CN115044616A - Adeno-associated virus vector for analyzing brain single-stage output neural network and application thereof - Google Patents
Adeno-associated virus vector for analyzing brain single-stage output neural network and application thereof Download PDFInfo
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Abstract
The invention provides an adeno-associated virus vector for analyzing a brain single-stage output neural network and application thereof, wherein the adeno-associated virus vector comprises a recombinant adenovirus core plasmid; the recombinant adenovirus core plasmid contains at least two promoters, and coding genes of a first marker protein capable of reaching the end of an axon and a second marker protein capable of reaching a cell nucleus, which are driven by different promoters respectively. The recombinant adenovirus core plasmid also encodes a transcription activation protein capable of improving the expression abundance of the first marker protein. The invention utilizes a plurality of promoters in the same recombinant AAV genome to drive a plurality of target genes to express, and analyzes the brain single-stage output neural network more accurately. The invention solves the problems of imprecise crossing single-stage synaptic system with rAAV1 cisoid, reverse marking with H129 and large application range limitation of toxicity, and avoids the defect of marking the crossing fiber by the common rAAV 9.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a virus vector for analyzing a brain central specific nucleus single-stage output network and application thereof.
Background
The neural circuit is the material basis for the brain to perform all functions, about 10 in the human brain 11 One neuron passes through about 10 15 The interweaving of synapses forms a three-dimensional neural network of the brain, and therefore, the analysis of neural circuit connections in the brain is the basis for understanding brain working mechanisms. The tool virus is used as the most efficient and widely applied nerve network tracing means at present, has the advantages of easiness in modification, specific propagation direction, portability of exogenous genes, continuous and stable expression and the like, and is a tool for drawing a brain connection map. With the innovation of molecular biology and gene targeting technology, although the existing neural loop marker tool library is continuously expanded, tool viruses for analyzing a direct output neural network are still in short supply.
Currently, the high titer recombinant adeno-associated virus type 1 (rAAV1) and Thymidine Kinase (TK) gene-deleted herpes simplex virus type 1 strain H129 (H129-delta TK-tdT) is a commonly used cis-trans-single-stage synaptic virus. High titer rAAV1 requires a step of amplification, usually using rAAV1-hSyn-Cre (R) ((R))>1×10 13 VG/mL). In addition, high titer rAAV1-hSyn-Cre invades the axonal terminals of the upper-level neurons in a retrograde fashion, making it less rigorous to use rAAV1 to resolve single-level output networks of specific nuclei in brain centers across a single-level synaptic system.The herpes simplex virus 1H 129 strain has the phenomenon of axonal terminal retrograde absorption and is high in virus toxicity, so that the result marked by H129-delta TK-tdT is not strict enough, and marked cells in a downstream brain region are easy to lesion, and the fluorescence intensity needs to be increased by means of immunohistochemical staining. Therefore, the development of a low-toxicity and strict virus vector for analyzing the brain single-stage output network is of great significance.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide an adeno-associated virus vector for analyzing a brain single-stage output neural network, wherein the virus can accurately analyze a direct output network of a brain specific nuclear neuron. The recombinant adeno-associated virus is used as a vector for researching the structure and the function of a neural network by utilizing the advantages that the recombinant adeno-associated virus has high safety, low immunogenicity and wide host range, can mediate long-term stable expression of genes in animal bodies and the like. The invention provides an adeno-associated virus vector through gene recombination, which simultaneously realizes the positioning of high-abundance fluorescent protein at the axon tail end of a neuron and the positioning of fluorescent protein of a cell nucleus through two groups of promoters, thereby shielding 'passing-through' fibers of the neuron and realizing the accurate marking of a direct output network of the neuron with a specific nucleus in a brain. The vector has great application value and market prospect in the field of neuroscience, particularly in the analysis of the neural output network of the non-human primate.
In order to achieve the above objects, one aspect of the present invention provides an adeno-associated virus vector for analyzing a brain single-stage output neural network, wherein the adeno-associated virus vector comprises a recombinant adenovirus core plasmid;
the recombinant adenovirus core plasmid contains at least two promoters, and coding genes of a first marker protein capable of reaching the end of an axon and a second marker protein capable of reaching a cell nucleus, which are driven by different promoters respectively.
Furthermore, the recombinant adenovirus core plasmid also encodes a transcription activator protein capable of improving the expression abundance of the first marker protein.
Further, the first marker protein is selected from a fusion protein of Synaptophysin and any one of eGFP, BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eFP, eYFP, eBFP, DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mOrange, mPlum, tagRFP, tagBFP and HRP, preferably a Synaptophysin-eGFP fusion protein.
Further, the second marker protein is selected from a fusion protein of sv40nls and any one of mCherry, BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eCFP, eYFP, eBFP, DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mbana, mCitrine, mrorange, mPlum, tagfp, tagBFP or HRP, preferably sv40 nl-mCherry fusion protein.
Further, the promoter of the first marker protein is different from the promoter of the second marker protein, wherein the promoter of the first marker protein is selected from trelight, UAS, LexAop or UAS, and the promoter of the second marker protein is selected from CMV, CAG, Ef1 α, nEf1 α, hSyn, CaMKII α, Vgat, Thy1, TRE, UAS, GFAP or gfaABC 1D.
Further, the transcriptional activator protein is selected from tTA transcriptional activator protein, GAL4, LexA and QF, and preferably the gene of the tTA transcriptional activator protein is shown as SEQ ID NO. 7.
Further, the TRExtight promoter is shown in SEQ ID NO.1, and the CMV promoter is shown in SEQ ID NO. 4.
Furthermore, the sequence of the Synaptophysin-eGFP fusion protein is shown as SEQ ID NO. 2.
Furthermore, the sequence of the sv40nls-mCherry fusion protein is shown in SEQ ID NO. 5.
In another aspect, the present invention provides a recombinant adenovirus core plasmid, which comprises at least two promoters, and genes encoding a first marker protein capable of reaching the axon terminal and a second marker protein capable of reaching the nucleus, which are driven by different promoters, respectively.
Further, the recombinant adenovirus core plasmid also comprises a transcription activator protein capable of improving the expression abundance of the first marker protein.
In another aspect, the present invention provides a recombinant adeno-associated virus vector, wherein the recombinant adeno-associated virus comprises the recombinant adeno-associated virus core plasmid.
In another aspect, the present invention provides the use of the recombinant adenovirus core plasmid or the recombinant adeno-associated virus in neural network markers.
Further, the neural network is labeled as a label for the neural single stage output neural network.
In a further aspect, the present invention provides the use of the recombinant adeno-associated virus as a neuroloop tracer virus.
Further, the neural loop tracing is to trace a neural single-stage output neural network.
Specifically, the construction method for analyzing the adeno-associated virus vector of the brain single-stage output neural network comprises the following steps:
1) construction of recombinant adenovirus core plasmid: the vector is characterized in that a TRETight promoter (shown in SEQ ID NO. 1), a Synaptophysin-eGFP fusion protein (shown in SEQ ID NO. 2), a cw3sl post-transcriptional regulatory element (shown in SEQ ID NO. 3), a CMV promoter (shown in SEQ ID NO. 4), an sv40nls-mCherry fusion protein (shown in SEQ ID NO. 5), a T2A connecting sequence (shown in SEQ ID NO. 6), a tTA gene (shown in SEQ ID NO. 7) and a cw3sl post-transcriptional regulatory element (shown in SEQ ID NO. 3) are sequentially inserted into the same recombinant AAV genome to construct a recombinant adeno-associated virus core plasmid pAAV-TRETight-Synaptophysin-eGFP-cw3sl-CMV-sv40 nls-hercM Cry-T2A-tTA-cw 3 sl.
2) The preparation method of the recombinant adeno-associated virus comprises the following steps: co-transfecting HEK293 cells with helper plasmids pAAV helper, type 5 serum AAV capsid plasmids and core plasmids pAAV-TRETight-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl required for packaging rAAV, respectively collecting supernatant and precipitate, and purifying the virus by using an iodixanol density gradient centrifugation method to obtain the recombinant adeno-associated virus.
3) The recombinant adeno-associated virus application comprises the following steps: the virus drives the expression of Synaptophysin-eGFP fusion protein through a TRExtight promoter, and carries the eGFP fluorescent protein to the end of an axon by utilizing the axon distribution property of the Synaptophysin, thereby achieving the effect of marking the end of the axon of a neuron; the expression of nuclear localization red fluorescent protein sv40nls-mCherry and tTA genes is driven by the promoter CMV, the nucleus of a neuron can be marked to be red by entering the nuclear of the mCherry through sv40nls signal peptide, and the tTA transcriptional activator protein acts on a TRTIght promoter so as to enable the Synaptophysin and the eGFP genes to be expressed in high abundance. Based on the above principle, the recombinant adeno-associated virus can be used to resolve the primary output network of a specific nucleus.
Compared with the prior art, the method has the following advantages:
1. the invention utilizes a plurality of promoters in the same recombinant AAV genome to drive a plurality of target genes to express, and analyzes the brain single-stage output neural network more accurately.
2. The invention solves the problems of imprecise crossing single-stage synaptic system with rAAV1 cisoid, reverse marking with H129 and large application range limitation of toxicity, and avoids the defect of marking the crossing fiber by the common rAAV 9.
3. The invention utilizes a TRE-tTA signal amplification system and a Synaptophysin positioning function to ensure that the eGFP protein is expressed at the tail end of the axon of the neuron in a high abundance manner, and avoids the phenomenon that the signal of the neuron marked by the common rAAV9 is gradually reduced along with the extension of the axon.
4. The invention can also be combined with a nerve cell specific promoter and used for the research of a single-stage output neural network of a specific type of neuron.
5. The invention has wide application value and prospect in the aspects of single-stage output neural network of specific type neurons in specific brain areas of mammals, loop function research and the like.
6. The invention has potential application value in the aspect of multi-gene delivery in gene therapy by using adeno-associated virus.
Drawings
FIG. 1 is a schematic diagram illustrating the principle of single-stage output neural network of brain for analyzing adeno-associated virus vectors and their operation.
FIG. 2 shows the result of infection of the motor cortex with rAAV5-TRETight-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl virus and control virus rAAV9-Ef1 alpha-eGFP-WPRE-pA, in which the cells of M1 in panel A are red-labeled with rAAV5-TRETight-Synaptophysin-eGFP-cw3sl-CMV-sv40 nls-mChery-T2A-tTA-cw 3sl, and the cells of M1 in panel A' are green-labeled with rAAV9-Ef1 alpha-eGFP-WPRE-pA. Panels B and C are partial fiber distribution displays of rAAV 5-trelight-Synaptophysin-eGFP-cw 3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl virus injected at M1, panels B 'and C' are fiber distribution displays of rAAV9-Ef1 α -eGFP-WPRE-pA virus injected at M1 with corresponding brain regions of B and C, a ', B', C ', d', e ', f' are cc, enlarged views of CPu, APT, ZI, cp brain regions at different positions, respectively. Neurons labeled with rAAV5-TRETIGHT-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl are limited to central CPu (c picture) and APT (d picture) with fiber distribution, while classical viral rAAV9-Ef1 alpha-eGFP-WPRE-pA labeled neuron green fluorescence appears not only in central CPu (c picture), APT (d picture) but also in cc (a 'picture), dorsal CPu (b' picture), ZI (e 'picture) and cp (f' picture), and green fluorescence appearing in cc, dorsal CPu, ZI and cp is a fluorescent protein remnant generated by projection of M1 neurons to axon pathways of downstream brain regions, and no projection of M1 neurons to these brain regions exists. The results show that the rAAV5-TRETight-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl can accurately analyze the single-stage output neural network of the brain and solve the defect that the traditional tool marks the bypass fibers.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, specific embodiments thereof are described in detail below, but the present invention is not to be construed as being limited to the implementable range thereof.
The technical scheme of the invention is the conventional technology in the field if not particularly stated.
Example 1: pAAV-TRETIGHT-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl vector construction and preparation of recombinant adeno-associated virus rAAV5-TRETIGHT-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3 sl.
The vector pAAV-TRAntight-Synaptophysin-eGFP-cw 3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl is in AAV vector plasmidA TRETight promoter (shown in SEQ ID NO. 1), a Synaptophysin-eGFP fusion protein gene (shown in SEQ ID NO. 2), a cw3sl post-transcriptional regulatory element (shown in SEQ ID NO. 3), a CMV promoter (shown in SEQ ID NO. 4), an sv40nls-mCherry fusion protein gene (shown in SEQ ID NO. 5), a T2A connecting sequence (shown in SEQ ID NO. 6), a tTA gene (shown in SEQ ID NO. 7) and a cw3sl post-transcriptional regulatory element (shown in SEQ ID NO. 3) are sequentially inserted among ITRs, and the construction of the vector is entrusted to Beijing Optimalaceae Biotechnology Limited. In order to improve rAAV packaging and infection efficiency, AAV5 serotype with larger packaging capacity is selected as a packaging serotype. Adopting a traditional method for packaging adeno-associated virus by three plasmids, carrying out virus packaging on recombinant expression vectors, concentrating and purifying by iodixanol gradient density centrifugation, and finally detecting the titer of the recombinant adeno-associated virus by SYBR Green qPCR (quantitative polymerase chain reaction) method to finally obtain the titer of the rAAV-TRentight-synaptophin-eGFP-cw 3sl-CMV-sv40 nls-mChery-T2A-tTA-cw 3sl virus of 1.14 multiplied by 10 12 vg/mL。
SEQ ID NO.1：315bp
gcgtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgatgtcgagtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgtatgtcgagtttatccctatcagtgatagagaacgtatgtcgagtttactccctatcagtgatagagaacgtatgtcgaggtaggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcc
SEQ ID NO.2：549aa
MDVVNQLVAGGQFRVVKEPLGFVKVLQWVFAIFAFATCGSYTGELRLSVECANKTESALNIEVEFEYPFRLHQVYFDAPSCVKGGTTKIFLVGDYSSSAEFFVTVAVFAFLYSMGALATYIFLQNKYRENNKGPMMDFLATAVFAFMWLVSSSAWAKGLSDVKMATDPENIIKEMPMCRQTGNTCKELRDPVTSGLNTSVVFGFLNLVLWVGNLWFVFKETGWAAPFMRAPPGAPEKQPAPGDAYGDAGYGQGPGGYGPQDSYGPQGGYQPDYGQPASGGGGGYGPQGDYGQQGYGQQGAPTSFSNQMGSIVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK*
SEQ ID NO.3：432bp
gataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttagttcttgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgtttatttgtgaaatttgtgatgctattgctttatttgtaaccatctagctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggagatgtgggaggttttttaaagcgg
SEQ ID NO.4：584bp
gacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagct
SEQ ID NO.5：242aa
PKKKRKVVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK
SEQ ID NO.6：54bp
gagggcagaggaagtctgctaacatgcggtgacgtcgaggagaatcctggccca
SEQ ID NO.7：747bp
atgtctagactggacaagagcaaagtcataaactctgctctggaattactcaatgaagtcggtatcgaaggcctgacgacaaggaaactcgctcaaaagctgggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctggcaatcgagatgctggacaggcatcatacccacttctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcattccgctgtgctctcctctcacatcgcgacggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagctcgcgttcctgtgtcagcaaggcttctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcgtattggaggatcaggagcatcaagtagcaaaagaggaaagagagacacctaccaccgattctatgcccccacttctgagacaagcaattgagctgttcgaccatcagggagccgaacctgccttccttttcggcctggaactaatcatatgtggcctggagaaacagctaaagtgcgaaagcggcgggccggccgacgcccttgacgattttgacttagacatgctcccagccgatgcccttgacgactttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccccgggtaa
Example 2: rAAV-TRETight-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl in vivo test:
300nL of rAAV-TRETIGHT-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl virus and control virus rAAV-Ef1 alpha-eGFP-WPRE-pA are respectively injected into a moving cortex M1 of a 2-month-old C57BL/6 mouse through brain stereotaxic, and the brain is perfused and imaged after three weeks. The results are shown in FIG. 2, in panel A, M1 cells are marked red by rAAV5-TRETIGHT-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl, and in panel A', M1 cells are marked green by rAAV9-ef1 a-eGFP-WPRE-pA. Panels B and C are partial fiber distribution displays of rAAV 5-trelight-Synaptophysin-eGFP-cw 3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl virus injected at M1, panels B 'and C' are fiber distribution displays of rAAV9-Ef1 α -eGFP-WPRE-pA virus injected at M1 with corresponding brain regions of B and C, a ', B', C ', d', e ', f' are cc, enlarged views of CPu, APT, ZI, cp brain regions at different positions, respectively. Neurons labeled with rAAV5-TRETIGHT-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl are limited to central CPu (c picture) and APT (d picture) with fiber distribution, while classical viral rAAV9-Ef1 alpha-eGFP-WPRE-pA labeled neuron green fluorescence appears not only in central CPu (c picture), APT (d picture) but also in cc (a 'picture), dorsal CPu (b' picture), ZI (e 'picture) and cp (f' picture), and green fluorescence appearing in cc, dorsal CPu, ZI and cp is a fluorescent protein remnant generated by projection of M1 neurons to axon pathways of downstream brain regions, and no projection of M1 neurons to these brain regions exists. The results show that the rAAV5-TRETight-Synaptophysin-eGFP-cw3sl-CMV-sv40nls-mCherry-T2A-tTA-cw3sl can accurately analyze the single-stage output neural network of the brain and solve the defect that the traditional tool marks the bypass fibers.
SEQUENCE LISTING
<110> Shenzhen advanced technology research institute of Chinese academy of sciences
<120> adeno-associated virus vector for analyzing brain single-stage output neural network and application thereof
<130> CP122010538C
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 315
<212> DNA
<213> Artificial sequence
<400> 1
gcgtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag 60
agaacgatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct 120
atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg 180
agtttatccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag 240
aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg 300
aaccgtcaga tcgcc 315
<210> 2
<211> 549
<212> PRT
<213> Artificial sequence
<400> 2
Met Asp Val Val Asn Gln Leu Val Ala Gly Gly Gln Phe Arg Val Val
1 5 10 15
Lys Glu Pro Leu Gly Phe Val Lys Val Leu Gln Trp Val Phe Ala Ile
20 25 30
Phe Ala Phe Ala Thr Cys Gly Ser Tyr Thr Gly Glu Leu Arg Leu Ser
35 40 45
Val Glu Cys Ala Asn Lys Thr Glu Ser Ala Leu Asn Ile Glu Val Glu
50 55 60
Phe Glu Tyr Pro Phe Arg Leu His Gln Val Tyr Phe Asp Ala Pro Ser
65 70 75 80
Cys Val Lys Gly Gly Thr Thr Lys Ile Phe Leu Val Gly Asp Tyr Ser
85 90 95
Ser Ser Ala Glu Phe Phe Val Thr Val Ala Val Phe Ala Phe Leu Tyr
100 105 110
Ser Met Gly Ala Leu Ala Thr Tyr Ile Phe Leu Gln Asn Lys Tyr Arg
115 120 125
Glu Asn Asn Lys Gly Pro Met Met Asp Phe Leu Ala Thr Ala Val Phe
130 135 140
Ala Phe Met Trp Leu Val Ser Ser Ser Ala Trp Ala Lys Gly Leu Ser
145 150 155 160
Asp Val Lys Met Ala Thr Asp Pro Glu Asn Ile Ile Lys Glu Met Pro
165 170 175
Met Cys Arg Gln Thr Gly Asn Thr Cys Lys Glu Leu Arg Asp Pro Val
180 185 190
Thr Ser Gly Leu Asn Thr Ser Val Val Phe Gly Phe Leu Asn Leu Val
195 200 205
Leu Trp Val Gly Asn Leu Trp Phe Val Phe Lys Glu Thr Gly Trp Ala
210 215 220
Ala Pro Phe Met Arg Ala Pro Pro Gly Ala Pro Glu Lys Gln Pro Ala
225 230 235 240
Pro Gly Asp Ala Tyr Gly Asp Ala Gly Tyr Gly Gln Gly Pro Gly Gly
245 250 255
Tyr Gly Pro Gln Asp Ser Tyr Gly Pro Gln Gly Gly Tyr Gln Pro Asp
260 265 270
Tyr Gly Gln Pro Ala Ser Gly Gly Gly Gly Gly Tyr Gly Pro Gln Gly
275 280 285
Asp Tyr Gly Gln Gln Gly Tyr Gly Gln Gln Gly Ala Pro Thr Ser Phe
290 295 300
Ser Asn Gln Met Gly Ser Ile Val Ser Lys Gly Glu Glu Leu Phe Thr
305 310 315 320
Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn Gly His
325 330 335
Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys
340 345 350
Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp
355 360 365
Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg
370 375 380
Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro
385 390 395 400
Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn
405 410 415
Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn
420 425 430
Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu
435 440 445
Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met
450 455 460
Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His
465 470 475 480
Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn
485 490 495
Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu
500 505 510
Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His
515 520 525
Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu Gly Met
530 535 540
Asp Glu Leu Tyr Lys
545
<210> 3
<211> 432
<212> DNA
<213> Artificial sequence
<400> 3
gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt 60
gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc 120
cgtatggctt tcattttctc ctccttgtat aaatcctggt tagttcttgc cacggcggaa 180
ctcatcgccg cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat 240
tccgtggtgt ttatttgtga aatttgtgat gctattgctt tatttgtaac catctagctt 300
tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca 360
agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggaga tgtgggaggt 420
tttttaaagc gg 432
<210> 4
<211> 584
<212> DNA
<213> Artificial sequence
<400> 4
gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60
catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120
acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420
ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480
ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 540
tgggcggtag gcgtgtacgg tgggaggtct atataagcag agct 584
<210> 5
<211> 242
<212> PRT
<213> Artificial sequence
<400> 5
Pro Lys Lys Lys Arg Lys Val Val Ser Lys Gly Glu Glu Asp Asn Met
1 5 10 15
Ala Ile Ile Lys Glu Phe Met Arg Phe Lys Val His Met Glu Gly Ser
20 25 30
Val Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro
35 40 45
Tyr Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro
50 55 60
Leu Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser
65 70 75 80
Lys Ala Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu
85 90 95
Ser Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp
100 105 110
Gly Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu
115 120 125
Phe Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly
130 135 140
Pro Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg
145 150 155 160
Met Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu
165 170 175
Lys Leu Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr
180 185 190
Lys Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile
195 200 205
Lys Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln
210 215 220
Tyr Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu
225 230 235 240
Tyr Lys
<210> 6
<211> 54
<212> DNA
<213> Artificial sequence
<400> 6
gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg ccca 54
<210> 7
<211> 747
<212> DNA
<213> Artificial sequence
<400> 7
atgtctagac tggacaagag caaagtcata aactctgctc tggaattact caatgaagtc 60
ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc 120
ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg ccctggcaat cgagatgctg 180
gacaggcatc atacccactt ctgccccctg gaaggcgagt catggcaaga ctttctgcgg 240
aacaacgcca agtcattccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat 300
ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg 360
tgtcagcaag gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt 420
acactgggct gcgtattgga ggatcaggag catcaagtag caaaagagga aagagagaca 480
cctaccaccg attctatgcc cccacttctg agacaagcaa ttgagctgtt cgaccatcag 540
ggagccgaac ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag 600
ctaaagtgcg aaagcggcgg gccggccgac gcccttgacg attttgactt agacatgctc 660
ccagccgatg cccttgacga ctttgacctt gatatgctgc ctgctgacgc tcttgacgat 720
tttgaccttg acatgctccc cgggtaa 747
Claims (10)
1. An adeno-associated virus vector for analyzing a brain single-stage output neural network, which is characterized in that the adeno-associated virus vector comprises a recombinant adenovirus core plasmid;
the recombinant adenovirus core plasmid contains at least two promoters, and coding genes of a first marker protein capable of reaching the end of an axon and a second marker protein capable of reaching a cell nucleus, which are driven by different promoters respectively.
2. The adeno-associated virus vector according to claim 1 wherein the recombinant adenoviral core plasmid further encodes a transcriptional activator protein that increases the abundance of expression of the first marker protein.
3. The adeno-associated viral vector according to claim 1, wherein the first marker protein is selected from the group consisting of fusion proteins of Synaptophysin with eGFP, BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eCFP, eYFP, eBFP, DsRed, dtomat, tdTomato, mCherry, mKate, mApple, mbana, mCitrine, mrorange, mPlum, tagRFP, tagBFP, HRP, preferably a Synaptophysin-eGFP fusion protein.
4. The adeno-associated viral vector according to claim 1, wherein the second marker protein is selected from the group consisting of fusion proteins of sv40nls with any of mCherry, BFP, CFP, GFP, YFP, RFP, iRFP, Cerulean, Venus, eGFP, eCFP, eYFP, eBFP, DsRed, dTomato, tdTomato, mCherry, mKate, mApple, mBanana, mCitrine, mrorange, mPlum, tagRFP, tagBFP or HRP, preferably sv40nls-mCherry fusion proteins.
5. The adeno-associated viral vector according to claim 1 wherein the promoter of the first marker protein is different from the promoter of the second marker protein, wherein the promoter of the first marker protein is selected from trelight, UAS, LexAop or UAS and the promoter of the second marker protein is selected from CMV, CAG, Ef1 α, nEf1 α, hsin, CaMKII α, Vgat, Thy1, TRE, UAS, GFAP or gfaABC 1D.
6. The adeno-associated viral vector according to claim 2, wherein the transcriptional activator protein is selected from the group consisting of tTA transcriptional activator protein, GAL4, LexA, QF, preferably the tTA transcriptional activator protein is represented by SEQ ID No. 7.
7. A recombinant adenovirus core plasmid is characterized in that the recombinant adenovirus core plasmid comprises at least two promoters, and coding genes of a first marker protein which can reach the end of an axon and a second marker protein which can reach the nucleus, which are driven by different promoters respectively;
preferably, the recombinant adeno-associated virus packaging plasmid further comprises a transcription activator protein capable of increasing the expression abundance of the first marker protein.
8. A recombinant adeno-associated virus vector, wherein the recombinant adeno-associated virus comprises the recombinant adeno-associated virus core plasmid.
9. Use of the recombinant adenoviral core plasmid of claim 7 or the adeno-associated viral vector of any one of claims 1-6 for neural network tagging;
preferably, the neural network is labeled as a label for a neural single stage output neural network.
10. Use of the adeno-associated viral vector according to any one of claims 1 to 6 as a neuroloop tracer virus;
preferably, the neural circuit is traced to trace a neural single stage output neural network.
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| CN114107231B (en) * | 2021-12-13 | 2023-08-18 | 重庆大学 | Recombinant adeno-associated virus for realizing whole brain postsynaptic neuron cell body marking and application thereof |
| CN117084934A (en) * | 2023-10-20 | 2023-11-21 | 深圳市维琪科技股份有限公司 | New use of polypeptide |
| CN117084934B (en) * | 2023-10-20 | 2024-01-19 | 深圳市维琪科技股份有限公司 | New use of polypeptide |
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