CN113025655B

CN113025655B - Gene vector

Info

Publication number: CN113025655B
Application number: CN201911349932.6A
Authority: CN
Inventors: 刘武青; 韩芳婷; 吴涛; 蔡丽娥; 唐小莉; 曾立; 卢孔鑫; 龙学
Original assignee: Shanghai Jikai Gene Medical Technology Co ltd
Current assignee: Shanghai Jikai Gene Medical Technology Co ltd
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2023-02-28
Anticipated expiration: 2039-12-24
Also published as: CN113025655A

Abstract

The invention relates to the technical field of biology, in particular to a gene vector. The invention provides a gene vector, which comprises an ITR mutant. The mutated ITR sequences provided by the invention are obviously different in a C-C' palindrome region, and compared with the influence of wild type and mutated AAV2-ITR (AAV 2-ITR-Mut) on the packaging and infectivity of different subtypes of AAV viruses, related experimental results show that the efficiency and titer of AAV2-ITR-Mut vector plasmid packaging viruses are obviously higher than those of complete plasmids of ITR, in the aspect of infection efficiency, the ability of the mutated AAV viruses to infect cells is also better than that of complete AAV virions, and the virus stability is not obviously influenced, which shows that the ITR sequence mutation obviously enhances the packaging and infectivity of the AAV viruses.

Description

Gene vector

Technical Field

The invention relates to the technical field of biology, in particular to a gene vector.

Background

The adeno-associated virus vector is a virus vector which is generated by transforming naturally-occurring adeno-associated virus by using genetic engineering and can be used for introducing foreign genes. Wherein, the ends of the genome of the adeno-associated virus are Inverted Terminal Repeat (ITR) and the Cap/Rep gene of two proteins encoded by the genome are expressed by independent plasmids. Because of the characteristics of good safety, low immunogenicity, long time for expressing exogenous genes and the like, the recombinant human immunodeficiency virus (HPV) is regarded as one of the most promising gene transfer vectors.

In the packaging vector, in addition to the cap gene for expressing the capsid protein of the virus and the rep gene involved in the replication and integration of the virus, in the overexpression plasmid, two inverted terminal repeats of the T type are the only reserved parts in the AAV genome, and the two ITRs are the origin of the viral DNA replication and the signal for triggering the viral packaging, and have decisive effect on the replication and packaging of the virus. The ITR sequence is modified to better play a role in guiding the replication of a genome and the assembly of a virus vector, so that the AAV vector is endowed with new functions and characteristics.

Among many AAV viruses, AAV type 2 is the most deeply studied, uses the most widely used gene vector, and becomes the first choice template for AAV subtype generation, and the currently known subtypes are basically based on AAV type 2 (AAV 2-ITR) and are obtained by hybridization with cap/rep genes of other subtypes.

The ITR sequences at both ends of wild type AAV2 type DNA have a total length of 145bp, wherein the first 125bp forms a T-shaped hairpin structure consisting of three palindromes: B-B ' and C-C ' form the top of the T, and A-A ' forms the long arm of the T. They play a very important role in the replication of DNA.

The modification of the ITR region is always a hotspot and difficulty in the AAV viral vector research. However, so far, only Self-complementary AAV viruses (scAAV) obtained by deleting Terminal melting sites (TRS) have been widely accepted and used.

Disclosure of Invention

In view of the above-described drawbacks of the prior art, it is an object of the present invention to provide a gene expression vector for solving the problems of the prior art.

To achieve the above and other related objects, according to one aspect of the present invention, there is provided a gene vector comprising an ITR mutant having a polynucleotide sequence comprising:

a) A polynucleotide sequence as shown in SEQ ID NO. 1; or the like, or, alternatively,

b) A polynucleotide sequence having a sequence similarity of more than 95% to SEQ ID NO.1 and having the function of the polynucleotide sequence defined in a).

In some embodiments of the invention, the gene vector comprises a gene expression cassette for expression of a foreign gene, at least one end of the gene expression cassette comprising an ITR mutant.

In some embodiments of the present invention, the gene vector comprises a gene expression cassette for expressing a foreign gene, and both ends of the gene expression cassette comprise ITR mutants, respectively.

In some embodiments of the invention, the ITR mutant functions as an origin of viral DNA replication and a signal that triggers viral packaging.

In some embodiments of the invention, the ITR mutant is derived from a wild-type AAV type 2 adeno-associated virus.

In some embodiments of the invention, the genetic vector is an AAV expression vector having a serotype selected from AAV1, AAV2, AAV3b-st, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVAnc80L65, aav2.7m8, AAVDJ, retroAAV, vpaahp. Eb, aahp.s, aaven, or AAVMG.

In some embodiments of the present invention, the gene expression cassette comprises a combination of one or more of a transcription initiation element, an enhanced gene expression element, a reporter gene, a post-transcriptional regulatory element, and a transcription termination element, and preferably, the transcription initiation element, the enhanced gene expression element, the reporter gene, the post-transcriptional regulatory element, and the transcription termination element are sequentially distributed in the gene expression cassette.

In some embodiments of the invention, the transcription initiation element is selected from the group consisting of a CMV promoter, and preferably, the polynucleotide sequence of the CMV promoter includes the sequence shown in SEQ ID NO. 9.

In some embodiments of the present invention, the gene expression enhancer element is selected from β -Globin, and preferably, the polynucleotide sequence of the gene expression enhancer element comprises a sequence shown as SEQ ID No. 2.

In some embodiments of the present invention, the reporter gene is selected from the group consisting of EGFP and luciferase, and preferably, the polynucleotide sequence of the reporter gene comprises a sequence as shown in one of SEQ ID nos. 3 to 4.

In some embodiments of the invention, the post-transcriptional regulatory element is selected from the group consisting of WPREs, and preferably, the polynucleotide sequence of the post-transcriptional regulatory element comprises the sequence shown as SEQ ID No. 5.

In some embodiments of the invention, the transcription termination element is selected from hGH polyA, and preferably, the polynucleotide sequence of the transcription termination element comprises a sequence as set forth in SEQ ID NO. 6.

In some embodiments of the invention, the genetic vector further comprises a replication initiator and a resistance gene.

In another aspect, the present invention provides an adeno-associated virus vector system, comprising the gene vector;

optionally, helper plasmids are included.

In another aspect, the present invention provides an adeno-associated virus packaging system, which comprises the gene vector or the adeno-associated virus vector system, preferably, the packaging cell of the adeno-associated virus packaging system is selected from insect cell and/or mammalian cell.

In another aspect, the present invention provides a method for preparing adeno-associated virus, comprising: culturing the adeno-associated virus packaging system under suitable conditions.

The invention also provides the adeno-associated virus which is prepared by the preparation method of the adeno-associated virus.

Drawings

FIG. 1a shows a schematic representation of the genomic structure of AAV2 ITRs of the invention (cited in the literature: the effect of ITR deficiency on AAV viral packaging and infectivity: cao Zuowu, 2008).

FIG. 1b shows a schematic representation of an alignment of AAV 2-ITRs and AAV2-ITR-Mut sequences of the invention.

FIG. 2a is a schematic diagram showing an AAV2-ITR-Mut expression plasmid map of the present invention.

FIG. 2b shows a schematic diagram of an AAV2-ITR-Mut expression plasmid map of the invention.

FIG. 3 is a schematic representation of the AAV2-ITR and AAV2-ITR-Mut serotype 2 and 9 titers comparison of the present invention.

FIG. 4 shows a schematic representation of the infectivity analysis of AAV2-ITR and AAV2-ITR-Mut viruses of the invention.

FIG. 5 is a schematic representation of luciferase expression in AAV2-ITR and AAV2-ITR-Mut serotype 9 infected cells of the invention.

FIG. 6 shows a schematic representation of the in vivo imaging luciferase expression of AAV 2-ITRs and AAV2-ITR-Mut serotype 9 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.

The modified AAV expression vector has Rep and Cap gene sequence eliminated and other target gene expression frame inserted between the ITRs in two ends of naturally existing AAV virus genome. The rAAV viral expression vector is released and packaged into a new virion by virtue of the presence of two self-ITR structures. However, the 145bp AAV2-ITR sequence is a self-complementary and inverted T-hairpin structure and plays a very critical role in AAV viral replication and packaging. As the content of GC in the AAV-ITR sequence is up to more than 80 percent, the T-shaped structure is unstable, and the AAV-ITR sequence is easy to mutate or lose in the replication process, and the AAV expression vector with the mutate or lack of the ITR can influence the packaging capacity, the stability and the infection rate.

The inventor of the invention discovers a new AAV mutant strain through a great deal of research, the AAV mutant strain has the characteristics of stable structure, high virus yield, strong infectivity and the like, and further discovers that ITRs of the AAV mutant strain have obvious mutation, so that a new gene vector is provided by utilizing the mutated ITRs, an adeno-associated virus vector system comprising the gene vector is further provided, and the gene vector comprising the mutated ITRs and the adeno-associated virus vector system have better packaging capacity, stability and infectivity compared with a wild type ITR sequence, and the invention is completed on the basis.

In a first aspect, the present invention provides a genetic vector comprising an ITR mutant, the polynucleotide sequence of which comprises:

a) A polynucleotide sequence as shown in SEQ ID NO. 1; or the like, or a combination thereof,

b) A polynucleotide sequence having a sequence similarity of more than 95% to SEQ ID NO.1 and having the function of the polynucleotide sequence defined in a). Specifically, the polynucleotide sequence in b) specifically refers to: the polynucleotide sequence shown in SEQ ID No.1 is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) nucleotides to the polynucleotide sequence shown in SEQ ID No.1, or one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) nucleotides to the N-terminal and/or C-terminal, and the polynucleotide fragment having the function of the DNA fragment shown in SEQ ID No.1 can function as an origin of viral DNA replication and a signal triggering viral packaging, for example. The polynucleotide sequence in b) may have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% similarity to SEQ ID No. 1.

In the present invention, the ITR mutant is derived from the ITR sequence of wild type AAV type 2 adeno-associated virus (GenBank: AF 043303.1), has obvious deletions at positions B 'and C, has obvious mutations at A, B and C', e.g., C63G, C G, G A, G C at position C ', e.g., 74-80 at position C, e.g., G85C, G C, C3291G, T3535C, T C at position B', e.g., T97G at position B ', 98-101 at position B', e.g., A130 zxft 4284 53131 5325 zxft 3523 zxft 6256 zxft 3456, 3226 zxft 5756, 3238 zxft 5756, 3226 zxft 27 zxft 5756, 3446 zxft, 3456, 3462 zxft 5756, 3248, 3446, and 3438 zxft 5756 at position A.

In the gene vector provided by the present invention, the gene vector is usually an adeno-associated virus vector (AAV vector), and the adeno-associated virus expression vector can be usually matched with helper plasmids (for example, a plasmid including a cap gene and a rep gene, and further for example, an adenovirus helper plasmid phelprer, etc.) to package and prepare adeno-associated virus. The gene vector can be used to form adeno-associated viruses of various serotypes, for example, the serotype of the gene vector can be selected from AAV1, AAV2, AAV3b-st, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVAnc80L65, aav2.7m8, AAVDJ, retroAAV, vpaahp. Eb, aavphp.s, aaven, and AAVMG, and the like. In a specific embodiment of the invention, the genetic vector is a serotype AAV2 vector, or a serotype AAV9 vector.

In the gene vector provided by the invention, the gene vector usually comprises a gene expression cassette for expressing a foreign gene, at least one end of the gene expression cassette can comprise an ITR mutant, or two ends of the gene expression cassette can respectively comprise ITR mutants, for example, the gene expression cassette can be usually positioned between a first ITR sequence and a second ITR sequence, and the polynucleotide sequence of the first ITR sequence and/or the second ITR sequence is the same as the sequence of the ITR mutant. The gene expression cassette can include various expression elements suitable for use in adeno-associated virus technology, for example, the gene expression cassette can include a combination of one or more of a transcription initiation element, an enhanced gene expression element, a reporter gene, a post-transcriptional regulatory element, a transcription termination element, and the like. In a specific embodiment of the present invention, a transcription initiation element, an enhanced gene expression element, a reporter gene, a post-transcriptional regulatory element, and a transcription termination element are sequentially distributed in the gene expression frame.

In the gene vector provided by the present invention, the transcription initiation element is usually a DNA fragment of the foreign gene for recognition, binding and initiation of transcription by RNA polymerase. The transcription initiation element may be various transcription initiation elements that can be suitably used in adeno-associated virus technology, and for example, the transcription initiation element may be selected from a CMV promoter, EF1a, SV40, ubc, CAG, and the like. In one embodiment of the invention, the polynucleotide sequence of the transcription initiation element comprises the sequence shown in SEQ ID NO. 9.

In the gene vector provided by the invention, the gene expression enhancing element is generally used for enhancing the expression of a target gene. The gene expression enhancer can be any gene expression enhancer suitable for adeno-associated virus technology, and for example, the gene expression enhancer can be selected from beta-Globin, enhancer, and the like. In one embodiment of the present invention, the polynucleotide sequence of the gene expression enhancer element comprises the sequence shown in SEQ ID NO. 2.

In the gene vector provided by the present invention, the reporter gene generally refers to a type of gene that is expressed in a specific condition of a cell, tissue/organ or individual and allows them to produce a trait that is easy to detect and would not otherwise be produced by the test material. The reporter gene may be any of a variety of reporter genes suitable for use with adeno-associated viruses, for example, the reporter gene may be selected from the group consisting of EGFP, luciferase, and the like, in combination with one or more of them. In one embodiment of the invention, the polynucleotide sequence of the reporter gene comprises a sequence as shown in one of SEQ ID No.3 to 4.

In the gene vectors provided by the present invention, the post-transcriptional regulatory element is generally used to improve the stability of post-transcriptional mRNA of the gene of interest. The post-transcriptional regulatory element may be any of a variety of post-transcriptional regulatory elements suitable for use with adeno-associated viruses, for example, the post-transcriptional regulatory element may be selected from WPRE and the like. In one embodiment of the present invention, the polynucleotide sequence of the post-transcriptional regulatory element comprises the sequence shown in SEQ ID No. 5.

In the gene vector provided by the present invention, the transcription termination element is generally used for termination of transcription after the target gene and the tag. The transcription termination element may be any suitable transcription termination element for adeno-associated viruses, for example, the transcription termination element may be generally selected from hGH polyA, SV40 polyA, HSVtk polyA, and the like. In one embodiment of the invention, the polynucleotide sequence of the transcription termination element comprises the sequence shown in SEQ ID NO. 6.

In the gene vector provided by the present invention, the gene vector further includes various conventional plasmid backbones and other elements, and specifically, these elements may be, for example, replication initiators, resistance genes, and the like. In one embodiment of the present invention, the replication initiator may comprise one or more of f1 Origin, pUC ori, and the like. In another embodiment of the present invention, the resistance gene may include AmpR or the like.

In a preferred embodiment of the present invention, the full-length sequence of the gene vector includes the sequence shown in SEQ ID NO.8.

In a second aspect, the present invention provides an adeno-associated viral vector system comprising the gene vector of the first aspect of the present invention, which is generally used for preparing adeno-associated viruses. The adeno-associated virus vector system can further comprise various other materials which cooperate with the gene vector provided by the first aspect of the invention to form adeno-associated virus. For example, the adeno-associated viral vector system can further include helper plasmids including phepper plasmid (adenovirus helper plasmid phepper), RC plasmid (plasmid including cap gene and rep gene).

In a third aspect, the present invention provides an adeno-associated virus packaging system, which comprises the gene vector provided in the first aspect of the present invention or the adeno-associated virus vector system provided in the second aspect of the present invention. The adeno-associated virus packaging system can be obtained by transfecting a packaging cell, which can be a host cell suitable for packaging adeno-associated virus, usually a cell type susceptible to baculovirus infection, with the above-mentioned gene vector, plasmid, or the like. For example, the host cell to be used may be an insect cell or the like, and more specifically, highfive, sf9, se301, seIZD2109, seUCR1, sf9, sf900+, sf21, BTI-TN-5B1-4, MG-1, tn368, hzAm1, BM-N, ha2302, hz2E5, ao38 or the like; as another example, the host cell to be used may be a mammalian cell, and more specifically, HEK293, heLa, CHO, NS0, SP2/0, PER. C6, vero, RD, BHK, HT1080, A549, cos-7, ARPE-19, MRC-5 cell and the like.

In a fourth aspect, the present invention provides a method for producing AAV, suitably using an adeno-associated virus packaging system, which method should be known to those skilled in the art. For example, the method for producing the AAV virus may comprise: culturing the AAV virus packaging system provided by the third aspect of the present invention under suitable conditions to express the desired protein and packaging to obtain AAV, followed by isolation and purification to provide AAV. In a specific embodiment of the invention, the AAV virus can be recovered from a cell culture supernatant of adeno-associated virus packaging cells, which typically contains a mixture of cells of the recombinant adeno-associated virus vector and the culture supernatant.

In a fifth aspect, the present invention provides an AAV virus prepared by the method for producing an AAV virus provided in the fourth aspect of the present invention.

The mutated ITR sequences provided by the invention are obviously different in a C-C' palindrome region, and compared with the influence of wild type and mutated AAV2-ITR (AAV 2-ITR-Mut) on the packaging and infectivity of different subtypes of AAV viruses, related experimental results show that the efficiency and titer of AAV2-ITR-Mut vector plasmid packaging viruses are obviously higher than those of complete plasmids of ITR, in the aspect of infection efficiency, the ability of the mutated AAV viruses to infect cells is also better than that of complete AAV virions, and the virus stability is not obviously influenced, which shows that the ITR sequence mutation obviously enhances the packaging and infectivity of the AAV viruses. Therefore, the AAV vector plasmid and the adeno-associated virus vector system comprising ITR sequence mutation provided by the invention have good industrialization prospect.

The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application. Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, sambrook et al: a LABORATORY MANUAL, second edition, cold Spring Harbor LABORATORY Press,1989and Third edition,2001; ausubel et al, current PROTOCOLS IN MOLECULAR BIOLOGY, john Wiley & Sons, new York,1987 and periodic updates; the series METHODS IN ENZYMOLOGY, academic Press, san Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, third edition, academic Press, san Diego,1998; (iii) METHODS IN ENZYMOLOGY, vol.304, chromatin (P.M.Wassarman and A.P.Wolffe, eds.), academic Press, san Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, totowa,1999, etc.

Example 1

Based on random mutation analysis, a plurality of AAV virus mutant strains with stable structure, high virus yield and strong infectivity are obtained by screening, wherein one AAV virus mutant strain is sequenced to find that ITR of the AAV virus mutant strains has obvious deletion at B 'and C positions and A, B and C' have obvious mutation (as shown in figure 1).

According to the sequencing result, synthesizing an AAV2-ITR-Mut sequence (SEQ ID NO. 1), and constructing an expression vector containing the AAV2-ITR-Mut, namely, an EGFP gene (SEQ ID NO. 3) or a luciferase gene (SEQ ID NO. 4) is contained between the AAV2-ITR-Mut at two ends, CMV (SEQ ID NO. 9) and beta-Globin (SEQ ID NO. 2) are contained at the upstream, a WPRE sequence (SEQ ID NO. 5) and a hGH polyA sequence (SEQ ID NO. 6) are contained at the downstream (shown in FIG. 2), and the full-length sequence of the vector is referred to SEQ ID NO.7. Simultaneously, a control AAV expression vector containing AAV2-ITR elements is constructed, and the full-length sequence of the vector refers to SEQ ID NO.8.

Example 2

Packaging AAV virus: HEK-293T cells were co-transfected using a three plasmid system to generate AAV viruses, and 3 plasmids included the vector plasmid pHelper (Stratagene), RC plasmid (Stratagene), expression vector (AAV 2-ITR-GOI and AAV2-ITR-Mut-GOI, prepared in example 1). The gene vectors for the two different ITRs, both containing EGFP or luciferase elements, were prepared separately for two different types of AAV viruses to compare virus titer and infectivity. The RC plasmid contains cap and rep genes. The specific packaging process is as follows:

1) HEK-293T cell culture:

the HEK293T cells were recovered into 100mm dishes and cultured in a 37 ℃ C., 5% CO2-based incubator in DMEM with 10% FBS. When the cell confluency reaches 90%, according to the specific experimental arrangement, the cells can be passaged according to the proportion of 1:3 or 1:6-1:8, and plasmid transfection can be carried out when the cell confluency reaches 70% -80%.

2) Transfection of HEK-293T cells:

preparing a transfection system: to a 50mL centrifuge tube was added OMEM medium (750. Mu.L/dish), followed by Vector target plasmid (5. Mu.g/dish), pHelper Vector plasmid KL4195 (5. Mu.g/dish) and "RC" Vector plasmid (5. Mu.g/dish), gently mixed using a vortex shaker, after mixing, 1 XPEI solution (PEI: DNA = 3:1) was added, and gently mixed again on a vortex shaker. Standing at room temperature for 15min. Checking the cells to be transfected before transfection, wherein the confluence needs to reach 70-80%; the prepared transfection system was added dropwise to the petri dish in an amount of 1 mL/dish, mixed well and then placed in an incubator at 37 ℃ with 5% CO2 for cultivation. 6h after transfection, the medium in the dish was replaced with 10ml of fresh medium; the dish was returned to the incubator and incubated for an additional 66-72 hours.

3) And (3) toxin collection:

viral particles are present in both the packaging cells and the culture supernatant. Both cells and culture supernatant were collected for best yield. After transfection for 48h, the medium supernatant was collected by an electronic pipette into a 50mL centrifuge tube and stored temporarily at 4 ℃.

After 96h of transfection, the mixture of medium supernatant and cells was collected by an electropipettor into a 50mL centrifuge tube.

Purification of AAV viral particles: AAV2-ITR (AAV 2) represents AAV2 ITR and serotype 2 viruses, AAV2-ITR (AAV 9) represents AAV2 ITR and serotype 9 viruses, AAV2-ITR-Mut (AAV 2) represents AAV2 ITR Mut and serotype 2 viruses, AAV2-ITR-Mut (AAV 9) represents AAV2 ITR Mut and serotype 2 viruses, and AAV2-ITR-Mut (AAV 9) represents AAV2 ITR Mut and serotype 9 viruses. The specific process is as follows:

1) Concentration of AAV viruses

1. 40% of PEG8000 was added to the supernatant until the final concentration was 8%, and after standing on ice for 2 hours (mixing once every 15 minutes), centrifuged at 2,500 Xg for 30 minutes. Removing the supernatant, resuspending the precipitate with PBS, and mixing with cell lysis supernatant;

2.3,000 xg for 30 minutes and the supernatant transferred to another clean tube. There should be visible cell debris in the supernatant at this point. If partial debris still exists, centrifuging again;

3. residual plasmid DNA (final concentration of 50U/ml) was removed by digestion with Benzonase nuclease. Invert several times to mix thoroughly. Incubation at 37 ℃ for 30 min;

4. the mixture was filtered through a 0.45 μm filter head, and the filtrate was taken out.

2) Purification of AAV

1. Adding solid CsCl into the virus concentrated solution until the density is 1.41g/ml (the refractive index is 1.372), adding 6.5g CsCl into about 10ml virus solution, shaking to dissolve, and absorbing heat and cooling CsCl when dissolved;

2. adding a sample into an ultracentrifuge tube, and filling the residual space of the centrifuge tube with a pre-prepared 1.41g/ml CsCl solution;

3. centrifugation was carried out at 175,000g for 24 hours to form a density gradient. Samples of different densities were collected in sequential steps, as line titer determinations. Collecting the fraction enriched in AAV particles;

4. the above process is repeated once.

3) Desalination by ultrafiltration

1. Leaching: 4ml of deionized water is added into an Amicon-15 ultrafiltration device;

2. the virus solution obtained by density gradient centrifugation is added into an ultrafiltration device. Adding PBS to make the total volume be 4ml, and covering the cover;

note that if an angular rotor is used, the maximum volume cannot exceed 3.5ml

3.1,500 × g are centrifuged for about 5 to 10 minutes, and the number of remaining volumes is checked every 5 minutes until the tissue volume is 200-250 μ L;

4. collecting the filtrate for sterilization, and returning the filter membrane to the device;

5. the concentrated virus was diluted with 1 XPBS to a volume of 4ml;

6. repeating the above process for 3 times;

7. the ultrafiltration tube was centrifuged to a final volume of approximately 0.5ml.

4) Preservation of viruses

Glycerol was added to the virus concentrate to make the tissue concentration 5%. Subpackaging and storing at-80 ℃.

AAV virus titer assay: the titer detection is carried out on the purified and concentrated AAV virus liquid by using a fluorescent quantitative PCR method, and the specific process is as follows:

and (3) establishing a standard curve by using the plasmid as a standard substance, and comparing the sample to be detected with the standard curve to obtain the titer of the sample to be detected.

The specific operation steps are as follows:

1. sample processing 20. Mu.L of virus was taken into a clean 1.5mL centrifuge tube, 1. Mu.L of totipotent enzyme was added, mixed well and placed in a 37 ℃ water bath for 30min. Centrifuge at 12000rpm for 10min at 4 ℃. After centrifugation, 10. Mu.L of the supernatant was added to another clean centrifuge tube, 90. Mu.L of dilution buffer was added, and after mixing, the mixture was placed in a 37 ℃ water bath for 30min. Naturally cooling, adding 1 mu L of protease K, uniformly mixing, and placing in a water bath at 65 ℃ for incubation for 1h. The cells were incubated in a water bath at 95 ℃ for 20min to inactivate proteinase K. And taking out the sample tube, and putting the sample tube into a refrigerator at 4 ℃ for cooling.

2. Dilution of the sample: and (3) sucking 10 mu L of AAV sample stock solution to be detected into a first centrifuge tube, uniformly mixing for 2-3s by using a vortex oscillator, taking 10 mu L of AAV sample stock solution out of the first centrifuge tube, adding the AAV sample stock solution into a second centrifuge tube, and repeating the steps to obtain 4 diluted samples, and taking all samples as templates to enter quantitative PCR. Aiming at gradient dilution of the same sample, the mode of sucking the sample each time and the time of shaking, uniformly mixing and centrifuging are consistent.

3. Dilution of plasmid standards: in the same dilution mode of the sample, the plasmid standard substance is diluted by 10 times in a gradient manner from 101 times to 109 times, and 8 plasmid samples are taken as templates to enter quantitative PCR. (Standard calculation formula = Conc/(MW per bp Vector Size). Avogadro constant/Dilution Factor)

4, preparing a PCR reaction system according to the specification of an SYBR Green kit;

5. putting the sample tubes into a fluorescent quantitative PCR instrument according to a certain sequence by a computer, and setting according to the following program: 10min at 95 ℃; 1, 10, 5,0, 72 and 40 cycles at 95 ℃;95 ℃ for 1min,55 ℃ for 30S,95 ℃ for 30S.

6. And obtaining a standard curve according to the logarithm value of the concentration of the standard substance and the Ct average value after obtaining Ct value data. The concentration of other samples can be calculated according to a standard curve;

7. the final value of the concentration of the sample to be tested is obtained by dividing the measurement by the dilution and multiplying by 2, where 2 is multiplied because the standard is double stranded and the AAV viral particles are single stranded.

8. And averaging the titers obtained by measuring the viruses with different dilutions to obtain the final concentration of the viruses.

Titer results are shown in figure 3, and are used to guide subsequent infectivity tests. As can be seen in fig. 3, the mutant virus titers were higher in the two serotypes of AAV virus compared to the wild type virus.

Example 3

Comparison of the ability of AAV comprising different ITRs to infect cells: the infectivity of different ITRs and different serotypes of rAAV virus is determined by HEK-293T cells and animal level expression. AAV viruses of two different serotypes, AAV2 and AAV9 (prepared in example 2), were infected with HEK293T cells at MOI of 1E3 and 2E4, respectively, and EGFP expression was recorded 72 hours after infection by photographing by fluorescence microscopy, and the results are shown in fig. 4. As can be seen from FIG. 4, the mutated viruses have a stronger infection ability on HEK-293T cells in both serotypes of AAV than in wild type viruses.

Example 4

AAV2-ITR (AAV 9) and AAV2-ITR-Mut (AAV 9) (prepared in example 2) infected HEK293T cells at an MOI of 1E5, and luciferase expression was quantitatively analyzed 72 hours after infection by a microplate reader, and the results are shown in FIG. 5. As can be seen from FIG. 5, the mutant virus had a higher infectivity than the wild-type virus.

Example 5

AAV2-ITR (AAV 9) and AAV2-ITR-Mut (AAV 9) (prepared in example 2) were injected into C57BL/6 mice by tail vein administration in a virus injection amount of 1E11, and luciferase expression was quantitatively analyzed by in vivo imager in Day7 and Day14, and the results are shown in FIG. 6. As can be seen from FIG. 6, the mutant virus had a higher infectivity than the wild-type virus.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be accomplished by those skilled in the art without departing from the spirit and scope of the present invention as set forth in the appended claims.

Sequence listing

<110> Shanghai Ji Kai Gen chemical technology GmbH

<120> a gene vector

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 130

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc 120

tgcctgcagg 130

<210> 2

<211> 604

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga 60

agacaccggg accgatccag cctccgcgga ttcgaatccc ggccgggaac ggtgcattgg 120

aacgcggatt ccccgtgcca agagtgacgt aagtaccgcc tatagagtct ataggcccac 180

aaaaaatgct ttcttctttt aatatacttt tttgtttatc ttatttctaa tactttccct 240

aatctctttc tttcagggca ataatgatac aatgtatcat gcctctttgc accattctaa 300

agaataacag tgataatttc tgggttaagg caatagcaat atttctgcat ataaatattt 360

ctgcatataa attgtaactg atgtaagagg tttcatattg ctaatagcag ctacaatcca 420

gctaccattc tgcttttatt ttatggttgg gataaggctg gattattctg agtccaagct 480

aggccctttt gctaatcatg ttcatacctc ttatcttcct cccacagctc ctgggcaacg 540

tgctggtctg tgtgctggcc catcactttg gcaaagaatt gggattcgaa catcgattga 600

attc 604

<210> 3

<211> 717

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717

<210> 4

<211> 1653

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatccgct ggaagatgga 60

accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120

gcttttacag atgcacatat cgaggtggac atcacttacg ctgagtactt cgaaatgtcc 180

gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240

tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300

gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgggcatt 360

tcgcagccta ccgtggtgtt cgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420

aaaaagctcc caatcatcca aaaaattatt atcatggatt ctaaaacgga ttaccaggga 480

tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540

tttgtgccag agtccttcga tagggacaag acaattgcac tgatcatgaa ctcctctgga 600

tctactggtc tgcctaaagg tgtcgctctg cctcatagaa ctgcctgcgt gagattctcg 660

catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720

gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780

cgagtcgtct taatgtatag atttgaagaa gagctgtttc tgaggagcct tcaggattac 840

aagattcaaa gtgcgctgct ggtgccaacc ctattctcct tcttcgccaa aagcactctg 900

attgacaaat acgatttatc taatttacac gaaattgctt ctggtggcgc tcccctctct 960

aaggaagtcg gggaagcggt tgccaagagg ttccatctgc caggtatcag gcaaggatat 1020

gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080

gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140

acgctgggcg ttaatcaaag aggcgaactg tgtgtgagag gtcctatgat tatgtccggt 1200

tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260

ggagacatag cttactggga cgaagacgaa cacttcttca tcgttgaccg cctgaagtct 1320

ctgattaagt acaaaggcta tcaggtggct cccgctgaat tggaatccat cttgctccaa 1380

caccccaaca tcttcgacgc aggtgtcgca ggtcttcccg acgatgacgc cggtgaactt 1440

cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500

tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560

gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 1620

aaggccaaga agggcggaaa gatcgccgtg taa 1653

<210> 5

<211> 589

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360

ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540

cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589

<210> 6

<211> 477

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60

gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 120

ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 180

acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg 240

ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg 300

ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg 360

ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct 420

tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtcctt 477

<210> 7

<211> 6090

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60

gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120

actccatcac taggggttcc tgcggccgca cgcgtgtgtc tagaacgcgt ggagctagtt 180

attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta 240

cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt 300

caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg 360

tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta 420

cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga 480

ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg 540

tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 600

caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 660

ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 720

gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc 780

cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgc ggattcgaat 840

cccggccggg aacggtgcat tggaacgcgg attccccgtg ccaagagtga cgtaagtacc 900

gcctatagag tctataggcc cacaaaaaat gctttcttct tttaatatac ttttttgttt 960

atcttatttc taatactttc cctaatctct ttctttcagg gcaataatga tacaatgtat 1020

catgcctctt tgcaccattc taaagaataa cagtgataat ttctgggtta aggcaatagc 1080

aatatttctg catataaata tttctgcata taaattgtaa ctgatgtaag aggtttcata 1140

ttgctaatag cagctacaat ccagctacca ttctgctttt attttatggt tgggataagg 1200

ctggattatt ctgagtccaa gctaggccct tttgctaatc atgttcatac ctcttatctt 1260

cctcccacag ctcctgggca acgtgctggt ctgtgtgctg gcccatcact ttggcaaaga 1320

attgggattc gaacatcgat tgaattcggt accggaattc ggaactggag gtggaggtag 1380

tggaatggat cccgccacca tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc 1440

catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg 1500

cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct 1560

gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg 1620

ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg aaggctacgt 1680

ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa 1740

gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga 1800

cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat 1860

ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga 1920

cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg acggccccgt 1980

gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag accccaacga 2040

gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat 2100

ggacgagctg tacaaggcta gcgactacaa ggatgacgat gacaaggatt acaaagacga 2160

cgatgataag gactataagg atgatgacga caaaaagctt taaaccggtt atcgataatc 2220

aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt 2280

ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg 2340

ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc 2400

ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt 2460

ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg 2520

ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg 2580

gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct 2640

gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc 2700

cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc 2760

ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgagcgc 2820

tgctcgagag atctacgggt ggcatccctg tgacccctcc ccagtgcctc tcctggccct 2880

ggaagttgcc actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt 2940

gtctgactag gtgtccttct ataatattat ggggtggagg ggggtggtat ggagcaaggg 3000

gcaagttggg aagacaacct gtagggcctg cggggtctat tgggaaccaa gctggagtgc 3060

agtggcacaa tcttggctca ctgcaatctc cgcctcctgg gttcaagcga ttctcctgcc 3120

tcagcctccc gagttgttgg gattccaggc atgcatgacc aggctcagct aatttttgtt 3180

tttttggtag agacggggtt tcaccatatt ggccaggctg gtctccaact cctaatctca 3240

ggtgatctac ccaccttggc ctcccaaatt gctgggatta caggcgtgaa ccactgctcc 3300

cttccctgtc cttctgattt tgtaggtaac cacgtgcgga ccgagcggcc gcaggaaccc 3360

ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 3420

ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 3480

agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca 3540

caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 3600

gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 3660

tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 3720

gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 3780

atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 3840

cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 3900

ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 3960

aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 4020

ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 4080

acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 4140

gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 4200

aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 4260

taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 4320

gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 4380

tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 4440

ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 4500

taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 4560

gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 4620

aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 4680

gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 4740

ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 4800

ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 4860

acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 4920

taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 4980

tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 5040

cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 5100

ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 5160

gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 5220

gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 5280

aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 5340

aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 5400

actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 5460

gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 5520

atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 5580

atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 5640

ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 5700

gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 5760

cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 5820

tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 5880

cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 5940

ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 6000

gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 6060

tggccttttg ctggcctttt gctcacatgt 6090

<210> 8

<211> 6094

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc 120

gagcgcgcag agagggagtg gccgcggccg cacgcgtgtg tctagaacgc gtggagctag 180

ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt 240

tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac 300

gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg 360

ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc atatgccaag 420

tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg cccagtacat 480

gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg ctattaccat 540

ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact cacggggatt 600

tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa atcaacggga 660

ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta ggcgtgtacg 720

gtgggaggtc tatataagca gagctcgttt agtgaaccgt cagatcgcct ggagacgcca 780

tccacgctgt tttgacctcc atagaagaca ccgggaccga tccagcctcc gcggattcga 840

atcccggccg ggaacggtgc attggaacgc ggattccccg tgccaagagt gacgtaagta 900

ccgcctatag agtctatagg cccacaaaaa atgctttctt cttttaatat acttttttgt 960

ttatcttatt tctaatactt tccctaatct ctttctttca gggcaataat gatacaatgt 1020

atcatgcctc tttgcaccat tctaaagaat aacagtgata atttctgggt taaggcaata 1080

gcaatatttc tgcatataaa tatttctgca tataaattgt aactgatgta agaggtttca 1140

tattgctaat agcagctaca atccagctac cattctgctt ttattttatg gttgggataa 1200

ggctggatta ttctgagtcc aagctaggcc cttttgctaa tcatgttcat acctcttatc 1260

ttcctcccac agctcctggg caacgtgctg gtctgtgtgc tggcccatca ctttggcaaa 1320

gaattgggat tcgaacatcg attgaattcg gtaccggaat tcggaactgg aggtggaggt 1380

agtggaatgg atcccgccac catggtgagc aagggcgagg agctgttcac cggggtggtg 1440

cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag 1500

ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag 1560

ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 1620

cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac 1680

gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg 1740

aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 1800

gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc 1860

atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag 1920

gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 1980

gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac 2040

gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc 2100

atggacgagc tgtacaaggc tagcgactac aaggatgacg atgacaagga ttacaaagac 2160

gacgatgata aggactataa ggatgatgac gacaaaaagc tttaaaccgg ttatcgataa 2220

tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc 2280

ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat 2340

ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg 2400

gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg 2460

ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat 2520

tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt 2580

gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc 2640

ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa 2700

tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg 2760

ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgagc 2820

gctgctcgag agatctacgg gtggcatccc tgtgacccct ccccagtgcc tctcctggcc 2880

ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaaattaag ttgcatcatt 2940

ttgtctgact aggtgtcctt ctataatatt atggggtgga ggggggtggt atggagcaag 3000

gggcaagttg ggaagacaac ctgtagggcc tgcggggtct attgggaacc aagctggagt 3060

gcagtggcac aatcttggct cactgcaatc tccgcctcct gggttcaagc gattctcctg 3120

cctcagcctc ccgagttgtt gggattccag gcatgcatga ccaggctcag ctaatttttg 3180

tttttttggt agagacgggg tttcaccata ttggccaggc tggtctccaa ctcctaatct 3240

caggtgatct acccaccttg gcctcccaaa ttgctgggat tacaggcgtg aaccactgct 3300

cccttccctg tccttctgat tttgtaggta accacgtgcg gaccgagcgg ccgcggccac 3360

tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc 3420

gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 3480

catcactagg ggttcctggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 3540

ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 3600

gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 3660

cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 3720

aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 3780

cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 3840

ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 3900

aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 3960

ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 4020

acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 4080

cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 4140

tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 4200

ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 4260

ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 4320

atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 4380

taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 4440

cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 4500

aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 4560

aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 4620

tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 4680

ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 4740

catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 4800

aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 4860

ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 4920

gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 4980

aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 5040

gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 5100

gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 5160

gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 5220

gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 5280

gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 5340

atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 5400

ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 5460

ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 5520

ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 5580

ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 5640

ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 5700

tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 5760

tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 5820

tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 5880

tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 5940

gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 6000

tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 6060

ttcctggcct tttgctggcc ttttgctcac atgt 6094

<210> 9

<211> 508

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 360

atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 420

ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 480

acggtgggag gtctatataa gcagagct 508

Claims

1. A gene vector comprises an ITR mutant, and the polynucleotide sequence of the ITR mutant comprises the polynucleotide sequence shown in SEQ ID NO. 1.

2. The gene vector of claim 1, wherein the gene vector comprises a gene expression cassette for expressing a foreign gene, at least one end of the gene expression cassette comprising an ITR mutant;

and/or the gene vector comprises a gene expression frame for expressing a foreign gene, wherein two ends of the gene expression frame respectively comprise ITR mutants;

and/or the ITR mutant has the functions of acting as an origin of viral DNA replication and a signal triggering viral packaging;

and/or, the ITR mutant is derived from a wild-type AAV type 2 adeno-associated virus.

3. The genetic vector of claim 2, wherein the genetic vector is an AAV expression vector, and the serotype of the genetic vector is selected from the group consisting of AAV1, AAV2, AAV3b-st, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVAnc80L65, aav2.7m8, aavet, AAVDJ, retroAAV, aavphp. Eb, aavphp.s, and AAVMG.

4. The gene vector of claim 2, wherein the gene expression cassette comprises a combination of one or more of a transcription initiation element, an enhanced gene expression element, a reporter gene, a post-transcriptional regulatory element, and a transcription termination element.

5. A gene vector as claimed in claim 4, wherein the gene expression cassette has distributed therein, in order, a transcription initiation element, an enhanced gene expression element, a reporter gene, a post-transcriptional regulatory element and a transcription termination element.

6. A gene vector according to claim 4 or 5, wherein the transcription initiation element is selected from the group consisting of a CMV promoter;

and/or, the gene expression-enhancing element is selected from β -Globin;

and/or the reporter gene is selected from one or more combinations of EGFP and luciferase;

and/or, the post-transcriptional regulatory element is selected from WPRE; and/or, the transcription termination element is selected from hGH polyA.

7. The genetic vector of claim 6, wherein the polynucleotide sequence of the CMV promoter comprises the sequence shown as SEQ ID No. 9;

and/or the polynucleotide sequence of the gene expression enhancing element comprises a sequence shown as SEQ ID NO. 2;

and/or the polynucleotide sequence of the reporter gene comprises a sequence shown as one of SEQ ID NO. 3-4;

and/or the polynucleotide sequence of the post-transcriptional regulatory element comprises a sequence shown as SEQ ID No. 5;

and/or the polynucleotide sequence of the transcription termination element comprises a sequence shown as SEQ ID NO. 6.

8. The genetic vector of any one of claims 2-5 or 7 further comprising a replication initiator and a resistance gene.

9. An adeno-associated viral vector system comprising the gene vector according to any one of claims 1 to 8.

10. The adeno-associated viral vector system of claim 9 further comprising a helper plasmid.

11. An adeno-associated virus packaging system comprising a gene vector according to any one of claims 1 to 8 or an adeno-associated virus vector system according to claim 9 or 10.

12. The adeno-associated viral packaging system according to claim 11 wherein the packaging cells of the adeno-associated viral packaging system are selected from insect cells and/or mammalian cells.

13. A method for producing an adeno-associated virus, comprising: culturing the adeno-associated virus packaging system of claim 11 or 12 under suitable conditions.

14. An adeno-associated virus produced by the method according to claim 13.