CN112695032B - Promoter pLRRK2 and application thereof - Google Patents

Promoter pLRRK2 and application thereof Download PDF

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CN112695032B
CN112695032B CN202011434481.9A CN202011434481A CN112695032B CN 112695032 B CN112695032 B CN 112695032B CN 202011434481 A CN202011434481 A CN 202011434481A CN 112695032 B CN112695032 B CN 112695032B
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plrrk2
recombinant adeno
associated virus
paav
promoter
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CN112695032A (en
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林剑邦
陈晔菲
路中华
杨立新
朱钰媛
徐冬冬
李梦奇
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Shenzhen Institute of Advanced Technology of CAS
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    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
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    • C12N2800/00—Nucleic acids vectors
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Abstract

The invention discloses a promoter pLRRK2 and application thereof, and the nucleotide sequence of the promoter pLRRK2 is shown as SEQ ID NO: 1. The promoter pLRRK2 can be applied to recombinant adeno-associated virus, so that the exogenous gene can be efficiently expressed in a new cortex, and has wide application prospects in the fields of nerve loop tracing and gene therapy.

Description

Promoter pLRRK2 and application thereof
Technical Field
The invention belongs to the technical field of nerve engineering, and particularly relates to a promoter pLRRK2 and application thereof.
Background
Neocortex is a part of the mammalian central nervous system that is involved in a variety of important functions including sensation, perception, voluntary movement, learning, cognition, and the like. Analysis of the neural loops connecting the neocortex with each nucleus can help us to better understand the advanced functions of the brain. Research on new cortex can also help us understand the pathogenesis of cortex-related neurological diseases, and find a treatment method thereof. However, there is currently no viral tool for efficient labeling of new cortex, which hampers research on new cortex.
Adeno-associated virus is a non-enveloped single-stranded DNA virus. Recombinant adeno-associated virus (rAAV) is a recombinant virus engineered on the basis of wild-type AAV. The recombinant adeno-associated virus has the advantages of long-term stable expression of the mediated gene in a mammal body, low immunogenicity, wide host range and the like, thus being a widely used virus tool in the field of neuroscience and being the most widely applied gene therapy vector tool in the clinical experiments of central nervous system diseases at present.
In the application of rAAV in neuroscience research and clinical treatment, how to improve the spatiotemporal specificity and expression level of gene expression is the key to gene delivery. Strategies to regulate exogenous gene expression directly affect the efficiency and safety of gene delivery. For neuroscience research, different brain regions or nuclei are labeled if labeling specific neurons in a particular neural circuit is to be achieved. For gene therapy of neurological diseases, the manipulation of neurons of the whole brain or of different subtypes is required in order to intervene in different neurological diseases. One important approach is to select rAAV viruses of different serotypes. Among the currently available AAV serotypes that are 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. Viruses of different serotypes have different infection efficiencies and diffusion capacities for different brain regions, so that different rAAV serotypes can be selected as gene delivery vectors according to study subjects or treatment targets. However, due to the complexity of the central nervous system, these serotypes far from meet the current demand for gene delivery specificity. Another approach is to use tissue specific promoters in the rAAV genome. Promoters are cis-acting elements necessary for the regulation of gene expression. Under the control of a promoter with expression specificity, the foreign gene is generally expressed only in certain specific cell types or brain regions. The specific promoter is used for driving gene expression in rAAV, so that the efficiency of gene expression can be improved, and the immune response generated in the rAAV infection process can be reduced.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a promoter pLRRK2 for high-efficiency expression of a new cortex and application thereof. The promoter pLRRK2 can be suitable for recombinant adeno-associated virus, so that the exogenous gene can be efficiently expressed in a new cortex, and has wide application prospects in the fields of nerve loop tracing and gene therapy.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: in one aspect, the invention provides a promoter pLRRK2, the nucleotide sequence of the promoter pLRRK2 is shown in SEQ ID NO: 1.
Further, the promoter pLRRK2 is a partial sequence of a human leucine-rich repeat kinase 2 (LRRK 2) gene, wherein the sequence selects 2201bp sequence near the transcription initiation site of the LRRK2 gene, and ATG sequence contained in the sequence is replaced by TTG sequence to be used as a final promoter sequence.
Further, the promoter pLRRK2 is highly expressed in the neocortex.
In another aspect, the invention provides a recombinant adeno-associated viral vector comprising the promoter described above.
Further, the recombinant adeno-associated viral vector comprises a Paav-pLRRK 2-fluorescent protein, a Paav-pLRRK 2-functional protein, or a Paav-pLRRK 2-therapeutic protein.
Further, the fluorescent protein comprises eYFP, tdTomato;
preferably, the functional protein comprises a optogenetic related protein or a chemical genetic related protein.
The Paav-pLRRK 2-fluorescent protein (fluorescent proteins include eYFP, tdTomato) can enable multi-color tracing of the nerve loop. Paav-pLRRK 2-functional proteins, including optogenetically related proteins or chemically genetically related proteins, can produce recombinant adeno-associated viruses that activate or inhibit specific neurons. The Paav-pLRRK 2-therapeutic protein can be used in gene therapy.
Further, the recombinant adeno-associated virus vector Paav-pLRRK2-eYFP adopts Paav-hSyn-eYFP as a skeleton vector, and replaces hSyn with pLRRK2 promoter in the vector to obtain the recombinant adeno-associated virus vector Paav-pLRRK2-eYFP.
Further, the preparation method of the recombinant adeno-associated virus vector Paav-pLRRK2-eYFP comprises the following steps: cloning the promoter pLRRK2 into a PUC-57 vector to obtain a PUC57-pLRRK2 vector; treating the Paav-hSyn-eYFP vector with restriction enzymes XbalI-HF and kpnI-HF, while treating the PUC57-pLRRK2 vector with restriction enzymes XbalI-HF and kpnI-HF; and (3) recovering, purifying and connecting the two enzyme digestion products to obtain the recombinant adeno-associated virus vector Paav-pLRRK2-eYFP.
In another aspect, the invention provides a recombinant adeno-associated virus comprising a recombinant adeno-associated virus vector as described in any one of the preceding.
Further, the recombinant adeno-associated virus comprises AAVDJ-retro-pLRRK 2-fluorescent protein, AAVDJ-retro-pLRRK 2-functional protein or AAVDJ-retro-pLRRK 2-therapeutic protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the functional protein comprises a optogenetic related protein or a chemical genetic related protein.
Recombinant adeno-associated viruses AAVDJ-retro-pLRRK2-eYFP, AAVDJ-retro-pLRRK2-tdTomato can effectively reverse the nerve loop of synaptically labeled substantia nigra reticula to cortex and express high fluorescence intensity eYFP or tdTomato.
In yet another aspect, the invention provides a kit comprising the promoter pLRRK2 described above, the recombinant adeno-associated vector described above, or the recombinant adeno-associated virus described above.
In a further aspect, the invention provides the use of the promoter plrk 2, any of the recombinant adeno-associated vectors, any of the recombinant adeno-associated viruses or the kit for the preparation of a neural loop tracer and/or nerve cell manipulation and/or gene therapy agent.
In yet another aspect, the present invention provides a neural network tracer comprising a Paav-pLRRK 2-fluorescent protein and/or a recombinant adeno-associated virus comprising a Paav-pLRRK 2-fluorescent protein;
preferably, the fluorescent protein comprises eYFP, tdTomato;
preferably, the recombinant adeno-associated virus comprises AAVDJ-retro-pLRRK2-eYFP, AAVDJ-retro-pLRRK2-tdTomato.
In yet another aspect, the present invention provides the use of a neural network tracer as described above in a substantia nigra reticula-cortex loop tracer.
Compared with the prior art, the invention has the following beneficial effects:
(1) The promoter pLRRK2 has high expression efficiency in a new cortex, can realize high-efficiency expression of exogenous genes in the new cortex, improves the expression specificity after virus infection, and has wide application prospect in the field of nerve loop tracing and gene therapy;
(2) The promoter pLRRK2 of the invention is non-cytotoxic in bacteria and mammalian cells;
(3) The recombinant adeno-associated virus vector adopts pLRRK2 as a promoter, and has high expression efficiency in a new cortex;
(4) The recombinant adeno-associated virus can efficiently reversely mark the nerve loop from the substantia nigra compact part to the cortex in the brain, and has high marking efficiency and strong infection capability.
Drawings
FIG. 1 is a flow chart of the construction of the Paav-pLRRK2-eYFP vector of the present invention;
FIG. 2 is a graph showing the expression efficiency of AAVDJ-retro-pLRRK2-eYFP of the invention in black reticular regions and cortex.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
EXAMPLE 1 construction of the PUC57-pLRRK2 vector
The pLRRK2 promoter is a partial sequence of a human leucine-rich repeat kinase 2 (LRRK 2) gene, the sequence selects 2201bp sequence near the transcription initiation site of the LRRK2 gene, and the ATG sequence contained in the sequence is replaced by a TTG sequence to be used as a final promoter sequence, and the nucleotide sequence of the promoter pLRRK2 is shown as SEQ ID NO: 1.
And cloning the promoter pLRRK2 on a PUC-57 vector after artificial synthesis to obtain the PUC57-pLRRK2 vector.
EXAMPLE 2 construction of recombinant adeno-associated Virus vector Paav-pLRRK2-eYFP
In this example, since YFP is used as a reporter gene of a promoter, a recombinant gland-related vector Paav-hSyn-eYFP containing the YFP gene is used as a vector backbone to connect pLRRK2 promoter in constructing a recombinant vector, and the steps are as follows:
as shown in FIG. 1, the Paav-hSyn-eYFP vector was treated with restriction enzymes XbalI-HF and kpnI-HF, and the PUC57-pLRRK2 vector was treated with restriction enzymes XbalI-HF and kpnI-HF, and digested at 37℃for 3 hours, the digested systems were as shown in tables 1 and 2, and after recovering the digested products, ligation was performed at 16℃for 30 minutes using a ligation premix (TAKARA), the system was as shown in Table 3, to give a successfully ligated Paav-hSyn-eYFP vector.
TABLE 1 Paav-hSyn-Eyfp vector cleavage System
Reagent(s) Dosage of
Enzyme digestion Buffer 10×CutSmart Buffer 5μL
XbaI-HF 1μL
KpnI-HF 1μL
Paav-hSyn-Eyfp 1μg
ddH2O Supplement to 50 mu L
TABLE 2 cleavage System for PUC57-pLRRK2 vector
Table 3 connection system
Reagent(s) Dosage of
Connecting premix 2 x ligation premix 5μL
Paav-Eyfp connection framework 0.5μL
pLRRK2 linker fragment 4.5μL
Example 3 preparation of recombinant adeno-associated Virus AAVDJretro-pLRRK2-eYFP
The present example uses a three plasmid co-transfection method to prepare the virus. Prior to virus preparation, plasmids required for packaging the virus need to be extracted, including recombinant adeno-associated viral vectors Paav-pLRRK2-eYFP, packaging plasmids AAVDJ-retro-RCB and helper plasmids. The method comprises the following specific steps:
preparation of cells: 293T cells were plated in culture dishes containing whole medium (10% fetal bovine serum, 1% diabody) and cultured in an incubator.
Preparation of transfection reagents: 5.25mL of ultrapure water, 75. Mu.g of the packaging plasmid, 75. Mu.g of the recombinant plasmid, 75. Mu.g of the helper plasmid and 800. Mu.L of the calcium chloride solution were pipetted and gently mixed. To the above reagent, an equal volume of 2×hbs was added, and the mixture was allowed to stand for 30min after vortexing.
Transfected cells: mu.L of chloroquine was added to each dish of cells, and the cells were incubated after the addition of transfection reagent.
And (3) virus collection: after 72h, the transfected cells were collected in a centrifuge tube and centrifuged at 3000rpm for 30min. After discarding the supernatant, the cell lysate is added, followed by cell lysis by repeated freeze thawing. To obtain a virus-containing cell disruption solution.
Purifying virus: the above cell disruption solution was centrifuged at 11500rpm for 30min, the supernatant was discarded, 200. Mu.L of HBS 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 cooled overnight 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 30min. Subsequently, 30. Mu.L of chloroform was added thereto and centrifuged at 12000rpm for 5 minutes. After purification, the mixture was stored in a refrigerator at-80 ℃.
Titer was measured: the purified virus was subjected to titer measurement using AAV titer dye method fluorescent quantitative kit (TAKARA Co.), and the titer was 1.0X10 13 VG/mL。
EXAMPLE 4 recombinant adeno-associated Virus marks the mouse substantia nigra reticula-cortex neural loop
The purified recombinant adeno-associated virus was injected into the bilateral substantia nigra reticula of mice (Bregma-3.40 mm;.+ -. 1.40mm; D:4.70 mm) using stereotactic. The recombinant adeno-associated virus capsid is AAVDJ-retro, has the ability to reverse infect neurons of the cortex upstream of the injected substantia nigra reticula. Three weeks later, mice were perfused to the brain, stained with immunohistochemical sections, and brain slice results were observed. As shown in FIG. 2, the eYFP has high expression efficiency in the new cortex of the mouse.
In summary, the invention is verified on mice, and proves that the promoter pLRRK2 has high-efficiency expression level in new cortex.
The foregoing is merely a specific embodiment of the present invention and not all embodiments, and any equivalent modifications of the technical solution of the present invention that will be obvious to those skilled in the art from reading the present specification are intended to be encompassed by the claims of the present invention.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute of China academy of sciences
<120> a promoter pLRRK2 and use thereof
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<170> PatentIn version 3.3
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ctttggggtt atagacaccc atactcataa gtatttctga ataccaagag aagagtattt 60
aggtttgctt ctctcaactt ttcacctttc atttcatgta ccctgtcctt tgtctcagct 120
ctaatagctc tgagagctga ttacttttcg ggtgtcccaa gtatcaggat cctgcctagt 180
gcaactcaaa tttccaaaag ttaatttagt ggccttttgg tgaccagagc ttcagataac 240
tcacagggaa acaatgttta tttcctctcc cactaacagt cacaaaaaat cataaaaaag 300
agtagcgggg gcagttttga tggctaaccc ctctttccat cctttggggg aaaattgctc 360
atctccctat aggtggaact ctaaagacaa tgtattccta aaaggggcca tctgggcggt 420
gtcctctttt cccagcgccc tgatttctat tcttagatct ggagataggc ggctttcatt 480
tttcctgctc ccagttccca gaccttccgt ggggccgcag gatccccggc tggcgggtcg 540
cggagggtgg ccggccgggc tgcgcactgc gcgcctccgc tgcggggctc cgggcctgtg 600
gactcagcgg agtccgctga gtcagtttct tcccgcgcga ctcccggccg cgccgccgct 660
gcggtggaat ctggtcccag gaggcggcgt ccgcccgggg tccggtctag gcgtgcgtgg 720
gggccacggt cacggtcatc ccagccaggc ccggctccag cagccccacg gccgccgcca 780
gagttctgcg cggcccgtcg cctcggcgga gcctctggca ggcccctgag ctcgtttttg 840
gggcctgagt gggggaggag gaagccgagc aggagggctc cggagaggga gggcaacgcg 900
gggcggggag ctgcctcctt cctcataaac aggcgggcgt gggcgccgat ggggcccgcg 960
gggagcgctg gctgcgggcg gtgagctgag ctcgcccccg gggagctgtg gccggcgccc 1020
ctgccggttc cctgagcagc ggacgttctt gctgggaggg cggcgggttg gaagcaggtg 1080
ccaccttggc tagtggcagc tgtcaggggt gcgaagagga cgaggaaact ctgaagaagt 1140
tgatagtcag gctgaacatt gtccaggaag gaaaacagat agaaacgctg gtccaaatcc 1200
tggaggatct gctggtgttc acgtactccg agcgcggtaa tcacttgaaa ataaactgtg 1260
cttttatttt tgcaaacttt ctccccctcc ttacatttgc aaattttgtc ctcctcccct 1320
tgaccctgct caaacccgga ctcttaagga gccgcaaact cccatatcct ttccttaggg 1380
cagaaagcag ctgagaattt caggaaggtc ttcacctttt tgacttttct ccccgtttca 1440
gactaaaaag gagagggggt gctgtggatt gtgactttgc ttcttttccc cacccacttg 1500
ttttccagcc tccaagttat ttcaaggcaa aaatatcctt gtgcctctgt tgatcgtctt 1560
ggactcctat ttgagagtcg cgagtgtgca gcaggtaaag gcattgtttt cacttcaact 1620
cattctccct tctgtttgga aggagacgtt ttactggcaa tgttaatata gccgagagtt 1680
cttggttatt cccaaaattt ggcttgagga acctctgact gtgattttaa gatgggaata 1740
ttgttaaatc attacgcaat gtaaacggga tgaagagccc cagtatgtgt tccctgagtg 1800
tctttaagaa gtaactttat aaaaccaaca gtatggatgg tggtagaagg aggataaaaa 1860
tgggttcggt tttagtctcg ttattggcaa gatgaattca ttagtgttta gacttgacta 1920
ttccaagtat cttcccaata cagagcatgt cctagatgag aagattatga atagtttgga 1980
aaaggggaat aattaatagt gaagaatacg gcttgctttt gtttctgggt aagcatttta 2040
gggtatcatt ttgttgtata accattgttt acaagtgaga taagcatcta ttccactaag 2100
attgaagaga ttcatgtttg actgagtatg ctctattaac attctttaaa acatgtgaat 2160
atatgtcttt cttgttttca ggtgggttgg tcacttctgt g 2201

Claims (4)

1. An application of a recombinant adeno-associated virus vector or recombinant adeno-associated virus in preparing a reagent for tracing and/or controlling nerve cells in a nerve loop, which is characterized in that the recombinant adeno-associated virus vector is a Paav-pLRRK 2-fluorescent protein, the Paav-pLRRK 2-fluorescent protein takes Paav-hSyn-eYFP as a skeleton vector, and the promoter hSyn is replaced by the promoter pLRRK2;
the nucleotide sequence of the promoter pLRRK2 is shown in SEQ ID NO:1 is shown in the specification;
the fluorescent protein is eYFP or tdTomato;
the recombinant adeno-associated virus is obtained by packaging the Paav-pLRRK 2-fluorescent protein recombinant adeno-associated virus vector;
the nerve loop is a substantia nigra reticula to cortex nerve loop;
the nerve cells are those of the neocortex.
2. The use according to claim 1, wherein the recombinant adeno-associated virus is AAVDJ-retro-pLRRK2-eYFP, which is packaged by a recombinant adeno-associated virus vector Paav-pLRRK2-eYFP, a packaging plasmid AAVDJ-retro-RCB and a helper plasmid.
3. A neural network tracer comprising the Paav-pLRRK 2-fluorescent protein of claim 1 and/or a recombinant adeno-associated virus obtained from the Paav-pLRRK 2-fluorescent protein package of claim 1;
the neural network tracers are used to label the substantia nigra reticula-cortex neural loop.
4. A neural network tracer according to claim 3, wherein the recombinant adeno-associated virus is AAVDJ-retro-pLRRK2-eYFP, which is packaged from recombinant adeno-associated virus vector Paav-pLRRK2-eYFP, packaging plasmid AAVDJ-retro-RCB and helper plasmid.
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