CN117143203A - Capsid protein mutants that can improve the ability of AAV virus to infect the whole eye and its applications - Google Patents
Capsid protein mutants that can improve the ability of AAV virus to infect the whole eye and its applications Download PDFInfo
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Abstract
本发明公开了可提高AAV病毒全眼感染能力的衣壳蛋白突变体及其应用。发明人研究发现使用特定的肽替换野生型AAV2病毒衣壳蛋白的561至588位氨基酸，可以有效提高AAV病毒全眼感染能力，从根本上解决AAV2对眼睛感染效率的问题。
The invention discloses a capsid protein mutant that can improve the ability of AAV virus to infect the whole eye and its application. The inventor's research found that using specific peptides to replace amino acids 561 to 588 of the wild-type AAV2 virus capsid protein can effectively improve the ability of AAV virus to infect the whole eye and fundamentally solve the problem of AAV2's efficiency in eye infection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a capsid protein mutant capable of improving the full-eye infection capacity of AAV virus.
Background
Adeno-associated virus (AAV) is a common vector for gene therapy delivery, and the principle is that the sequence between ITRs of AAV genome is replaced by a target gene sequence by a genetic engineering method, and the target gene is delivered to target cells through cell infection, so as to achieve the purpose of gene therapy. Based on the characteristics of safety, high efficiency, stability, persistence, specificity, low integration and the like of recombinant AAV, AAV becomes one of main delivery means in the field of gene therapy. In the course of gene therapy, high dose, high purity AAV virus is often required for in vivo injection in animals, and high AAV production costs become one of the bottlenecks in the course of gene therapy.
Along with the maturation of downstream development techniques for gene therapy, limitations in upstream AAV production throughput become more and more apparent. To solve this problem, the existing strategies focus on the following two aspects, namely, optimizing the existing AAV production process and improving the yield; secondly, the AAV capsid protein is modified, so that the targeting and the infection capability of the AAV capsid protein to specific tissues are improved, and the AAV capsid protein can be used for realizing lower and safer administration dosage. The AAV production process is more in that the aim of improving the virus yield is achieved by optimizing the plasmid proportion, the transfection reagent proportion, the cell culture condition, the amplified production specification and the like, and the AAV production process does not involve the transformation of AAV. Based on the structural characteristics of AAV viruses, the viral properties such as tissue targeting, immunogenicity, packaging yield, etc., are mainly determined by the surface capsid proteins. Previous studies showed that mutation at amino acid positions 561 to 588 of Cap2 can optimize the infection ability to mouse eye tissue. However, it remains unknown which mutations can increase the full-eye infectivity of AAV viruses.
Disclosure of Invention
The present invention aims to overcome at least one of the disadvantages of the prior art and provide capsid protein mutants that can enhance the full-eye infection capacity of AAV viruses.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided:
capsid protein mutant capable of improving full-eye infection ability of AAV virus, which is obtained by using one of the following polypeptides to replace 561 to 588 amino acids of wild AAV virus capsid protein:
1)DEHEIKTTNPVATEGYGEVATNWQRGNR
2)DEEEIRTTNPVATEQYGSVSTNLQRGNTGRSAGLGTGLSR
3)DEQEIAATNPVATEQYGSVSTNLQRGNR
4)DEEEIRTTNPVATEQYGSVSTNLQRGNTGGMVLVSAKSGLSR
5)DEEEIRTTNPVATEQYGSVSTNLQRGNTGGRILVATTGLSR
6)DEEEIRTTNPVATEQYGSVSTNLQRGNTGPLLDGTKGLSR
7)DEEEIRTTNPVATEQYGSVSTNLQRGNTGSSPIKDGTKGLSR。
in some examples of capsid protein mutants, the amino acid sequence of the wild-type AAV2 viral capsid protein is as set forth in SEQ ID No.: 1.
In a second aspect of the invention, there is provided:
a gene encoding a mutant capsid protein according to the first aspect of the invention.
In some examples of genes, codon optimization is performed according to the expression system.
In a third aspect of the invention, there is provided:
an expression system into which the gene according to the second aspect of the present invention is inserted.
In some examples of expression systems, the expression system is a recombinant AAV vector.
In some examples of expression systems, the AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, and variants thereof.
In a fourth aspect of the invention, there is provided:
a composition comprising an expression system according to the third aspect of the invention.
In some examples of compositions, adjuvants are also added.
In a fifth aspect of the invention, there is provided:
the use of a composition according to the fourth aspect of the invention in the preparation of a gene therapy formulation or a transgene formulation.
In some examples of applications, the methods are used to prepare eye-targeted gene therapy formulations or transgenic formulations.
In a fifth aspect of the invention, there is provided:
a method of constructing a transgenic animal model comprising introducing into an animal a composition according to the fourth aspect of the invention.
In some examples of the construction methods, the construction of transgenic animal models for ocular diseases is used.
The beneficial effects of the invention are as follows:
the capsid protein mutants of some examples of the invention can effectively improve the full-eye infection capacity of AAV virus, and fundamentally solve the problem of the infection efficiency of AAV2 to eyes.
Drawings
FIG. 1 is a diagram showing agarose gel electrophoresis identification of mutant plasmids constructed in the test examples of the present invention.
FIG. 2 is a diagram showing the agarose gel electrophoresis identification of the expression plasmid pAAV-CAG-EGFP-barmod-HGH constructed in the test example of the present invention.
FIG. 3 is a map of pAAV2-Rep2/Cap2 packaging plasmid in an example of the present invention.
FIG. 4 is a map of pAAV2-7M8-Rep2/Cap2-7M8 packaging plasmid in an example of the present invention.
FIG. 5 is a map of pAAV2-Mut-Rep2/Cap2-Mut packaging plasmid constructed in the examples of the present invention.
FIG. 6 is a diagram of pAAV-CAG-EGFP-barmod-HGH expression plasmid of a virus packaging three plasmid system in an embodiment of the present invention.
FIG. 7 is a pHelper helper plasmid map of a three plasmid viral packaging system in an embodiment of the present invention.
FIG. 8 shows the results of the performance of the adenovirus and wild type virus cynomolgus monkey infection prepared by the protocol of the test examples of the present invention.
FIG. 9 is an alignment of wild-type, 7M8 and AAV 2-EYE-01-EYE-07 amino acid sequences according to an embodiment of the invention.
Detailed Description
In a first aspect of the invention, there is provided:
capsid protein mutant capable of improving full-eye infection ability of AAV virus, which is obtained by using one of the following polypeptides to replace 561 to 588 amino acids of wild AAV virus capsid protein:
1)DEHEIKTTNPVATEGYGEVATNWQRGNR
2)DEEEIRTTNPVATEQYGSVSTNLQRGNTGRSAGLGTGLSR
3)DEQEIAATNPVATEQYGSVSTNLQRGNR
4)DEEEIRTTNPVATEQYGSVSTNLQRGNTGGMVLVSAKSGLSR
5)DEEEIRTTNPVATEQYGSVSTNLQRGNTGGRILVATTGLSR
6)DEEEIRTTNPVATEQYGSVSTNLQRGNTGPLLDGTKGLSR
7)DEEEIRTTNPVATEQYGSVSTNLQRGNTGSSPIKDGTKGLSR。
wild type AAV viral capsid proteins have similar structures and, after the same modification, have the same or similar functions.
In some examples of capsid protein mutants, the amino acid sequence of the wild-type AAV2 viral capsid protein is as set forth in SEQ ID No.: 1. Experimental data show that after the wild AAV2 virus capsid protein is modified, the full-eye infection capability of AAV virus can be effectively improved.
In a second aspect of the invention, there is provided:
a gene encoding a mutant capsid protein according to the first aspect of the invention.
In some examples of genes, codon optimization is performed according to the expression system.
In a third aspect of the invention, there is provided:
an expression system into which the gene according to the second aspect of the present invention is inserted.
In some examples of expression systems, the expression system is a recombinant AAV vector.
In some examples of expression systems, the AAV is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, and variants thereof.
In a fourth aspect of the invention, there is provided:
a composition comprising an expression system according to the third aspect of the invention.
In some examples of compositions, adjuvants are also added. The auxiliary materials are the auxiliary materials commonly used in AAV expression systems.
In a fifth aspect of the invention, there is provided:
the use of a composition according to the fourth aspect of the invention in the preparation of a gene therapy formulation or a transgene formulation.
In some examples of applications, the methods are used to prepare eye-targeted gene therapy formulations or transgenic formulations.
In a fifth aspect of the invention, there is provided:
a method of constructing a transgenic animal model comprising introducing into an animal a composition according to the fourth aspect of the invention.
In some examples of the construction methods, the construction of transgenic animal models for ocular diseases is used.
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Examples
This example provides a variety of adeno-associated virus variants (AAV 2-EYE-01 to AAV 2-EYE-07) based on AI design and previous studies, the preparation of which comprises the steps of:
1. plasmid construction
Cloning the synthesized sequence into pAAV2-Rep2/Cap2 plasmid by gene synthesis (Jin Weizhi biotechnology Co., ltd.) of AAV 2-EYE-01-EYE-07 amino acid sequence (shown in Table 1) corresponding nucleic acid sequence, and replacing Cap2 561-588 sequence with mutant sequence to construct pAAV-mutant plasmid vector; the ligation product is transformed into escherichia coli competent Stbl3, single colony extracted plasmids are selected for sequencing verification and enzyme digestion verification, and agarose gel electrophoresis identification is carried out, and the result is shown in figure 1, so that the mutant plasmids are successfully constructed.
The nucleic acid sequence of Barcode41-Barcode49 (shown in table 2) is synthesized by gene synthesis (Jin Weizhi biotechnology Co., ltd.), the synthesized sequence is cloned into pAAV-CAG-EGFP-hGH plasmid, a nucleic acid sequence with the length of 15bp is inserted between EGFP protein stop codon TAA and hGH-polyA, pAAV-CAG-EGFP-Barcode-hGH plasmid is constructed, the connection product is transformed, single colony is picked, plasmid extraction is sequenced and enzyme digestion verification is carried out, agarose gel electrophoresis identification is carried out, and if the construction of the Barcode plasmid is successful as shown in figure 2.
2. AAV2-WT, AAV mutant-Barcode virus library packaging
AAV2-WT or AAV 2-mutant packaging plasmid, together with pAAV-CAG-EGFP-Barcode-hGH expression plasmid and pHelper helper plasmid three plasmids, were co-transfected into suspension HEK293T cells. The packaging plasmid corresponds one-to-one to the unique Barcode (Barcode sequence) pAAV-CAG-EGFP-Barcode-hGH expression plasmid as shown in Table 3. AAV2-WT, AAV 2-mutant, pAAV-CAG-EGFP-Barcode-hGH and pHelper plasmid maps are shown in FIGS. 3-7, respectively.
The virus package comprises the following steps:
A. cell culture
(1) Taking out a suspension 293T cell from a liquid nitrogen tank, placing into a water bath at 37 ℃ for rapid shaking to defrost the cell, adding into a centrifuge tube filled with 5ml of Wayne 293 serum-free medium, centrifuging for 5min at 200 Xg, discarding the supernatant, re-suspending into 10ml of Wayne 293 serum-free medium, placing into a 125ml triangular shake flask, supplementing the Wayne 293 serum-free medium to 20ml, and adding 5% CO at 120rpm and 37 DEG C 2 Culturing in medium;
(2) After 48-72 hours of culture, the cell density reaches 2-3E+6cell/ml, and the cells can be subjected to subculture;
(3) Taking out seed cells to an ultra-clean workbench, uniformly shaking, absorbing about 500 mu l of samples, taking 20 mu l of samples, adding 20 mu l of trypan blue, uniformly mixing, absorbing 20 mu l of mixed liquid, adding a cell counting plate, reading 3 times of data in an automatic cell counter, and taking an average value to obtain the cell density;
(4) Appropriate amount of cells were taken, diluted to 0.6-0.65E+6cell/ml with fresh Wayne 293 serum-free culture, and placed in 125ml triangular shake flasks for culture, each flask having a culture volume of 25ml. On oscillating CO 2 Culturing in a cell culture box under the following culture conditions: 120rpm,37 ℃,5% CO 2 。
B. Cell transfection
(1) The cells are cultured for 48 hours, the cell density reaches 3-3.8E+6 cells/ml, and the cells can be transfected. 1 bottle of cells were transfected with each packaging plasmid;
(2) Each bottle of cells is added with 37.5 mug of packaging plasmid, pHelper, pAAV-CAG-EGFP-Barcode-hGH in sequence into 1mL of DMEM culture medium, and the mixture is fully and uniformly mixed to obtain plasmid diluent;
(3) Taking 1mL of DMEM culture medium, adding 112.5 mu g of PEI, and uniformly mixing to obtain PEI diluent;
(4) Pouring PEI diluent into plasmid diluent, quickly and uniformly mixing, and standing at room temperature for 25 minutes to form a transfection complex; adding the transfection complex into the cell sap dropwise while gently shaking;
(5) Placing the flask in shaking CO 2 Culturing in a cell culture box under the following culture conditions: 120rpm,37 ℃,5% CO 2 。
C. Virus harvesting
(1) The cells are cultured for 72 hours after transfection, and virus harvesting is started;
(2) Mixing and collecting cell suspensions in a centrifugal bottle, centrifuging 1000g for 5min, collecting supernatant in 1 new centrifugal bottle, adding 50% PEG8000 solution (containing 0.5mol/L NaCl) of 0.245mL per mL supernatant, mixing thoroughly, and standing at 2-8deg.C overnight;
(3) Adding a proper amount of 0.5% TritonX-100 cell lysis buffer (containing totipotent nuclease) into the cell precipitate, treating for 1-2h at 37 ℃, then adding 1/10 volume of 5mol/L NaCl, fully mixing uniformly, centrifuging at 4 ℃ and 3000g for 15min, and collecting the supernatant, namely the virus crude extract temporarily storing at 2-8 ℃;
(4) Centrifuging the supernatant after PEG8000 concentration at 4deg.C for 15min at 3000g, discarding the supernatant, and re-suspending with the virus crude extract in step (3) for use.
D. Virus purification
(1) Taking an overspeed centrifuge tube, and slowly adding 15 ml of virus crude extract, 9 ml of 15% iodixanol, 6 ml of 25% iodixanol, 5ml of 40% iodixanol and 5ml of 60% iodixanol into each overspeed centrifuge tube from the bottom by using a stainless steel needle; sealing with a heat sealing instrument after trimming;
(2) Ultracentrifuge, 340000 ×g (57500 rpm), 16 ℃, centrifuge for 2h;
(3) Carefully extracting 4-5ml of the interface liquid layer (namely virus liquid) of 40-60% of the iodixanol of the overspeed centrifuge tube by using a syringe, and avoiding sucking the interface liquid layer to a white membrane layer;
(4) Taking an ultrafiltration tube, rinsing a filter membrane with 4mL PBS buffer solution, and introducing the virus liquid collected in the step 3) into the ultrafiltration tube;
(5) Adding a proper amount of PBS buffer solution, blowing uniformly, centrifuging for 3-5 min at 3000g, and repeating the steps for 5-7 times until iodixanol is removed;
(6) Adding 1mL PBS buffer solution, blowing 40-50 to form virus suspension, and transferring to an EP tube;
(7) The virus suspension in the EP tube is sucked by a 1mL syringe and filtered by a 0.22 mu m filter, 20 mu L of virus liquid is collected as a detection sample, and the residual virus is split into 100 mu L of virus per tube, so that the AAV-CAG-EGFP-Barcode virus library is obtained.
3. Virus yield assay
A. Virus lysis
(1) Taking 20 mu L of Barcode virus library sample, respectively adding 10% SDS, 0.5mol/L EDTA and 1 mu L of proteoaseK, and uniformly mixing;
(2) Incubating for 1 hour at 56 ℃ in a constant temperature mixing instrument, and then incubating for 10min at 90 ℃;
(3) 10ul of virus lysate is taken, and 10 times of virus lysate is diluted to 1000 times in a gradient manner, so that the virus lysate is the virus diluent for standby.
B. PCR amplification
PCR amplification was performed on the inserted Bacode sequence using the "virus dilution" in step (1) as a DNA template. The PCR system is that
(1) PCR amplification primer:
forward primer 5'-GAGTTCGTGACCGCCGCC-3' (SEQ ID No. 21);
reverse primer 5'-TTTATTAGGACAAGGCTGGT-3' (SEQ ID NO. 22).
(2) The PCR amplification system is shown in Table 4.
(3) PCR amplification conditions were
Pre-denaturation: 98 ℃ for 3 min;
25 x cycle: 98 ℃ for 15s;60 ℃ for 20s;72℃for 24s.
Extension: 72℃for 2min.
C. PCR product gel recovery
And (3) carrying out 2% agarose gel electrophoresis on the PCR product, observing by a gel imaging system, cutting out gel blocks with correct strips, and carrying out gel recovery by using a gel recovery kit.
D. NGS sequencing
And sending the gel recovery product to Huada genes for NGS sequencing. And analyzing the sequencing result, namely analyzing the reading and the sequence ratio of different Barcode sequences, and carrying out standardization treatment on the sequencing result (RPM, wherein the sample is not influenced by the length of a gene), wherein each packaging plasmid corresponds to the Barcode expression plasmid one by one, so that the RPM value of each Barcode sequence in the Barcode virus library is in direct proportion to the yield of the corresponding packaging plasmid. RPM was used as a yield performance index for each mutant virus.
Comparative example 1
This comparative example provides a wild-type adeno-associated virus (AAV 2-WT) which was prepared as in example 1, except that the sequence of the adeno-associated virus capsid protein was the wild-type AAV2 capsid protein sequence (SEQ ID NO. 1).
Comparative example 2
This comparative example provides an AAV2 adeno-associated virus mutant (AAV 2/7M 8) having a high retinal targeting ability, which has been used in clinical trials, and which is prepared as described in example 1, except that the adeno-associated virus capsid protein sequence employs the mutant AAV2/7M8 capsid protein sequence (SEQ ID NO. 2).
Test case
NHP full-eye infection effect test
Injecting AAV-CAG-EGFP-Barcode virus library into a cynomolgus monkey body, taking whole eye tissues, extracting DNA (deoxyribonucleic acid) to carry out PCR (polymerase chain reaction) amplification on a Barcode region of a viral genome, sending and measuring NGS (non-human immunodeficiency Virus), and evaluating the infection performance of viruses on the whole eye tissues according to the readings of different Barcode amplification products.
The specific operation steps are as follows:
A. virus injection
After anaesthetizing the cynomolgus monkey, taking out an AAV-CAG-EGFP-Barcode virus library, injecting into vitreous cavities, injecting 100 mu l into each eye, treating the eyes with triamcinolone acetonide every day, and observing the eye condition.
B. Drawing materials
(1) After virus injection for 3W, euthanizing the cynomolgus monkey, picking eyeballs, and placing the eyeballs in a pre-cooled DMEM culture medium on ice;
(2) Taking 1 culture dish of 10cm, pouring about 2ml of DMEM culture medium on ice, and removing muscle and connective tissue around eyes;
(3) The insulin syringe needle point pierces the cornea, the Venus scissors cut along the cornea and choroid boundary (white), the cornea, iris and lens are removed, and the rest tissue is taken as whole eye tissue, and the average is divided into 10 parts.
C. Genomic DNA extraction, PCR amplification and gel recovery
(1) Placing the whole eye tissue in a homogenizing tube, adding zirconium beads and Buffer GA in a blood/cell/tissue genome DNA extraction kit, operating at room temperature and 65Hz for 30s, homogenizing for 30s, and repeating for 4 times;
(2) Extracting tissue genome DNA from the tissue homogenate by using a blood/cell/tissue genome DNA extraction kit, and measuring the DNA concentration by using an ultra-micro ultraviolet spectrophotometer;
(3) The tissue DNA was diluted to 100ng/ml and used as a DNA template for PCR amplification, agarose gel electrophoresis, gel recovery and concentration measurement, and the specific experimental procedure and virus yield verification were the same.
D. NGS sequencing and analysis
And sending the amplified product to the Hua megagene for NGS sequencing. And analyzing the sequencing result, and analyzing different Barcode sequence reads and the ratio thereof, wherein the higher the ratio is, the higher the ratio of the corresponding mutant virus in the tissue is. The test results were normalized (RPM, the samples were not affected by the length of the gene) and the effect of sequencing depth on the different samples was excluded. The RPM of the Barcode sequence in the tissue was then divided by the RPM in the viral library (see for details the viral yield test in the examples) as its corresponding mutant viral tissue targeting performance index.
3 weeks after virus injection, genomic DNA was obtained and NGS was performed, and the results were shown in FIG. 8, in which AAV2-7M8 had 4.3 times the full-eye infection capacity of AAV 2-WT. The infection capacity of the whole EYE of the gland-related mutant is 1.1-6.2 times of AAV2-WT, wherein the AAV2-EYE-04 has the strongest infection capacity.
In conclusion, the adeno-associated virus variant prepared by the scheme of the invention can obviously enhance the full-eye infection capacity of AAV2 viruses compared with a wild type.
The sequence alignment of adeno-associated virus variant capsid proteins is shown in figure 9. The sequence of the adeno-associated virus variants EYE-01-EYE-07 capsid protein differs from that of wild-type AAV2 capsid protein in that it replaces the sequence of wild-type capsid proteins D561-R588 (shown in SEQ ID NO. 3) with a polypeptide sequence of 28 to 42 amino acids, wherein EYE-01 and EYE-03 are partial amino acid substitutions, and EYE-02, EYE-04, EYE-05, EYE-06, EYE-07 are multiple amino acid insertions.
TABLE 1 AAV2 mutant amino acid sequence information at Cap 2D 561 to R588 positions replaced
| Sequence name | Sequence information | SEQ ID NO.： |
| AAV2-WT | DEEEIRTTNPVATEQYGSVSTNLQRGNR | 3 |
| AAV2-7M8 | DEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPAR | 4 |
| AAV2-EYE-01 | DEHEIKTTNPVATEGYGEVATNWQRGNR | 5 |
| AAV2-EYE-02 | DEEEIRTTNPVATEQYGSVSTNLQRGNTGRSAGLGTGLSR | 6 |
| AAV2-EYE-03 | DEQEIAATNPVATEQYGSVSTNLQRGNR | 7 |
| AAV2-EYE-04 | DEEEIRTTNPVATEQYGSVSTNLQRGNTGGMVLVSAKSGLSR | 8 |
| AAV2-EYE-05 | DEEEIRTTNPVATEQYGSVSTNLQRGNTGGRILVATTGLSR | 9 |
| AAV2-EYE-06 | DEEEIRTTNPVATEQYGSVSTNLQRGNTGPLLDGTKGLSR | 10 |
| AAV2-EYE-07 | DEEEIRTTNPVATEQYGSVSTNLQRGNTGSSPIKDGTKGLSR | 11 |
TABLE 2 pAAV-CAG-EGFP-Barcode-hGH plasmid Barcode nucleic acid sequence
| Sequence name | Barcode sequence information | SEQ ID NO.： |
| Barcode41 | GAGCGTAATTGTGAG | 12 |
| Barcode42 | GTCGACTTCATGGCA | 13 |
| Barcode43 | GGGCCCTAGCGCGTG | 14 |
| Barcode44 | CGTGACCCAGGAAGT | 15 |
| Barcode45 | GACTTTGACATGTCA | 16 |
| Barcode46 | GTCCCGACTAGGACT | 17 |
| Barcode47 | GGCCACCGTGTGTGA | 18 |
| Barcode48 | TTGGACTCACAGATG | 19 |
| Barcode49 | CAATCCGGCGCGGGT | 20 |
TABLE 3 correspondence between packaging plasmids and expression plasmids
| Experimental group | Packaging plasmid | Expression plasmid |
| 1 | AAV2-WT | pAAV-CAG-EGFP-Barcode41 |
| 2 | AAV2-7M8 | pAAV-CAG-EGFP-Barcode42 |
| 3 | AAV2-EYE-01 | pAAV-CAG-EGFP-Barcode43 |
| 4 | AAV2-EYE-02 | pAAV-CAG-EGFP-Barcode44 |
| 5 | AAV2-EYE-03 | pAAV-CAG-EGFP-Barcode45 |
| 6 | AAV2-EYE-04 | pAAV-CAG-EGFP-Barcode46 |
| 7 | AAV2-EYE-05 | pAAV-CAG-EGFP-Barcode47 |
| 8 | AAV2-EYE-06 | pAAV-CAG-EGFP-Barcode48 |
| 9 | AAV2-EYE-07 | pAAV-CAG-EGFP-Barcode49 |
| 10 | AAV2-EYE-08 | pAAV-CAG-EGFP-Barcode50 |
| 11 | AAV2-EYE-09 | pAAV-CAG-EGFP-Barcode51 |
| 12 | AAV2-EYE-10 | pAAV-CAG-EGFP-Barcode52 |
TABLE 4 PCR amplification System
| Reagent name | Volume added |
| Q5 High-Fidelity 2X Master Mix | 25ul |
| 10. Mu M forward primer (SEQ ID NO. 21) | 2.5ul |
| 10. Mu M reverse primer (SEQ ID NO. 22) | 2.5ul |
| Template DNA | 10µl |
| Non-nucleic acid water | To 50ul |
Related Cap2 protein sequence:
wild-type adeno-associated virus capsid protein Cap2:
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL(Genbank ID：YP_680426.1) (SEQ ID NO.1)。
adeno-associated virus variant 7M8 capsid protein Cap2:
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVTTTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSINVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO.2)。
the above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to make simple deductions or substitutions without departing from the concept of the present invention.
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| CN118027157A (en) * | 2024-01-16 | 2024-05-14 | 广州译码基因科技有限公司 | Capsid protein mutant for improving retina penetration capability of AAV virus and application thereof |
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| CN117866054A (en) * | 2024-01-16 | 2024-04-12 | 广州译码基因科技有限公司 | Capsid protein mutants for improving AAV virus production and whole-eye infection ability and their applications |
| CN118027157A (en) * | 2024-01-16 | 2024-05-14 | 广州译码基因科技有限公司 | Capsid protein mutant for improving retina penetration capability of AAV virus and application thereof |
| CN118420723A (en) * | 2024-01-16 | 2024-08-02 | 广州译码基因科技有限公司 | Capsid protein mutant for improving retina penetration capability of AAV virus and application thereof |
| CN118459557A (en) * | 2024-01-16 | 2024-08-09 | 广州译码基因科技有限公司 | Capsid protein mutants for improving the retinal penetration ability of AAV virus and their applications |
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