CN114686521A - Recombinant adeno-associated virus vector and preparation method and application thereof - Google Patents
Recombinant adeno-associated virus vector and preparation method and application thereof Download PDFInfo
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- CN114686521A CN114686521A CN202011580751.7A CN202011580751A CN114686521A CN 114686521 A CN114686521 A CN 114686521A CN 202011580751 A CN202011580751 A CN 202011580751A CN 114686521 A CN114686521 A CN 114686521A
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Abstract
The invention provides a recombinant adeno-associated virus vector and a preparation method and application thereof. Specifically, the invention provides a recombinant gene expression vector for treating retinal detachment, which is an AAV vector, wherein an expression cassette of a therapeutic gene for treating retinal detachment is inserted or carried; the therapeutic genes include: a gene encoding a protein fragment useful for binding the N-terminal domain of Gasdermin D (Gsdmd) protein. The recombinant gene expression vector can obviously reduce the focal death of the retina outer nuclear layer cells and increase the survival rate of the mouse photoreceptor cells.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a recombinant adeno-associated virus vector and a preparation method and application thereof.
Background
Retinal Detachment (RD) is a common pathological feature of many retinochoroidal diseases such as poriferous Retinal detachment, age-related macular degeneration, diabetic retinopathy, and the like. Retinal detachment, i.e., separation of the neural epithelium layer of the retina from the RPE and choroid complex, is clinically manifested as decreased vision, obstruction of visual objects, anterior floating objects, and glistening sensation, and when the detachment range of the neural epithelium is expanded to the macular region, the vision of the patient is rapidly decreased. Because RPE and choroid complex are the main sources of oxygen, blood and energy substances of the outer retinal nerve fiber layer/photoreceptor cells, and the oxidative metabolism in the photoreceptor cells is active to maintain the normal visual chemistry signal-electric signal conversion to form the visual function, after the retinal detachment occurs, the supply of oxygen and energy substances of the photoreceptor cells is interrupted, so that a large number of photoreceptor cells are rapidly killed, which is the key pathological basis of the visual impairment caused by the photoreceptor cells, and the apoptosis plays an important role in photoreceptor injury as a highly proinflammatory cell death form.
Gasdermin D is a protein that is the executive of the process of cellular apoptosis, a newly discovered mode of cell death, that exhibits severe morphological features of cellular explosions, with intense inflammatory factor release and inflammatory responses. Unlike apoptosis, autophagy, and other forms of cell death, it has unique morphological features and pathway mechanisms for cell death. For a long time, its terminal effector proteins have not been known and studied. Recent studies have found that a family of functionally unknown proteins, called Gasdermin, are cleaved by inflammatory Caspase proteins under the precondition of activation of the inflammasome to complete the focal death process. The activated Caspase-1 protein can be sheared at 276 site of the GSDMD protein to form a Gsdmd amino segment (Gsdmd-N) with the molecular weight of about 31kda, the GSDMD-N is transferred and anchored on a cell membrane to form a non-selective ion channel, the osmotic pressure inside and outside the cell is damaged, the intracellular substances are promoted to be released to the outside, and then the cell apoptosis process is completed.
At present, no report is found about a method for inhibiting the Gsdmd-N protein and the application of the Gsdmd-N protein in treating retinal detachment photoreceptor cell injury death.
Therefore, there is an urgent need in the art to develop a method for effectively inhibiting retinal detachment caused by photoreceptor cell damage and scorching.
Disclosure of Invention
The invention aims to provide a method for effectively inhibiting retinal detachment caused by photoreceptor cell damage and focal death.
Specifically, the first objective of the present invention is to provide a recombinant adeno-associated virus vector, which addresses the deficiencies of the prior art.
The second purpose of the invention is to provide a preparation method of the recombinant adeno-associated virus vector.
The third purpose of the invention is to provide a pharmaceutical composition.
The fourth purpose of the invention is to provide an application of the recombinant adeno-associated virus vector.
It is a fifth object of the present invention to provide a method for treating retinal detachment photoreceptor cell apoptosis.
In order to achieve the first purpose, the invention adopts the technical scheme that:
provides a recombinant adeno-associated virus vector, which contains an enhancer, a specific or non-specific promoter and a gene sequence for expressing Gsdmd-C-HA protein, wherein the amino acid sequence of the Gsdmd-C-HA protein is shown as SEQ ID NO. 2.
Preferably, the gene sequence of the Gsdmd-C-HA protein is selected from nucleotide sequences having at least 96%, 97%, 98%, 99%, 100% homology with SEQ ID NO. 1.
More preferably, the gene sequence of the Gsdmd-C-HA protein is shown in SEQ ID NO. 1.
Preferably, the enhancer/promoter is CMV selected from nucleotide sequences having at least 95% homology with SEQ ID NO. 4.
More preferably, the enhancer/promoter is CMV and has the sequence shown in SEQ ID NO 4.
Preferably, the recombinant adeno-associated viral vector further comprises a shortened chimeric intron sequence selected from the group consisting of nucleotide sequences having at least 90% homology to SEQ ID No. 5.
Preferably, the recombinant adeno-associated virus vector contains a CMV enhancer/promoter, a gene sequence for expressing Gsdmd-C-HA, a beta-globin intron, hGHpA (human growth hormone poly (A) tail), L-ITR, R-ITR, Ampicillin resistance gene, and f1 ori.
More preferably, the gene sequence of the Gsdmd-C-HA protein is shown as SEQ ID NO. 1, the L-ITR sequence is shown as SEQ ID NO. 3, the CMV enhancer/promoter sequence is shown as SEQ ID NO. 4, the beta-globin intron sequence is shown as SEQ ID NO. 5, the hGHpA sequence is shown as SEQ ID NO. 7, the R-ITR sequence is shown as SEQ ID NO. 8, the Ampicillin resistance sequence is shown as SEQ ID NO. 9, and the f1 ori sequence is shown as SEQ ID NO. 11.
Preferably, the recombinant adeno-associated virus vector sequence comprises SEQ ID NO 10.
More preferably, the recombinant adeno-associated virus vector has a sequence shown in SEQ ID NO. 12.
In order to achieve the second object, the invention adopts the technical scheme that:
a method for preparing the recombinant adeno-associated virus vector comprises the following steps:
1) constructing a recombinant plasmid AAV _ Gsdmd-C-HA, wherein the main elements comprise a CMV enhancer/promoter and a Gsdmd-C-HA gene sequence;
2) co-transfecting an AAV _ Gsdmd-C-HA plasmid and an HEK 293T cell to preliminarily form a recombinant adeno-associated virus vector, wherein the helper plasmid and the AAV _ Gsdmd-C-HA plasmid contain AAV coat protein genes and genes capable of helping AAV replication;
3) after the recombinant adeno-associated virus vector is primarily purified by iodic butanol, the recombinant adeno-associated virus vector is further purified by a fast protein liquid chromatograph with 5ml-Hitrp Q sepharose as a filler through ion exchange chromatography;
4) then eluting the agarose gel column by using NaCl with the pH value of 8.0 and the concentration of 215mM, and collecting the recombinant adeno-associated virus vector with the peak value;
5) after the collected liquid passes through a concentrator, eluting the concentrator by using Tween 20 containing 0.014% to concentrate the recombinant adeno-associated virus vector, digesting DNA (deoxyribonucleic acid) except virus particles in the concentrated liquid by using DNase I (deoxyribose nucleic acid), and determining the titer of the virus by using a real-time fluorescent quantitative PCR (polymerase chain reaction) method;
6) finally, silver nitrate staining-SDS polyacrylamide gel electrophoresis is used to ensure that the recombinant adeno-associated virus vector particles are not polluted and do not contain endotoxin, and the recombinant adeno-associated virus vector particles are subpackaged at minus 80 ℃ for storage.
In order to achieve the third object, the invention adopts the technical scheme that:
a pharmaceutical composition for treating retinal detachment photoreceptor cell damage and apoptosis comprises the recombinant adeno-associated virus vector and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition expresses Gsdmd-C-HA protein.
In order to achieve the fourth object, the invention adopts the technical scheme that:
the use of the adeno-associated virus vector as defined above in the preparation of a medicament for the treatment of retinal detachment photoreceptor cell damage and apoptosis.
In order to achieve the fifth object, the invention adopts the technical scheme that:
a method of treating retinal detachment photoreceptor cell damage burn and death by administering the adeno-associated viral vector as described above to an individual in need thereof.
Preferably, the method of administration is intraocular injection.
More preferably, the intraocular injection is a subretinal injection or a vitreous cavity injection.
In a first aspect of the present invention, there is provided a recombinant gene expression vector for treating retinal detachment, the expression vector being an adeno-associated virus (AAV) vector, wherein an expression cassette for a therapeutic gene for treating retinal detachment is inserted into or carried by the AAV vector;
wherein the therapeutic gene comprises: a gene encoding a protein fragment useful for binding the N-terminal domain of Gasdermin D (Gsdmd) protein; wherein the N-terminal domain of the Gsdmd protein is the domain from position 1 to position 276 of the amino acid sequence of the Gsdmd protein, and the positions 1 and 276 are the positions 1 and 276 corresponding to the sequence shown in SEQ ID NO. 13.
In another preferred embodiment, the retinal detachment treatment is retinal detachment caused by photoreceptor cell damage and apoptosis.
In another preferred example, the protein fragment that can be used to bind the N-terminal domain of the Gsdmd protein is a fragment of the C-terminal domain of the Gsdmd protein.
In another preferred embodiment, the fragment of the C-terminal domain of the Gsdmd protein comprises an amino acid sequence corresponding to positions 277 to 484 (preferably positions 275 to 484; more preferably positions 276 to 484) of the sequence shown in SEQ ID NO: 13.
In another preferred embodiment, the amino acid sequence of the C-terminal domain fragment of the Gsdmd protein is shown in SEQ ID NO. 14.
In another preferred embodiment, the protein tag sequence is also included in the protein fragment used to bind the N-terminal domain of the Gsdmd protein.
In another preferred embodiment, the protein tag is selected from the group consisting of: HA tag, His tag, GST tag, c-Myc tag, Flag tag, mCherry tag, Avi tag, or a combination thereof.
In another preferred embodiment, the protein tag is an HA tag.
In another preferred example, the amino acid sequence of said protein fragment for binding the N-terminal domain of the Gsdmd protein is as set forth in SEQ ID NO:2, respectively.
In another preferred embodiment, the adeno-associated virus is selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV2-AAV3, AAVrh.10, AAVhu.14, AAV3a/3b, AAVvh32.33, AAVHSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15/17, AAVM41, AAV9.45, AAV6(Y445F/Y731F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, AAVShH10, AAV2(Y- > F), AAV8(Y733F), AAV2.15, AAV2.4, AAV 41, AAVrh 3.2, AAV5, or AAV 865.
In another preferred embodiment, the adeno-associated virus is AAV2.
In another preferred embodiment, the adeno-associated virus is single stranded AAV2.
In another preferred embodiment, the recombinant gene expression vector comprises the following elements: a therapeutic gene expression cassette, inverted Terminal Repeat (TR) sequences, resistance genes and an F1 ori sequence which are respectively positioned at two sides of the expression cassette.
In another preferred embodiment, the therapeutic gene expression cassette comprises the following elements: enhancer/promoter, therapeutic gene sequence, hGHpA (human growth hormone poly (A) tail), beta-globin intron.
In another preferred embodiment, the inverted Terminal Repeats (TR) flanking each side of the expression cassette include L-ITR and R-ITR.
In another preferred embodiment, the enhancer/promoter is a CMV enhancer/promoter.
In another preferred embodiment, the therapeutic gene has the sequence shown in SEQ ID NO. 1 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology thereto.
In another preferred embodiment, the CMV enhancer/promoter has the sequence shown in SEQ ID NO 4 or is a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology thereto.
In another preferred embodiment, the sequence of the beta-globin intron is as shown in SEQ ID NO. 5, or a nucleotide sequence that is at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologous thereto.
In another preferred embodiment, the hGHpA sequence is as shown in SEQ ID NO. 7 or is a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology thereto.
In another preferred embodiment, the L-ITR has a sequence as shown in SEQ ID NO 3 or a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology thereto.
In another preferred embodiment, the R-ITR has a sequence as shown in SEQ ID NO. 8 or a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology thereto.
In another preferred embodiment, the resistance gene is an Ampicillin (Ampicillin) -resistant gene, the sequence of which is shown in SEQ ID NO 9, or a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology thereto.
In another preferred embodiment, the sequence of the F1 ori is as shown in SEQ ID NO. 11, or a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology thereto.
In another preferred embodiment, the recombinant gene expression vector contains a nucleotide sequence shown as SEQ ID NO. 10.
In another preferred embodiment, the sequence of the recombinant gene expression vector is shown in SEQ ID NO. 12.
In a second aspect of the invention, there is provided a pharmaceutical composition comprising:
(i) a recombinant gene expression vector according to the first aspect of the present invention;
(ii) a pharmaceutically acceptable carrier.
In another preferred embodiment, the component (i) is 0.1 to 99.9 wt%, preferably 10 to 99.9 wt%, more preferably 70 to 99 wt% of the total weight of the pharmaceutical composition.
In another preferred embodiment, the pharmaceutical composition is in a liquid dosage form.
In another preferred embodiment, the pharmaceutical composition is in the form of injection or drops.
In another preferred embodiment, the pharmaceutical composition is in an injectable dosage form for intraocular injection.
In another preferred embodiment, the intraocular injection is a sub-retinal injection or a vitreous cavity injection.
In another preferred embodiment, the pharmaceutical composition is a drop for instillation into the eye.
In another preferred embodiment, the carrier is selected from the group consisting of: water, bbs (balanced salt solution) phosphate buffered saline, ringer solution, physiological saline, balanced salt solution, glycerol, sorbitol, or a combination thereof.
In another preferred embodiment, the carrier may further comprise: a lubricant, glidant, wetting or emulsifying agent, pH buffering substance, stabilizer, or combination thereof.
In another preferred embodiment, the carrier is an injection carrier, and preferably, the carrier is one or more selected from the group consisting of: normal saline, dextrose saline, or combinations thereof.
In another preferred embodiment, the carrier is a drop carrier, preferably the carrier is one or more selected from the group consisting of: sodium hyaluronate and distilled water.
In another preferred embodiment, the pharmaceutical compositions are used alone or in combination in the treatment of retinal detachment.
In another preferred embodiment, the combination comprises: it can be used in combination with other drugs for treating retinal detachment.
In another preferred embodiment, the other drugs for treating retinal detachment include: lingqi macular granules, pachyma cocos granules, or a combination thereof.
In a third aspect of the invention, there is provided the use of a recombinant gene expression vector according to the first aspect of the invention for the preparation of a formulation or pharmaceutical composition for the treatment of retinal detachment.
In another preferred embodiment, the preparation or the pharmaceutical composition is used for treating patients with retinal detachment caused by photoreceptor cell damage and focal death.
In a fourth aspect of the present invention, there is provided a method for treating retinal detachment, characterized by administering the recombinant gene expression vector according to the first aspect of the present invention or the pharmaceutical composition according to the third aspect of the present invention to a subject in need thereof.
In another preferred embodiment, the mode of administration is intraocular injection.
In another preferred embodiment, the administration is by sub-retinal or intravitreal injection.
In another preferred embodiment, the dosage for administration is 1 × 1010-1×1014Viral particles formed by recombinant gene expression vectors.
In a fifth aspect of the present invention, there is provided a method of preparing the gene expression vector according to the first aspect of the present invention, comprising the steps of: an expression cassette of a therapeutic gene for treating retinal detachment is ligated into an AAV vector, thereby obtaining a gene expression vector according to the first aspect of the present invention.
In another preferred example, the method comprises the steps of:
(i) constructing a recombinant plasmid AAV _ Gsdmd-C-HA, wherein the main elements comprise a CMV enhancer/promoter and a Gsdmd-C-HA gene sequence;
(ii) co-transfecting an AAV _ Gsdmd-C-HA plasmid and an HEK 293T cell to preliminarily form a recombinant adeno-associated virus vector, wherein the helper plasmid and the AAV _ Gsdmd-C-HA plasmid contain AAV coat protein genes and genes capable of helping AAV replication;
(iii) after the recombinant adeno-associated virus vector is primarily purified by ioxol, the recombinant adeno-associated virus vector is further purified by a fast protein liquid chromatograph with 5ml-Hitrp Q sepharose as a filler through ion exchange chromatography;
(iv) then eluting the agarose gel column by using NaCl with the pH value of 8.0 and the concentration of 215mM, and collecting the recombinant adeno-associated virus vector with the peak value;
(v) after the collected liquid passes through a concentrator, eluting the concentrator by using 0.014% Tween 20 to concentrate the recombinant adeno-associated virus vector, digesting DNA (deoxyribonucleic acid) except virus particles in the concentrated liquid by using DNase I (deoxyribose nucleic acid), and determining the titer of the virus by using a real-time fluorescent quantitative PCR (polymerase chain reaction) method; and
(vi) and (3) using a silver nitrate staining-SDS polyacrylamide gel electrophoresis method to ensure that the recombinant adeno-associated virus vector particles are not polluted and contain no endotoxin, and subpackaging at-80 ℃ for storage.
In a sixth aspect of the invention, there is provided a kit comprising:
(i) a first container, and an active ingredient (a) in the first container, wherein the active ingredient (a) is the recombinant gene expression vector according to the first aspect of the invention or the pharmaceutical composition according to the third aspect of the invention;
(ii) optionally a second container, and an active ingredient (b) in the second container, said active ingredient (b) being another drug for treating retinal detachment or an active ingredient thereof; and
(iii) optionally a third container, and a syringe located in the third container.
In another preferred embodiment, the kit further comprises instructions describing instructions for administering the active ingredients (a) and (b) to treat retinal detachment.
In another preferred embodiment, the first container and the second container are the same or different containers.
In another preferred embodiment, the syringe is an injection applicator, preferably a syringe needle.
In another preferred embodiment, the first and second containers are the same container, preferably the active ingredient (a) is located in the syringe in a dose of one dose.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows a characteristic map of the viral vector AAV _ Gsdmd-C-HA and the start-stop sites of the respective elements.
FIG. 2 is a plot of immunofluorescent staining of retinal sections three weeks after subretinal injection of AAV _ Gsdmd-C-HA in Brown-Norway rats.
FIG. 3 is a PI staining pattern of retinas 1 day after retinal detachment, 3 weeks after subretinal injection of AAV _ Gsdmd-C-HA or AAV-GFP in Brown-Norway rats.
FIG. 4 is a graph of PI staining of retinas after 3 weeks of subretinal injection of AAV _ Gsdmd-C-HA or AAV-GFP in Brown-Norway rats with viscoelastic injection to cause retinal detachment 1 day after subretinal injection, and is graphically presented as a result of Joule count and viability analysis after PI staining thereof.
Detailed Description
The inventor develops a recombinant gene expression vector capable of effectively inhibiting photoreceptor cell damage and scorch for the first time through extensive and intensive research and a large amount of screening. Specifically, the invention optimizes the human Gsdmd-C-HA gene sequence, selects a proper vector and related elements, and constructs a recombinant adeno-associated virus vector to construct Gsdmd-C-HA. Animal experiments show that AAV _ Gsdmd-C-HA HAs excellent infection effect in rat photoreceptor cells, is stably expressed in cell nucleus, is a stable recombinant adeno-associated virus vector, and can remarkably reduce epiretinal cell apoptosis and increase the survival rate of mouse photoreceptor cells. The present invention has been completed based on this finding.
Therapeutic genes of the invention
As used herein, the term "therapeutic gene" refers to a therapeutic gene in a recombinant gene expression vector for the treatment of retinal detachment provided in the present invention, which encodes a protein that can bind to the N-terminal domain of Gasdermin D (Gsdmd) protein or a fragment thereof.
As used herein, the terms "protein encoded by a therapeutic gene of the invention", "recombinant protein of the invention", "fusion protein" are used interchangeably and all refer to the aforementioned proteins or fragments thereof that bind to the N-terminal domain of Gasdermin D (Gsdmd) protein.
In one embodiment of the invention, the protein fragment that can be used to bind the N-terminal domain of the Gsdmd protein is a fragment of the C-terminal domain of the Gsdmd protein. Preferably, the C-terminal domain fragment of the Gsdmd protein comprises an amino acid sequence corresponding to positions 277 to 484 (preferably positions 275 to 484; more preferably positions 276 to 484) of the sequence shown in SEQ ID NO: 13. More preferably, the amino acid sequence of the C-terminal domain fragment of the Gsdmd protein is shown as SEQ ID NO. 14.
Protein tag sequences are also included in the protein fragments used to bind the N-terminal domain of the Gsdmd protein. In one embodiment, the protein tag is selected from the group consisting of: HA tag, His tag, GST tag, c-Myc tag, Flag tag, mCherry tag, Avi tag, or a combination thereof. Preferably, the protein tag is an HA tag.
In a preferred embodiment of the invention, the amino acid sequence of said protein fragment for binding the N-terminal domain of the Gsdmd protein is as set forth in SEQ ID NO:2, respectively.
It is to be understood that although the genes provided in the examples of the present invention are partially human, amino acid sequences derived from other similar species (especially mammals) having some homology (conservation) to the protein sequence encoded by the therapeutic gene of the present invention (preferably, the sequence shown in SEQ ID NO: 2) are also included within the scope of the present invention, as long as the sequence can be readily isolated from other species (especially mammals) by one skilled in the art after reading the present application, based on the information provided herein.
The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA forms include: DNA, genomic DNA or synthetic DNA, DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The therapeutic gene sequence may be identical to the sequence of the coding region shown in SEQ ID NO. 1 or may be a degenerate variant.
The polynucleotide encoding the fusion protein includes a coding sequence encoding only the fusion protein; the coding sequence of the fusion protein and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences of the fusion protein.
The term "polynucleotide encoding a fusion protein" or "therapeutic gene of the invention" may be a polynucleotide comprising a polynucleotide encoding said fusion protein, or may be a polynucleotide further comprising additional coding and/or non-coding sequences. The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polyglycosides or polypeptides having the same amino acid sequence as the invention. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the fusion protein encoded thereby.
The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) methyl phthalein amine, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more.
The full-length nucleotide sequence encoding the fusion protein of the present invention or a fragment thereof can be obtained by PCR amplification, recombinant methods, or synthetic methods. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the sequences can be amplified using a commercially available DNA library or a cDNA library prepared by conventional methods known to those skilled in the art as a template. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice together the amplified fragments in the correct order. Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. Usually, it is cloned into a vector, transferred into a cell, and then isolated from the propagated host cell by a conventional method to obtain the relevant sequence.
In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. At present, DNA sequences encoding the fusion proteins of the present invention (or fragments or derivatives thereof) have been obtained entirely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the fusion protein sequences of the invention by chemical synthesis.
The invention relates to a therapeutic gene for treating retinal detachment, and in a preferred embodiment of the invention, the amino acid sequence of a fusion protein encoded by the therapeutic gene is shown as SEQ ID NO. 2. The fusion protein coded by the therapeutic gene can be effectively combined with the protein fragment of the N-terminal structural domain of Gasderm D (Gsdmd) protein, so that the retinal detachment caused by photoreceptor cell damage and focal death is inhibited.
The present invention also includes polypeptides or proteins having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more, e.g., 99%) homology to the sequence of SEQ ID NO. 2 of the present invention, and having the same or similar functions.
The "same or similar functions" mainly refer to: the function of "effectively binding protein fragments of the N-terminal domain of the Gasderm min D (Gsdmd) protein, thereby inhibiting retinal detachment due to photoreceptor cell damage and apoptosis".
The fusion protein of the invention can be recombinant polypeptide, natural polypeptide and synthetic polypeptide. The fusion proteins of the invention can be naturally purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, plant, insect, and mammalian cells). Depending on the host used in the recombinant production protocol, the fusion protein of the invention may be glycosylated or may be non-glycosylated. The fusion protein of the present invention may or may not also include an initial methionine residue.
The invention also includes other polypeptide fragments and analogs having the activity of the fusion proteins of the invention. As used herein, the terms "fragment" and "analog" refer to a polypeptide that retains substantially the same biological function or activity as the fusion protein of the invention.
The polypeptide fragment, derivative or analogue of the invention may be: (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, e.g., polyethylene glycol; or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein.
In the present invention, the fusion protein variant is an amino acid sequence shown in SEQ ID NO. 2, a derivative sequence obtained by several (usually 1-10, preferably 1-8, more preferably 1-4, and most preferably 1-2) substitutions, deletions, or additions of at least one amino acid, and one or several (usually less than 10, preferably less than 5, and more preferably less than 3) amino acids added at the C-terminal and/or N-terminal. For example, substitutions in the protein with amino acids of similar or analogous properties will not generally alter the function of the protein, nor will the addition of one or more (e.g., 1-3) amino acids at the C-terminus and/or N-terminus. These conservative changes are best made by making substitutions according to table 1.
TABLE 1
The invention also includes analogs of the claimed proteins. These analogs may differ from the native SEQ ID NO. 2 by amino acid sequence differences, by modifications that do not affect the sequence, or by both. Analogs of these proteins include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, site-directed mutagenesis, or other known molecular biological techniques. Analogs also include analogs having residues other than the natural L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, gamma-amino acids). It will be appreciated that the proteins of the invention are not limited to the representative proteins listed above.
Modified (generally without altering primary structure) forms include: chemically derivatized forms of the protein such as acetoxylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those performed during protein synthesis and processing. Such modification may be accomplished by exposing the protein to an enzyme that performs glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine).
Recombinant gene therapy vector
Adeno-associated virus (AAV), a member of the parvoviridae (Parvovirus) family, is a small, non-enveloped, icosahedral virus with a single-stranded linear DNA genome of 4.7 kilobases (kb) to 6 kb. Since the virus is found as a contaminant of purified adenovirus stocks, AAV is designated as a dependent virus (dependently). The life cycle of AAV includes a latent phase (AAV genome site-specifically integrates into the host chromosome after infection), and an infectious phase (following infection with adenovirus or herpes simplex virus, the integrated genome is then rescued, replicated, and packaged into the infected virus). The properties of non-pathogenicity, broad host range (including non-dividing cells), infectivity, and potential site-specific chromosomal integration make AAV an attractive tool for gene transfer.
Recombinant adeno-associated virus (rAAV) still suffers from disadvantages, such as instability of the recombinant virus, low viral titers, and limited capacity to accept therapeutic genes (typically only a maximum of about 2000 base pairs (bp) of an exogenous gene fragment can be inserted, otherwise rAAV stability will be compromised). Therefore, a reasonable recombinant adeno-associated virus (rAAV) vector needs to be designed to meet the needs of practical applications.
The invention provides a recombinant gene expression vector for treating retinal detachment, which is an AAV vector, wherein an expression cassette of a therapeutic gene for treating retinal detachment is inserted or carried in the AAV vector; wherein the therapeutic gene comprises: a gene encoding a protein fragment useful for binding the N-terminal domain of Gasdermin D (Gsdmd) protein; wherein the N-terminal domain of the Gsdmd protein is the domain from position 1 to position 276 of the amino acid sequence of the Gsdmd protein, and the positions 1 and 276 are the positions 1 and 276 corresponding to the sequence shown in SEQ ID NO. 13.
Preferably, the treatment of retinal detachment is treatment of retinal detachment caused by photoreceptor cell damage and apoptosis.
In addition, the recombinant gene expression vector of the present invention comprises the following elements: a therapeutic gene expression cassette, inverted Terminal Repeat (TR) sequences, resistance genes and F1 ori sequences respectively positioned at two sides of the expression cassette. Wherein the therapeutic gene expression cassette comprises the following elements: enhancer/promoter, therapeutic gene sequence, hGHpA (human growth hormone poly (A) tail), beta-globin intron.
Wherein the inverted Terminal Repeats (TR) flanking each of the expression cassettes comprise L-ITR and R-ITR. And, the enhancer/promoter is a CMV enhancer/promoter.
In a preferred embodiment of the invention, the therapeutic gene has the sequence shown in SEQ ID NO. 1 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology thereto.
In one embodiment, the CMV enhancer/promoter has the sequence shown in SEQ ID NO. 4, or is a nucleotide sequence that is at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologous thereto.
In one embodiment, the beta-globin intron has the sequence shown in SEQ ID NO. 5, or a nucleotide sequence at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homologous thereto.
In one embodiment, the hGHpA sequence is as set forth in SEQ ID NO. 7, or is a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) homology thereto.
In one embodiment, the L-ITR has a sequence as set forth in SEQ ID NO 3 or a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology thereto.
In one embodiment, the R-ITR has the sequence shown in SEQ ID NO. 8, or a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology thereto.
In one embodiment, the resistance gene is an Ampicillin (Ampicillin) -resistant gene, the sequence of which is shown in SEQ ID NO 9, or a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology thereto.
In one embodiment, the sequence of the F1 ori is as set forth in SEQ ID NO. 11, or is a nucleotide sequence having at least 90% (preferably, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) homology thereto.
In a preferred embodiment, the recombinant gene expression vector contains a nucleotide sequence shown as SEQ ID NO. 10.
In a more preferred embodiment, the sequence of the recombinant gene expression vector is shown in SEQ ID NO. 12.
The gene therapy vectors of the present invention are viral expression vectors, according to the present invention, the viral expression vectors are adeno-associated virus (AAV) vectors, such as AAV vectors selected from the group consisting of serotype AAV1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, or chimeric AAV derived therefrom, such as AAV2-AAV3, aavrh.10, aavhu.14, AAV3a/3b, aavrh32.33, AAVHSC15, AAV-HSC17, aavhu.37, aavrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC 9/17, AAVM41, AAV9.45, AAV6(Y445F/Y F), AAV2.5T, AAV-HAE1/2, AAV clone 32/83, aahhh 10, AAV2(Y- > F), vrh F (Y445), AAV 8), AAV 3842, AAV-AAV 464.3875, which are more preferably of interest in AAV tissues transduced by AAV.
Upon transfection, AAV elicits only a mild immune response (if any) in the host. In a preferred embodiment of the invention, the gene therapy vector is an AAV serotype 2 or 5 vector. In a further preferred embodiment, the gene therapy vector is an AAV2 vector.
The AAV vector of the invention is a single-stranded AAV, and a recombinant viral vector can be produced according to standard techniques. For example, recombinant adeno-associated virus vectors can be transmitted in human 293 cells (which provide the properties of trans E1A and E1B) to reach 107-1013Titers in the range of individual virus particles/mL.
Prior to in vivo use, the viral vector may be desalted by gel filtration methods (such as agarose columns) and purified by subsequent filtration. Purification reduces potential deleterious effects in the host of the administration vehicle. The administered virus is substantially free of wild-type and replication-competent viruses.
The purity of the virus can be demonstrated by a suitable method, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by silver staining.
In a preferred embodiment, a suitable dosage of AAV for use in humans is about 1X 1010-1×1014Individual viral particles.
The gene therapy vector can be administered by intraocular injection, and can be administered by subretinal space or intravitreal injection.
Pharmaceutical composition and kit
As used herein, the term "comprising" includes "comprising," "consisting essentially of … …, and" consisting of … ….
As used herein, the term "consisting essentially of … …" means that in addition to the active ingredient or adjunct ingredient, a small amount of minor ingredients and/or impurities which do not affect the active ingredient may be included in the pharmaceutical composition.
For convenience of clinical application, the pharmaceutical composition of the present invention may be contained in an administration device for injection (e.g., a needle for injection), in which the pharmaceutical composition may be contained in an amount administered at one time. The administration device for injection may be contained in a cartridge for convenient storage and use. The transportation requires placing a tiny container filled with the drug suspension in dry ice. It should be stored in a refrigerator at-80 deg.C.
The kit or kit of the present invention may further comprise instructions for use, which will facilitate the use of the kit or kit in a proper manner by those skilled in the art.
As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.
The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.
As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity), i.e., with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents, i.e., carriers for such agents: they are not essential active ingredients per se and are not unduly toxic after administration.
Suitable pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. Sufficient description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack pub. co., n.j.1991); pharmaceutically acceptable carriers in the compositions may comprise liquids such as water, BBS (balanced salt solution) phosphate buffer, ringer's solution, physiological saline, balanced salt solution, glycerol or sorbitol, and the like.
In addition, auxiliary substances, such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers, etc., may also be present in these carriers.
In an embodiment of the invention, the pharmaceutical composition is in a liquid dosage form; preferably in the form of injections or drops.
In one embodiment, the pharmaceutical composition is an injectable dosage form for intraocular injection; for example, the intraocular injection is a subretinal injection or a vitreous injection. In one embodiment, the pharmaceutical composition is a drop for instillation into an eye.
The pharmaceutical compositions of the present invention may be used alone or in combination in the treatment of retinal detachment. The combined use comprises: it can be used in combination with other drugs for treating retinal detachment. Wherein, the other medicine for treating the retinal detachment comprises: lingqi macular granules, pachyma cocos granules, or a combination thereof.
The main advantages of the invention include:
(1) the Gsdmd-C-HA gene sequence is optimized, and HAs a proper carrier and related elements so as to efficiently express the Gsdmd-C-HA.
(2) The AAV _ Gsdmd-C-HA of the invention HAs excellent infection effect in rat photoreceptor cells, is stably expressed in cell nucleus, is a stable recombinant adeno-associated virus vector, and can obviously reduce the apoptosis of retina outer nuclear layer cells and increase the survival rate of mouse photoreceptor cells.
(3) The recombinant gene expression vector can be used for preparing the focal death for treating retinal detachment photoreceptor cell damage apoptosis and has wide market prospect.
(4) The apoptosis inhibiting protein Gsdmd-C-HA can play an inhibiting role in the peak period of photoreceptor cell death after retinal detachment for one day, and can save damaged retinal tissues in time.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
Example 1: construction, separation and purification of recombinant adeno-associated virus vector
Gene sequences
SEQ ID NO. 1 shows a Gsdmd-C-HA nucleotide sequence;
SEQ ID NO. 2 shows a Gsdmd-C-HA amino acid sequence;
the nucleotide sequence of Left ITR is shown in SEQ ID NO. 3;
SEQ ID NO. 4 shows the CMV sequence;
SEQ ID NO. 5 shows a human beta-globin intron sequence;
the nucleotide sequence of MCS shown as SEQ ID NO. 6;
SEQ ID NO. 7 shows the hGHpA nucleotide sequence;
shown in SEQ ID NO 8 is the Right ITR sequence;
SEQ ID NO 9 shows an Ampicillin resistance sequence;
10 is the complete nucleotide sequence shown in SEQ ID NO;
SEQ ID NO. 11 shows the ori sequence of f 1;
SEQ ID NO 12 shows a recombinant sequence.
The construction, characteristic map and start-stop sites of each element of the plasmid AAV _ Gsdmd-C-HA are shown in figure 1, the main elements comprise a CMV enhancer/promoter (SEQ ID NO:4) and a Gsdmd-C-HA sequence (SEQ ID NO:1), the CMV enhancer can enhance the expression of a transferred gene, a hGHpA (SEQ ID NO:7) is arranged immediately after a target gene, an inverted Terminal Repeat (TR) is arranged at two sides of an expression cassette, namely, a virus vector comprises an L-ITR (SEQ ID NO:3) and an R-ITR (SEQ ID NO:8), and further comprises an Ampicillin resistance gene sequence (SEQ ID NO:9) and a f1 ori sequence (SEQ ID NO: 11).
Viral vectors are obtained by plasmid cotransfection methods. Co-transfecting an AAV2 coat protein gene, an AAV _ Gsdmd-C-HA plasmid and HEK 293T cells to preliminarily form a recombinant adeno-associated virus vector, wherein the AAV _ Gsdmd-C-HA plasmid and the helper plasmid contain the AAV2 coat protein gene and the gene capable of helping AAV replication; after preliminary purification with ioxobutanol, further purification by ion exchange chromatography on a fast protein liquid chromatograph using 5ml-Hitrp Q sepharose as packing using an instrument of Pharmacia AKTA FPLC system (Amersham Biosciences, Piscataway, N.J.); then eluting the agarose gel column by using NaCl with pH8.0 and 215mM, and collecting the recombinant adeno-associated virus vector with the peak value; after the collected liquid passed through a concentrator (100K concentrator, Millipore), the recombinant adeno-associated virus vector was concentrated by rinsing the concentrator with Tween 20 containing 0.014%; then digesting DNA except the virus particles by DNase I, and determining the titer of the virus by a real-time fluorescent quantitative PCR method; finally, silver nitrate staining-SDS polyacrylamide gel electrophoresis is used to ensure that the recombinant adeno-associated virus vector particles are not polluted and do not contain endotoxin, and the recombinant adeno-associated virus vector particles are subpackaged at minus 80 ℃ for storage.
Example 2: treatment of retinal detachment model mice with recombinant adeno-associated virus vectors
2.1 materials of the experiment
2.1.1 Experimental animals
Normal Brown-Norway male rats were purchased from Beijing Wintounli Hua laboratory animals, Inc., with a light cycle of 12h light-12 h dark, with free food intake and free water intake, and all animal studies were conducted strictly in accordance with the Experimental animals regulations issued by the national Committee for science and technology.
2.1.2 Experimental reagent and consumables
Physiological saline (Zhejiang Tianrui pharmaceutical Co., Ltd.), a disposable syringe needle (Becton Dickinson and Company, USA), HA antibody (Cell signaling technology), PI (Sigma Aldrich Co., Ltd.), secondary antibody goat anti-rabbit IgG (Jackson Immuno), goat serum (Sigma), polyethylene glycol octyl phenyl ether (Triton X-100) (Sigma), paraformaldehyde (Sigma).
2.1.3 Experimental instruments
Ophthalmic experimental operating microscope (Nikon corporation, japan); ophthalmic microscopy instruments (suzhou mingren medical instruments ltd); microsyrinths (Hamilton, usa); confocal microscopy (come, germany).
2.2 construction of rat retinal detachment model
The tropicamide eye drop is used for dilating the right eye of a mydriasis rat twice, each time is separated by 10min, and the rat is anesthetized by intraperitoneal injection; dripping surface anesthesia eye drops on the anesthetized rat cornea, and placing the rat under a micro-operation lens; cutting a temporoconjunctiva, performing sclera puncture by a 34G needle at a position 3mm behind a corneoscleral limbus to form a full-layer sclera incision, and withdrawing the needle after a needle tip inclined plane completely enters a vitreous cavity under direct vision under a mirror; the glass needle head connected with the micro-syringe enters the vitreous cavity through the scleral incision, slowly enters the subretinal space under direct vision, slightly pushes the syringe, and after slight separation of the neuroepithelium and the pigment epithelium is observed, the needle head is confirmed to be positioned under the retina under direct vision but not under the choroid or in the vitreous cavity, and slowly injects 0.05ml of sodium hyaluronate to the subretinal space, so that the gray retina further bulges to form 3-4 retina bulges. Observing the formation of retinal detachment under a microscope, wherein the height and the range of the retinal detachment exceed 80% of the whole retina, dripping biological tissue glue to the scleral incision for sealing, and finally spreading the dianbizu eye ointment on the corneal surface of the rat surgery eye. Rats were resuscitated on a thermostatically heated pad until anaesthesia was recovered.
2.3 treatment method
2.3.1 subretinal injection
The 34G needle is used for inserting needle and puncturing at the position close to the junction of the retina edge and the corneosclera, when the front end of the needle enters the retina, injection is carried out, the needle inserting angle and depth need to be noticed during injection, and the injury to the lens or other intraocular tissues is avoided.
2.3.2 tissue treatment
Animals were perfused systemically first with PBS and then sequentially with 4% Paraformaldehyde (PFA) at a perfusion rate of 5 mL/min. The eyeball and optic nerve were removed and then soaked in 4% PFA solution for about 2 hours. The tissue should be stored in 0.1M PBS at 4 ℃ before staining; the tissue was soaked overnight in a 30% sucrose solution before cryosectioning.
2.4 adeno-associated virus infection and detection of Gsdmd-HA protein expression
After injecting AAV _ Gsdmd-C-HA into rat retina successfully molded for two weeks, pretreating eyeball according to the above tissue treatment method, clamping eyeball bottom with micro-forceps, lifting up, shearing conjunctiva and muscle tissue around eyeball with scissors, keeping eyeball tissue intact, shearing cornea, iris and crystalline lens to make into eye cup, soaking in 30% sucrose solution overnight, and making into 10 μm thick tissue section.
And (3) immunofluorescence staining: selecting retinas, sealing in 20% goat serum solution for 1h, incubating the subsequent antibody overnight, washing with antibody HA and PBS for three times, each time for 10min, incubating the subsequent antibody for 1h, washing with PBS for three times, and sealing after each time for 10 min; the retinal structure changes were observed using a confocal microscope and photographed.
As a result:
two weeks after injection of AAV _ Gsdmd-C-HA into rats, immunofluorescence staining of retinal sections revealed that a large amount of AAV _ Gsdmd-C-HA was expressed in the cell membrane, and was localized around DAPI, indicating that AAV _ Gsdmd-C-HA had an excellent infectious effect (FIG. 2).
2.5 photoreceptor cell Focus mortality detection
8 weeks old rats single side eye subretinal injection AAV _ Gsdmd-C-HA (experimental group), AAV _ GFP (control group), 3 weeks later to make retinal detachment model, retinal detachment 3 days later, according to the tissue processing method processing eyeball and retina, immunofluorescence staining, using confocal microscope to observe retinal structure change and take pictures, each eyeball selecting 5 visual field counting PI positive cells (proportional bar: 25 μm), and calculating photoreceptor cell survival rate.
As a result:
grouping | Number of PI |
Blank group | 0.00±0.00 |
Model set | 854.0±62.04 |
Control group | 835.3±51.15 |
Experimental group | 415.0±10.39 |
As can be seen from the above table, the numbers of positive photoreceptor cells PI in the model group, the control group and the experimental group are increased and statistically different (P <0.05) compared with the blank group, the numbers of positive photoreceptor cells PI in the control group are not statistically different (P >0.05) compared with the model group, and the numbers of positive photoreceptor cells PI in the experimental group are significantly reduced and statistically different (P <0.05) compared with the control group.
Quantitative analysis proves that the photoreceptor cells reach the peak of focal death in 1 day after the retinal detachment, and the thickness of the photoreceptor cell layer is obviously reduced (a model group and a control group); in contrast, rat photoreceptor cell apoptosis was not significant when AAV _ Gsdmd-C-HA was injected, indicating that AAV _ Gsdmd-C-HA HAs protective effect on photoreceptor cells (FIGS. 3 and 4).
The experiments show that the recombinant adeno-associated virus AAV _ Gsdmd-C-HA can significantly reduce the apoptosis of the photoreceptor cells of mice and effectively treat the photoreceptor cell injury caused by retinal detachment.
All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> first-person hospital in Shanghai City
<120> recombinant adeno-associated virus vector, preparation method and application thereof
<130> P2019-1588
<160> 14
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atgggaacca atgaggaaga agtaatcagg gaagacttcc aaggcctgcg tgcggaggtg 60
gaggcaggct cttcagaact gcggagctta gaaatggagt tgagacaaca gctactagta 120
gacatcggga ggattttaca ggaccagccc agcatggaag ccttagaggc ctcactagag 180
cagggtctgt gcagtggtga gcaggtagag cctctggaag gcccagcagg cagcatcctt 240
gagtgtctgg tgctcgactc tggagaactg gtgccggaac ttgcagcccc cgtcttctac 300
ctgttgggag cactggctgg gttgagtgaa acccagcagc agctactggc tactaaggct 360
ctggaggcaa cggtgctgtc aaaggagcta gagttggtga agcacgtctt ggaacagagc 420
accccatggc aggagcagag atctgtgccc ctgccctcca ggctccttgg ggacagctgg 480
gatgaggagg ctctcacctg ggtcttgcta gaagaatgtg gcctaacgct gcaggtggaa 540
cccccccagg tatactggga accaacatct cagggcccca tatgtgcact ctatgcctcc 600
ctggccctat tgtcaagtct aggccagaaa tcgtgttacc catacgatgt tccagattac 660
gcttcg 666
<210> 2
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<213> Artificial sequence (artificial sequence)
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Met Gly Thr Asn Glu Glu Glu Val Ile Arg Glu Asp Phe Gln Gly Leu
1 5 10 15
Arg Ala Glu Val Glu Ala Gly Ser Ser Glu Leu Arg Ser Leu Glu Met
20 25 30
Glu Leu Arg Gln Gln Leu Leu Val Asp Ile Gly Arg Ile Leu Gln Asp
35 40 45
Gln Pro Ser Met Glu Ala Leu Glu Ala Ser Leu Glu Gln Gly Leu Cys
50 55 60
Ser Gly Glu Gln Val Glu Pro Leu Glu Gly Pro Ala Gly Ser Ile Leu
65 70 75 80
Glu Cys Leu Val Leu Asp Ser Gly Glu Leu Val Pro Glu Leu Ala Ala
85 90 95
Pro Val Phe Tyr Leu Leu Gly Ala Leu Ala Gly Leu Ser Glu Thr Gln
100 105 110
Gln Gln Leu Leu Ala Thr Lys Ala Leu Glu Ala Thr Val Leu Ser Lys
115 120 125
Glu Leu Glu Leu Val Lys His Glu Leu Val Lys His Val Leu Glu Gln
130 135 140
Ser Thr Pro Trp Gln Glu Leu Pro Ser Arg Leu Leu Gly Asp Ser Trp
145 150 155 160
Asp Glu Glu Ala Leu Thr Trp Val Leu Leu Glu Glu Cys Gly Leu Thr
165 170 175
Leu Gln Val Glu Pro Pro Gln Val Tyr Trp Glu Pro Thr Ser Gln Gly
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Pro Ile Cys Ala Leu Tyr Ala Ser Leu Ala Leu Leu Ser Ser Leu Gly
195 200 205
Gln Lys Ser Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
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<212> DNA
<213> Artificial sequence (artificial sequence)
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cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc t 141
<210> 4
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<212> DNA
<213> Artificial sequence (artificial sequence)
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acgcgtggaa ttcgctagtt attaatagta atcaattacg gggtcattag ttcatagccc 60
atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 120
cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgt caatagggac 180
tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca 240
agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg 300
gcattatgcc cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt 360
agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg 420
gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg 480
caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 540
ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag 600
atcgcctgga gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc 660
agc 663
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<212> DNA
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gattcgaatc ccggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac 60
gtaagtaccg cctatagagt ctataggccc acaaaaaatg ctttcttctt ttaatatact 120
tttttgttta tcttatttct aatactttcc ctaatctctt tctttcaggg caataatgat 180
acaatgtatc atgcctcttt gcaccattct aaagaataac agtgataatt tctgggttaa 240
ggcaatagca atatttctgc atataaatat ttctgcatat aaattgtaac tgatgtaaga 300
ggtttcatat tgctaatagc agctacaatc cagctaccat tctgctttta ttttatggtt 360
gggataaggc tggattattc tgagtccaag ctaggccctt ttgctaatca tgttcatacc 420
tcttatcttc ctcccacagc tcctgggcaa cgtgctggtc tgtgtgctgg cccatcactt 480
tggcaaagaa ttg 493
<210> 6
<211> 76
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
atcgattgaa ttccccgggg atcctctaga gtcgacctgc agaagcttgc ctcgagcagc 60
gctgctcgag agatct 76
<210> 7
<211> 479
<212> DNA
<213> Artificial sequence (artificial sequence)
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acgggtggca tccctgtgac ccctccccag tgcctctcct ggccctggaa gttgccactc 60
cagtgcccac cagccttgtc ctaataaaat taagttgcat cattttgtct gactaggtgt 120
ccttctataa tattatgggg tggagggggg tggtatggag caaggggcaa gttgggaaga 180
caacctgtag ggcctgcggg gtctattggg aaccaagctg gagtgcagtg gcacaatctt 240
ggctcactgc aatctccgcc tcctgggttc aagcgattct cctgcctcag cctcccgagt 300
tgttgggatt ccaggcatgc atgaccaggc tcagctaatt tttgtttttt tggtagagac 360
ggggtttcac catattggcc aggctggtct ccaactccta atctcaggtg atctacccac 420
cttggcctcc caaattgctg ggattacagg cgtgaaccac tgctcccttc cctgtcctt 479
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<212> DNA
<213> Artificial sequence (artificial sequence)
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aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60
ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120
gagcgcgcag ctgcctgcag g 141
<210> 9
<211> 861
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 9
atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240
cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300
gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360
tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540
cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840
tcactgatta agcattggta a 861
<210> 10
<211> 4654
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 10
atgggaacca atgaggaaga agtaatcagg gaagacttcc aaggcctgcg tgcggaggtg 60
gaggcaggct cttcagaact gcggagctta gaaatggagt tgagacaaca gctactagta 120
gacatcggga ggattttaca ggaccagccc agcatggaag ccttagaggc ctcactagag 180
cagggtctgt gcagtggtga gcaggtagag cctctggaag gcccagcagg cagcatcctt 240
gagtgtctgg tgctcgactc tggagaactg gtgccggaac ttgcagcccc cgtcttctac 300
ctgttgggag cactggctgg gttgagtgaa acccagcagc agctactggc tactaaggct 360
ctggaggcaa cggtgctgtc aaaggagcta gagttggtga agcacgtctt ggaacagagc 420
accccatggc aggagcagag atctgtgccc ctgccctcca ggctccttgg ggacagctgg 480
gatgaggagg ctctcacctg ggtcttgcta gaagaatgtg gcctaacgct gcaggtggaa 540
cccccccagg tatactggga accaacatct cagggcccca tatgtgcact ctatgcctcc 600
ctggccctat tgtcaagtct aggccagaaa tcgtgttacc catacgatgt tccagattac 660
gcttcggtaa caactccgcc ccattgacgc aatgggcggt aggcgtgtac ggtgggaggt 720
taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg 780
acctccatag aagacaccgg gaccgatcca gcctccgcgg attcgaatcc cggccgggaa 840
cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc 900
tataggccca caaaaaatgc tttcttcttt taatatactt ttttgtttat cttatttcta 960
atactttccc taatctcttt ctttcagggc aataatgata caatgtatca tgcctctttg 1020
caccattcta aagaataaca gtgataattt ctgggttaag gcaatagcaa tatttctgca 1080
tataaatatt tctgcatata aattgtaact gatgtaagag gtttcatatt gctaatagca 1140
gctacaatcc agctaccatt ctgcttttat tttatggttg ggataaggct ggattattct 1200
gagtccaagc taggcccttt tgctaatcat gttcatacct cttatcttcc tcccacagct 1260
cctgggcaac gtgctggtct gtgtgctggc ccatcacttt ggcaaagaat tgggattcga 1320
acatcgattg aattccccgg ggatcctcta gagtcgacct gcagaagctt gcctcgagca 1380
gcgctgctcg agagatctac gggtggcatc cctgtgaccc ctccccagtg cctctcctgg 1440
ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta agttgcatca 1500
ttttgtctga ctaggtgtcc ttctataata ttatggggtg gaggggggtg gtatggagca 1560
aggggcaagt tgggaagaca acctgtaggg cctgcggggt ctattgggaa ccaagctgga 1620
gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc 1680
tgcctcagcc tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt 1740
tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc aactcctaat 1800
ctcaggtgat ctacccacct tggcctccca aattgctggg attacaggcg tgaaccactg 1860
ctcccttccc tgtccttctg attttgtagg taaccacgtg cggaccgagc ggccgcagga 1920
acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg 1980
gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc 2040
gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat 2100
ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct gtagcggcgc attaagcgcg 2160
gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct 2220
cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta 2280
aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa 2340
cttgatttgg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct 2400
ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc 2460
aaccctatct cgggctattc ttttgattta taagggattt tgccgatttc ggcctattgg 2520
ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgttt 2580
acaattttat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc 2640
cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct 2700
tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca 2760
ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg 2820
ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct 2880
atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga 2940
taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc 3000
cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg 3060
aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc 3120
aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact 3180
tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc 3240
ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag 3300
catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat 3360
aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt 3420
ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa 3480
gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc 3540
aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg 3600
gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt 3660
gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca 3720
gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat 3780
gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca 3840
gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg 3900
atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg 3960
ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt 4020
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg 4080
ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata 4140
ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca 4200
ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag 4260
tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc 4320
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga 4380
tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg 4440
tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac 4500
gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg 4560
tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg 4620
ttcctggcct tttgctggcc ttttgctcac atgt 4654
<210> 11
<211> 306
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 11
cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa 60
gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc 120
aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt 180
cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca 240
acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc 300
tattgg 306
<210> 12
<211> 6124
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 12
cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 60
gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 120
actccatcac taggggttcc tgcggccgca cgcgtggaat tcgctagtta ttaatagtaa 180
tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg 240
gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg 300
tatgttccca tagtaacgtc aatagggact ttccattgac gtcaatgggt ggagtattta 360
cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt 420
gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac 480
tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt 540
tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac 600
cccattgacg tcaatgggag tttgttttgc accaaaatca acgggacttt ccaaaatgtc 660
gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 720
taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg 780
acctccatag aagacaccgg gaccgatcca gcctccgcgg attcgaatcc cggccgggaa 840
cggtgcattg gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc 900
tataggccca caaaaaatgc tttcttcttt taatatactt ttttgtttat cttatttcta 960
atactttccc taatctcttt ctttcagggc aataatgata caatgtatca tgcctctttg 1020
caccattcta aagaataaca gtgataattt ctgggttaag gcaatagcaa tatttctgca 1080
tataaatatt tctgcatata aattgtaact gatgtaagag gtttcatatt gctaatagca 1140
gctacaatcc agctaccatt ctgcttttat tttatggttg ggataaggct ggattattct 1200
gagtccaagc taggcccttt tgctaatcat gttcatacct cttatcttcc tcccacagct 1260
cctgggcaac gtgctggtct gtgtgctggc ccatcacttt ggcaaagaat tgggattcga 1320
acatcgattg aattccacca tgatggtcag aaagcctgtt gtgtccacca tctccaaagg 1380
aggttacctg cagggaaatg ttaacgggag gctgccttcc ctgggcaaca aggagccacc 1440
tgggcaggag aaagtgcagc tgaagaggaa agtcacttta ctgaggggag tctccattat 1500
cattggcacc atcattggag caggaatctt catctctcct aagggcgtgc tccagaacac 1560
gggcagcgtg ggcatgtctc tgaccatctg gacggtgtgt ggggtcctgt cactatttgg 1620
agctttgtct tatgctgaat tgggaacaac tataaagaaa tctggaggtc attacacata 1680
tattttggaa gtctttggtc cattaccagc ttttgtacga gtctgggtgg aactcctcat 1740
aatacgccct gcagctactg ctgtgatatc cctggcattt ggacgctaca ttctggaacc 1800
attttttatt caatgtgaaa tccctgaact tgcgatcaag ctcattacag ctgtgggcat 1860
aactgtagtg atggtcctaa atagcatgag tgtcagctgg agcgcccgga tccagatttt 1920
cttaaccttt tgcaagctca cagcaattct gataattata gtccctggag ttatgcagct 1980
aattaaaggt caaacgcaga actttaaaga cgccttttca ggaagagatt caagtattac 2040
gcggttgcca ctggcttttt attatggaat gtatgcatat gctggctggt tttacctcaa 2100
ctttgttact gaagaagtag aaaaccctga aaaaaccatt ccccttgcaa tatgtatatc 2160
catggccatt gtcaccattg gctatgtgct gacaaatgtg gcctacttta cgaccattaa 2220
tgctgaggag ctgctgcttt caaatgcagt ggcagtgacc ttttctgagc ggctactggg 2280
aaatttctca ttagcagttc cgatctttgt tgccctctcc tgctttggct ccatgaacgg 2340
tggtgtgttt gctgtctcca ggttattcta tgttgcgtct cgagagggtc accttccaga 2400
aatcctctcc atgattcatg tccgcaagca cactcctcta ccagctgtta ttgttttgca 2460
ccctttgaca atgataatgc tcttctctgg agacctcgac agtcttttga atttcctcag 2520
ttttgccagg tggcttttta ttgggctggc agttgctggg ctgatttatc ttcgatacaa 2580
atgcccagat atgcatcgtc ctttcaaggt gccactgttc atcccagctt tgttttcctt 2640
cacatgcctc ttcatggttg ccctttccct ctattcggac ccatttagta cagggattgg 2700
cttcgtcatc actctgactg gagtccctgc gtattatctc tttattatat gggacaagaa 2760
acccaggtgg tttagaataa tgtcagagaa aataaccaga acattacaaa taatactgga 2820
agttgtacca gaagaagata agttatgaca gcgctgctcg agagatctac gggtggcatc 2880
cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca 2940
gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata 3000
ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg 3060
cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa 3120
tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc 3180
aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca 3240
tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca 3300
aattgctggg attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg 3360
taaccacgtg cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc 3420
tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt 3480
tgcccgggcg gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg 3540
gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 3600
tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 3660
gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 3720
acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 3780
agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 3840
ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 3900
ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta 3960
taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt 4020
aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 4080
tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 4140
tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 4200
tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg 4260
atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc 4320
acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 4380
atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag 4440
agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt 4500
cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt 4560
gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc 4620
cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta 4680
tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac 4740
ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa 4800
ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg 4860
atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc 4920
cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg 4980
atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta 5040
gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg 5100
cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg 5160
tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc 5220
tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt 5280
gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt 5340
gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc 5400
atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 5460
atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 5520
aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 5580
aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 5640
ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 5700
ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 5760
tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 5820
ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 5880
acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 5940
gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 6000
cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 6060
aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 6120
atgt 6124
<210> 13
<211> 484
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 13
Met Gly Ser Ala Phe Glu Arg Val Val Arg Arg Val Val Gln Glu Leu
1 5 10 15
Asp His Gly Gly Glu Phe Ile Pro Val Thr Ser Leu Gln Ser Ser Thr
20 25 30
Gly Phe Gln Pro Tyr Cys Leu Val Val Arg Lys Pro Ser Ser Ser Trp
35 40 45
Phe Trp Lys Pro Arg Tyr Lys Cys Val Asn Leu Ser Ile Lys Asp Ile
50 55 60
Leu Glu Pro Asp Ala Ala Glu Pro Asp Val Gln Arg Gly Arg Ser Phe
65 70 75 80
His Phe Tyr Asp Ala Met Asp Gly Gln Ile Gln Gly Ser Val Glu Leu
85 90 95
Ala Ala Pro Gly Gln Ala Lys Ile Ala Gly Gly Ala Ala Val Ser Asp
100 105 110
Ser Ser Ser Thr Ser Met Asn Val Tyr Ser Leu Ser Val Asp Pro Asn
115 120 125
Thr Trp Gln Thr Leu Leu His Glu Arg His Leu Arg Gln Pro Glu His
130 135 140
Lys Val Leu Gln Gln Leu Arg Ser Arg Gly Asp Asn Val Tyr Val Val
145 150 155 160
Thr Glu Val Leu Gln Thr Gln Lys Glu Val Glu Val Thr Arg Thr His
165 170 175
Lys Arg Glu Gly Ser Gly Arg Phe Ser Leu Pro Gly Ala Thr Cys Leu
180 185 190
Gln Gly Glu Gly Gln Gly His Leu Ser Gln Lys Lys Thr Val Thr Ile
195 200 205
Pro Ser Gly Ser Thr Leu Ala Phe Arg Val Ala Gln Leu Val Ile Asp
210 215 220
Ser Asp Leu Asp Val Leu Leu Phe Pro Asp Lys Lys Gln Arg Thr Phe
225 230 235 240
Gln Pro Pro Ala Thr Gly His Lys Arg Ser Thr Ser Glu Gly Ala Trp
245 250 255
Pro Gln Leu Pro Ser Gly Leu Ser Met Met Arg Cys Leu His Asn Phe
260 265 270
Leu Thr Asp Gly Val Pro Ala Glu Gly Ala Phe Thr Glu Asp Phe Gln
275 280 285
Gly Leu Arg Ala Glu Val Glu Thr Ile Ser Lys Glu Leu Glu Leu Leu
290 295 300
Asp Arg Glu Leu Cys Gln Leu Leu Leu Glu Gly Leu Glu Gly Val Leu
305 310 315 320
Arg Asp Gln Leu Ala Leu Arg Ala Leu Glu Glu Ala Leu Glu Gln Gly
325 330 335
Gln Ser Leu Gly Pro Val Glu Pro Leu Asp Gly Pro Ala Gly Ala Val
340 345 350
Leu Glu Cys Leu Val Leu Ser Ser Gly Met Leu Val Pro Glu Leu Ala
355 360 365
Ile Pro Val Val Tyr Leu Leu Gly Ala Leu Thr Met Leu Ser Glu Thr
370 375 380
Gln His Lys Leu Leu Ala Glu Ala Leu Glu Ser Gln Thr Leu Leu Gly
385 390 395 400
Pro Leu Glu Leu Val Gly Ser Leu Leu Glu Gln Ser Ala Pro Trp Gln
405 410 415
Glu Arg Ser Thr Met Ser Leu Pro Pro Gly Leu Leu Gly Asn Ser Trp
420 425 430
Gly Glu Gly Ala Pro Ala Trp Val Leu Leu Asp Glu Cys Gly Leu Glu
435 440 445
Leu Gly Glu Asp Thr Pro His Val Cys Trp Glu Pro Gln Ala Gln Gly
450 455 460
Arg Met Cys Ala Leu Tyr Ala Ser Leu Ala Leu Leu Ser Gly Leu Ser
465 470 475 480
Gln Glu Pro His
<210> 14
<211> 212
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 14
Met Gly Thr Asn Glu Glu Glu Val Ile Arg Glu Asp Phe Gln Gly Leu
1 5 10 15
Arg Ala Glu Val Glu Ala Gly Ser Ser Glu Leu Arg Ser Leu Glu Met
20 25 30
Glu Leu Arg Gln Gln Leu Leu Val Asp Ile Gly Arg Ile Leu Gln Asp
35 40 45
Gln Pro Ser Met Glu Ala Leu Glu Ala Ser Leu Glu Gln Gly Leu Cys
50 55 60
Ser Gly Glu Gln Val Glu Pro Leu Glu Gly Pro Ala Gly Ser Ile Leu
65 70 75 80
Glu Cys Leu Val Leu Asp Ser Gly Glu Leu Val Pro Glu Leu Ala Ala
85 90 95
Pro Val Phe Tyr Leu Leu Gly Ala Leu Ala Gly Leu Ser Glu Thr Gln
100 105 110
Gln Gln Leu Leu Ala Thr Lys Ala Leu Glu Ala Thr Val Leu Ser Lys
115 120 125
Glu Leu Glu Leu Val Lys His Glu Leu Val Lys His Val Leu Glu Gln
130 135 140
Ser Thr Pro Trp Gln Glu Leu Pro Ser Arg Leu Leu Gly Asp Ser Trp
145 150 155 160
Asp Glu Glu Ala Leu Thr Trp Val Leu Leu Glu Glu Cys Gly Leu Thr
165 170 175
Leu Gln Val Glu Pro Pro Gln Val Tyr Trp Glu Pro Thr Ser Gln Gly
180 185 190
Pro Ile Cys Ala Leu Tyr Ala Ser Leu Ala Leu Leu Ser Ser Leu Gly
195 200 205
Gln Lys Ser Cys
210
Claims (10)
1. A recombinant gene expression vector for treating retinal detachment is characterized in that the expression vector is an AAV vector, wherein an expression cassette of a therapeutic gene for treating retinal detachment is inserted or carried in the AAV vector;
the therapeutic genes include: a gene encoding a protein fragment useful for binding the N-terminal domain of Gasdermin D (Gsdmd) protein; wherein the N-terminal domain of the Gsdmd protein is the domain from position 1 to position 276 of the amino acid sequence of the Gsdmd protein, and the positions 1 and 276 are the positions 1 and 276 corresponding to the sequence shown in SEQ ID NO. 13.
2. The recombinant gene expression vector of claim 1, wherein the treatment of retinal detachment is a treatment of retinal detachment resulting from photoreceptor cell damage and apoptosis.
3. The recombinant gene expression vector according to claim 1 wherein the fragment of the C-terminal domain of the Gsdmd protein comprises an amino acid sequence corresponding to positions 277 to 484 (preferably positions 275 to 484; more preferably positions 276 to 484) of the sequence shown in SEQ ID NO: 13.
4. The recombinant gene expression vector according to claim 1, wherein the amino acid sequence of the protein fragment for binding the N-terminal domain of the Gsdmd protein is shown as SEQ ID NO. 2.
5. The recombinant gene expression vector of claim 1, wherein the recombinant gene expression vector comprises a nucleotide sequence as set forth in SEQ ID NO. 10.
6. A pharmaceutical composition, comprising:
(i) the recombinant gene expression vector of claim 1;
(ii) a pharmaceutically acceptable carrier.
7. Use of the recombinant gene expression vector of claim 1 for the preparation of a formulation or pharmaceutical composition for the treatment of retinal detachment.
8. A method for preparing the gene expression vector of claim 1, comprising the steps of: the gene expression vector of claim 1 obtained by ligating an expression cassette of a therapeutic gene for treating retinal detachment into an AAV vector.
9. A kit, comprising:
(i) a first container, and an active ingredient (a) in the first container, wherein the active ingredient (a) is the recombinant gene expression vector of claim 1 or the pharmaceutical composition of claim 6;
(ii) optionally a second container, and an active ingredient (b) in the second container, said active ingredient (b) being another drug for treating retinal detachment or an active ingredient thereof; and
(iii) optionally a third container, and a syringe located in the third container.
10. Kit according to claim 9, wherein the syringe is an injection applicator, preferably a needle.
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CN115820444A (en) * | 2022-11-11 | 2023-03-21 | 苏州大学 | Cell model for researching human Gasderm protein function and construction method and application thereof |
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2020
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN115820444A (en) * | 2022-11-11 | 2023-03-21 | 苏州大学 | Cell model for researching human Gasderm protein function and construction method and application thereof |
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