CN112725342A - Promoter pCALM2 and application thereof - Google Patents
Promoter pCALM2 and application thereof Download PDFInfo
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Abstract
The invention discloses a promoter pCALM2 and application thereof, wherein the nucleotide sequence of the promoter pCALM2 is shown as SEQID NO: 1 is shown. The promoter pCALM2 can be applied to recombinant adeno-associated virus, so that exogenous genes can be efficiently expressed in forebrain, and the promoter pCALM2 has wide application prospect in the field of neural circuit tracing and gene therapy.
Description
Technical Field
The invention belongs to the technical field of nerve engineering, and particularly relates to a promoter pCALM2 and application thereof.
Background
The forebrain belongs to the highest part of the brain and is the most complex and important central nervous system in mammals. It is mainly composed of two parts: telencephalon and diencephalon. The telencephalon mainly includes the whole cerebral cortex, limbic system and basal ganglia, etc., and the diencephalon mainly includes the thalamus and hypothalamus, etc. It participates in the regulation of a variety of important physiological functions including movement, sensation, perception, autonomic nerves, endocrine, food intake, learning, memory, cognition, and the like. The analysis of the forebrain neural circuit and the function thereof can help us to better understand the higher functions of the brain. The research on forebrain can help us to better understand the pathogenic mechanism of nervous system diseases, and further search for more effective treatment methods.
In recent years, with the application of novel neural loop tracers, particularly neurotropic virus tracers, research related to the analysis of the structure and function of the neural loop has been rapidly developed. Adeno-associated virus (rAAV) and lentivirus are the most commonly used, wherein recombinant adeno-associated virus (rAAV) is a gene vector modified on the basis of non-pathogenic wild AAV, and the rAAV is considered to be one of the most promising gene research and gene therapy vectors due to various types, extremely low immunogenicity, high safety, wide host cell range, strong diffusion capacity, long in-vivo gene expression time and the like. However, recently, it has been found that there are many obvious defects in the application of such vectors, including that the number of viral receptors on some cell membranes is very small, site-specific integration of rAAV vectors is insufficient, and AAV capsid components and transgene products cause host immune response, etc., which limits the high-efficiency and safe application of rAAV in neuroscience research and clinical gene therapy. Therefore, there is a need for better improvement of AAV vectors, such that the new generation of rAAV vectors have the necessary safety, efficiency, and targeting properties for gene therapy, and are expected to be more widely used in clinical applications.
The key to the successful application of rAAV in neuroscience research and clinical gene therapy is to improve the space-time specificity and expression level of gene expression. One important method is to select different serotypes of rAAV virus, which have different affinities for different tissues and show certain organ targeting specificity. AAV serotypes currently reported as being suitable for use in the nervous system are: type 1, type 2, type 5, type 6, type 8, type 9, DJ, PHP.B, PHP.eB, PHP.S, Retro, rh10, etc. We can select different serotypes according to different experimental purposes and therapeutic targets. However, due to the complexity of the central nervous system, these serotypes are far from meeting the current requirements of gene delivery and specificity of gene manipulation. Another approach is to use tissue-specific promoters in the rAAV genome. The promoter is a cis-acting element necessary in the regulation of gene expression, and the promoter element of the viral vector determines the specificity of viral expression. For targeting studies or gene therapy, we need to continuously develop promoters suitable for specific cells or tissues.
Disclosure of Invention
In order to solve the problems of the background art, the invention aims to provide a promoter pCALM2 for high-efficiency expression of forebrain and application thereof. The starting pCALM2 can be applied to recombinant adeno-associated virus, so that exogenous genes can be efficiently expressed in forebrain, and the starting pCALM2 has wide application prospect in the field of neural circuit 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 pCALM2, wherein the nucleotide sequence of the promoter pCALM2 is as shown in SEQ ID NO: 1 is shown.
Further, the promoter pCALM22 is a partial sequence of calmodulin 2(CALM2) gene, which is selected from a partial sequence near the transcription start site of CALM2 gene, and a partial sequence of the first exon, in total, 3020.
Further, the promoter pCALM2 was highly expressed in the forebrain.
In another aspect, the present invention provides a recombinant adeno-associated viral vector comprising the promoter pCALM2 described above.
Further, the recombinant adeno-associated viral vector includes pAAV-pCALM 2-fluorescent protein, pAAV-pCALM 2-functional protein or pAAV-pCALM 2-therapeutic protein.
Further, the fluorescent protein includes eYFP, tdTomato;
preferably, the functional protein comprises a optogenetically-related protein or a chemically genetically-related protein.
pAAV-pCALM 2-fluorescent proteins (fluorescent proteins include eYFP, tdTomato) can realize multi-color tracing of the neural circuit. pAAV-pCALM 2-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 2-therapeutic protein can be used for gene therapy.
Further, the recombinant adeno-associated virus vector pAAV-pCALM2-eYFP adopts pAAV-hSyn-eYFP as a skeleton vector, hSyn is replaced in the vector to be a pCALM2 promoter, and the recombinant adeno-associated virus vector pAAV-pCALM2-eYFP is obtained.
Further, the preparation method of the recombinant adeno-associated virus vector pAAV-pCALM2-eYFP comprises the following steps: cloning the promoter pCALM2 into a pUC-57 vector to obtain a pUC57-pCALM2 vector; treating the pAAV-hSyn-eYFP vector by using restriction enzymes MluI-HF and KpnI-HF, and treating the pUC57-pCALM2 vector by using the restriction enzymes MluI-HF and KpnI-HF; and (3) recovering, purifying and connecting the two enzyme digestion products to obtain the recombinant adeno-associated virus vector pAAV-pCALM 2-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-retroro-pCALM 2-fluorescent protein, AAV-retroro-pCALM 2-functional protein or AAV-retroro-pCALM 2-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-retro-pCALM2-eYFP and AAV-retro-pCALM2-tdTomato can effectively reverse mark cortical to striatal nerve loops and express eYFP or tdTomato with high fluorescence intensity.
In still another aspect, the present invention provides a kit comprising the promoter pCALM2 described above, the recombinant adeno-associated viral vector described above, or the recombinant adeno-associated virus described above.
In another aspect, the invention provides an application of the promoter pCALM2, the recombinant adeno-associated virus 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 invention provides a neural network tracer comprising pAAV-pCALM 2-fluorescent protein and/or a recombinant adeno-associated virus comprising pAAV-pCALM 2-fluorescent protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the recombinant adeno-associated virus includes AAV-retro-pCALM2-eYFP, AAV-retro-pCALM 2-tdTomato.
In still another aspect, the present invention provides a use of the above neural network tracer in cortical-striatal neural circuit tracing.
Compared with the prior art, the invention has the following beneficial effects:
(1) the promoter pCALM2 has high-efficiency expression efficiency in the forebrain, can realize high-efficiency expression of exogenous genes in the forebrain, 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 pCALM2 of the invention has no cytotoxicity in bacteria and mammalian cells;
(3) the recombinant adeno-associated virus vector adopts pCALM2 as a promoter, and has high-efficiency expression efficiency in forebrain;
(4) the recombinant adeno-associated virus can efficiently mark the cortical to striatal nerve loop reversely in the brain, and has high marking efficiency and strong infection capacity.
Drawings
FIG. 1 is a flow chart showing the construction of the pAAV-pCALM2-eYFP vector of the present invention;
FIG. 2 is a graph showing the expression efficiency of AAV-retro-pCALM2-eYFP in cortex and striatum.
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-pCALM2 vector
The pCALM2 promoter is a partial sequence of the calmodulin 2(CALM2) gene, which is selected from the partial sequence near the transcription start site of the CALM2 gene, and the first exon partial sequence, in total, 3020. The nucleotide sequence of the promoter pCALM2 is shown as SEQ ID NO: 1 is shown.
The promoter pCALM2 was artificially synthesized and cloned in pUC-57 vector to obtain pUC57-pCALM2 vector.
Example 2 construction of recombinant adeno-associated Virus vector pAAV-pCALM2-eYFP
In this example, because eYFP is used as a reporter gene of a promoter, when constructing a recombinant vector, a recombinant adeno-associated vector pAAV-hSyn-eYFP containing eYFP gene is selected as a vector backbone to connect pCALM2 promoter, and the following steps are performed:
as shown in FIG. 1, the vector pAAV-hSyn-eYFP was treated with restriction enzymes MluI-HF and KpnI-HF, while the vector pUC57-pCALM2 was treated with restriction enzymes MluI-HF and KpnI-HF, and digested at 37 ℃ for 3 hours in the system shown in tables 1 and 2, after recovering the digested products, ligation was performed at 16 ℃ for 30min with a ligation premix (2 × ligation premix, TAKARA) in the system shown in Table 3, to obtain a successfully ligated vector pAAV-hSyn-eYFP.
TABLE 1 pAAV-hSyn-eYFP vector restriction system
| Reagent | Dosage of |
| Enzyme digestion Buffer solution 10 XCutSmart Buffer | 5μL |
| MluI-HF | 1μL |
| KpnI-HF | 1μL |
| pAAV-hSyn-eYFP | 1μg |
| ddH2O | Make up to 50 μ L |
TABLE 2 pUC57-pCALM2 vector restriction system
| Reagent | Dosage of |
| Enzyme digestion Buffer solution 10 XCutSmart Buffer | 5μL |
| MluI-HF | 1μL |
| KpnI-HF | 1μL |
| pUC57-pCALM2 | 1μg |
| ddH2O | Make up to 50 μ L |
TABLE 3 connection System
| Reagent | Dosage of |
| Connecting the premix 2 × ligation premix | 5μL |
| pAAV-eYFP connection skeleton | 0.5μL |
| pCALM2 ligation fragment | 4.5μL |
Example 3 preparation of recombinant adeno-associated Virus AAV-retro-pCALM2-eYFP
This example uses a three plasmid co-transfection method to prepare the virus. Before virus preparation, plasmids required for packaging virus are subjected to middle-mentioned steps, and comprise a recombinant adeno-associated virus vector pAAV-pCALM2-eYFP, a packaging plasmid AAV-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: fluorescent quantitative kit (TAKARA) by using AAV titer determination dye methodCo.) the titer of the purified virus was determined to be 1.53X 1013VG/mL。
Example 4 recombinant adeno-associated Virus labeling mouse cortical-striatal neural circuits
The purified recombinant adeno-associated virus was injected into the right striatum of mice (AP: +0.62mm, ML: +1.75mm, D/V: -3.5 mm). The recombinant adeno-associated virus capsid is AAV-retro, has a reverse infectivity, and is capable of reversely tracing cortical neurons upstream of the striatum. 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 forebrain.
In conclusion, the invention is verified on mice, and the promoter pCALM2 is proved to have high expression level in forebrain.
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 pCALM2 and application thereof
<130> CP121010013C
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 3020
<212> DNA
<213> Artificial sequence
<400> 1
gctcatgtgt ggcaggcatg gtattcaagg ctacactctt cagtgtggga cctttggagg 60
tgattggatc acgatggctc tgacttcatg aatagattga ttcctccgtg gcctcatgga 120
ttaatgagtt ggggaggtgg ggagaggctt ggttataaca ttaagttctc taagagcttt 180
gctctttccc ctgacgtttc caggatgctt tcctccacac aggcctggaa gtgacagaga 240
caagtgagtg tcagccccag ctgcagctgc catggtgctt tcttgaatcg aggtgtttcg 300
tcacagtgat ggaaagctaa ctgtcgcctc atcttagtcc tgctttgcct cattaaagga 360
aaaacagcgt aaccttctca gccagggcga cgagtctgaa ccgtggaaga agatggcttc 420
cgtcctacct ccccaattcc attctagatc atagaagaga aattgactca ttgcatagga 480
tcaatgccct ggaaaaagta ggacttaatt ttttcaattt gttcagaaac ccatagacta 540
tattcaagaa tggtgttatg tgatgttctg tttgcttgtt tcactgctaa ctagcaaaac 600
tgggtaaaag agagagaagc tccggaaagg aagctatcca gagataaggc ggtattatct 660
caattcagaa aatgaggaag aagcagcgat ggggaagaac ttgcttgttt tttttctttc 720
ccccaagatg gacaaaggta gagaactcta agatctcacc gtgggaacca gacagaagaa 780
aaaaaaagat atttccaagg ctctgataca cagtcttcgc tgactttgcg tttcactgac 840
aactgccagg gttaggcgga gatctctgtc atatttcgga agacaagtgc tctataaata 900
aattgacttg gaacagtctc aaatacacat aaaccgcaat ggctttaaat gtaacccttg 960
acttttcttt gtcttcttaa gaggtcaaca aggaagatta tgcaagaaag gcgttaccaa 1020
aacccaacct acgaaacctg gaagagcttt cctatattct ctatttaaaa aaaaaatata 1080
tttctatata cttgttagca agcactaccg cgtctctcta ggcacgagtc agtgcgtact 1140
tatcatacat tctgcaccaa tcctcttaaa aattccttcc cttgaagagg tgattattca 1200
ttttgtcatt caatttccca ccattaaaaa tttcatatgt aaaatcaccg catgctaatg 1260
gggtactgtg taaggagaga atatttttac tgagtcagcg gaaagacaaa aaaaaaaatt 1320
agtaggtctt ccatctgaag atctgaggag gagtcacaga caaatttctg aggtatatct 1380
tggaaaaact ttgaatttac cagaatgtag tgcactggaa cggaacggtg gcgtcagatt 1440
cttggctgga ggtgcccatt ttaaagtaat attcccaaat aatgatattt atctcatttg 1500
tggggtaagg ttaattagct ctcctccctt tcagtattat cagggcacca cattggcaag 1560
gataatagaa acagaaatct gaggtttggt ttggtttggt attcccatcg agtgggcatt 1620
cgttcgaaag ggtgtcagct ttgttagtgc taattagaag gccgcttaat gtctggtttc 1680
catgactgaa agagggccca gctctggctt cattaaaaga tatgtttaaa ggtgtgaggg 1740
atgtctaact taggtaaagg aggttaaaat ggactctccg agtgggtgca tatagctgtt 1800
agcaccgaaa tgttcttgct gaacggggaa actatcctga aggcaggctt ctttctgaag 1860
ttgaggtttc atcttgcctt gctttttaca aagtgattgc tctccacggt ttgagggttt 1920
gcatggtggt gctttttcaa ctgggaagtt ctttctgcct tcgccatctt cctaattctc 1980
tctgggccgt ggctgttcca tcaatcttct ctgggcactg tattttgagg atggaatcct 2040
tattttcctc tctaaccccc atcatccttt tctggttgtc tactttgggc ttctaatttc 2100
agaggggctc cttggggaga ccgacagcac acactctcgg atacagtggg tttgagtagc 2160
tttctgataa attactgcta ggaaagagaa gcgaagggta ggcataaacg gaagcccctt 2220
ttttgattag cttgtcagct gagctgcatg ggttcggcac cgtgtaatga aaaacgaccg 2280
gcctccctat gcagcaacgc tgagaagctg ggcgcgctgg gtgggtgttt actggggggg 2340
gggggtgtcc ttcagtttta tttatttttt gttttggccc gtgttgtttt tgtcagcagc 2400
acctccatac ggggcgggaa ggtgagtgtg tgcgtgtgtc tgtggcaggc aggacgggtg 2460
gaagaaggtc ctccagccga gcgcagagag agccacgctg ccgcctggcg cagttgccag 2520
gacagcgcgg cgcgagcaag caacggcaga agccgcaacc gcgttccggc tgcgagagaa 2580
agtgacgcat tgcaggtaca aatcagccaa tcagcagcaa gctcaacgag gttcgcctgg 2640
tcgaccgcca ccgcggtgct cctccgcgcc gcaaagtagc tcctgccagg cgcggttccc 2700
ccggcctttt aacggcttgc ctgggagtgg aacccactta cgtcatccgt ccccctgctc 2760
ccctttctct cttgcgtgtc tgcccagtga ctgcggagaa agggggtctc ccggagagtt 2820
gggggggggg agggggcggg ggctggccgg gactcgttcg ctaggttccg ttatctggat 2880
gaggcggagg gatctggcgg agggaggcgg agggaggtgt ttatgaggag ctgggggcgg 2940
aggaggagaa ttagtccgag tggagcgagc gagtcgagcg gttgtctggt cgcgtctcgg 3000
aaacccgtag cccttgcagc 3020
Claims (10)
1. The promoter pCALM2, wherein the nucleotide sequence of the promoter pCALM2 is shown as SEQ ID NO: 1 is shown.
2. The promoter pCALM2 of claim 1, wherein the promoter pCALM2 is highly expressed in the forebrain.
3. A recombinant adeno-associated viral vector comprising the promoter pCALM2 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 pAAV-pCALM 2-fluorescent protein, pAAV-pCALM 2-functional protein or pAAV-pCALM 2-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 an AAV-retro-pCALM 2-fluorescent protein, an AAV-retro-pCALM 2-functional protein or an AAV-retro-pCALM 2-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 pCALM2 of any one of claims 1-2, the recombinant adeno-associated viral 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 pCALM2 of any one of claims 1-2, the recombinant adeno-associated virus vector of any one of claims 3-4, the recombinant adeno-associated virus of any one of claims 5-6, or the kit of claim 7 for the preparation of a neural circuit tracking and/or manipulating nerve cells and/or gene therapy reagents.
9. A neural network tracer, which is characterized by comprising pAAV-pCALM 2-fluorescent protein and/or recombinant adeno-associated virus comprising pAAV-pCALM 2-fluorescent protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the recombinant adeno-associated virus includes AAV-retro-pCALM2-eYFP, AAV-retro-pCALM 2-tdTomato.
10. Use of a neural network tracer according to claim 9 in cortical-striatal loop tracing.
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| CN113652427A (en) * | 2021-08-20 | 2021-11-16 | 深圳市恩辑生物科技有限公司 | Mini promoter pATP1B1 and application thereof |
| 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 |
| 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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| 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 |
| CN113652427A (en) * | 2021-08-20 | 2021-11-16 | 深圳市恩辑生物科技有限公司 | Mini promoter pATP1B1 and application thereof |
| 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 |
| CN113667671B (en) * | 2021-08-20 | 2023-08-29 | 深圳市恩辑生物科技有限公司 | Mini promoter pRTN1 and application thereof |
| CN113652427B (en) * | 2021-08-20 | 2023-08-29 | 深圳市恩辑生物科技有限公司 | Mini promoter pATP1B1 and application thereof |
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