CN113667671A - Mini promoter pRTN1 and application thereof - Google Patents

Mini promoter pRTN1 and application thereof Download PDF

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CN113667671A
CN113667671A CN202110963597.XA CN202110963597A CN113667671A CN 113667671 A CN113667671 A CN 113667671A CN 202110963597 A CN202110963597 A CN 202110963597A CN 113667671 A CN113667671 A CN 113667671A
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recombinant adeno
prtn1
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路中华
林剑邦
李田
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Shenzhen Enji Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of nerve engineering, and particularly relates to a mini promoter pRTN1 specifically and efficiently expressed in neuronal cells. The invention provides a mini promoter pRTN1 and application thereof, wherein the nucleotide sequence of the promoter pRTN1 is shown as SEQ ID NO: 1 is shown. The invention also provides application of the promoter pRTN1 in recombinant adeno-associated virus vectors, recombinant adeno-associated viruses, kits of recombinant adeno-associated viruses and gene therapy vectors, and in preparation of nerve cell manipulation, gene editing and/or gene therapy reagents. The mini promoter pRTN1 provided by the invention enables an exogenous gene to be efficiently expressed in neurons in the whole brain range, and has wide application prospects in the fields of gene editing and gene therapy.

Description

Mini promoter pRTN1 and application thereof
Technical Field
The invention belongs to the technical field of nerve engineering, and particularly relates to a mini promoter pRTN1 and application thereof.
Background
Adeno-associated virus (AAV) 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 is a gene delivery vector widely used in the neuroscience field and in clinical gene therapy for nervous system diseases due to the advantages of long-term stable expression of mediated genes in mammals, low immunogenicity, wide host range and the like.
In the process of applying rAAV to neuroscience research and clinical treatment, the key of gene delivery is to improve the space-time specificity and the expression level of target gene expression. Strategies to regulate foreign gene expression directly affect the efficiency and safety of gene delivery. 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 reports available today, rAAV serotypes suitable for use in the nervous system include: 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 affinities, infection efficiencies, and spreading capabilities for different brain regions, and therefore, different rAAV serotypes can be selected as gene delivery vectors according to the study subject and the therapeutic target. However, given 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. The gene expression is driven by using a specific promoter in the rAAV, so that the gene expression efficiency can be improved, and the immune response generated in the rAAV infection process can be reduced.
rAAV is a reliable viral vector for delivering therapeutic gene elements to the central nervous system, but there are two important factors that prevent its development and use in gene therapy strategies. Firstly, the maximum packaging capacity of rAAV is 5.2kb, greatly limiting the packaging of large therapeutic gene elements in a single AAV vector; secondly, the low targeting of rAAV is also a problem limiting the tools for development of rAAV viruses. The size of the currently common neuron-specific promoter hSyn is 485bp, and the length of the promoter further limits the packaging capacity of functional proteins on the premise of a single AAV vector.
Disclosure of Invention
The invention aims to provide a mini promoter pRTN1 and application thereof. The mini promoter pRTN1 is only 120bp in length, can be applied to recombinant adeno-associated virus, enables an exogenous gene to be efficiently expressed in neuronal cells, and has wide application prospects in the fields of gene editing and gene therapy.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first purpose of the invention is to provide a mini promoter pRTN1 specifically and efficiently expressed in neuronal cells, wherein the length of the promoter pRTN1 is 120bp, and the nucleotide sequence is shown as SEQ ID NO: 1 is shown.
CGCTGATTCGCTGCCGGCCGCGCAGACTGCGGCGCCAGCGACGCTCCCGCCCCTCACCGCGCGCCGCCTGGTGCAGCCTCAGTCTCTCCGCAGCCGCGCGGCCACTGCGAGCCCAGCCGC(SEQ ID NO：1)
The second purpose of the invention is to provide a recombinant adeno-associated virus vector containing the promoter.
Preferably, the recombinant adeno-associated virus vector adopts pAAV-hSyn-X as a framework vector, hSyn in the vector is replaced by pRTN1 promoter, and the recombinant adeno-associated virus vector pAAV-pRTN1-X is obtained and is a protein sequence to be expressed.
Preferably, the recombinant adeno-associated viral vector is one of pAAV-pRTN 1-fluorescent protein, pAAV-pRTN 1-functional protein or pAAV-pRTN 1-therapeutic protein; the fluorescent protein is eYFP or tdTomato; the functional protein is a protein related to chemical genetics.
The pAAV-pRTN 1-fluorescent protein can realize specific cell infection tracing; the pAAV-pRTN 1-functional protein can be used for preparing recombinant adeno-associated viruses which activate or inhibit specific neurons; the pAAV-pRTN 1-therapeutic protein can be used for gene therapy.
Preferably, the preparation method of the recombinant adeno-associated virus vector pAAV-pRTN1-X comprises the following steps:
(1) adding restriction enzyme MluI-HF and BamHI-HF recognition sites to both ends of a promoter pRTN1 gene fragment respectively, and then artificially synthesizing;
(2) treating the pAAV-hSyn-X vector by using restriction enzymes MluI-HF and BamHI-HF, and treating the pRTN1 fragment by using the restriction enzymes MluI-HF and BamHI-HF;
(3) and (4) recovering, purifying and connecting the two enzyme digestion products to obtain the recombinant adeno-associated virus vector pAAV-pRTN 1-X.
Still another object of the present invention is to provide a recombinant adeno-associated virus comprising the promoter or the recombinant adeno-associated virus vector.
The recombinant adeno-associated virus AAV-PHP.B-pRTN1-eYFP and AAV-PHP.B-pRTN1-tdTomato can specifically infect neuron cells and express eYFP or tdTomato with high fluorescence intensity.
The invention also aims to provide a kit of the promoter or the recombinant adeno-associated virus vector or the recombinant adeno-associated virus.
Further, the invention also provides application of the promoter or the recombinant adeno-associated virus vector or the recombinant adeno-associated virus kit in preparation of a reagent for controlling nerve cells, gene editing and/or gene therapy.
Compared with the prior art, the invention has the following beneficial effects:
(1) the mini promoter pRTN1 has the length of 120bp, has high-efficiency expression efficiency in neuron cells, can realize high-efficiency expression of foreign genes in the neurons, improves the expression specificity after virus infection, and has wide application prospect in the fields of gene editing and gene therapy;
(2) the promoter pRTN1 of the invention has no cytotoxicity in bacteria and mammalian cells;
(3) the recombinant adeno-associated virus vector adopts pRTN1 as a promoter, has higher packaging capacity, and has high-efficiency expression efficiency in excitatory and inhibitory neurons;
(4) the recombinant adeno-associated virus can infect neuron cells in the whole brain range, and the high-level expression of eYFP can be driven by cortex, hippocampus and midbrain.
Drawings
FIG. 1 is a flow chart showing the construction of pAAV-pRTN1-eYFP vector;
FIG. 2 is a graph showing the expression efficiency of AAV-PHP.B-pRTN1-eYFP in mouse brain.
Detailed Description
The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.
The promoter pRTN1 gene fragment is synthesized by Suzhou Jinweizhi biotechnology limited;
the nucleases are available from NEB corporation under accession numbers R3198S, R3136S;
the AAV titer determination dye method fluorescent quantitative kit is purchased from TAKARA;
the PFA was purchased from Leagene, Cat DF 0135.
Example 1 Synthesis of pRTN1 Gene fragment
The promoter pRTN1 is a partial sequence of a human reticulin 1(Reticulon 1) gene, the sequence selects a core promoter region sequence from-80 to +40 bits of a transcription start site of an RTN1 gene, a sequence of 120bp in total is used as a final promoter sequence, and the nucleotide sequence of the promoter pRTN1 is shown as SEQ ID NO: 1 is shown.
Restriction enzyme MluI-HF and BamHI-HF recognition sites were added to both ends of the pRTN1 gene fragment and synthesized by Jinweizhi Biotech, Suzhou.
Example 2 construction of recombinant adeno-associated Virus vector pAAV-pRTN1-eYFP
In this example, because YFP is used as a reporter gene of a promoter, constructing a recombinant vector is to select a recombinant adeno-associated vector pAAV-hSyn-eYFP containing YFP gene as a vector backbone to connect with a pRTN1 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 BamHI-HF, while the vector pRTN1 was treated with restriction enzymes MluI-HF and BamHI-HF, and digested at 37 ℃ for 3 hours in the digestion system shown in tables 1 and 2, and the digested products were recovered and ligated with a ligation premix (2x ligation premix, TAKARA) at 16 ℃ for 30 minutes in the digestion system shown in Table 3, to obtain a successfully ligated vector pAAV-pRTN 1-eYFP.
TABLE 1pAAV-hSyn-eYFP vector restriction 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 2pRTN1 fragment cleavage system
Reagent Dosage of
Enzyme digestion Buffer solution 10 XCutSmart Buffer 5μL
MluI-HF 1μL
BamHI-HF 1μL
pRTN1 fragment 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
pRTN1 ligation fragment 4.5μL
Example 3 preparation of recombinant adeno-associated Virus AAV-PHP.B-pRTN1-eYFP
This example uses a three plasmid co-transfection method to prepare the virus. Before virus preparation, plasmids required for packaging virus are extracted by using QIAGEN Plasmid Plus Midi Kit (QIAGEN company, cat 12943), including recombinant adeno-associated virus vector pAAV-pRTN1-eYFP, packaging Plasmid AAV-PHP.B and helper Plasmid pHelper. The method comprises the following specific steps:
preparing cells: 293T cells were plated in a dish containing complete medium (DMEM, 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 2M calcium chloride solution were aspirated and gently mixed. To the reagent, an equal volume of 2 × HBS was added, vortexed and allowed to stand for 30 minutes.
Transfection of cells: mu.L of chloroquine (25mM) was added to each dish of cells, and the cells were incubated after addition of transfection reagent.
And (3) virus collection: after 72 hours, the transfected cells were collected in a centrifuge tube and centrifuged at 3000rpm for 30 min. Discarding the supernatant, adding 2ml of cell lysate (100mM Tris-HCl,150mM NaCl, pH8.0), then performing four-round repeated freeze thawing to lyse the cells, performing quick-freezing and thawing for 10 minutes respectively by a refrigerator at-80 ℃ and a water bath at 37 ℃ in each round, and performing brief vortex after each thawing to promote the lysis, thereby finally obtaining the cell disruption solution containing the virus.
And (3) purifying the virus: the cell disruption solution was centrifuged at 11500rpm for 30min, the supernatant was discarded and then 200. mu.L of LHBS was added and mixed well, 200. mu.L of chloroform was added and centrifuged at 12000rpm for 5min, the supernatant was taken, 100. mu.L of 2.5mM NaCl and 100. mu.L of 40% PEG8000 were added and mixed well by vortexing, and the mixture was left to stand 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 2.0X 1013vg/ml。
Example 4 specific expression of recombinant adeno-associated Virus in mouse Whole brain neurons
The purified recombinant adeno-associated virus was stereotactically injected into the lateral ventricle of mouse (Bregma: +0.02 mm; R: -0.80 mm; D:2.40mm) and the expression characteristics of pRTN1 were studied over the whole brain. Three weeks later, mice were perfused with 4% PFA to take whole brains, fixed with 4% PFA and dehydrated with 30% sucrose solution, cryosectioning and immunofluorescence staining were performed after brain tissue was completely dehydrated, and brain slice results were observed.
The experimental results are shown in fig. 2, eYFP has very high expression level in neurons in the whole brain range of mice, which indicates that php.b serotype virus can cross the brain-cerebrospinal fluid barrier and infect cells in the whole brain. pRTN1 was transcriptionally active throughout the brain, and was able to drive high levels of eYFP expression in the cortex, hippocampus, and midbrain. Notably, at high power microscope, pRTN1 was observed to drive only eYFP expression in neurons, but not in glial cells, thus demonstrating that promoter pRTN1 has neuron-specific and high expression levels.
In conclusion, the invention is verified on mice, and the promoter pRTN1 is proved to have neuron specificity and high expression level.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Sequence listing
<110> Shenzhen Shen En edition Biotech Limited
<120> mini promoter pRTN1 and application thereof
<130> 2021.8.20
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 120
<212> DNA
<213> Promoter pRTN1 nucleotide sequence (Promoter pRTN1 nucleotide sequence)
<400> 1
cgctgattcg ctgccggccg cgcagactgc ggcgccagcg acgctcccgc ccctcaccgc 60
gcgccgcctg gtgcagcctc agtctctccg cagccgcgcg gccactgcga gcccagccgc 120

Claims (8)

1. The mini promoter pRTN1 specifically and efficiently expressed in neuronal cells is characterized in that the length of the promoter pRTN1 is 120bp, and the nucleotide sequence is shown as SEQ ID NO: 1 is shown.
2. A recombinant adeno-associated virus vector comprising the promoter of claim 1.
3. The recombinant adeno-associated virus vector according to claim 2, wherein the recombinant adeno-associated virus vector employs pAAV-hsin-X as a backbone vector, and hsin in the vector is replaced by pRTN1 promoter to obtain the recombinant adeno-associated virus vector pAAV-pRTN1-X, wherein X is a protein sequence to be expressed.
4. The recombinant adeno-associated viral vector according to claim 2 wherein the recombinant adeno-associated viral vector is one of pAAV-pRTN 1-fluorescent protein, pAAV-pRTN 1-functional protein or pAAV-pRTN 1-therapeutic protein; the fluorescent protein is eYFP or tdTomato; the functional protein is a protein related to chemical genetics.
5. The method for producing the recombinant adeno-associated viral vector pAAV-pRTN1-X according to claim 2, comprising the steps of:
(1) adding restriction enzyme MluI-HF and BamHI-HF recognition sites to both ends of a promoter pRTN1 gene fragment respectively, and then artificially synthesizing;
(2) treating the pAAV-hSyn-X vector by using restriction enzymes MluI-HF and BamHI-HF, and treating the pRTN1 fragment by using the restriction enzymes MluI-HF and BamHI-HF;
(3) and (4) recovering, purifying and connecting the two enzyme digestion products to obtain the recombinant adeno-associated virus vector pAAV-pRTN 1-X.
6. A recombinant adeno-associated virus comprising the promoter according to claim 1 or the recombinant adeno-associated virus vector according to any one of claims 2 to 5.
7. A kit comprising the promoter of claim 1, the recombinant adeno-associated virus vector of any one of claims 2 to 5, or the recombinant adeno-associated virus of any one of claim 6.
8. Use of the promoter according to claim 1 or the recombinant adeno-associated virus vector according to any one of claims 2 to 5 or the recombinant adeno-associated virus according to claim 6 or the recombinant adeno-associated virus according to claim 7 in the preparation of a reagent for nerve cell manipulation, gene editing and/or gene therapy.
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Citations (7)

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