M-Slc18a2 gene overexpression vector for targeting intracardiac neurons and construction method of m-Slc18a2 gene overexpression vector

Abstract

The invention discloses an m-Slc18a2 gene overexpression vector and a construction method thereof, and belongs to the technical field of biology. The m-Slc18a2 gene overexpression vector comprises a vector which is operably connected with the following sequence elements: a promoter sequence, an inverted ZsGreen sequence, an inverted m-Slc18a2 gene sequence and a DIO element sequence. The m-Slc18a2 gene overexpression vector can realize accurate expression regulation and control of the m-Slc18a2 gene in a specific intracardiac neuron through a Cre-loxP recombination system, and can be used for constructing an animal model for overexpressing the m-Slc18a2 gene.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an m-Slc18a2 gene overexpression vector targeting intracardiac neurons and a construction method thereof. Background Art

[0002] The m-Slc18a2 gene is expressed in the body as type 2 vesicular monoamine transporter VMAT2, which is responsible for the transport, storage and release of monoamine neurotransmitters in neurons and is crucial for maintaining the normal function of the intracardiac nervous system.

[0003] Adeno-associated virus (AAV), as a gene therapy vector, has the advantages of high safety, low immunogenicity, and a wide host range, and has been widely used in the field of gene therapy.

[0004] The loxP site is a 34-base-pair palindrome consisting of two 13-base inverted repeats and an 8-base spacer. This spacer determines the orientation of the loxP site. The loxP site was first discovered in the P1 bacteriophage and is specifically recognized by the Cre recombinase.

[0005] In the Cre-loxP recombination system, LoxP sites act as markers and locations, and are usually located on both sides of the DNA sequence that requires genetic manipulation. When Cre recombinase is present, it recognizes and binds to the LoxP sites, mediating the recombination of the DNA sequence between the two LoxP sites, achieving operations such as gene knockout, insertion, flipping, or translocation. Generally speaking, there are several possibilities: (1) If two LoxP sites are located on the same DNA chain but in opposite directions, Cre recombinase can cause sequence flipping between the two LoxP sites; (2) If two LoxP sites are located on the same DNA chain and in the same direction, Cre recombinase can effectively remove the sequence between the two LoxP sites; (3) If two LoxP sites are located on two different DNA chains or chromosomes, Cre enzyme can mediate the exchange of the two DNA chains or chromosome translocation; (4) If four LoxP sites are located on two different DNA chains or chromosomes, Cre recombinase may induce sequence exchange between LoxPs. Summary of the Invention

[0006] To solve the above technical problems, on the one hand, the present invention constructs an m-Slc18a2 gene overexpression vector and introduces it into the body, which can effectively increase the expression level of the m-Slc18a2 gene, thereby improving the synthesis and release of neurotransmitters, and providing a new strategy for the study of cardiac neuronal plasticity. On the other hand, the present invention can introduce the m-Slc18a2 gene recombinant overexpression vector into the body of Cre mice, and the AAV2 / 9-php.s serotype adeno-associated virus specifically targets cardiac neuronal cells. The Cre recombinase in the Cre mice flips the DNA sequence between the two Lox-P sites in the m-Slc18a2 gene overexpression vector, thereby achieving specific expression of the m-Slc18a2 gene in cardiac neuronal (especially Chat neuronal) cells.

[0007] In a first aspect, the present invention provides an m-Slc18a2 gene overexpression vector.

[0008] A m-Slc18a2 gene overexpression vector comprises: a vector operably connected with the following sequence elements: a promoter sequence, an inverted ZsGreen sequence, an inverted m-Slc18a2 gene sequence and a DIO element sequence.

[0009] In some embodiments, the DIO element sequence is two sequence fragments (DIO element sequence-1 and DIO element sequence-2); the ZsGreen sequence and the m-Slc18a2 gene sequence are operably linked between the two sequences of the DIO element sequence.

[0010] In some embodiments, each of the two sequence fragments of the DIO element sequence contains a LoxP site, and the two LoxP sites are in opposite directions.

[0011] In some embodiments, the promoter sequence is an h-Syn promoter sequence.

[0012] In some embodiments, the h-Syn promoter sequence comprises the sequence of SEQ ID NO. 4 or a functional variant thereof having at least 80% identity to SEQ ID NO. 4.

[0013] In some embodiments, the inverted ZsGreen sequence comprises the sequence of SEQ ID NO. 5 or a functional variant having at least 80% identity to SEQ ID NO. 5. In some embodiments, the inverted ZsGreen sequence comprises the sequence of SEQ ID NO. 5 or a functional variant having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO. 5.

[0014] In some embodiments, the inverted m-Slc18a2 gene sequence comprises the sequence of SEQ ID NO. 6 or a functional variant having at least 80% identity to SEQ ID NO. 6. In some embodiments, the inverted m-Slc18a2 gene sequence comprises the sequence of SEQ ID NO. 6 or a functional variant having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO. 6.

[0015] In some embodiments, the DIO element sequence comprises: (1) a sequence of SEQ ID NO. 7 or a functional variant thereof having at least 80% identity thereto, and (2) a sequence of SEQ ID NO. 8 or a functional variant thereof having at least 80% identity thereto.

[0016] In some embodiments, the DIO element sequence comprises: (1) a sequence of SEQ ID NO. 7 or a functional variant thereof having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto, and (2) a sequence of SEQ ID NO. 8 or a functional variant thereof having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto.

[0017] In some embodiments, the DIO element sequence comprises: (1) a sequence of SEQ ID NO. 7 or a functional variant thereof having at least 90% identity to SEQ ID NO. 7, and (2) a sequence of SEQ ID NO. 8 or a functional variant thereof having at least 90% identity to SEQ ID NO. 8.

[0018] In some embodiments, the DIO element sequence comprises: (1) a sequence of SEQ ID NO. 7 or a functional variant thereof having at least 95% identity to SEQ ID NO. 7, and (2) a sequence of SEQ ID NO. 8 or a functional variant thereof having at least 95% identity to SEQ ID NO. 8.

[0019] In some embodiments, the DIO element sequence comprises: (1) a sequence of SEQ ID NO. 7 or a functional variant thereof having at least 98% identity to SEQ ID NO. 7, and (2) a sequence of SEQ ID NO. 8 or a functional variant thereof having at least 98% identity to SEQ ID NO. 8.

[0020] In some embodiments, the DIO element sequence comprises: (1) a sequence of SEQ ID NO. 7 or a functional variant thereof having at least 99% identity to SEQ ID NO. 7, and (2) a sequence of SEQ ID NO. 8 or a functional variant thereof having at least 99% identity to SEQ ID NO. 8.

[0021] In some embodiments, the m-Slc18a2 gene overexpression vector further comprises a terminator sequence.

[0022] In some embodiments, the terminator sequence in the m-Slc18a2 gene overexpression vector is a sequence of SEQ ID NO. 11 or a functional variant thereof having at least 80% identity to SEQ ID NO. 11. In some embodiments, the terminator sequence in the m-Slc18a2 gene overexpression vector is a sequence of SEQ ID NO. 11 or a functional variant thereof having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO. 11.

[0023] In some embodiments, the terminator sequence in the m-Slc18a2 gene overexpression vector is operably linked to the inverted m-Slc18a2 gene sequence.

[0024] In some embodiments, the vector is an adeno-associated virus.

[0025] In some embodiments, the adeno-associated virus is an adeno-associated virus of serotype AAV2 / 9-php.s.

[0026] In some embodiments, the sequence of the m-Slc18a2 gene overexpression vector comprises a sequence of SEQ ID NO. 1 or a functional variant thereof having at least 80% identity with SEQ ID NO. 1. In some embodiments, the sequence of the m-Slc18a2 gene overexpression vector comprises a sequence of SEQ ID NO. 1 or a functional variant thereof having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO. 1.

[0027] In a second aspect, the present invention provides an expression cassette.

[0028] An expression cassette comprising the promoter sequence, the inverted ZsGreen sequence, the inverted m-Slc18a2 gene sequence and the DIO element sequence in the m-Slc18a2 gene overexpression vector described in the first aspect;

[0029] Wherein, the promoter sequence, the inverted ZsGreen sequence and the DIO element sequence are operably connected to the inverted m-Slc18a2 gene sequence.

[0030] In some embodiments, the expression cassette further comprises a terminator sequence.

[0031] In some embodiments, the terminator sequence in the expression cassette is a sequence of SEQ ID NO. 11 or a functional variant thereof having at least 80% identity to SEQ ID NO. 11. In some embodiments, the terminator sequence in the expression cassette is a sequence of SEQ ID NO. 11 or a functional variant thereof having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO. 11.

[0032] In some embodiments, the terminator sequence in the expression cassette is operably linked to the inverted m-Slc18a2 gene sequence.

[0033] In a third aspect, the present invention provides an in vitro method for expressing genes in Chat neuronal cells.

[0034] An in vitro method for expressing a gene in Chat neuron cells, comprising transfecting a cell line or cell population with the m-Slc18a2 gene overexpression vector of the first aspect or the expression cassette of the second aspect, wherein the cell line or cell population comprises Chat neuron cells.

[0035] In a fourth aspect, the present invention provides an isolated cell.

[0036] An isolated cell, comprising the m-Slc18a2 gene overexpression vector of the first aspect or the expression cassette of the second aspect.

[0037] In some embodiments, the isolated cell is a Chat neuron cell.

[0038] In a fifth aspect, the present invention provides a reagent.

[0039] A reagent comprising the m-Slc18a2 gene overexpression vector described in the first aspect or the expression cassette described in the second aspect.

[0040] In a sixth aspect, the present invention provides a use of the m-Slc18a2 gene overexpression vector described in the first aspect, the expression cassette described in the second aspect, or the reagent described in the third aspect.

[0041] A use of the m-Slc18a2 gene overexpression vector of the first aspect, the expression cassette of the second aspect, or the reagent of the third aspect in constructing an animal model overexpressing the m-Slc18a2 gene.

[0042] In some embodiments, the animal model used is a transgenic animal in which Cre recombinase is inserted into the cholineacetyltransferase (Chat) gene locus.

[0043] In some embodiments, the animal model used is a transgenic mouse with Cre recombinase inserted into the cholineacetyltransferase (Chat) gene locus.

[0044] In some embodiments, the animal model used is a transgenic rat in which Cre recombinase is inserted into the cholineacetyltransferase (Chat) gene locus.

[0045] In a seventh aspect, the present invention provides a primer pair.

[0046] A primer pair, comprising SEQ ID NO. 2 and SEQ ID NO. 3.

[0047] In an eighth aspect, the present invention provides a method for constructing the aforementioned m-Slc18a2 gene overexpression vector.

[0048] A method for constructing the m-Slc18a2 gene overexpression vector according to the first aspect, characterized by comprising the following steps:

[0049] (1) Enzymatic digestion of the vector;

[0050] (2) Obtaining and PCR amplifying the m-Slc18a2 gene fragment;

[0051] (3) The m-Slc18a2 gene fragment is connected to the vector obtained after enzyme digestion in step (1).

[0052] In some embodiments, the step (1) comprises: mixing the vector, buffer, water and endonuclease, performing an enzyme digestion reaction, and then performing agarose gel electrophoresis to recover the target fragment.

[0053] In some embodiments, step (2) comprises: mixing primer pair 1 and primer pair 2 described in the seventh aspect, template DNA, DNA polymerase, 2'-deoxynucleotide-5'-triphosphate mixture and buffer, performing PCR amplification to obtain the amplified m-Slc18a2 gene fragment.

[0054] In some embodiments, step (3) comprises: mixing the target fragment obtained in step (1), the m-Slc18a2 gene fragment amplified in step (2), a ligase and water, and reacting to obtain an m-Slc18a2 gene overexpression vector.

[0055] In some embodiments, the construction method further comprises: (4) virus packaging and purification.

[0056] In some embodiments, the viral serotype of the m-Slc18a2 gene overexpression vector obtained after the virus packaging is AAV2 / 9-php.s.

[0057] Beneficial effects

[0058] Compared with the prior art, a certain embodiment of the present invention has at least one of the following beneficial effects:

[0059] The m-Slc18a2 gene overexpression vector provided by the present invention can be specifically expressed in cardiac neurons (especially Chat neurons), and can be used to construct an animal model that specifically overexpresses the m-Slc18a2 gene in cardiac neurons (especially Chat neurons), and is used for screening and researching drugs for cardiovascular diseases targeting cardiac neurons or Chat neurons.

[0060] Terminology

[0061] In the description of the present invention, the term "room temperature" means ambient temperature, which refers to a temperature of about 10°C to about 30°C, or about 20°C to 30°C, or about 25°C.

[0062] The term "wt%" means percentage by mass.

[0063] The term "W / W" means mass ratio.

[0064] The term "kb" means kilobase pairs.

[0065] The term "ChAT neuron" refers to a neuron of the acetylcholinesterase phenotype.

[0066] The "Gene ID" numbers described herein (eg, Gene ID: 6010) are numbers provided by the gene-specific database of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0067] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0068] In the following disclosure, all numerical values ​​disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. The numerical value of each number may vary by 1%, 2%, 5%, 7%, 8%, 10%, 15%, or 20%. Whenever a number having a value of N is disclosed, any number having a value of N+ / -1%, N+ / -2%, N+ / -3%, N+ / -5%, N+ / -7%, N+ / -8%, N+ / -10%, N+ / -15%, or N+ / -20% is expressly disclosed, where "+ / -" means plus or minus.

[0069] When used in this article, term " nucleic acid " or " polynucleotide " refer to the polymeric form of nucleotide of any length, and described nucleotide can be ribonucleotide or deoxyribonucleotide.Therefore, this term includes but not limited to single-stranded, double-stranded or multi-chain DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid or the polymer that comprises the non-natural or derivatized nucleotide base of purine and pyrimidine base or other natural, chemical or biochemical modification.The backbone of described polynucleotide can comprise sugar and phosphate group (as can be present in RNA or DNA usually) or sugar or phosphate group that modify or replace.Perhaps, the backbone of described polynucleotide can comprise the polymer of synthetic subunit for example phosphoramidate, and therefore can be oligodeoxynucleoside phosphoramidate (P-NH ) or mixed phosphoramidate-phosphodiester oligomer.Nucleic acid of the present invention can be prepared by any technology well known to persons skilled in the art, comprises chemical synthesis, reorganization and sudden change.In a preferred embodiment, nucleic acid of the present invention is a DNA molecule, and it is preferably synthesized by recombinant method well known to persons skilled in the art.

[0070] The term "promoter" refers to a regulatory element that directs the transcription of a nucleic acid to which it is operably linked. A promoter can regulate both the rate and efficiency of transcription of an operably linked nucleic acid. A promoter can also be operably linked to other regulatory elements that enhance ("enhancer") or repress ("repressor") the promoter-dependent transcription of a nucleic acid.

[0071] The term "promoter activity" refers to the ability of a promoter to initiate transcription of a nucleic acid to which it is operably linked. Promoter activity can be measured using procedures known in the art or described in the Examples. For example, promoter activity can be measured as the amount of transcribed mRNA using, for example, Northern blotting or polymerase chain reaction (PCR). Alternatively, promoter activity can be measured as the amount of translated protein product, for example, by Western blotting, ELISA, colorimetric assays, and various activity assays, including reporter gene assays and other procedures known in the art or described in the Examples.

[0072] The term "operably linked" as used herein refers to the association of nucleic acid sequences on a single nucleic acid molecule such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when the promoter is capable of affecting the expression of the coding sequence, i.e., the coding sequence is under the transcriptional control of the promoter.

[0073] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. These polymers of amino acid residues may contain natural or non-natural amino acid residues and include, but are not limited to, dimers, trimers and multimers of peptides, oligopeptides, amino acid residues. Both full-length proteins and fragments thereof are encompassed by this definition. The term also includes post-translational modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of the present invention, "polypeptide" may refer to proteins comprising modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), as long as the protein maintains the desired activity. These modifications may be carefully designed by site-directed mutagenesis, or may be accidental, such as by mutations in the host producing the protein or errors caused by PCR amplification.

[0074] As used herein, the term "sequence identity" or "identity" refers to the number (%) of position matches (same nucleic acid residues) obtained from the comparison of two polynucleotide sequences. Sequence identity is determined by comparing the sequences when aligning to maximize overlap and identity and simultaneously minimizing sequence gaps. In particular, sequence identity can be determined using any of a variety of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (such as the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) that optimally aligns the sequences over the entire length, while sequences of significantly different lengths are preferably aligned using a local alignment algorithm (such as the Smith and Waterman algorithm (Smith and Waterman, 1981) or the Altschul algorithm (Altschul et al., 1997; Altschul et al., 2005)). Comparison for determining nucleotide sequence identity percentage purpose can be realized in various ways within the art, for example, using the publicly available computer software that can be obtained on the Internet website for example http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Those skilled in the art can determine the suitable parameters for measuring the comparison, be included in any algorithm required for obtaining maximum comparison within the full length of the sequence being compared. For purposes of the present invention, nucleotide sequence identity percentage values ​​refer to the values ​​produced using the paired sequence alignment program EMBOSS Needle, which uses the Needleman-Wunsch algorithm to produce the best global comparison of two sequences, wherein all search parameters are set to default values, i.e., scoring matrix=BLOSUM62, gap opening=10, gap extension=0.5, end gap penalty=error, end gap opening=10, end gap extension=0.5.

[0075] As used herein, the term "variant" refers to a nucleotide sequence that is different from the original sequence but retains its essential properties. In general, a variant is closely similar to the original polynucleotide overall and is identical in many regions. The sequence of a variant may differ in nucleotide substitutions, deletions, or insertions of one or more nucleotides in the sequence that do not impair the promoter activity. A variant may have the same length as the original sequence, or may be shorter or longer.

[0076] The term "functional variant" refers to a variant that exhibits the activity of the corresponding nucleotide sequence, for example: "a functional variant having at least 80% identity with SEQ ID NO. 8" means that the functional variant exhibits the activity of SEQ ID NO. 8.

[0077] The term "expression cassette" refers to a nucleic acid construct comprising a coding sequence and one or more control sequences required for the expression of the coding sequence. In particular, one of these control sequences is a promoter of the present invention. Typically, the expression cassette comprises the coding sequence required for expressing the selected gene product and regulatory sequences before (5' non-coding sequences) and after (3' non-coding sequences) the coding sequence. Therefore, an expression cassette typically comprises a promoter sequence, a coding sequence, and a 3' non-translated region typically containing a polyadenylation site and / or a transcription terminator. The expression cassette may also comprise other regulatory elements, such as enhancer sequences, polylinker sequences and / or splicing signal sequences that facilitate insertion of the DNA fragment in the vector. An expression cassette is typically contained in a vector for ease of cloning and transformation.

[0078] The term "vector" refers to a nucleic acid molecule used as a vehicle to transfer genetic material in vitro or in vivo, particularly to deliver nucleic acids into a host cell. Vectors include, but are not limited to, plasmids, phasmids, cosmids, transposable elements, viruses, and artificial chromosomes (e.g., YACs).

[0079] The term "DIO" stands for "double-floxed inverse orientation." "Double-floxed" refers to the insertion of a specific DNA sequence into the genome. This sequence contains two LoxP sites, which, through the action of the Cre recombinase, can delete or insert specific genes. "Inverse orientation" refers to the orientation of the two LoxP sites, meaning one is upstream or downstream of the other.

[0080] The human parvovirus adeno-associated virus (AAV) is a dependent virus that is naturally defective in replication and can integrate into the genome of infected cells to establish a latent infection. The last property appears to be unique among mammalian viruses because the integration occurs at a specific site in the human genome located on chromosome 19, known as AAVS1 (19q13.3-qter). Therefore, AAV has attracted considerable interest as a potential vector for human gene therapy. The favorable properties of the virus include its lack of association with any human disease, its ability to infect both dividing and non-dividing cells, and the ability to infect a wide range of cell lines derived from different tissues.

[0081] The promoter or expression cassette of the present invention can be introduced into the vector by any method known to the skilled person.

[0082] The expression cassette or vector of the present invention can be transferred into the host cell using any known technique, including but not limited to calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, microinjection, gene gun, lipofection or viral infection, and the expression cassette or vector of the present invention can be maintained in the host cell in an ectopic form or can be integrated into the genome.

[0083] These compositions comprise a therapeutically effective amount of a therapeutic agent (an expression cassette, vector, virion or cell of the present invention) and a pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable" means approved for use in animals and / or humans by a regulatory agency or a recognized pharmacopoeia, such as the European Pharmacopoeia. The term "excipient" refers to a diluent, adjuvant, carrier or vehicle with which the therapeutic agent is administered.

[0084] As is well known in the art, pharmaceutically acceptable excipients are relatively inert substances that facilitate the administration of pharmacologically effective substances and can be provided as liquid solutions or suspensions, emulsions, or solid forms suitable for dissolving or suspending in liquids before use. For example, excipients can provide shape or consistency, or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for changing osmotic pressure, encapsulating agents, pH buffer substances, and buffers. Suitable excipients include any medicament suitable for direct delivery to the eye that can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various Tween compounds, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may be included, such as inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences, 15th edition. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 This is the plasmid map of the target plasmid of Example 1 of the present invention. DETAILED DESCRIPTION

[0086] In order to enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to further illustrate the present invention in detail.

[0087] The reagents used in the present invention can be purchased from the market or prepared according to conventional methods or can be prepared by the method described in the present invention.

[0088] The m-Slc18a2 gene overexpression vector provided by the present invention can be prepared according to the following examples, or according to conventional methods in the art. The sequence of the obtained m-Slc18a2 gene overexpression vector is SEQ ID NO. 1. Example 1: Adeno-associated virus vector construction and transformation

[0089] 1. Vector digestion

[0090] Each reagent was added in the order listed in Table 1, and the mixture was mixed by gentle pipetting. The mixture was placed in a 37°C water bath for 2 h. After the enzyme digestion was completed, agarose gel electrophoresis was performed to recover the vector fragment (about 5 kb).

[0091] Table 1: Vector enzyme digestion system

[0092] Reagents Volume (μL) Vector DNA (1ug / uL) 2 10*buffer 4 <![CDATA[ddH2O]]> 32 SpeI 1 EcoRI 1 total 40

[0093] 2. Obtaining m-Slc18a2 gene fragments and PCR amplification

[0094] (1) Primer design

[0095] The primers shown in Table 2 were designed.

[0096] Table 2: Primer sequences

[0097]

[0098] (2) PCR amplified fragment

[0099] Prepare the system shown in Table 3, mix gently, place in a PCR instrument and perform PCR reaction according to the PCR program shown in Table 4 to obtain the amplified m-Slc18a2 gene fragment.

[0100] Table 3: PCR system

[0101]

[0102] (3) PCR procedure:

[0103] Table 4: PCR program

[0104]

[0105] (4) Ligation reaction between target fragment and vector

[0106] The components shown in Table 5 were mixed and reacted in a water bath at 50°C for 30 minutes to obtain a ligation product (AAV-DIO-m-slc18a2-zsgreen).

[0107] Table 5: Reaction system

[0108]

[0109] (5) Conversion

[0110] (5-1) After taking the DH5α competent cells out of the -80°C freezer, place them on ice to thaw.

[0111] (5-2) After the competent medium has thawed, aliquot the solution into 50 μL aliquots per tube. Add 5 μL of the ligation product obtained in step (4) and place on ice for 20 min.

[0112] (5-3) Heat shock at 42°C for 90 seconds. Immediately place on ice and incubate for 3 minutes.

[0113] (5-4) In a clean bench, add 500 μl of LB medium (make sure it is antibiotic-free LB medium) and gently invert the tube five times.

[0114] (5-5) Incubate at 37°C, 230 rpm, shaking for 45 min;

[0115] (5-6) Apply the bacterial solution evenly to an Amp-resistant solid plate, then incubate the plate upside down at 37°C for 12 hours.

[0116] (6) Bacteria selection sequencing

[0117] (6-1) Bacterial liquid PCR identification system

[0118] The components were mixed as shown in Table 6 to obtain a bacterial liquid PCR identification system, and then the reaction was carried out according to the bacterial liquid PCR identification procedure shown in Table 7.

[0119] Table 6: Bacterial liquid PCR identification system

[0120] Component name Volume (μL) 2xHieff PCR Master MIX(Dye) 5 F3 primer (SEQ ID NO.9) 0.5 R3 primer (SEQ ID NO.10) 0.5 The bacterial solution obtained in step (5) 2 <![CDATA[ddH2O]]> 2 Total volume 10

[0121] (6-2) Bacterial liquid PCR identification procedure

[0122] Table 7: Bacterial liquid PCR identification procedure

[0123]

[0124] Note: The F3 primer sequence is (SEQ ID NO.9) GGCGCGACCATCTGCGCT, and the R3 primer sequence is (SEQ ID NO.10) CAAAGGCATTAAAGCAGCG

[0125] (6-3) Sequencing

[0126] The bacterial solution obtained after the PCR reaction was sequenced, and the sequencing results were compared with the target sequence (SEQ ID NO.1). The results showed that the sequencing results were consistent with the target sequence, and the target plasmid was successfully constructed.

[0127] Example 2: Virus packaging

[0128] 1. Adeno-associated vectors, packaging cells, and strains

[0129] Virus packaging system: three-plasmid system, pAAV-RC, pHelper, AAV-DIO-m-slc18a2-zsgreen (ligation product obtained in Example 1).

[0130] Packaging cell line: AAV-293 cells, stored long-term by Hanbio Biotechnology Co., Ltd., cultured under the conditions of DMEM+10% FBS (Thermo, 11965118), 37°C, 5% CO2, and 95% relative humidity.

[0131] Bacterial strain: Escherichia coli strain Stbl3, used for amplification of adeno-associated vectors and auxiliary packaging vector plasmids.

[0132] 2. Experimental principles and steps

[0133] Adeno-associated virus shuttle plasmid and its auxiliary packaging vector plasmid were prepared. The three plasmid vectors were extracted with high purity and endotoxin-free, and co-transfected into AAV-293 cells. The culture medium was replaced with complete culture medium 6 hours after transfection. After 72 hours, AAV-293 cells rich in adeno-associated virus particles were collected. After repeated freezing and thawing of the cells, the supernatant containing the virus particles was collected and treated with a universal nuclease and column purification method to obtain high-titer purified adeno-associated virus particles.

[0134] Test Example 1: Titer Detection

[0135] The high titer purified adeno-associated virus particles obtained in Example 2 were detected, and the titer was 1.2×10 12 vg / mL.

[0136] Experimental Example 2: Construction of an animal model overexpressing the m-Slc18a2 gene

[0137] 1. Experimental Materials

[0138] Chat-cre mice (a transgenic mouse model with Cre recombinase inserted into the choline acetyltransferase (Chat) locus).

[0139] 2. Intracardiac targeted injection experimental steps

[0140] (1) Six Chat-cre mice were selected as the experimental group. The left chest was opened between the 3rd and 4th intercostal spaces to expose the heart. A total of 100 μl of virus (1.2×10 12After completion, the chest was closed promptly, and the air in the thoracic cavity was aspirated with a syringe to maintain negative pressure in the thoracic cavity of Chat-cre mice, thereby obtaining mice in the experimental group.

[0141] (2) Take another 6 Chat-cre mice as the control group and replace the virus with 100ul of normal saline in the same steps as step (1) to obtain control group mice.

[0142] (3) The mice in the experimental group and the control group were raised normally for 21 days after injection.

[0143] (4) Fluorescence imaging

[0144] After the experimental group mice and the control group mice were raised normally for 21 days after injection, the hearts were harvested and perfused for in vitro imaging; the steps included 4% PFA fixation, sucrose dehydration, sample embedding and frozen sectioning, and the sections were washed and then subjected to fluorescence imaging.

[0145] Results: In the heart slices of the experimental group mice, the fluorescence was only partially expressed in the intracardiac neurons, but not in the myocardium. In the heart slices of the control group mice, there was no fluorescence signal.

[0146] Conclusion: The m-Slc18a2 gene overexpression vector provided by the present invention can be specifically expressed in cardiac neurons (especially Chat neurons), and can be used to construct an animal model that specifically overexpresses the m-Slc18a2 gene in cardiac neurons (especially Chat neurons), and is used for screening and research of drugs for cardiovascular diseases targeting cardiac neurons or Chat neurons.

[0147] The methods of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art will be able to modify or appropriately alter and combine the methods and applications described herein within the scope, spirit, and spirit of the present invention to implement and apply the technology of the present invention. Those skilled in the art may refer to the disclosure herein and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications apparent to those skilled in the art are considered encompassed by the present invention.

Claims

1. A m-Slc18a2 gene overexpression vector, characterized in that: include: A vector operably linked to the following sequence elements: a promoter sequence, an inverted ZsGreen sequence, an inverted m-Slc18a2 gene sequence and a DIO element sequence.

2. The m-Slc18a2 gene overexpression vector according to claim 1, wherein the DIO element sequence is two sequence fragments; the ZsGreen sequence and the m-Slc18a2 gene sequence are operably connected between the two sequences of the DIO element sequence; and / or There is one LoxP site in each of the two sequence fragments of the DIO element sequence, and the two LoxP sites are in opposite directions; and / or The promoter sequence is an h-Syn promoter sequence; and / or The h-Syn promoter sequence comprises the sequence of SEQ ID NO.4 or a functional variant having at least 80% identity with SEQ ID NO.4; and / or The inverted ZsGreen sequence comprises the sequence of SEQ ID NO.5 or a functional variant having at least 80% identity with SEQ ID NO.5; and / or The inverted m-Slc18a2 gene sequence comprises the sequence of SEQ ID NO.6 or a functional variant having at least 80% identity with SEQ ID NO.6; and / or The DIO element sequence includes: (1) a sequence of SEQ ID NO.7 or a functional variant thereof having at least 80% identity with SEQ ID NO.7, and (2) a sequence of SEQ ID NO.8 or a functional variant thereof having at least 80% identity with SEQ ID NO.8; and / or Optionally, the expression cassette further comprises a terminator sequence; Optionally, the terminator sequence in the expression cassette is the sequence of SEQ ID NO.11 or a functional variant having at least 80% identity with SEQ ID NO.11; Optionally, the terminator sequence in the expression cassette is operably linked to the inverted m-Slc18a2 gene sequence; Optionally, the vector is an adeno-associated virus; and / or Optionally, the adeno-associated virus is an adeno-associated virus of serotype AAV2 / 9-php.s.

3. The m-Slc18a2 gene overexpression vector according to any one of claims 1 to 2, wherein the sequence of the m-Slc18a2 gene overexpression vector comprises a sequence of SEQ ID NO.1 or a functional variant having at least 80% identity with SEQ ID NO.

1.

4. An expression cassette, characterized in that It comprises the promoter sequence, the inverted ZsGreen sequence, the inverted m-Slc18a2 gene sequence and the DIO element sequence in the m-Slc18a2 gene overexpression vector according to any one of claims 1 to 3; wherein the promoter sequence, the inverted ZsGreen sequence and the DIO element sequence are operably connected to the inverted m-Slc18a2 gene sequence; Optionally, the expression cassette further comprises a terminator sequence; Optionally, the terminator sequence in the expression cassette is the sequence of SEQ ID NO.11 or a functional variant having at least 80% identity with SEQ ID NO.11; Optionally, the terminator sequence in the expression cassette is operably linked to the inverted m-Slc18a2 gene sequence.

5. An in vitro method for expressing a gene in Chat neuron cells, characterized in that: The method comprises transfecting a cell line or a cell group with the m-Slc18a2 gene overexpression vector according to any one of claims 1 to 3 or the expression cassette according to claim 4, wherein the cell line or the cell group comprises Chat neuron cells.

6. An isolated cell, characterized in that The isolated cell comprises the m-Slc18a2 gene overexpression vector according to any one of claims 1 to 3 or the expression cassette according to claim 4; Optionally, the isolated cells are Chat neuron cells.

7. A reagent, characterized in that The reagent comprises the m-Slc18a2 gene overexpression vector according to any one of claims 1 to 3 or the expression cassette according to claim 4.

8. Use of the m-Slc18a2 gene overexpression vector according to any one of claims 1 to 3, the expression cassette according to claim 4, the cell according to claim 6 or the reagent according to claim 7 in constructing an animal model overexpressing the m-Slc18a2 gene.

9. A primer pair, characterized in that: The primer pair is SEQ ID NO.2 and SEQ ID NO.

3.

10. A method for constructing the m-Slc18a2 gene overexpression vector according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Vector digestion; (2) Obtaining m-Slc18a2 gene fragments and PCR amplification; (3) connecting the m-Slc18a2 gene fragment to the vector obtained after restriction digestion in step (1); Optionally, the step (1) comprises: mixing the vector, buffer, water and endonuclease, performing an enzyme digestion reaction, and then performing agarose gel electrophoresis to recover the target fragment; Optionally, the step (2) comprises: mixing the primer pair 1 and the primer pair 2 according to claim 9, template DNA, DNA polymerase, 2'-deoxynucleotide-5'-triphosphate mixture and buffer, performing PCR amplification to obtain an amplified m-Slc18a2 gene fragment; Optionally, the step (3) comprises: mixing the target fragment obtained in step (1), the m-Slc18a2 gene fragment amplified in step (2), a ligase and water, and reacting them to obtain an m-Slc18a2 gene overexpression vector; Optionally, the construction method further comprises: (4) virus packaging and purification; Optionally, the virus serotype of the m-Slc18a2 gene overexpression vector obtained after the virus packaging is AAV2 / 9-php.s.

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