CN114231564B - rAAV vector core plasmid and application thereof - Google Patents

Abstract

The invention discloses a rAAV vector core plasmid and application thereof, wherein the rAAV vector core plasmid mainly comprises a double-report fusion gene of a fluorescent protein gene and a luciferase gene, and a specific AAV serotypeCapA recognition DNA sequence corresponding to the gene. The rAAV vector core plasmid is utilized to pack rAAV infected cells, and the expression of fluorescent protein or luciferase reporter genes is detected, so that the infection activity of a plurality of different serotypes of rAAV is rapidly determined; in addition, it is also possible to treat a specific AAV serotype in infected cellsCapThe identification DNA sequence or transcription product corresponding to the gene is detected and analyzed in abundance, and is used for evaluating the infection efficiency difference of various serotypes of rAAV.

Description

rAAV vector core plasmid and application thereof

Technical Field

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

Background

The recombinant adeno-associated virus vector (rAAV) is modified by wild adeno-associated virus (AAV), the rAAV genome only comprises Terminal Repeated Sequences (ITRs) at two ends of the AAV, and the exogenous gene is contained to completely replace the encoding gene of the virus. rAAV can be prepared by trans-compensating the replication gene Rep, the structural gene Cap and the packaging auxiliary gene of AAV in a packaging cell line. Because of the characteristics of high safety, low immunogenicity, wide host range, multiple virus serotypes, capability of infecting dividing and non-dividing cells, capability of mediating long-term stable expression of exogenous genes in animals, and the like, the virus 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, there are 3 gene rAAV gene therapeutic drugs on the market.

Currently, AAV isolated from nature has predominantly 13 serotypes and more than 100 mutant subtypes. The diversity of AAV viral capsid proteins (caps) of different serotypes determines AAV infectivity differences. These infection characteristics involve adsorption and entry of AAV-infected cells, intracellular transport, viral capsid removal within the nucleus, and transgene expression. AAV-specific receptors have been found on the surface of some cells, such as: heparan Sulfate Proteoglycan (HSPG), integrins, and the like. The differences in the capsid proteins of AAV of different serotypes and their cell surface receptors have demonstrated a variety of different infection efficiencies, chemotaxis and immunological properties of tissues or cell types. By mutating AAV capsid protein Cap gene, several rAAV vectors with excellent new characteristics can be obtained. For example: AAV-DJ type with remarkably improved transduction activity in liver, AAV9-PHP.eB type capable of efficiently crossing the brain blood brain barrier of mice, etc.

Determining the infection activity of the rAAV of different serotypes and the difference of the infection efficiency of the rAAV on different tissue cells is very important for better application. The packaging and purifying method process, production batch, storage conditions and the like can greatly influence the infection activity of the rAAV virus vector, and the total particle number of the rAAV vector is less influenced by the storage conditions and the like. Currently, the physical titer (viral particle concentration) of a widely used rAAV is typically determined by fluorescence real-time quantitative PCR (Q-PCR) methods, whereby the rAAV viral particles contain only a single copy of genomic DNA and thus the physical titer of the rAAV can be defined as the copy number per milliliter of rAAV viral genome, i.e., (vector genome per ml, vg/ml). Infection activity of rAAV is often expressed in terms of transduction titer (Transducing titer in TU/ml) or infection titer (IU/ml). The method for measuring the infection activity of the rAAV2 standard substance is to infect a Hela RC32 cell line integrating Cap and Rep genes of AAV2 with a gradient diluted rAAV sample under the auxiliary infection of adenovirus type 5 (AdV 5), and the method is to obtain the AAV genome copy number calculation TCID50 by observing the expression of a fluorescent protein reporter gene or quantitative PCR (refer to Lock M et al, 2010Human Gene Therapy,21 (10): 1273-85). The method requires a special Hela RC32 cell line and auxiliary infection of type 5 adenovirus with a certain safety risk, and is difficult to popularize in common. In recent years, some researchers have also used luciferase (Luc) carried in the rAAV genome to determine the infection activity of rAAV, but often limited to the detection of the activity of a few serotypes of rAAV, which is not highly versatile and the data obtained lacks comparability.

Therefore, a novel method suitable for detecting the infection activity of the rAAV with various serotypes, multiple parameters and comparability is developed, and the method has important application value for the quality control of the rAAV products and the rAAV in gene therapy basis and clinical research.

Disclosure of Invention

Aiming at the defects or improvement demands of the prior art, the novel rAAV vector core plasmid provided by the invention contains a dual-report fusion gene of a fluorescent protein gene and a luciferase gene and a sexual DNA sequence corresponding to a specific AAV serotype Cap gene, can be used for rapidly measuring the infection activities of a plurality of different serotypes of rAAV by detecting the expression of the fluorescent protein gene or the luciferase reporter gene, and can also evaluate the infection efficiency difference of a plurality of different serotypes of rAAV simultaneously.

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

a rAAV vector core plasmid, wherein a rAAV vector core plasmid expression element integrates a CMV promoter, a dual-reporter fusion gene of a fluorescent protein gene and a luciferase gene in sequence, a post-transcriptional regulatory sequence WPRE, a human growth hormone polyA element, and a recognition DNA sequence corresponding to a specific AAV serotype capsid protein Cap gene interposed between the WPRE and the human growth hormone polyA element. The rAAV vector core plasmid efficiently expresses a double-report fusion gene containing fluorescent protein genes and luciferase genes through a broad-spectrum strong promoter CMV. NanoLuc is a novel luciferase developed in recent years, and has brighter luminescence and smaller molecular weight (only contains 171 amino acids and has the molecular weight of 19.1 kDa) compared with other luciferases, is one of bioluminescence reporter genes with the best performance at present, and is connected with the C end of a green fluorescent protein (EGFP) gene through a connecting sequence, so that an EGFP-NanoLuc double-reporter fusion gene with the advantages of the two genes is obtained, the expression of EGFP can be observed through a fluorescence microscope, and the expression of NanoLuc can be quantitatively detected through a luciferase detector, so that the novel luciferase is suitable for tissue cells cultured in vitro and is also suitable for animal living body detection. The expression level of the dual-reporter fusion gene was further increased by the human growth hormone polyA (HGHpA) element using the post-transcriptional regulatory sequence WPRE. In particular, the recognition DNA sequence corresponding to the specific AAV serotype Cap gene can be flexibly introduced between the WPRE and HGHpA elements through a polyclonal enzyme cutting site (MCS).

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

Preferably, the pFast.Bac.Dual shuttle plasmid is used as the vector backbone. The rAAV can be prepared by directly packaging HEK293 cells through a plasmid transfection method, or by constructing recombinant Baculovirus (BEV) and using the method of infecting insect Sf9 cells through the BEV.

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

(1) Preparing rAAV of a specific serotype corresponding to the carrying recognition DNA sequence by using the rAAV vector core plasmid;

(2) Infecting cells cultured in a 96-well plate with the specific serotype rAAV prepared in the step (1) after gradient dilution according to a certain proportion;

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

The application of the rAAV vector core plasmid in simultaneous detection and analysis of infection efficiency differences of a plurality of different serotypes of rAAV comprises the following steps:

(1) Preparing rAAV of a specific serotype corresponding to the carrying recognition DNA sequence by using the rAAV vector core plasmid;

(2) Mixing the plurality of different serotypes of 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, the difference of infection efficiency of a plurality of different serotypes of rAAV can be evaluated simultaneously by detecting and analyzing the recognition DNA sequences corresponding to specific AAV serotypes of Cap genes or the transcription products thereof in infected cells.

Compared with the prior art, the invention has the following advantages:

the rAAV vector core plasmid disclosed by the invention provides an efficient 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 specific AAV serotype Cap gene by utilizing gene design and optimization, can package rAAV of almost all serotypes by utilizing a plurality of rAAV preparation methods, is suitable for rapid and flexible detection of infection activity of rAAV viruses of different serotypes on in-vitro tissue cell culture level and animal living body level, can evaluate infection efficiency difference of rAAVs of different serotypes simultaneously, and can conveniently screen AAV serotypes with optimal infection efficiency on specific tissue cells from AAV virus libraries of different serotypes.

Drawings

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

-schematic construction of HGHpA.

FIG. 2 analysis of purity and infection activity of 3 serotypes of rAAV obtained by rAAV core vector plasmid packaging and purification, wherein the 3 rAAV are respectively as follows: rAAV2-CMV-EGFP-nanoLuc-WPRE- (Cap 2-ID) -HGHpA, rAAV9-CMV-EGFP-nanoLuc-WPRE- (Cap 9-ID) -HGHpA and rAAV13-CMV-EGFP-nanoLuc-WPRE- (Cap 13-ID) -HGHpA. Silver staining patterns of 3 rAAV viruses; 2 days after infection of HEK293T cells by 3 rAAV viruses, luciferase (nanoLuc) activity is detected; after infection of HEK293T cells with the C.3 rAAV viruses for 2 days, the cells were observed with a green fluorescent protein (EGFP) fluorescent microscope.

FIG. 3 viral activity assay for HEK293T cells infected with gradient dilutions of 3 serotypes of rAAV virus. After HEK293T cells are infected by rAAV2-CMV-EGFP-nanoLuc-WPRE- (Cap 2-ID) -HGHpA virus in a 5-fold gradient dilution manner for 2 days, green fluorescent protein (EGFP) is observed by a fluorescent microscope (A), and luciferase (nanoLuc) activity is detected (B); rAAV9-CMV-EGFP-nanoLuc-WPRE- (Cap 9-ID) -HGHpA virus 5-fold gradient dilution HEK293T cells were infected for 2 days, green fluorescent protein (EGFP) fluorescence microscopy (C), luciferase (nanoLuc) activity detection (D);

rAAV13-CMV-EGFP-nanoLuc-WPRE- (Cap 13-ID) -HGHpA virus 5-fold gradient infection HEK293T cells 2 days later, green fluorescent protein (EGFP) fluorescence microscopy (E), luciferase (nanoLuc) activity assay (F).

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

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

First, we utilized the pfast.bac.dual (pFD) shuttle vector in insect expression system Bac to Bac as a backbone. In the embodiment, ITR is connected with a pFD vector skeleton through a 5 '-end connecting nucleic acid fragment and a 3' -end connecting nucleic acid fragment, an ITR nucleic acid sequence of type 2 AAV is selected, a sequence shown as SEQ ID No.1 is adopted, a broad-spectrum strong promoter CMV is adopted to control the expression of EGFP-NanoLuc double-report fusion gene, a post-transcriptional regulatory sequence WPRE is adopted, and a polyclonal enzyme cutting site sequence (MCS) consisting of BamH1, xba1, afe1 and Xho1 enzyme cutting sites is adopted, and then a human growth hormone polyA (HGHpA) element is adopted. In order to keep the functions of EGFP and nanoLuc genes unaffected, the junction sequence between them employs a sequence encoding a flexible polypeptide (Asp-Ile-Ser-Gly-Gly). The pFD/ITR-CMV-EGFP-NanoLuc-WPRE- (MCS) -HGHpA vector plasmid is successfully constructed by adopting the conventional molecular cloning operation methods such as gene fragment artificial synthesis, PCR amplification, enzyme digestion connection and the like, and is shown in figure 1. The result is correct through the verification of nucleic acid sequencing, and the sequence is shown as SEQ ID No. 2.

For the recognition DNA sequence corresponding to the Cap gene of a specific AAV serotype, the differences of the Cap gene sequences of the rAAVs of different serotypes are considered to exist in a variable region exposed on the surface of capsid protein, and particularly, the 587 amino acid accessory region of the capsid protein Cap of AAV mutants (such as AAV9-PHP.eB, AAV 2-retrotype and the like) with excellent performances is obtained by commonly used surface insertion polypeptide display. Therefore, we prefer the 1760 base of the specific AAV serotype capsid gene Cap as the central site, and extend 250-400bp to the 3 'end and 5' end of the central site, respectively, to be used as the recognition DNA sequence corresponding to the specific AAV serotype Cap gene. In this example, we selected 3 conventional natural serotypes 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; the recognition DNA sequence Cap9-ID of AAV9 serotype Cap gene is 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 used plasmids containing Cap2, cap9 and Cap13 genes as templates, amplified by PCR to obtain a recognition DNA sequence fragment, and then inserted into a Multiple Cloning Site (MCS) between WPRE and HGHpA by enzyme ligation, as shown in FIG. 1. Finally, the corresponding pFD/ITR-CMV-EGFP-nanoLuc-WPRE- (Cap 2-ID) -HGHpA, pFD/ITR-CMV-EGFP-nanoLuc-WPRE- (Cap 9-ID) -HGHpA and pFD/ITR-CMV-EGFP-nanoLuc-WPRE- (Cap 13-ID) -HGHpA vector plasmids were obtained.

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

rAAV core carrier plasmid based on pFast.Bac.Dual skeleton can be directly packaged and prepared by a method of transfecting HEK293 cells with the plasmid; rAAV can also be prepared by constructing recombinant Baculoviruses (BEVs) by infecting insect Sf9 cells with the BEVs. Thus, higher titers of various serotypes of rAAV virus can be packaged. In this example we used the method of the new and versatile OneBac system established in this laboratory (cf. Wu Y et al, current Gene Therapy,2021.21 (2): 167-176.) to construct the 3 serotypes pFD/ITR-CMV-EGFP-nanoLuc-WPRE- (Cap 2-ID) -HGHpA, pFD/ITR-CMV-EGFP-nanoLuc-WPRE- (Cap 9-ID) -HGHpA and pFD/ITR-CMV-EGFP-nanoLuc-WPRE- (Cap 13-ID) -HGHpA vector plasmids based on the construction of the 3 serotypes obtained in example 1, then infection of suspension cultured Sf9 cells to encapsulate rAAV, and then using the operations of iodixanol ultracentrifugation purification to prepare the corresponding rAAV2, rAAV9, rAAV13 viruses, the titres of which can reach 1E+13/ml, and the silver staining detection can see the corresponding capsid protein subunits (1, 3, VP 3) with higher purity than VP2, as shown in FIG. 3.

Then, we infected HEK293T cells cultured in 96-well plates with high titers of rAAV2, rAAV9, rAAV13, respectively. After 2 days of infection, we added luciferase substrate to 96-well plates of HEK293T in culture, and then detected strong chemiluminescent signals generated by substrate catalysis using a multifunctional microplate reader, as shown in fig. 2B, and the results indicate that 3 rAAV infections can all be followed by efficient expression of luciferase (NanoLuc). After 2 days of infection, we observed 96-well plate cultured HEK293T cells with fluorescence microscopy, all of which can see significant green fluorescence, as shown in fig. 2C, and the results indicate that 3 rAAV's can efficiently Express Green Fluorescent Protein (EGFP) after infection. This shows that EGFP-NanoLuc double-report fusion genes carried by 3 rAAVs can be effectively expressed and have functional activity.

Example 3: rAAV infection Activity assay and simultaneous detection of infection efficiency differences in multiple different serotypes of rAAV

We tested the high titers (1E+13VG/ml) of rAAV2, rAAV9, rAAV13 prepared in example 2 for infection activity on HEK293T cells. We infected HEK293T cells cultured in 96-well plates with 5-fold gradient dilutions of 3 rAAVs at the unused multiplicity of infection (MOI), respectively. 2 days after infection, the expression of EGFP and nanoLuc genes was examined, respectively. For the rAAV2, rAAV9, rAAV13 experimental groups, we can see a proportional decrease in EGFP fluorescence with gradient dilution (fig. 3a,3c,3 e); the NanoLuc signal also decreased proportionally with the gradient dilution (fig. 3b,3d,3 f). Only a few HEK293 cells expressing EGFP in 96-well plates at one dilution were present, while HEK293 cells expressing EGFP were not present in 96-well plates at the next dilution. The titer of the rAAV to be detected can be comprehensively calculated by counting the number of HEK293 cells expressing EGFP in a certain 96-well plate with high dilution and the dilution times of rAAV corresponding to the 96-well plate and the volume of the initial infection rAAV, and the titer is expressed by the fluorescence focus unit (FFU/ml) of the virus. By detecting the expression change of EGFP and nanoLuc reporter genes corresponding to the rAAV gradient dilution infection MOI, the infection activity of the rAAV relative to HEK293T cells can be approximately analyzed.

Whereas the rAAV2, rAAV9, rAAV13 genomes carry non-coding, recognition DNA sequences (Cap 2-ID, cap9-ID, cap 13-ID) corresponding to the respective serotype Cap genes, respectively. We mixed rAAV2, rAAV9, rAAV13 of high titer (1E+13VG/ml) prepared in example 2 in the same ratio, and then infected HEK293T cells cultured in 96 wells with the mixed virus of 3 rAAVs at a certain MOI. 2 days after infection we can detect the expression of EGFP and nanoLuc genes. Because of the difference of activity of rAAV2, rAAV9 and rAAV13 in infecting HEK293T cells, specific primers are designed by utilizing recognition DNA sequences (Cap 2-ID, cap9-ID and Cap 13-ID), and the primers aiming at rAAV2 are Cap2-ID-F and Cap2-ID-R (the sequences are shown as SEQ ID No.6 and SEQ ID No. 7); primers for rAAV9 are Cap9-ID-F and Cap9-ID-R (sequences are shown as SEQ ID No.8 and SEQ ID No. 9); the primers for rAAV13 are Cap13-ID-F and Cap13-ID-R (the sequences are shown as SEQ ID No.10 and SEQ ID No. 11), the nucleic acid extract of HEK293T cells is detected and analyzed by a quantitative PCR method or a high-throughput sequencing method, and the relative content ratio of rAAV2, rAAV9 and rAAV13 in the HEK293T cells after infection can be calculated to be about 200:1:4. the method realizes the simultaneous detection of infection efficiency difference of various serotypes of rAAV on HEK293T cells.

It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.

Sequence listing

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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 (6)

1. A rAAV vector core plasmid is characterized in that a CMV promoter, a dual-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 recognition DNA sequence corresponding to an AAV2 or AAV9 or AAV13 serotype capsid protein Cap gene inserted between the WPRE and the polyA element are sequentially integrated into a rAAV vector core plasmid expression element, wherein the recognition DNA sequence of the AAV2 serotype Cap gene is shown as SEQ ID No.3, the recognition DNA sequence of the AAV9 serotype Cap gene is shown as SEQ ID No.4, and the recognition DNA sequence of the AAV13 serotype Cap gene is shown as SEQ ID No. 5.

2. The rAAV vector core plasmid according to claim 1, wherein the pfast. Bac. Dual shuttle plasmid is used as a vector backbone.

3. Use of the rAAV vector core plasmid of claim 1 or 2 for detecting the infectious activity of rAAV of different serotypes.

4. The use according to claim 3, characterized by the following steps:

(1) Preparing and obtaining rAAV of a specific serotype corresponding to the sequence carrying the recognition DNA by using the rAAV vector core plasmid of claim 1;

(2) Infecting cells cultured in a 96-well plate with the specific serotype rAAV prepared in the step (1) after gradient dilution according to a certain proportion;

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

5. Use of the rAAV vector core plasmid of claim 1 or 2 for simultaneous detection and analysis of differences in infection efficiency of a plurality of different serotypes of rAAV.

6. The use according to claim 5, characterized by the steps of:

(1) Preparing and obtaining rAAV of a specific serotype corresponding to the sequence carrying the recognition DNA by using the rAAV vector core plasmid of claim 1;

(2) Mixing the several different serotypes of rAAV virus prepared in step (1) in equal proportion, and infecting cells cultured in a 96-well plate;

(3) After 2 days of infection, the difference of infection efficiency of a plurality of different serotypes of rAAV can be evaluated simultaneously by detecting and analyzing the recognition DNA sequences corresponding to specific AAV serotypes of Cap genes or the transcription products thereof in infected cells.

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