CN113584032B - Mini promoter pAPP and application thereof - Google Patents

Abstract

The invention belongs to the technical field of neural engineering, and in particular relates to a mini-promoter pAPP specifically and highly expressed in neuron cells. The present invention provides a mini-promoter pAPP and its application. The nucleotide sequence of the promoter pAPP is shown in SEQ ID NO:1. Another aspect of the present invention provides the use of the promoter pAPP in recombinant adeno-associated virus vectors, recombinant adeno-associated virus, kits for recombinant adeno-associated virus, gene therapy vectors, and in the preparation and manipulation of nerve cells, gene editing and/or gene Applications in therapeutic reagents. The mini-promoter pAPP provided by the present invention enables highly efficient expression of exogenous genes in neurons in the whole brain, and has broad application prospects in the fields of gene editing and gene therapy.

Description

A kind of mini promoter pAPP and its application

technical field

The invention belongs to the technical field of neural engineering, and in particular relates to a mini-promoter pAPP and its application.

Background technique

Adeno-associated virus (AAV) is a non-enveloped, single-stranded DNA virus. Recombinant adeno-associated virus (rAAV) is a recombinant virus modified on the basis of wild-type AAV. Recombinant adeno-associated virus is a widely used gene delivery vector in the field of neuroscience and clinical gene therapy of nervous system diseases due to its advantages of long-term stable expression of genes in mammals, low immunogenicity, and wide host range.

In the process of applying rAAV in neuroscience research and clinical treatment, improving the spatiotemporal specificity and expression level of target gene expression is the key to gene delivery. Strategies to regulate the expression of exogenous genes directly affect the efficiency and safety of gene delivery. For gene therapy of neurological diseases, to intervene in different neurological diseases, it is necessary to manipulate the whole brain or different subtypes of neurons. One of the important methods is to select rAAV viruses of different serotypes. In the existing reports, rAAV serotypes applicable to 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 and more than a dozen. Different serotypes of viruses have different affinities, infection efficiencies, and diffusion capabilities for different brain regions. Therefore, we can choose different rAAV serotypes as gene delivery vehicles according to the research objects and therapeutic targets. However, given the complexity of the CNS, these serotypes are far from meeting the current need for gene delivery specificity. Another approach is to use tissue-specific promoters in the rAAV genome. Promoters are essential cis-acting elements in the regulation of gene expression. Under the regulation of expression-specific promoters, foreign genes are generally only expressed in certain cell types or brain regions. The use of specific promoters in rAAV to drive gene expression can improve the efficiency of gene expression and reduce the immune response generated during rAAV infection.

rAAV is a reliable viral vector for delivering therapeutic genetic elements to the central nervous system, but two important factors hinder its development and application in gene therapy strategies. First, the maximum packaging capacity of rAAV is 5.2kb, which greatly limits the packaging of large therapeutic gene elements in a single AAV vector; second, the low targeting of rAAV is also a problem that limits the development of rAAV viral tools. The currently commonly used neuron-specific promoter hSyn has a size of 485bp, which further limits the packaging capacity of functional proteins under the premise of a single AAV vector.

Contents of the invention

The present invention aims to provide a mini-promoter pAPP and its application. The length of the mini-promoter pAPP is only 120bp, and it can be used in recombinant adeno-associated virus, so that foreign genes can be efficiently expressed in neuron cells, and has broad application prospects in the fields of gene editing and gene therapy.

In order to achieve the above object, the present invention adopts the following technical solutions:

The first object of the present invention is to provide a mini-promoter pAPP specifically and efficiently expressed in neuron cells, the length of the promoter pAPP is 120bp, and the nucleotide sequence is shown in SEQ ID NO:1.

CTCTCGGGTGCCGAGCGGGGTGGGCCGGATCAGCTGACTCGCCTGGCTCTGAGCCCCGCCGCCGCGCTCGGGCTCCGTCAGTTTCCTCGGCAGCGGTAGGCGAGAGCACGCGGAGGAGCG (SEQ ID NO: 1)

The second object of the present invention is to provide a recombinant adeno-associated virus vector containing the above-mentioned promoter.

Preferably, the recombinant adeno-associated virus vector uses pAAV-hSyn-X as the backbone vector, and hSyn in the vector is replaced by the pAPP promoter to obtain the recombinant adeno-associated virus vector pAAV-pAPP-X, where X is the protein sequence to be expressed .

Preferably, the recombinant adeno-associated virus vector is one of pAAV-pAPP-fluorescent protein, pAAV-pAPP-functional protein or pAAV-pAPP-therapeutic protein; the fluorescent protein is eYFP or tdTomato; the function The sex protein is an optogenetically-associated protein or a chemogenetically-associated protein.

The pAAV-pAPP-fluorescent protein can realize specific cell infection tracking; the pAAV-pAPP-functional protein can be used to prepare a recombinant adeno-associated virus that activates or inhibits specific neurons; the pAAV-pAPP-therapeutic protein can be used for Gene therapy.

Preferably, the preparation method of the recombinant adeno-associated virus vector pAAV-pAPP-X comprises the following steps:

(1) artificially synthesize the two ends of the promoter pAPP gene fragment by adding restriction endonuclease MluI-HF and BamHI-HF recognition sites;

(2) Treat the pAAV-hSyn-X vector with restriction enzymes MluI-HF and BamHI-HF, and simultaneously treat the pAPP fragment with restriction enzymes MluI-HF and BamHI-HF;

(3) Recover, purify, and connect the two digested products to obtain the recombinant adeno-associated virus vector pAAV-pAPP-X.

Another object of the present invention is to provide a recombinant adeno-associated virus containing the above-mentioned promoter or the above-mentioned recombinant adeno-associated virus vector.

The recombinant adeno-associated virus AAV-PHP.B-pAPP-eYFP and AAV-PHP.B-pAPP-tdTomato can specifically infect neuron cells and express eYFP or tdTomato with high fluorescence intensity.

Another object of the present invention is to provide a kit containing the above-mentioned promoter or the above-mentioned recombinant adeno-associated virus vector or the above-mentioned recombinant adeno-associated virus.

Further, another aspect of the present invention provides the application of the promoter or recombinant adeno-associated virus vector or recombinant adeno-associated virus or recombinant adeno-associated virus kit in the preparation of manipulation of nerve cells, gene editing and/or gene therapy reagents .

Compared with the prior art, the present invention has the following beneficial effects:

(1) The mini-promoter pAPP of the present invention is 120bp long, and has high expression efficiency in neuron cells, can realize high-efficiency expression of exogenous genes in neurons, and improves expression specificity after virus infection, which is useful in gene editing and gene therapy The field has broad application prospects;

(2) The promoter pAPP of the present invention has no cytotoxicity in bacteria and mammalian cells;

(3) The recombinant adeno-associated virus vector of the present invention adopts pAPP as a promoter, has greater packaging ability, and has high expression efficiency in both excitatory and inhibitory neurons;

(4) The recombinant adeno-associated virus of the present invention can infect neuron cells in the whole brain, including the cortex, hippocampus and midbrain, and can drive high-level expression of eYFP.

Description of drawings

Figure 1 is a flow chart of the construction of the pAAV-pAPP-eYFP vector.

Figure 2 is a graph showing the expression efficiency of AAV-PHP.B-pAPP-eYFP in the mouse brain.

Detailed ways

The above-mentioned content of the present invention will be further described in detail through specific implementation in the form of examples below. However, it should not be construed that the scope of the above-mentioned subject matter of the present invention is limited to the following examples.

The promoter pAPP gene fragment was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.;

The nuclease was purchased from NEB Company, product number R3198S, R3136S;

The AAV titer determination dye method fluorescence quantitative kit was purchased from TAKARA company;

The PFA was purchased from Leagene Company, product number DF0135.

Embodiment 1 pAPP gene fragment synthesis

The pAPP promoter is a partial sequence of the amyloid beta precursor protein (APP, amyloid beta precursor protein) gene. The sequence selects the core promoter region sequence from -80 to +40 of the transcription start site of the APP gene, with a total of 120bp The sequence of the promoter is used as the final promoter sequence, and the nucleotide sequence of the promoter pAPP is shown in SEQ ID NO:1.

The two ends of the promoter pAPP gene fragment were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. after adding restriction endonuclease MluI-HF and BamHI-HF recognition sites.

Example 2 Construction of recombinant adeno-associated virus vector pAAV-pAPP-eYFP

In this example, since YFP is used as the reporter gene of the promoter, when constructing the recombinant vector, the recombinant adeno-associated vector pAAV-hSyn-eYFP containing the YFP gene is selected as the carrier backbone to connect the pAPP promoter, and the steps are as follows:

As shown in Figure 1, the pAAV-hSyn-eYFP vector was treated with restriction enzymes MluI-HF and BamHI-HF, and the pAPP fragment was treated with restriction enzymes MluI-HF and BamHI-HF at the same time, digested at 37°C For 3 hours, the enzyme digestion system is shown in Table 1 and Table 2. After recovering the enzyme digestion product, the ligation premix (2x ligation premix, TAKARA) was used for ligation at 16°C for 30 minutes. The system is shown in Table 3, and the ligation was successful. pAAV-pAPP-eYFP vector.

Table 1 pAAV-hSyn-eYFP vector restriction enzyme digestion system

Reagent Dosage Digestion buffer 10×CutSmart Buffer 5μL MluI-HF 1μL BamHI-HF 1μL pAAV-hSyn-eYFP 1μg ddH2O Make up to 50μL

Table 2 pAPP fragment digestion system

Reagent Dosage Digestion buffer 10×CutSmart Buffer 5μL MluI-HF 1μL BamHI-HF 1μL pAPP fragment 1μg ddH2O Make up to 50μL

Table 3 connection system

Example 3 Preparation of recombinant adeno-associated virus AAV-PHP.B-pAPP-eYFP

In this example, a three-plasmid co-transfection method was used to prepare viruses. Before virus preparation, the plasmids required for packaging viruses need to be extracted using QIAGEN Plasmid Plus Midi Kit (QIAGEN Company, Cat. No. 12943), including recombinant adeno-associated virus vector pAAV-pAPP-eYFP, packaging plasmid AAV-PHP.B and helper plasmids pHelper. Specific steps are as follows:

Preparation of cells: spread 293T cells in a petri dish containing full medium (DMEM, 10% fetal calf serum, 1% double antibody), and culture in an incubator.

Prepare transfection reagent: pipette 5.25ml ultrapure water, 75μg packaging plasmid, 75μg recombinant plasmid, 75μg helper plasmid and 800μL 2M calcium chloride solution, and mix gently. Add an equal volume of 2×HBS to the aforementioned reagents, vortex and let stand for 30 minutes.

Transfected cells: Add 20 μL of chloroquine (25 mM) to each plate of cells, add transfection reagent and culture.

Virus collection: 72 hours later, the aforementioned transfected cells were collected in a centrifuge tube and centrifuged at 3000 rpm for 30 min. After discarding the supernatant, add 2ml of cell lysate (100mM Tris-HCL, 150mM NaCl, pH8.0), and then lyse the cells through four rounds of repeated freezing and thawing, each round using a -80°C refrigerator and a 37°C water bath for 10 minutes each Quick freezing and thawing, with brief vortexing after each thaw, to facilitate lysis and eventually yield virus-containing cell lysates.

Purify the virus: centrifuge the above cell disruption solution at 11500rpm for 30min, discard the supernatant, add 200μL HBS and mix well, add 200μL chloroform and centrifuge at 12000rpm for 5min, take the supernatant, add 100μL 2.5mM NaCl and 100μL 40% PEG8000, vortex mix , 4 ° C refrigerator overnight. Centrifuge the aforementioned overnight sample at 12,000 rpm for 30 min, discard the supernatant, add 30 μL of HBS, 0.5 μL of nuclease, and let stand for 30 min. Subsequently, 30 μL of chloroform was added, and centrifuged at 12000 rpm for 5 min. Store in a -80°C refrigerator after purification.

Determination of titer: AAV titer determination dye method fluorescence quantitative kit (TAKARA company) was used to measure the titer of the aforementioned purified virus, and the titer result was 2.0×10 ¹³ vg/ml.

Example 4 The specific expression of recombinant adeno-associated virus in mouse whole brain neurons

The purified recombinant adeno-associated virus was injected stereotaxically into the lateral ventricle of mice (Bregma: +0.02mm; R: -0.80mm; D: 2.40mm), and the expression characteristics of pAPP were studied in the whole brain. Three weeks later, the mice were perfused with 4% PFA to take the whole brain, fixed with 4% PFA and dehydrated with 30% sucrose solution. After the brain tissue was completely dehydrated, frozen sections and immunofluorescent staining were performed, and the results of the brain slices were observed.

The experimental results are shown in Figure 2. eYFP has a high expression level in neurons in the whole brain of mice, indicating that the PHP.B serotype virus can cross the brain-cerebrospinal fluid barrier and infect cells in the whole brain. pAPP is transcriptionally active throughout the brain and can drive high-level expression of eYFP in the cortex, hippocampus, and midbrain. It is worth noting that under a high-power microscope, it can be observed that pAPP only drives the expression of eYFP in neurons, but not in glial cells, which proves that the promoter pAPP has neuron specificity and high expression level.

In summary, the present invention is verified on mice, which proves that the promoter pAPP has neuron specificity and high expression level.

The above-mentioned embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed in the present invention should still be covered by the claims of the present invention.

sequence listing

<110> Shenzhen Enji Biotechnology Co., Ltd.

<120> A mini-promoter pAPP and its application

<130> 2021.8.20

<160> 1

<170> SIP Sequence Listing 1.0

<210> 1

<211> 120

<212>DNA

<213> Promoter pAPP nucleotide sequence (Promoter pAPP nucleotide sequence)

<400> 1

ctctcgggtg ccgagcgggg tgggccggat cagctgactc gcctggctct gagccccgcc 60

gccgcgctcg ggctccgtca gtttcctcgg cagcggtagg cgagagcacg cggaggagcg 120

Claims (8)

1. A mini-promoter pAPP specifically and efficiently expressed in neuron cells, characterized in that the length of the promoter pAPP is 120 bp, and the nucleotide sequence is as shown in SEQ ID NO:1. 2. A recombinant adeno-associated virus vector containing the promoter of claim 1. 3. The recombinant adeno-associated virus vector according to claim 2, characterized in that, the recombinant adeno-associated virus vector uses pAAV-hSyn-X as the backbone vector, hSyn in the vector is replaced by the pAPP promoter to obtain a recombinant adeno-associated virus vector Viral vector pAAV-pAPP-X, X is the protein sequence to be expressed. 4. The recombinant adeno-associated virus vector according to claim 2, wherein the recombinant adeno-associated virus vector is pAAV-pAPP-fluorescent protein, pAAV-pAPP-functional protein or pAAV-pAPP-therapeutic protein One; the fluorescent protein is eYFP or tdTomato; the functional protein is an optogenetically related protein or a chemically genetically related protein. 5. the preparation method of recombinant adeno-associated virus vector pAAV-pAPP-X according to claim 2, is characterized in that, comprises the following steps: (1) artificially synthesize the promoter pAPP gene fragment according to claim 1 by adding recognition sites for restriction endonucleases MluI-HF and BamHI-HF respectively; (2) Treat the pAAV-hSyn-X vector with restriction enzymes MluI-HF and BamHI-HF, and treat the pAPP fragment with restriction enzymes MluI-HF and BamHI-HF at the same time; (3) Recover, purify, and connect the two digested products to obtain the recombinant adeno-associated virus vector pAAV-pAPP-X. 6. A recombinant adeno-associated virus containing the promoter according to claim 1 or the recombinant adeno-associated virus vector according to any one of claims 2-4. 7. A kit containing the promoter of claim 1 or the recombinant adeno-associated virus vector of any one of claims 2-4 or the recombinant adeno-associated virus of claim 6. 8. A reagent according to the promoter according to claim 1 or the recombinant adeno-associated virus vector according to any one of claims 2 to 4 or the recombinant adeno-associated virus according to claim 6 or the recombinant adeno-associated virus according to claim 7 Application of the cassette in preparation of reagents for manipulating nerve cells, gene editing and/or gene therapy.