CN114231564A - rAAV vector core plasmid and application thereof - Google Patents

Abstract

The rAAV carrier core plasmid mainly contains double-report fusion gene of fluorescent protein gene and luciferase gene, and specific AAV serotypeCapA discriminating DNA sequence corresponding to the gene. Cells are infected by rAAV packaged by the rAAV vector core plasmid, and the infection activity of various different serotype rAAV is rapidly determined by detecting the expression of fluorescent protein or luciferase reporter gene; in addition, the infection of cells by specific AAV serotypesCapDetection and enrichment of the corresponding discriminating DNA sequence of the gene or its transcription productAnd (3) degree analysis for simultaneously evaluating the infection efficiency difference of various different serotype rAAV.

Description

rAAV vector core plasmid and application thereof

Technical Field

The invention belongs to the technical field of viral vectors, and particularly relates to a rAAV vector for rapidly detecting the biological activity of the rAAV and application thereof.

Background

The recombinant adeno-associated virus (rAAV) is modified from wild adeno-associated virus (AAV), the rAAV genome only contains Terminal Repeat Sequences (ITRs) at two ends of the AAV, and the contained exogenous genes completely replace the virus self-encoding genes. rAAV can be prepared by compensating replication gene Rep, structural gene Cap and packaging auxiliary gene of AAV in trans in packaging cell line. Because of the characteristics of high safety, low immunogenicity, wide host range, multiple virus serotype types, capability of infecting divided and non-divided cells, capability of mediating the long-term stable expression of exogenous genes in animal bodies and the like, the recombinant adenovirus vector is an important virus tool widely used for carrying exogenous gene expression in the field of biomedical research and is a very important virus vector in the field of disease gene therapy (Wang Det al, Nat Rev Drug Discov.2019; 18(5): 358-378). To date, gene therapy drugs of 3 kinds of gene rAAV have been marketed.

Currently, there are 13 serotypes of AAV isolated from nature and over 100 mutant subtypes. The diversity of the capsid proteins (caps) of different serotypes of AAV viruses determines the differences in AAV infectivity. These infectious properties are involved in the adsorption and entry of AAV-infected cells, intracellular transport, viral uncoating within the nucleus, and transgene expression. Several cell surface AAV-specific receptors have been discovered, such as: heparan Sulfate Proteoglycans (HSPGs), integrins, etc. The capsid proteins of different serotypes of AAV and their cell surface receptors differ, exhibiting a variety of different infection efficiencies, tropism and immunological properties for tissues or cell types. Through mutational transformation of the Cap gene of the AAV capsid protein, a plurality of rAAV vectors with excellent and new characteristics can be obtained. For example: the AAV-DJ type with obviously improved transduction activity in the liver, the AAV9-PHP.eB type capable of efficiently crossing the brain blood brain barrier of mice and the like.

The determination of the infection activity of different serotype rAAV and the difference of the infection efficiency of different histiocyte is very important for better application of rAAV. The infection activity of the rAAV virus vector can be greatly influenced due to the differences of packaging and purification methods, production batches, storage conditions and the like, and the total particle number of the rAAV vector is less influenced by the storage conditions and the like. Currently, the physical titer of rAAV (viral particle concentration) widely used is typically determined using a fluorescent real-time quantitative PCR method (Q-PCR), in which rAAV viral particles contain only a single copy of genomic DNA, and thus the physical titer of rAAV can be defined as the number of copies of rAAV viral genome per ml, i.e. (vector genome per ml, vg/ml). Infectious activity of rAAV is often expressed as transduction titer (in TU/ml) or Infectious titer (IU/ml). The method for determining the infection activity of the rAAV2 standard is to use a rAAV sample with gradient dilution to infect a Hela RC32 cell line integrating Cap and Rep genes of AAV2 under the auxiliary infection of adenovirus type 5 (AdV5), and to calculate TCID50 by observing the expression of a fluorescent protein reporter Gene or obtaining AAV genome copy number through quantitative PCR (refer to Lock M et al, 2010Human Gene Therapy,21(10): 1273-85). The method needs a special Hela RC32 cell line and auxiliary infection of adenovirus type 5 with certain safety risk, and is difficult to popularize generally. In recent years, some researchers also measure the infection activity of rAAV by using luciferase (Luc) carried in rAAV genome, but the detection is limited to the detection of the activity of a few serotype rAAV, the universality is not high, and the obtained data is poor in comparability.

Therefore, the development of a novel method which is suitable for various serotypes, has multiple parameters and is comparable, and can quickly and flexibly detect the infection activity of the rAAV has important application values for the quality control of rAAV products and the rAAV in gene therapy basis and clinical research.

Disclosure of Invention

Aiming at the defects or the improvement requirements of the prior art, the novel rAAV vector core plasmid provided by the invention contains a double-report fusion gene of a fluorescent protein gene and a luciferase gene and a corresponding DNA sequence of a Cap gene of a specific AAV serotype, can be used for rapidly determining the rAAV infection activity of various serotypes by detecting the expression of the fluorescent protein gene or the luciferase reporter gene, and can simultaneously evaluate the infection efficiency difference of various rAAV serotypes.

In order to achieve the purpose, the invention adopts the following technical scheme:

a core plasmid of rAAV vector, the core plasmid expression element of rAAV vector integrates CMV promotor, double-reporting fusion gene of fluorescent protein gene and luciferase gene, post-transcriptional regulatory sequence WPRE, human growth hormone polyA element, and recognition DNA sequence corresponding to special AAV serotype capsid protein Cap gene inserted between WPRE and human growth hormone polyA element. The rAAV vector core plasmid efficiently expresses a double-report fusion gene containing a fluorescent protein gene and a luciferase gene through a broad-spectrum strong promoter CMV. The NanoLuc is a novel luciferase developed in recent years, has brighter luminescence and smaller molecular weight (only 171 amino acids and the molecular weight of 19.1kDa) compared with other luciferases, is one of bioluminescent reporter genes with the best performance at present, is connected with the C end of a green fluorescent protein (EGFP) gene through a connecting sequence, obtains an EGFP-NanoLuc double-reporter fusion gene with the advantages of the two genes, can observe the expression of the EGFP through a fluorescence microscope, can quantitatively detect the expression of the NanoLuc through a luciferase detector, and is suitable for tissue cells cultured in vitro and animal living body detection. The expression level of the double-report fusion gene is further improved by utilizing a post-transcriptional regulatory sequence WPRE and a human growth hormone polyA (HGHpA) element. In particular, the corresponding recognition DNA sequence of the Cap gene of a particular AAV serotype can be flexibly introduced between the WPRE and HGHpA elements via a multiple cloning enzyme cleavage site (MCS).

Preferably, the identifying DNA sequence corresponding to the Cap gene of the specific AAV serotype capsid protein is specifically: the 400bp extension is carried out by taking 1760 th base (about 587 th amino acid of Cap capsid protein coded by Cap gene) of Cap gene of capsid protein of specific AAV serotype as central site, extending 250 and 5' end of the central site respectively, and is basically positioned in main variable region of Cap gene sequence. Specifically, the recognition DNA sequence of the AAV2 serotype Cap gene is shown as a sequence in SEQ ID No. 3; the recognition DNA sequence of AAV9 serotype Cap gene is shown as SEQ ID No. 4; the recognition DNA sequence of AAV13 serotype Cap gene is shown as SEQ ID No. 5.

Preferably, the shuttle plasmid pfast. bac. dual is used as the vector backbone. The rAAV can be prepared by packaging directly by a method of transfecting HEK293 cells with plasmids, or can be prepared by constructing a recombinant Baculovirus (BEV) and infecting insect Sf9 cells by the BEV.

The application of the rAAV vector core plasmid in detecting the infection activity of different serotype rAAV comprises the following steps:

(1) preparing rAAV of specific serotype corresponding to the carried recognition DNA sequence by using the rAAV carrier core plasmid;

(2) diluting the specific serotype rAAV virus prepared in the step (1) according to a certain proportion in a gradient manner, and then infecting cells cultured in a 96-well plate;

(3) after 2 days of infection, the infectious activity of rAAV was determined by observing the expression of a fluorescent protein reporter, or detecting the expression of a luciferase reporter.

The application of the rAAV vector core plasmid in simultaneously detecting and analyzing the infection efficiency difference of a plurality of different serotype rAAV comprises the following steps:

(1) preparing rAAV of specific serotype corresponding to the carried recognition DNA sequence by using the rAAV carrier core plasmid;

(2) mixing a plurality of different serotype rAAV viruses prepared in the step (1) in equal proportion, and infecting cells cultured in a 96-well plate;

(3) after 2 days of infection, through detecting and analyzing the recognition DNA sequence corresponding to the Cap gene of a specific AAV serotype in the infected cell or the transcription product thereof, the infection efficiency difference of a plurality of different serotype rAAV can be simultaneously evaluated.

Compared with the prior art, the invention has the following advantages and beneficial effects:

the rAAV carrier core plasmid disclosed by the invention provides a high-efficiency expression frame of a double-report fusion gene of a fluorescent protein gene and a luciferase gene and a recognition DNA sequence corresponding to a Cap gene of a specific AAV serotype by utilizing the novel rAAV carrier core plasmid through gene design and optimization, can be used for packaging almost all different serotypes of rAAV by utilizing a plurality of rAAV preparation methods, is suitable for rapid and flexible detection of the infection activity of a plurality of different serotypes of rAAV viruses in vitro tissue cell culture level and animal living body level, can simultaneously evaluate the infection efficiency difference of a plurality of different serotypes of rAAV, and can conveniently screen the AAV serotype with the best infection efficiency on a specific tissue cell from a plurality of different serotype of AAV libraries.

Drawings

FIG. 1 rAAV core vector plasmid pFD/ITR-CMV-EGFP-NanoLuc-WPRE- (CapX-ID)

-schematic construction of HGHpA.

Fig. 2 shows the purity and infection activity analysis of 3 serotype rAAV viruses obtained by packaging and purifying rAAV core vector plasmids, wherein the 3 rAAV viruses are: rAAV2-CMV-EGFP-NanoLuc-WPRE- (Cap2-ID) -HGHpA, rAAV9-CMV-EGFP-NanoLuc-WPRE- (Cap9-ID) -HGHpA and rAAV13-CMV-EGFP-NanoLuc-WPRE- (Cap13-ID) -HGHpA. A.3 kinds of rAAV virus silver staining pattern; detecting luciferase (NanoLuc) activity 2 days after HEK293T cells are infected by 3 rAAV viruses; and (3) observing by a green fluorescent protein (EGFP) fluorescence microscope after HEK293T cells are infected by C3 rAAV viruses for 2 days.

FIG. 3 virus activity assay of HEK293T cells infected with 3 serotype rAAV virus gradient dilutions. After 5-fold gradient dilution of rAAV2-CMV-EGFP-NanoLuc-WPRE- (Cap2-ID) -HGHpA virus to infect HEK293T cells for 2 days, green fluorescent protein (EGFP) fluorescence microscope observation (A) and luciferase (NanoLuc) activity detection (B); after 5-fold gradient dilution of rAAV9-CMV-EGFP-NanoLuc-WPRE- (Cap9-ID) -HGHpA virus to infect HEK293T cells for 2 days, green fluorescent protein (EGFP) fluorescence microscope observation (C) and luciferase (NanoLuc) activity detection (D);

after 5-fold gradient dilution of rAAV13-CMV-EGFP-NanoLuc-WPRE- (Cap13-ID) -HGHpA virus to infect HEK293T cells for 2 days, green fluorescent protein (EGFP) fluorescence microscope observation (E) and luciferase (NanoLuc) activity detection (F) are carried out.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1: construction of rAAV core vector plasmid pFD/CMV-EGFP-NanoLuc-WPRE- (CapX-ID) -HGHpA

First, we used the pfast. Bac. dual (pFD) shuttle vector in the insect expression system Bac to Bac as the backbone. In the embodiment, an ITR is connected with a pFD vector framework through a 5 'end connecting nucleic acid fragment and a 3' end connecting nucleic acid fragment, an ITR nucleic acid sequence of AAV type 2, such as a sequence shown in SEQ ID No.1, is selected, a broad-spectrum strong promoter CMV is used for controlling the expression of EGFP-NanoLuc double-reporter fusion gene, a post-transcriptional regulatory sequence WPRE is adopted, a polyclonal enzyme cutting site sequence (MCS) is formed by BamH1, Xba1, Afe1 and Xho1 enzyme cutting sites, and a human growth hormone polyA (HGHpA) element is adopted. In order to keep various functions of EGFP and NanoLuc genes unaffected, a connecting sequence between the EGFP and the NanoLuc genes adopts a sequence for coding flexible polypeptide (Asp-Ile-Ser-Gly-Gly). The pFD/ITR-CMV-EGFP-NanoLuc-WPRE- (MCS) -HGHpA carrier plasmid is successfully constructed and obtained by adopting a conventional molecular cloning operation method such as gene fragment artificial synthesis, PCR amplification, enzyme digestion connection and the like, as shown in figure 1. The result is correct through nucleic acid sequencing verification, and the sequence is shown as SEQ ID No. 2.

Regarding the corresponding recognition DNA sequence of the Cap gene of a specific AAV serotype, considering that the Cap gene sequence difference of different serotypes rAAV exists in the variable region exposed on the surface of the capsid protein, particularly the 587 th amino acid attachment region of the capsid protein Cap which is commonly used for surface insertion polypeptide display to obtain a plurality of AAV mutants with excellent performance (such as AAV9-PHP. eB, AAV 2-retrotype and the like). Therefore, we prefer that the 1760 th base of the Cap gene of the specific AAV serotype capsid protein is the central site, and extend 250-400bp to the 3 'end and 5' end of the central site, respectively, to serve as the recognition DNA sequence corresponding to the Cap gene of the specific AAV serotype. In this example, we selected 3 conventional natural serotypes of rAAV2, rAAV9, rAAV13 for technical validation. Wherein the recognition DNA sequence Cap2-ID of AAV2 serotype Cap gene is preferably the sequence shown in SEQ ID No. 3; a recognition DNA sequence Cap9-ID of AAV9 serotype Cap gene, preferably the sequence shown in SEQ ID No. 4; the recognition DNA sequence Cap13-ID of AAV13 serotype Cap gene is preferably the sequence shown in SEQ ID No. 5. We obtained a recognition DNA sequence fragment by PCR amplification using plasmids containing Cap2, Cap9, and Cap13 genes as templates, and then inserted into the Multiple Cloning Site (MCS) between WPRE and HGHpA by enzymatic ligation, as shown in FIG. 1. Finally, corresponding pFD/ITR-CMV-EGFP-NanoLuc-WPRE- (Cap2-ID) -HGHpA, pFD/ITR-CMV-EGFP-NanoLuc-WPRE- (Cap9-ID) -HGHpA and pFD/ITR-CMV-EGFP-NanoLuc-WPRE- (Cap13-ID) -HGHpA carrier plasmids are obtained.

Example 2: packaging purification of rAAV virus and detection of reporter gene expression after infection

The rAAV core carrier plasmid based on the pFast. Bac.Dual framework can be directly packaged to prepare rAAV by a method of transfecting HEK293 cells by the plasmid; rAAV can also be prepared by constructing recombinant Baculovirus (BEV) and infecting insect Sf9 cells with BEV. Thus, higher titers of various serotype rAAV viruses can be packaged. In this example, we used the established method of the flexible and universal OneBac system in the laboratory (refer to Wu Y et al, Current Gene Therapy,2021.21(2):167-, the silver staining detection can clearly see 3 electrophoresis bands of the corresponding capsid protein subunits (VP1, VP2, VP3) respectively, and the purity is higher, as shown in FIG. 2A.

Then, we infected HEK293T cells cultured in 96-well plates with high titers of rAAV2, rAAV9, rAAV13, respectively. After 2 days of infection, luciferase substrate is added into a 96-well plate of cultured HEK293T, and then a chemiluminescence signal generated by catalysis of the substrate is detected by using a multifunctional microplate reader, as shown in FIG. 2B, the result shows that 3 rAAV can effectively express luciferase (NanoLuc) after infection. After 2 days of infection, the HEK293T cells cultured in a 96-well plate can be observed by using a fluorescence microscope to see obvious green fluorescence, and as shown in FIG. 2C, the results show that the green fluorescent protein (EGFP) can be effectively expressed after 3 rAAV infections. This indicates that EGFP-NanoLuc double-reporter fusion genes carried by 3 rAAV can be expressed effectively and have functional activity.

Example 3: rAAV infection activity test and simultaneous detection of infection efficiency differences of various different serotypes of rAAV

We performed the test of the infectious activity on HEK293T cells for rAAV2, rAAV9 and rAAV13 with high titer (1E +13VG/ml) prepared in example 2. We performed 5-fold gradient dilution of 3 raavs with different multiplicity of infection (MOI) to infect HEK293T cells cultured in 96-well plates. 2 days after infection, the expression of EGFP and NanoLuc genes was examined, respectively. For the experimental groups of rAAV2, rAAV9, rAAV13, we can see a proportional decrease in EGFP fluorescence with gradient dilution (fig. 3A, 3C, 3E); the NanoLuc signal also decreased proportionally with gradient dilution (fig. 3B, 3D, 3F). Until a dilution of 96-well plates appeared, there were only a few EGFP-expressing HEK293 cells, while the next dilution of 96-well plates did not express EGFP-expressing HEK293 cells. The titer of the rAAV to be detected can be comprehensively calculated by counting the number of only a few HEK293 cells expressing EGFP in a 96-well plate with high dilution, the rAAV dilution factor corresponding to the 96-well plate and the volume of the initially infected rAAV, and the titer is expressed by the fluorescence focus unit (FFU/ml) of the virus. Through the detection of the expression change of EGFP and NanoLuc reporter genes corresponding to MOI infection with rAAV gradient dilution, the infection activity of rAAV virus relative to HEK293T cells can be roughly analyzed.

As for the rAAV2, the rAAV9 and the rAAV13 genomes, non-coding recognition DNA sequences (Cap2-ID, Cap9-ID and Cap13-ID) corresponding to Cap genes of respective corresponding serotypes are respectively carried. High titer (1E +13VG/ml) rAAV2, rAAV9 and rAAV13 prepared in example 2 were mixed in the same ratio, and then the HEK293T cells cultured in 96 wells were infected with the mixed virus of 3 rAAV at a certain MOI. After 2 days of infection, we could detect the expression of EGFP and NanoLuc genes. Due to the difference of activities of rAAV2, rAAV9 and rAAV13 for infecting HEK293T cells, specific primers are designed by using recognition DNA sequences (Cap2-ID, Cap9-ID and Cap13-ID), and primers aiming at rAAV2 are Cap2-ID-F and Cap2-ID-R (sequences are shown as SEQ ID No.6 and SEQ ID No. 7); primers aiming at rAAV9 are Cap9-ID-F and Cap9-ID-R (sequences are shown as SEQ ID No.8 and SEQ ID No. 9); the primers aiming at the rAAV13 are Cap13-ID-F and Cap13-ID-R (the sequences are shown as SEQ ID No.10 and SEQ ID No. 11), a nucleic acid extract of the HEK293T cell is detected and analyzed by a quantitative PCR method or a high-throughput sequencing method, and the relative content proportion of rAAV2, rAAV9 and rAAV13 in the infected HEK293T cell is about 200 by calculation: 1: 4. the method realizes the simultaneous detection of the infection efficiency difference of various different serotypes of rAAV on HEK293T cells.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Sequence listing

<110> institute of precision measurement, science and technology innovation, of the Chinese academy of sciences

<120> rAAV vector core plasmid and application thereof

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aggcatttct gtcctggctg gcgaacgagc gcaaggtttc ggtctccacg catcgtcagg 3780

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ggaccgaacc ccgcgtttat gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt 4200

ttattgccgt catagcgcgg gttccttccg gtattgtctc cttccgtgtt tcagttagcc 4260

tcccccatct cccggtacct ccggacctct gacttgagcg tcgatttttg tgatgctcgt 4320

caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct 4380

tttgctggcc ttttgctcac atgtcctgca ggcagctgcg cgctcgctcg ctcactgagg 4440

ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca gtgagcgagc 4500

gagcgcgcag agagggagtg gccaactcca tcactagggg ttcctgcggc cgcacgcgtt 4560

aggaagatct tcaatattgg ccattagcca tattattcat tggttatata gcataaatca 4620

atattggcta ttggccattg catacgttgt atctatatca taatatgtac atttatattg 4680

gctcatgtcc aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat 4740

caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg 4800

taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 4860

atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 4920

ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg 4980

acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact 5040

ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 5100

ggcagtacac caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 5160

ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 5220

gtaataaccc cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 5280

taagcagagc tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc 5340

acagttaaat tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttacgataga 5400

gtctttggat taaattgaaa ctccgaattc gctagccacc atggtgagca agggcgagga 5460

gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 5520

gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt 5580

catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 5640

cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc 5700

cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta 5760

caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 5820

gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa 5880

cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa 5940

gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 6000

ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc 6060

cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 6120

cgccgggatc actctcggca tggacgagct gtacaaggat atctccggag gtatggtctt 6180

cacactcgaa gatttcgttg gggactggcg acagacagcc ggctacaacc tggaccaagt 6240

ccttgaacag ggaggtgtgt ccagtttgtt tcagaatctc ggggtgtccg taactccgat 6300

ccaaaggatt gtcctgagcg gtgaaaatgg gctgaagatc gacatccatg tcatcatccc 6360

gtatgaaggt ctgagcggcg accaaatggg ccagatcgaa aaaattttta aggtggtgta 6420

ccctgtggat gatcatcact ttaaggtgat cctgcactat ggcacactgg taatcgacgg 6480

ggttacgccg aacatgatcg actatttcgg acggccgtat gaaggcatcg ccgtgttcga 6540

cggcaaaaag atcactgtaa cagggaccct gtggaacggc aacaaaatta tcgacgagcg 6600

cctgatcaac cccgacggct ccctgctgtt ccgagtaacc atcaacggag tgaccggctg 6660

gcggctgtgc gaacgcattc tggcgtaagt cgacgatatc aagcttatcg ataatcaacc 6720

tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 6780

gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 6840

cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 6900

tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 6960

cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 7020

ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 7080

tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt 7140

tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 7200

ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 7260

ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatgga tccaccgtct 7320

agaagcgctg ctcgagagat ctacgggtgg catccctgtg acccctcccc agtgcctctc 7380

ctggccctgg aagttgccac tccagtgccc accagccttg tcctaataaa attaagttgc 7440

atcattttgt ctgactaggt gtccttctat aatattatgg ggtggagggg ggtggtatgg 7500

agcaaggggc aagttgggaa gacaacctgt agggcctgcg gggtctattg ggaaccaagc 7560

tggagtgcag tggcacaatc ttggctcact gcaatctccg cctcctgggt tcaagcgatt 7620

ctcctgcctc agcctcccga gttgttggga ttccaggcat gcatgaccag gctcagctaa 7680

tttttgtttt tttggtagag acggggtttc accatattgg ccaggctggt ctccaactcc 7740

taatctcagg tgatctaccc accttggcct cccaaattgc tgggattaca ggcgtgaacc 7800

actgctccct tccctgtcct tctgattttg taggtaacca cgtgcggacc gagcggccgc 7860

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 7920

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 7980

gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 8040

gtatttcaca ccgcatacgt caaagcaacc atagtaacta gagcctgcag tctcgacaag 8100

cttgtcgaga agtactagag gatcataatc agccatacca catttgtaga ggttttactt 8160

gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt 8220

gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 8280

ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 8340

gtatcttatc atgtctggat ctgatcactg cttgagccta ggagatccga accagataag 8400

tgaaatctag ttccaaacta ttttgtcatt tttaattttc gtattagctt acgacgctac 8460

acccagttcc catctatttt gtcactcttc cctaaataat ccttaaaaac tccatttcca 8520

cccctcccag ttcccaacta ttttgtccgc ccacagcggg gcatttttct tcctgttatg 8580

tttttaatca aacatcctgc caactccatg tgacaaaccg tcatcttcgg ctacttt 8637

<210> 3

<211> 803

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaccaccacg cagtcaaggc ttcagttttc tcaggccgga gcgagtgaca ttcgggacca 60

gtctaggaac tggcttcctg gaccctgtta ccgccagcag cgagtatcaa agacatctgc 120

ggataacaac aacagtgaat actcgtggac tggagctacc aagtaccacc tcaatggcag 180

agactctctg gtgaatccgg gcccggccat ggcaagccac aaggacgatg aagaaaagtt 240

ttttcctcag agcggggttc tcatctttgg gaagcaaggc tcagagaaaa caaatgtgga 300

cattgaaaag gtcatgatta cagacgaaga ggaaatcagg acaaccaatc ccgtggctac 360

ggagcagtat ggttctgtat ctaccaacct ccagagaggc aacagacaag cagctaccgc 420

agatgtcaac acacaaggcg ttcttccagg catggtctgg caggacagag atgtgtacct 480

tcaggggccc atctgggcaa agattccaca cacggacgga cattttcacc cctctcccct 540

catgggtgga ttcggactta aacaccctcc tccacagatt ctcatcaaga acaccccggt 600

acctgcgaat ccttcgacca ccttcagtgc ggcaaagttt gcttccttca tcacacagta 660

ctccacggga caggtcagcg tggagatcga gtgggagctg cagaaggaaa acagcaaacg 720

ctggaatccc gaaattcagt acacttccaa ctacaacaag tctgttaatg tggactttac 780

tgtggacact aatggcgtgt att 803

<210> 4

<211> 803

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

aacggttctg gacagaatca acaaacgcta aaattcagtg tggccggacc cagcaacatg 60

gctgtccagg gaagaaacta catacctgga cccagctacc gacaacaacg tgtctcaacc 120

actgtgactc aaaacaacaa cagcgaattt gcttggcctg gagcttcttc ttgggctctc 180

aatggacgta atagcttgat gaatcctgga cctgctatgg ccagccacaa agaaggagag 240

gaccgtttct ttcctttgtc tggatcttta atttttggca aacaaggaac tggaagagac 300

aacgtggatg cggacaaagt catgataacc aacgaagaag aaattaaaac tactaacccg 360

gtagcaacgg agtcctatgg acaagtggcc acaaaccacc agagtgccca agcacaggcg 420

cagaccggct gggttcaaaa ccaaggaata cttccgggta tggtttggca ggacagagat 480

gtgtacctgc aaggacccat ttgggccaaa attcctcaca cggacggcaa ctttcaccct 540

tctccgctga tgggagggtt tggaatgaag cacccgcctc ctcagatcct catcaaaaac 600

acacctgtac ctgcggatcc tccaacggcc ttcaacaagg acaagctgaa ctctttcatc 660

acccagtatt ctactggcca agtcagcgtg gagatcgagt gggagctgca gaaggaaaac 720

agcaagcgct ggaacccgga gatccagtac acttccaact attacaagtc taataatgtt 780

gaatttgctg ttaatactga agg 803

<210> 5

<211> 803

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ggaactcagc agtctcggct actgtttagc caagctggac ccaccagtat gtctcttcaa 60

gctaaaaact ggctgcctgg accttgctac agacagcagc gtctgtcaaa gcaggcaaac 120

gacaacaaca acagcaactt tccctggact ggtgccacca aatatcatct gaatggccgg 180

gactcattgg tgaacccggg ccctgctatg gccagtcaca aggatgacaa agaaaagttt 240

ttccccatgc atggaaccct gatatttggt aaagaaggaa caaatgccaa caacgcggat 300

ttggaaaatg tcatgattac agatgaagaa gaaatccgca ccaccaatcc cgtggctacg 360

gagcagtacg ggactgtgtc aaataatttg caaaactcaa acgctggtcc aactactgga 420

actgtcaatc accaaggagc gttacctggt atggtgtggc aggatcgaga cgtgtacctg 480

cagggaccca tttgggccaa gattcctcac accgatggac actttcatcc ttctccactg 540

atgggaggtt ttgggctcaa acacccgcct cctcagatca tgatcaaaaa cactcccgtt 600

ccagccaatc ctcccacaaa ctttagtgcg gcaaagtttg cttccttcat cacacagtac 660

tccacggggc aggtcagcgt ggagatcgag tgggagctgc agaaggagaa cagcaaacgc 720

tggaatcccg aaattcagta cacttccaac tacaacaaat ctgttaatgt ggactttact 780

gtggacacta atggtgtgta ttc 803

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cacacaaggc gttcttccag gc 22

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttgccgcact gaaggtggtc ga 22

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aaccaaggaa tacttccggg ta 22

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttgtccttgt tgaaggccgt tgg 23

<210> 10

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcaccaagga gcgttacctg gt 22

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ttgccgcact aaagtttgtg gg 22

Claims (8)

1. A rAAV vector core plasmid is characterized in that a CMV promoter, a double-report fusion gene of a fluorescent protein gene and a luciferase gene, a post-transcriptional regulatory sequence WPRE, a human growth hormone polyA element and a specific AAV serotype capsid protein inserted between the WPRE and the polyA element are sequentially integrated in an rAAV vector core plasmid expression elementCapThe corresponding recognition DNA sequence of the gene.

2. The rAAV vector core plasmid according to claim 1, wherein the rAAV vector core plasmid is a recombinant vectorThe specific AAV serotype capsid proteinsCapThe corresponding recognition DNA sequence of the gene is specifically as follows: capsid proteins from specific AAV serotypesCapThe 1760 th base of the gene is the central site, and the 3 'end and the 5' end of the central site are respectively extended by 250-400 bp.

3. The rAAV vector core plasmid of claim 1, wherein AAV2 serotype isCaThe identification DNA sequence of the p gene is shown as SEQ ID No. 3; AAV9 serotypeCapThe identification DNA sequence of the gene is shown as SEQ ID No. 4; AAV13 serotypeCapThe gene recognition DNA sequence is shown in SEQ ID No. 5.

4. The rAAV vector core plasmid according to claim 1, wherein the vector backbone is a pfast. bac. dual shuttle plasmid.

5. Use of the rAAV vector core plasmid of any one of claims 1 to 4 in the detection of infectious activity of different serotype raavs.

6. Use according to claim 5, characterized in that it comprises the following steps:

(1) preparing rAAV of a specific serotype corresponding to the recognition DNA sequence carried by the rAAV vector core plasmid of claim 1;

(2) diluting the specific serotype rAAV virus prepared in the step (1) according to a certain proportion in a gradient manner, and then infecting cells cultured in a 96-well plate;

(3) after 2 days of infection, the infectious activity of rAAV was determined by observing the expression of a fluorescent protein reporter, or detecting the expression of a luciferase reporter.

7. Use of the rAAV vector core plasmid of any one of claims 1 to 4 for simultaneous detection and analysis of differences in infection efficiency of a plurality of different serotype raavs.

8. Use according to claim 7, characterized in that it comprises the following steps:

(1) preparing rAAV of a specific serotype corresponding to the recognition DNA sequence carried by the rAAV vector core plasmid of claim 1;

(2) mixing a plurality of different serotype rAAV viruses prepared in the step (1) in equal proportion, and infecting cells cultured in a 96-well plate;

(3) 2 days after infection, by targeting specific AAV serotypes in infected cellsCapThe identification DNA sequence corresponding to the gene or the transcription product thereof is detected and analyzed in abundance, so that the infection efficiency difference of various different serotype rAAV can be evaluated simultaneously.

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