENHOOFD, S. L.: "Characterization of the human CALM2 calmodulin gene and comparison of the transcriptional activity of CALM1, CALM2 and CALMS", 《CELL CALCIUM》 * |
| 李嘉敏: "AAV神经环路示踪病毒的改造和应用", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022166413A1 (en) * | 2021-02-02 | 2022-08-11 | 中国科学院深圳先进技术研究院 | Promoter pcalm2 and application thereof |
| WO2023283749A1 (en) * | 2021-07-13 | 2023-01-19 | 深圳市恩辑生物科技有限公司 | Mini-promoter pcalm1 and application thereof |
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