New recombinant adeno-associated virus dual-vector system

Abstract

Disclosed in the present invention is a new recombinant adeno-associated virus dual-vector system, which comprises recombinant adeno-associated virus vector rAAV-T and recombinant adeno-associated virus vector rAAV-C, wherein the genome of rAAV-T contains two different recombinase recognition sequences I and II, and further contains an auxiliary functional sequence; and the genome of rAAV-C contains recombinase recognition sequences I' and II', and further contains a target gene sequence. By means of changing the positions of recombinase recognition sites I and II on rAAV-T, rAAV-T and rAAV-C can form a linear or circular expression cassette structure under the action of the recombinase. In the present invention, the target gene and other functional sequences are completely separately positioned on two rAAVs, which facilitates flexible modification and combined use, thereby expanding the loading capacity for the target gene and other functional elements.

Description

A novel recombinant adeno-associated virus dual vector system TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a novel recombinant adeno-associated virus dual vector system. BACKGROUND

[0002] Recombinant adeno-associated virus (rAAV) is a widely used tool virus and one of the most promising viral vectors in the field of gene therapy. By modifying the capsid, genome and developing new drug delivery routes of rAAV, the application of rAAV in different aspects can be enriched, and practical problems can be solved by researchers. The maximum capacity of the commonly used rAAV capsid can package about 4.7 kb of genome. When a single rAAV is used to express a target gene, a balance needs to be made between the length of the target gene and the length of the promoter, enhancer, post-transcriptional regulatory element and reporter gene to match the capacity of the capsid. In order to expand the loading capacity of rAAV, researchers have developed methods such as homologous recombination, mRNA trans-splicing and intein to express target genes using two rAAVs. The above methods are mainly used to express large fragment target genes, and need to be designed according to the target gene. Some methods need to add additional auxiliary sequences, leaving less space for the selection of promoters, enhancers, post-transcriptional regulatory elements and reporter genes. However, as researchers gain a deeper understanding of the structure and function of various tissues and organs, the demand for subdividing cell subtypes and analyzing fine structure and function increases. It is also particularly important to screen more specific functional sequences in a larger length range. Therefore, it is necessary to develop a rAAV expression vector that is more convenient to design and improve, and can simultaneously expand the loading capacity of various auxiliary elements and target genes.

[0003] The existing dual rAAV combination method for expressing target genes utilizes various intracellular and extracellular mechanisms: the homologous recombination method utilizes the cell's own homologous recombination mechanism at the pre-transcription level, resulting in the connection of two rAAV genomes into a long linear DNA expression cassette; the mRNA trans-splicing method utilizes the intron splicing mechanism at the post-transcription level, resulting in the connection of two mRNA precursors into a complete linear mRNA; the intein method utilizes the self-splicing peptide segment at the post-translation level, resulting in the connection of two peptide chains to form a complete protein. Different mechanisms or new auxiliary methods can be utilized at different levels to produce different structures of expression cassettes and expression intermediates. Given that the pre-transcription gene level is the most basic and easy-to-manipulate level, it is worth trying to utilize exogenous proteins to assist in producing different structures of expression cassettes at the pre-transcription DNA level.

[0004] SUMMARY

[0005] In order to solve the problems in the prior art, the application aims to provide a novel recombinant adeno-associated virus double vector system, which constructs a target gene and a regulatory sequence on two recombinant adeno-associated viruses respectively, and combines the two rAAVs into a complete linear or circular expression cassette by a recombinase to express the exogenous gene.

[0006] The specific technical solutions of the application are as follows:

[0007] The application provides a novel recombinant adeno-associated virus double vector system, which comprises a recombinant adeno-associated virus vector rAAV-T and a recombinant adeno-associated virus vector rAAV-C; the genome of the rAAV-T comprises a recombinase recognition sequence I, a recombinase recognition sequence II, a promoter sequence, a regulatory sequence and a reporter gene sequence, and the recombinase recognition sequence I and the recombinase recognition sequence II are different; the genome of the rAAV-C comprises a recombinase recognition sequence I', a recombinase recognition sequence II' and a target gene sequence, and the recombinase recognition sequence I' and the recombinase recognition sequence II' are located upstream and downstream of the target gene sequence; the recombinase recognition sequence I and the recombinase recognition sequence I' are recognition sequences of a recombinase and recombine under the mediation of the recombinase; the recombinase recognition sequence II and the recombinase recognition sequence II' are another pair of recognition sequences of the same recombinase and recombine under the mediation of the recombinase; only the recombinase recognition sequences I and I' and the recombinase recognition sequences II and II' can react with each other, and the reaction is irreversible, and any other combination cannot react; after the recombinase recognition sequences I and I' and the recombinase recognition sequences II and II' react, the functional sequences on the rAAV-T and the target gene on the rAAV-C form a correct expression cassette structure to express the target gene.

[0008] Further, the recombinase is a tyrosine recombinase Cre, and the combination of the recombinase recognition sequences I and I' includes but is not limited to lox66 and lox71, JT15 and JTZ17, lox2272 / 71 and lox66 / 2272, lox5171 / 71 and lox66 / 5171.

[0009] Further, the combination of the recombinase recognition sequences II and II' is selected from one of lox66 and lox71, JT15 and JTZ17, lox2272 / 71 and lox66 / 2272, lox5171 / 71 and lox66 / 5171.

[0010] Further, the promoter sequence is selected from one or more of CAG, CaMKIIa, CAR, CBA, CD68, c-fos, ChAT, CMV, CR, Ef1a, E-SARE, GAD67, GFAP, GFAP104, gfaABC1D, Grm6, hGRK1, hSyn, hUbC, LP1B, L7 / Pcp2, MBP, MCK, mD1X, mOXT, mTH, nEF, Nestin, NPY, Nrl, PGK, PV, RAM, RK, ROH, RPE65, SFRP2, SST, TBG, TCAP, TH, Thy1, TPH2, TRE, TRPV1, UAS, and Vgat.

[0011] Further, the target gene sequence is selected from a nucleotide coding sequence of a protein related to research on structure and function of tissue organs, overexpression of gene products, manipulation of nervous system, gene therapy, and / or a functional RNA product, etc.; the protein is preferably one or more of fluorescent protein, activating neuron protein, inhibiting neuron protein, calcium ion signal probe protein, small molecule signal probe protein, apoptosis mediating protein, disease related mutant protein, normal protein under physiological conditions, cytokine, anti-viral factor, viral infection auxiliary receptor, recombinase, and gene editing tool protein; the functional RNA is preferably one or more of small RNA, small interfering RNA, small hairpin RNA, small guide RNA, organelle localization RNA, and Barcode RNA for RNA sequencing or in situ hybridization analysis.

[0012] Further, the regulatory sequence comprises transcriptional and / or post-transcriptional regulatory sequence.

[0013] Preferably, the regulatory sequence is selected from one or more of WPRE, oPRE, cw3sl, SV40 polyA, hGH polyA, bGH polyA, rbGlob polyA, etc.

[0014] Further, the reporter gene sequence comprises a marker protein, an enzyme reaction color developing protein, and other molecular tag sequence for indication.

[0015] Further, the recombinant adeno-associated virus vector rAAV-T and the recombinant adeno-associated virus vector rAAV-C have the same or different serotypes and the ability to infect the same cells in a tissue or organ.

[0016] Further, the serotypes include AAV1, AAV2, AAV2-Retro, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9-Retro, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-PHP.eB, AAV-myo, and their derivative serotypes.

[0017] Further, the rAAV-T genome further comprises other helper function sequences

[0018] Further, the functional sequences on the rAAV-T form a correct expression cassette structure with the gene of interest on the rAAV-C, which is a linear expression cassette structure or a circular expression cassette structure.

[0019] Preferably, the positions of the recombinase recognition sites I and II on the rAAV-T are changed by the following way (1) or (2) to achieve different expression cassette structures formed by the rAAV-T and the rAAV-C under the action of the recombinase:

[0020] (1) the rAAV-T and the rAAV-C form a linear expression cassette structure under the action of the recombinase

[0021] The order of the elements in the designed rAAV-T genome comprises: ITR-promoter-reporter gene-recombinase recognition sequence I-recombinase recognition sequence II-regulatory sequence-ITR.

[0022] The order of the elements in the designed rAAV-C genome comprises: ITR-recombinase recognition sequence I'-gene of interest-recombinase recognition sequence II'-ITR.

[0023] The recombinase can mediate the recombination of I and I', II and II', and the sequence directions of I and I', II and II' satisfy that a linear expression cassette structure can be formed after recombination: (upper ITR)-promoter-reporter gene-gene of interest-regulatory sequence-(lower ITR).

[0024] (2) the rAAV-T and the rAAV-C form a circular expression cassette structure under the action of the recombinase

[0025] The order of the elements in the designed rAAV-T genome comprises: ITR-recombinase recognition sequence II-regulatory sequence-promoter-reporter gene-recombinase recognition sequence I-ITR.

[0026] The order of the elements in the designed rAAV-C genome comprises: ITR-recombinase recognition sequence I'-gene of interest-recombinase recognition sequence II'-ITR.

[0027] The recombinase can mediate the recombination of I and I', II and II', and the sequence direction of I and I', II and II' satisfies that the expression cassette structure of a ring can be formed after recombination: (upper junction regulatory sequence)-promoter-reporter gene-target gene-regulatory sequence-(lower junction promoter).

[0028] The application also provides a plasmid for packaging the novel recombinant adeno-associated virus double vector system, characterized in that the plasmid comprises a plasmid T for packaging a recombinant adeno-associated virus vector rAAV-T and a plasmid C for packaging a recombinant adeno-associated virus vector rAAV-C; the plasmid T comprises a recombinase recognition sequence I, a recombinase recognition sequence II, a promoter sequence, a regulatory sequence and a reporter gene sequence, and the recombinase recognition sequence I and the recombinase recognition sequence II are different; the plasmid C comprises a recombinase recognition sequence I', a recombinase recognition sequence II' and a target gene sequence, and the recombinase recognition sequence I' and the recombinase recognition sequence II' are located upstream and downstream of the target gene sequence respectively; the recombinase recognition sequence I and the recombinase recognition sequence I' are recognition sequences of a recombinase, and recombination occurs under the mediation of the recombinase; the recombinase recognition sequence II and the recombinase recognition sequence II' are another pair of recognition sequences of the same recombinase, and recombination occurs under the mediation of the recombinase; the recombinase recognition sequences I and I' and II and II' can only react with each other, and the reaction is irreversible, and any other combination cannot react; after the recombinase recognition sequences I and I' and the recombinase recognition sequences II and II' react, the functional sequences on the rAAV-T and the target gene on the rAAV-C form a correct expression cassette structure to express the target gene.

[0029] Further, in the plasmid, the recombinase corresponding to the recombinase recognition sequences is a tyrosine recombinase Cre, and the combination of the recombinase recognition sequence I and I' is selected from one of lox66 and lox71, JT15 and JTZ17, lox2272 / 71 and lox66 / 2272, and lox5171 / 71 and lox66 / 5171.

[0030] The combination of the recombinase recognition sequence II and II' is selected from one of lox66 and lox71, JT15 and JTZ17, lox2272 / 71 and lox66 / 2272, and lox5171 / 71 and lox66 / 5171.

[0031] Further, in the plasmid, the plasmid T for packaging the recombinant adeno-associated virus vector rAAV-T comprises elements in the order of: ITR-promoter-reporter gene-recombinase recognition sequence I-recombinase recognition sequence II-regulatory sequence-ITR; and the plasmid C for packaging the recombinant adeno-associated virus vector rAAV-C comprises elements in the order of: ITR-recombinase recognition sequence I'-target gene-recombinase recognition sequence II'-ITR.

[0032] Alternatively, in the plasmid, the plasmid T for packaging the recombinant adeno-associated virus vector rAAV-T comprises elements in the order of: ITR-recombinase recognition sequence II-regulatory sequence-promoter-reporter gene-recombinase recognition sequence I-ITR; and the plasmid C for packaging the recombinant adeno-associated virus vector rAAV-C comprises elements in the order of: ITR-recombinase recognition sequence I'-target gene-recombinase recognition sequence II'-ITR.

[0033] The present application also provides a method for expressing a target gene in a specific cell or animal tissue or organ using the novel recombinant adeno-associated virus dual vector system, which comprises: simultaneously injecting rAAV-T and rAAV-C into the specific cell or animal tissue or organ, and introducing the recombinase into the cell or animal through a transgene or a vector, so that the rAAV-T and the rAAV-C form a complete linear or circular expression cassette under the mediation of the recombinase to express the target gene.

[0034] Further, the recombinase is introduced into the cell or animal through a vector selected from one or more of an expression plasmid carrying a recombinase gene, a viral vector carrying a recombinase gene, and other chemical or biological vectors carrying a recombinase gene or protein.

[0035] Further, the tissue is selected from a neural tissue, a muscle, an epithelial tissue, or a connective tissue.

[0036] Preferably, the neural tissue is selected from a central nervous system and a tissue related to a neural circuit thereof.

[0037] Further, the organ is selected from a urinary bladder, an eye, an ear, a nose, a mouth, a tongue, a pharynx, a larynx, an organum vomeronasale, a salivary gland, a liver, a kidney, a spleen, a heart, an intestinal tract, a stomach, a pancreas, a lung, a trachea, a blood vessel, a lymph vessel, a lymph node, a limb, a pituitary, a thyroid, a parathyroid, an islet of Langerhans, an adrenal gland, or a genital organ.

[0038] In the present application, the rAAV-T and the rAAV-C can be used in physiological mechanism research, disease modeling, cell regulation, or neural circuit labeling in a conventional application scenario. Special application scenarios of the present application include long promoter screening, enhancer screening, post-transcriptional regulatory sequence screening, circular DNA expression cassette delivery, and special circuit labeling.

[0039] The present application provides a method for constructing a target gene and a regulatory sequence on two recombinant adeno-associated viruses respectively, and forming a complete expression cassette with different structures by recombinase to express the exogenous gene. Unlike the existing double rAAV expression method, the two rAAVs in the present application only have the target gene or the regulatory sequence, and the auxiliary functional sequence for reporting the expression of the target gene, and further contain two pairs of recombinase recognition sites for mediating the recombination between the two rAAVs. When the recombinase exists, the two rAAVs can be recombined into a complete expression cassette, and the expression cassette can form a linear or circular structure. Compared with the existing method, the present application has the following advantages:

[0040] 1. The present application completely separates the target gene and other functional sequences on two rAAVs, which can be conveniently and flexibly modified and combined, and further increases the length range of selectable genes and functional sequences, and expands the loading capacity of target genes and other functional elements.

[0041] 2. The rAAVs in the present application can be combined to produce different DNA expression cassette structures, which can be used to explore and compare the expression differences of linear and circular DNA structures.

[0042] 3. The present application uses exogenous enzyme-mediated recombination, which has higher efficiency than the intracellular mechanism, and is suitable for researches requiring rapid expression of target genes such as disease modeling, loop labeling, gene therapy, etc., and can shorten the experimental period. BRIEF DESCRIPTION OF DRAWINGS

[0043] FIG. 1 is a plasmid map for packaging recombinant adeno-associated virus AAV-T1.

[0044] FIG. 2 is a plasmid map for packaging recombinant adeno-associated virus rAAV-T2.

[0045] FIG. 3 is a plasmid map for packaging recombinant adeno-associated virus rAAV-C.

[0046] FIG. 4 is a plasmid construction method for packaging recombinant adeno-associated viruses AAV-T1 and rAAV-T2.

[0047] FIG. 5 is a plasmid construction method for packaging recombinant adeno-associated virus rAAV-C.

[0048] FIG. 6 is a model diagram and result display diagram of mixing rAAV-T1 with rAAV-C, rAAV-T2 with rAAV-C, and rAAV-Cre expressing Cre enzyme, and injecting into the LH brain area of C57BL / 6 strain mice. Scale bars: 250 μm. DETAILED DESCRIPTION

[0049] For a more complete understanding of the present application, reference is now made to the following examples and drawings in which: the examples are for purposes of illustration only and are not intended to limit the present application in any way. In the examples, each of the original reagent materials is commercially available, and the experimental methods, if not specified, are routine methods and routine conditions known in the art, or are according to the conditions recommended by the instrument manufacturer.

[0050] Example 1

[0051] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the drawings, but it should not be understood as limiting the scope of the present application. The technical solutions described in the present application are conventional technologies in the field of rAAV vectors, unless otherwise specified.

[0052] Example 1: Design and preparation of recombinant adeno-associated virus rAAV-T and rAAV-C vectors

[0053] Construction of plasmids T1 and T2 and C for packaging recombinant adeno-associated virus rAAV-T1, rAAV-T2 and rAAV-C:

[0054] T1: pAAV-CAG-NLS-eGFP-JT15-lox2272 / 71-WPRE-polyA, the nucleotide sequence is shown as SEQ ID NO. 1, the plasmid map is shown as FIG. 1, the promoter is CAG, the reporter gene is NLS-eGFP, the regulatory sequence is WPRE-polyA, and the recombinase recognition sites I and II are JT15 and lox2272 / 71, respectively.

[0055] T2: pAAV-lox2272 / 71-WPRE-polyA-CAG-NLS-eGFP-JT15, the nucleotide sequence is shown as SEQ ID NO. 2, the plasmid map is shown as FIG. 2, the promoter is CAG, the reporter gene is NLS-eGFP, the regulatory sequence is WPRE-polyA, and the recombinase recognition sites I and II are JT15 and lox2272 / 71, respectively;

[0056] C: pAAV-JTZ17-H2B-tdTomato-lox66 / 2272, the nucleotide sequence is shown as SEQ ID NO. 3, the plasmid map is shown as FIG. 3, the target gene is H2B-tdTomato, and the recombinase recognition sites I' and II' are JTZ17 and lox66 / 2272, respectively;

[0057] The construction process of each plasmid in the present application is shown in FIGS. 4-5.

[0058] Recombinant adeno-associated virus rAAV-T1, rAAV-T2 and rAAV-C were prepared using a three-plasmid packaging system. T1, T2 and C core plasmids were co-transfected into HEK-293T cells with helper plasmid pAd-Helper and AAV capsid plasmid pAAV-RC2 / 9 at the same number of plasmids. The cell culture was collected 72 hours after transfection, and concentrated and purified by iodixanol density gradient centrifugation. Finally, the rAAV titer was detected by SYBR Green qPCR method, and finally three kinds of rAAV were obtained:

[0059] rAAV-T1: rAAV-CAG-NLS-eGFP-JT15-lox2272 / 71-WPRE-polyA, titer 2.8 x 10 13 VG / mL.

[0060] rAAV-T2: rAAV-lox2272 / 71-WPRE-polyA-CAG-NLS-eGFP-JT15, titer 1 x 10 13 VG / mL.

[0061] rAAV-C: rAAV-JTZ17-H2B-tdTomato-lox66 / 2272, titer 3.2 x 10 13 VG / mL.

[0062] Example 2: Recombinant adeno-associated virus rAAV-T and rAAV-C are used in combination to express a target gene

[0063] Figure 6 is a schematic diagram of the use of recombinant adeno-associated virus rAAV-T1, rAAV-T2 and rAAV-C in combination. Figure 6A is rAAV-T1 + rAAV-C, and Figure 6C is rAAV-T2 + rAAV-C.

[0064] For each group of viruses, two rAAVs were mixed with rAAV-Cre (purchased from Shenzhen Blincas Biotechnology Co., Ltd.) expressing Cre enzyme at a ratio of 10:10:1 (total titer 2.15 x 10 13 VG / mL) according to the number of virions, and 200 nL of virus mixed solution was injected into the LH brain area of wild type C57 / BL6 mice by brain stereotactic injection method. rAAV-T can normally express NLS-eGFP fusion protein after entering the cell. rAAV-T and rAAV-C are recombined under the action of Cre, thereby expressing H2B-tdTomato fusion protein.

[0065] After three weeks, the mouse brain was taken by heart perfusion, fixed with paraformaldehyde, dehydrated with sucrose solution, and then cut into 40 μm thick sections using a freezing microtome for imaging.

[0066] The results of the live body detection are shown in Figures 6B and 6D. For the combination of rAAV-T1 and rAAV-C, there is green nuclear-localized eGFP signal expression in the LH brain region, proving that the rAAV-T1 infects cells and expresses in this region, and some of the eGFP-containing cells also contain red nuclear-localized tdTomato signal, proving that the rAAV-C recombines with the rAAV-T1 in the cells and expresses the target gene tdTomato. There are no cells that express tdTomato alone but not eGFP, proving that tdTomato cannot be expressed alone, which is rigorous. The labeling results of the combination of rAAV-T2 and rAAV-C are similar to those of rAAV-T1 and rAAV-C. It is worth noting that the tdTomato signal is slightly stronger when using rAAV-T2, which may be due to the structure of rAAV-T2 being more conducive to the recombination of rAAV-C, or the circular expression cassette formed by recombination being more stable.

[0067] The above results show that the rAAV-T and rAAV-C as examples can undergo recombination reactions under the action of the recombinase and express the target gene, and have the characteristics of high efficiency and expression rigor. Therefore, using two pairs of loxP recombinant rAAV-T and rAAV-C as expression cassettes of different structures and expressing the target gene is a feasible strategy.

[0068] SEQ ID NO. 1

[0069] SEQ ID NO. 2

[0070] SEQ ID NO. 3

[0071] It is apparent that the above embodiments are merely examples for the purpose of clarity, and are not limiting of the embodiments. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. Here, it is not necessary or possible to exhaust all embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A novel recombinant adeno-associated virus dual vector system, characterized in that, The double vector system comprises a recombinant adeno-associated virus vector rAAV-T and a recombinant adeno-associated virus vector rAAV-C; the rAAV-T genome comprises a recombinase recognition sequence I, a recombinase recognition sequence II, a promoter sequence, a regulatory sequence and a reporter gene sequence, the recombinase recognition sequence I and the recombinase recognition sequence II are different; the rAAV-C genome comprises a recombinase recognition sequence I', a recombinase recognition sequence II' and a target gene sequence, the recombinase recognition sequence I' and the recombinase recognition sequence II' are located upstream and downstream of the target gene sequence, respectively. The recombinase recognition sequence I and the recombinase recognition sequence I' are recognition sequences of a recombinase, and recombination occurs under the mediation of the recombinase; the recombinase recognition sequence II and the recombinase recognition sequence II' are another pair of recognition sequences of the same recombinase, and recombination occurs under the mediation of the recombinase; the recombinase recognition sequences I and I' and II and II' can only react with each other, and the reaction is irreversible, and any other combination cannot react; after the recombinase recognition sequences I and I' and II and II' react, the functional sequence on the rAAV-T and the target gene on the rAAV-C form a correct expression cassette structure to express the target gene.

2. The dual vector system of claim 1, wherein, The recombinase corresponding to the recombinase recognition sequence is a tyrosine recombinase Cre, and the combination of the recombinase recognition sequence I and I' is selected from one of lox66 and lox71, JT15 and JTZ17, lox2272 / 71 and lox66 / 2272, and lox5171 / 71 and lox66 / 5171. The combination of the recombinase recognition sequence II and II' is selected from one of lox66 and lox71, JT15 and JTZ17, lox2272 / 71 and lox66 / 2272, and lox5171 / 71 and lox66 / 5171.

3. The dual vector system of claim 1, wherein, The correct expression cassette structure formed by the functional sequence on the rAAV-T and the target gene on the rAAV-C is a linear expression cassette structure or a circular expression cassette structure.

4. The dual vector system of claim 3, wherein, The positions of the recombinase recognition sites I and II on the rAAV-T are changed by the following methods (1) or (2) to realize that the rAAV-T and the rAAV-C form different expression cassette structures under the action of the recombinase: (1) The rAAV-T and the rAAV-C form a linear expression cassette structure under the action of the recombinase The order of the elements in the designed rAAV-T genome comprises: ITR-promoter-reporter gene-recombinase recognition sequence I-recombinase recognition sequence II-regulatory sequence-ITR; The order of the elements in the designed rAAV-C genome comprises: ITR-recombinase recognition sequence I'-target gene-recombinase recognition sequence II'-ITR; The recombinase can mediate the recombination of I and I', II and II', and the sequence directions of I and I', II and II' satisfy that a linear expression cassette structure: promoter-reporter gene-target gene-regulatory sequence is formed after recombination; (2) The rAAV-T and the rAAV-C form a circular expression cassette structure under the action of the recombinase The order of elements in the rAAV-T genome is: ITR-recombinase recognition sequence II-regulatory sequence-promoter-reporter gene-recombinase recognition sequence I-ITR; The order of elements in the rAAV-C genome is: ITR-recombinase recognition sequence I'-target gene-recombinase recognition sequence II'-ITR; The recombinase can mediate the recombination of I and I', II and II', and the sequence directions of I and I', II and II' satisfy the formation of a circular expression cassette structure after recombination: (upper regulatory sequence)-promoter-reporter gene-target gene-regulatory sequence-(lower promoter).

5. The dual vector system of claim 1, wherein, The promoter sequence is selected from one or more of CAG, CaMKIIa, CAR, CBA, CD68, c-fos, ChAT, CMV, CR, Ef1a, E-SARE, GAD67, GFAP, GFAP104, gfaABC1D, Grm6, hGRK1, hSyn, hUbC, LP1B, L7 / Pcp2, MBP, MCK, mD1X, mOXT, mTH, nEF, Nestin, NPY, Nrl, PGK, PV, RAM, RK, ROH, RPE65, SFRP2, SST, TBG, TCAP, TH, Thy1, TPH2, TRE, TRPV1, UAS, and Vgat.

6. The dual vector system of claim 1, wherein, The regulatory sequence comprises a transcriptional and / or post-transcriptional regulatory sequence.

7. The dual vector system of claim 1, wherein, The reporter gene sequence is selected from one or more of a marker protein, an enzyme reaction color developing protein, and other molecular tag sequences for indication.

8. The dual vector system of claim 1, wherein, The rAAV-T genome further comprises other auxiliary functional sequences.

9. The dual vector system of claim 1, wherein, The recombinant adeno-associated viral vector rAAV-T and the recombinant adeno-associated viral vector rAAV-C have the same or different serotypes and the ability to infect the same cells in a tissue or organ.

10. The dual vector system of claim 9, wherein, The serotypes include AAV1, AAV2, AAV2-Retro, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9-Retro, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV-PHP.eB, AAV-myo, and their derived serotypes.

11. A plasmid for packaging the novel recombinant adeno-associated virus dual vector system of claim 1, characterized in that, The plasmids include a plasmid T for packaging the recombinant adeno-associated viral vector rAAV-T and a plasmid C for packaging the recombinant adeno-associated viral vector rAAV-C; the plasmid T comprises a recombinase recognition sequence I, a recombinase recognition sequence II, a promoter sequence, a regulatory sequence, and a reporter gene sequence, the recombinase recognition sequence I and the recombinase recognition sequence II are not the same; the plasmid C comprises a recombinase recognition sequence I', a recombinase recognition sequence II', and a target gene sequence, the recombinase recognition sequence I' and the recombinase recognition sequence II' are respectively located upstream and downstream of the target gene sequence; The recombinase recognition sequence I and the recombinase recognition sequence I' are recognition sequences of a recombinase, and recombination occurs under the mediation of the recombinase; the recombinase recognition sequence II and the recombinase recognition sequence II' are another pair of recognition sequences of the same recombinase, and recombination occurs under the mediation of the recombinase; the recombinase recognition sequences I and I' and II and II' can react with each other, and the reaction is irreversible, and any other combination cannot react; after the recombinase recognition sequences I and I' and II and II' react, the functional sequence on the rAAV-T and the target gene on the rAAV-C form a correct expression cassette structure to express the target gene.

12. The plasmid of claim 11, wherein, The recombinase corresponding to the recombinase recognition sequence is a tyrosine recombinase Cre, and the combination of the recombinase recognition sequence I and I' is selected from one of lox66 and lox71, JT15 and JTZ17, lox2272 / 71 and lox66 / 2272, and lox5171 / 71 and lox66 / 5171. The combination of the recombinase recognition sequence II and II' is selected from one of lox66 and lox71, JT15 and JTZ17, lox2272 / 71 and lox66 / 2272, and lox5171 / 71 and lox66 / 5171.

13. The plasmid of claim 11, wherein, The plasmid T for packaging the recombinant adeno-associated virus vector rAAV-T comprises the sequence of elements: ITR-promoter-reporter gene-recombinase recognition sequence I-recombinase recognition sequence II-regulatory sequence-ITR; and the plasmid C for packaging the recombinant adeno-associated virus vector rAAV-C comprises the sequence of elements: ITR-recombinase recognition sequence I'-target gene-recombinase recognition sequence II'-ITR. Alternatively, the plasmid T for packaging the recombinant adeno-associated virus vector rAAV-T comprises the sequence of elements: ITR-recombinase recognition sequence II-regulatory sequence-promoter-reporter gene-recombinase recognition sequence I-ITR; and the plasmid C for packaging the recombinant adeno-associated virus vector rAAV-C comprises the sequence of elements: ITR-recombinase recognition sequence I'-target gene-recombinase recognition sequence II'-ITR.

14. The method of expressing a gene of interest in a specific cell or animal tissue organ using the novel recombinant adeno-associated virus dual vector system of claim 1, characterized in that, The method comprises: simultaneously injecting rAAV-T and rAAV-C in a specific cell or animal tissue organ, and introducing the recombinase into the cell or animal through a transgene or a vector, so that the rAAV-T and the rAAV-C form a complete expression cassette under the mediation of the recombinase to express the target gene.

15. The method of claim 14, wherein, The recombinase is introduced into the cell or animal through a vector selected from one or more of an expression plasmid carrying a recombinase gene, a viral vector carrying a recombinase gene, and other chemical or biological vectors for introducing a recombinase gene or protein.

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