MYBPC3 mutant gene and application thereof

Abstract

The invention provides an MYBPC3 mutant gene and application thereof, and belongs to the technical field of biomedicine. The nucleotide sequence of the human MYBPC3 mutant gene is as shown in SEQ ID NO. 1. The MYBPC3 mutant gene can be used for screening of the hypertrophic cardiomyopathy, construction of a hypertrophic cardiomyopathy cell model and construction of a hypertrophic cardiomyopathy non-human animal model, and is beneficial to exploration of pathophysiological processes of the hypertrophic cardiomyopathy and exploration of therapeutic targets and drugs of the hypertrophic cardiomyopathy.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically involving... MYBPC3 Mutant genes and their applications. Background Technology

[0002] Hypertrophic cardiomyopathy (HCM) is a primary cardiomyopathy characterized by asymmetric hypertrophy of the left ventricle and interventricular septum. It can lead to heart failure, malignant arrhythmias, and even sudden cardiac death, and is mostly caused by gene mutations encoding sarcomere structural proteins. MYBPC3 The gene (NCBI ID NM_000256.3) is located at 11p11.2 and encodes cardiac myosin-binding protein C (cMyBP-C). MYBPC3 The gene is one of the main pathogenic genes for HCM. There are still unknown factors. MYBPC3 Gene mutation sites, further discovering new MYBPC3 Gene mutation sites are of great significance for studying the pathogenesis of hypertrophic cardiomyopathy, for the diagnosis of hypertrophic cardiomyopathy or to assist in clinical judgment, and for the discovery of drug targets for intervention. Summary of the Invention

[0003] In view of this, this application provides a MYBPC3 Mutant genes and their applications.

[0004] Firstly, this application provides human... MYBPC3 Mutant genes, the human MYBPC3 The nucleotide sequence of the mutated gene is shown in SEQ ID NO.1.

[0005] Secondly, this application provides a method for detecting the human described in the first aspect. MYBPC3 Application of reagents containing mutated genes in the preparation of kits for detecting hypertrophic cardiomyopathy.

[0006] Thirdly, this application provides a kit for detecting hypertrophic cardiomyopathy, including the detection of the human... MYBPC3 Reagents for mutated genes.

[0007] Fourthly, this application provides an sgRNA, the sequence of which is shown in SEQ ID NO.2.

[0008] Fifthly, this application provides the application of the sgRNA described in the fourth aspect in the preparation of hypertrophic cardiomyopathy cell models.

[0009] Sixthly, this application provides a method for preparing a hypertrophic cardiomyopathy cell model, comprising: preparing a hypertrophic cardiomyopathy cell model using the sgRNA described in the fourth aspect; or editing human embryonic stem cells to induce their directed differentiation into cardiomyocytes, wherein the cardiomyocytes... MYBPC3 A hypertrophic cardiomyopathy cell model was created by deleting the 3653rd base of the gene's coding sequence.

[0010] Seventhly, this application provides a targeting vector, including a vector encoding a non-human animal as a target. MYBPC3 Mutant gene or fragment thereof, which encodes the target animal's MYBPC3 The mutated gene or its fragment is similar to the human wild type. MYBPC3 The base corresponding to position 3653 of the coding sequence is missing, and the human wild type MYBPC3 The coding sequence of the gene is shown in SEQ ID NO.3.

[0011] Eighthly, this application provides the targeting vector described in the seventh aspect for constructing non-human animal models of hypertrophic cardiomyopathy or constructing models of... MYBPC3 Application of mutant genes in non-human animal models.

[0012] Ninthly, this application provides a method for constructing a non-human animal model of hypertrophic cardiomyopathy or constructing a non-human animal model of hypertrophic cardiomyopathy. MYBPC3 Methods for constructing non-human animal models of mutated genes include: constructing a non-human animal model of hypertrophic cardiomyopathy using the targeting vector described in aspect seven, or constructing a non-human animal model of hypertrophic cardiomyopathy. MYBPC3 Non-human animal models with mutated genes; or gene editing technology used to modify non-human animals. MYBPC3 The coding sequence is similar to that of the human wild type. MYBPC3 Deleting the base corresponding to position 3653 of the gene's coding sequence yields a non-human animal model of hypertrophic cardiomyopathy or a model of hypertrophic cardiomyopathy. MYBPC3 Non-human animal models of mutated genes, specifically the human wild-type. MYBPC3 The coding sequence of the gene is shown in SEQ ID NO.3.

[0013] In a tenth aspect, this application provides a hypertrophic cardiomyopathy cell model prepared by the method described in the sixth aspect, or a non-human animal model of hypertrophic cardiomyopathy prepared by the method described in the ninth aspect, or a non-human animal model of hypertrophic cardiomyopathy. MYBPC3 Application of non-human animal models of mutated genes in screening drugs for the prevention or treatment of hypertrophic cardiomyopathy or in preparing products for evaluating the efficacy of treatment for hypertrophic cardiomyopathy.

[0014] This application provides a novel approach related to hypertrophic cardiomyopathy. MYBPC3 Mutated genes, and corresponding information on this gene. MYBPC3Methods for detecting mutated genes enable timely disease screening and treatment intervention; and also, based on this... MYBPC3 The mutated gene provides targeting vectors for hypertrophic cardiomyopathy (HCM) cell models and non-human animal models of HCM, as well as methods for constructing non-human animal models. The resulting HCM cell models and non-human animal models can simulate the pathogenesis of HCM and more objectively reflect its progression. MYBPC3 The pathological characteristics of hypertrophic cardiomyopathy caused by mutated genes and in-depth exploration of the pathogenesis of hypertrophic cardiomyopathy caused by gene mutations are of great significance for further discovering therapeutic targets and drugs that can be intervened in hypertrophic cardiomyopathy. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. The specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0016] Figure 1 These are the results of gene sequencing.

[0017] Figure 2 The graph shows the differentiation efficiency of cardiomyocytes in each group. Figure 2 In the middle (a), the differentiation efficiency of undifferentiated human embryonic stem cells is shown. Figure 2 (b) represents the differentiation efficiency of the WT group. Figure 2 (c) represents the differentiation efficiency of the HE group. Figure 2 (d) represents the differentiation efficiency of the HO group.

[0018] Figure 3 This is a comparison chart of the surface area of ​​cardiomyocytes in each group, where... Figure 3 (a) shows the surface area detection results of cardiomyocytes in each group. Figure 3 (b) is a statistical graph showing the surface area detection results of myocardial cells in each group.

[0019] Figure 4 Immunofluorescence staining images of cardiomyocytes in each group are shown, with a scale bar of 20 μm.

[0020] Figure 5 This is a comparison diagram of the contractility of cardiomyocytes in different groups, where... Figure 5 (a) shows the results of the contractility test of myocardial cells in each group. Figure 5 (b) is a statistical graph showing the results of the contraction amplitude detection of myocardial cells in each group.

[0021] Figure 6 This is a comparison graph of calcium transient amplitudes, in which... Figure 6 (a) shows the results of calcium transient amplitude detection. Figure 6(b) is a statistical chart of the calcium transient amplitude detection results.

[0022] Figure 7 The result of the peak time test is shown in the figure.

[0023] Figure 8 The graph shows the results of the calcium transient decay time detection.

[0024] Figure 9 This is a comparison chart of the incidence of calcium alternation, in which... Figure 9 (a) shows the results of calcium alternation incidence detection. Figure 9 (b) is a statistical chart of the calcium alternation rate detection results. Detailed Implementation

[0025] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0026] This application provides a human MYBPC3 The mutated gene, whose nucleotide sequence is shown in SEQ ID NO.1. This human... MYBPC3 The mutated gene is associated with hypertrophic cardiomyopathy and can be used to construct cell models and non-human animal models of hypertrophic cardiomyopathy, which is of great significance for the pathogenesis, detection, intervention and treatment of hypertrophic cardiomyopathy.

[0027] The human beings provided in this application MYBPC3 The nucleotide sequence of the mutated gene was obtained from the human wild-type... MYBPC3 Obtained by deletion of guanine base G at position 3708 of the transcript sequence of the gene, human wild-type MYBPC3 The transcript number of the gene is NM_000256.3. The human [gene] provided in this application... MYBPC3 The nucleotide sequence of the mutated gene was obtained from the human wild-type... MYBPC3 Obtained by deletion of the guanine base G at position 3653 of the gene's coding sequence, human wild-type. MYBPC3 The coding sequence of the gene is shown in SEQ ID NO.3. "Wild-type" is a commonly used term in this field; a wild-type gene / amino acid sequence / nucleotide sequence refers to a mutated, original gene / amino acid sequence / nucleotide sequence. The transcript sequence includes the upstream untranslated region, the coding sequence region, and the downstream untranslated region.

[0028] This application provides a human MYBPC3The mutant protein, whose amino acid sequence is shown in SEQ ID NO.4, is a human mutant protein. MYBPC3 The amino acid sequence corresponding to the mutated gene is shown in SEQ ID NO.4. This human... MYBPC3 The mutant protein is associated with hypertrophic cardiomyopathy and can be used to construct cell models and non-human animal models of hypertrophic cardiomyopathy, which is of great significance for the pathogenesis, detection, intervention and treatment of hypertrophic cardiomyopathy.

[0029] The human beings provided in this application MYBPC3 Mutant genes, humans MYBPC3 The mutant protein has a base change of c. 3708del and an amino acid change of p. G1218A fs compared to transcript number NM_000256.3. 19. The human beings provided in this application MYBPC3 Mutant genes compared to human wild-type MYBPC3 The base change in the coding sequence of the gene is c. 3653del.

[0030] This application provides a non-human animal MYBPC3 Mutated genes, in non-human animals MYBPC3 The coding sequence of the mutated gene is similar to that of the human wild type. MYBPC3 The 3653rd base of the gene's coding sequence is missing, in the human wild-type... MYBPC3 The gene coding sequence is shown in SEQ ID NO.3. This non-human animal's... MYBPC3 Mutated genes can be used to construct mutation models in non-human animals, which is beneficial for research on hypertrophic cardiomyopathy. Understandably, by using mutation models from non-human animals... MYBPC3 The coding sequence is similar to that of wild-type humans. MYBPC3 By comparing the coding sequences of genes, the genetic information of non-human animals can be obtained based on the comparison results. MYBPC3 The coding sequence is similar to that of wild-type humans. MYBPC3 The base site corresponding to position 3653 of the gene's coding sequence.

[0031] This application provides for detecting the aforementioned humans. MYBPC3 Mutant genes, humans MYBPC3 Mutant proteins or non-human animals MYBPC3 Application of reagents containing mutated genes in the preparation of kits for detecting hypertrophic cardiomyopathy.

[0032] This application provides for the detection of human wild-type... MYBPC3 Application of reagents for the (NM_000256.3)c.3708del variant in the preparation of a kit for detecting hypertrophic cardiomyopathy.

[0033] This application provides a kit for detecting hypertrophic cardiomyopathy, including the detection of the above-mentioned human... MYBPC3 Reagents for detecting mutated genes in the above-mentioned humans MYBPC3 Reagents or detection methods for mutant proteins in the aforementioned non-human animals. MYBPC3 A reagent for mutated genes. This kit can be used to screen subjects who have or are suspected of having hypertrophic cardiomyopathy.

[0034] Subjects can be humans or non-human animals, and the type of biological sample is not particularly limited, as long as a sample reflecting the presence of mutations in the biological sample can be extracted from it. For example, the biological sample can be at least one selected from blood, skin, or subcutaneous tissue, such as peripheral blood. The sample can be any nucleic acid sample that can reflect the presence of mutations in the biological sample; for example, it can be whole-genome DNA extracted directly from the biological sample, or a portion of the whole genome containing the coding sequence of a pathogenic gene, or total RNA extracted from the biological sample, or mRNA extracted from the biological sample. Detection of hypertrophic cardiomyopathy can include screening for hypertrophic cardiomyopathy risk. When the subject is a human, the reagents in this kit can detect mutations in the subject's biological sample. MYBPC3 Whether the gene coding sequence has a deletion mutation at position 3653, or whether the reagents in this kit can detect the presence of this mutation in the subject's biological sample. MYBPC3 Does the transcript sequence contain the c.3708del mutation or the p.G1218A fs mutation? 19 mutations. When the subject is a non-human animal, the reagents in the kit can detect the mutation in the subject's biological sample. MYBPC3 The gene coding sequence is similar to that of the human wild type. MYBPC3 Does the 3653rd base of the gene's coding sequence correspond to a missing base?

[0035] In one embodiment of this application, the reagent can be a reagent for detecting coding genes based on nucleic acid detection technology. Specifically, the reagent can be, but is not limited to, DNA polymerases, primers, probes, reporter labels (such as fluorescent labels), etc., involved in nucleic acid sequencing. In one embodiment of this application, the reagent includes at least one of a primer set and a probe. The sequences of exemplary primer sets are shown in SEQ ID NO. 5 and SEQ ID NO. 6. In one embodiment of this application, the reagent can be a reagent for detecting target proteins based on protein detection technology. Specifically, the reagent can be, but is not limited to, specific antibodies, etc.

[0036] This application provides a method for detecting hypertrophic cardiomyopathy in humans, including obtaining a human biological sample and detecting [the following] in the human biological sample. MYBPC3 Does the gene's coding sequence have a deletion mutation at position 3653, or MYBPC3Does the transcript sequence contain the c.3708del mutation, or MYBPC3 Does the transcript sequence possess p.G1218A fs? 19 mutation. If the above mutation is present in a human biological sample, hypertrophic cardiomyopathy is confirmed.

[0037] This application provides a method for detecting hypertrophic cardiomyopathy in non-human animals, including obtaining a non-human animal biological sample and detecting [the following] in the non-human animal biological sample. MYBPC3 The gene coding sequence is similar to that of the human wild type. MYBPC3 The gene's coding sequence is checked for a missing base at position 3653. If this mutation is present in a non-human animal sample, hypertrophic cardiomyopathy is confirmed.

[0038] In one embodiment of this application, nucleic acid detection technology or protein detection technology can be used to detect substances in biological samples. MYBPC3 Does the gene have the above-mentioned mutations?

[0039] This application provides a method for treating hypertrophic cardiomyopathy, including obtaining human biological samples or non-human animal biological samples; Detection of human biological samples MYBPC3 A mutation with a deletion at position 3653 in the gene coding sequence, or in non-human animal biological samples. MYBPC3 The gene coding sequence is similar to that of the human wild type. MYBPC3 The 3653rd base of the gene's coding sequence is deleted; a therapeutic agent is administered. Specifically, when the biological sample... MYBPC3 When a gene has the above-mentioned mutations, it is confirmed that the patient has hypertrophic cardiomyopathy and can be treated with therapeutic agents. Alternatively, other detection methods, such as the physiological indicators of the subject, can be used to determine which therapeutic agent to administer to the subject.

[0040] In one embodiment of this application, the therapeutic agent is selected from drugs for treating hypertrophic cardiomyopathy. In one embodiment of this application, the therapeutic agent can be a β-receptor blocker, such as propranolol or metoprolol. In another embodiment of this application, the therapeutic agent can be a non-dihydropyridine calcium channel blocker, such as verapamil or diltiazem.

[0041] This application provides a carrier, the carrier comprising the aforementioned human... MYBPC3 Mutant genes or those of the aforementioned non-human animals MYBPC3 Mutant gene.

[0042] This application provides a host cell, the host cell comprising the aforementioned human... MYBPC3 Mutant genes or those of the aforementioned non-human animals MYBPC3 Mutant gene or the aforementioned vector.

[0043] This application provides the aforementioned human... MYBPC3 Mutated genes, the aforementioned human MYBPC3 Mutant proteins, and the aforementioned non-human animals MYBPC3 The application of the mutated gene, the aforementioned vector, or the aforementioned host cell in constructing hypertrophic cardiomyopathy cell models and animal models. The hypertrophic cardiomyopathy cell model in this application can be a human hypertrophic cardiomyopathy cell model or a non-human animal hypertrophic cardiomyopathy cell model; the animal model in this application is a non-human animal model.

[0044] This application provides an sgRNA, the sequence of which is shown in SEQ ID NO.2.

[0045] This application provides a plasmid that is transcribed to yield the aforementioned sgRNA.

[0046] This application provides the preparation of the above-mentioned sgRNA or plasmid. MYBPC3 Applications in mutant cell models.

[0047] This application provides a MYBPC3 Methods for preparing mutant cell models include: preparing hypertrophic cardiomyopathy cell models using the aforementioned sgRNA or plasmid; or editing the cells to induce mutations within the cells. MYBPC3 The 3653rd base of the gene's coding sequence was deleted to produce... MYBPC3 Mutant cell model.

[0048] This application provides the application of the above-mentioned sgRNA or plasmid in the preparation of hypertrophic cardiomyopathy cell models.

[0049] This application provides a method for preparing a hypertrophic cardiomyopathy cell model, comprising: preparing a hypertrophic cardiomyopathy cell model using the above-mentioned sgRNA or plasmid; or editing human embryonic stem cells to induce their directed differentiation into cardiomyocytes, wherein the cardiomyocytes... MYBPC3 A hypertrophic cardiomyopathy cell model was created by deleting the 3653rd base of the gene's coding sequence.

[0050] In one embodiment of this application, gene editing can be performed using the aforementioned sgRNA or plasmid via CRISPR / Cas9 gene editing technology. MYBPC3 Preparation of mutant cell models or hypertrophic cardiomyopathy cell models.

[0051] This application provides a targeting vector, comprising encoding a target non-human animal. MYBPC3 Mutant genes or fragments thereof encode the target animal's MYBPC3 The mutated gene or its fragment is similar to the human wild type. MYBPC3 The 3653rd base of the coding sequence is missing, human wild type MYBPC3 The coding sequence of the gene is shown in SEQ ID NO.3. In other words, the targeting vector includes the coding sequence... MYBPC3 The nucleotide sequence or fragment of a mutated gene can be used to replace the corresponding gene sequence in the genome of a target non-human animal, thereby enabling the target non-human animal to... MYBPC3 The corresponding person in the encoded sequence MYBPC3 The deletion of the 3653rd base in the coding sequence is beneficial for constructing an animal model of hypertrophic cardiomyopathy, which in turn helps in the study of hypertrophic cardiomyopathy.

[0052] In one embodiment of this application, the targeting vector further comprises homologous sequences upstream and downstream of the target non-human animal gene target site and components of the vector itself. In one embodiment of this application, the targeting vector further comprises positive and negative selection markers, which is beneficial for subsequent screening. In one embodiment of this application, the targeting vector may include a plasmid vector.

[0053] This application provides information on the use of the aforementioned targeting vector in constructing non-human animal models of hypertrophic cardiomyopathy or in constructing models of hypertrophic cardiomyopathy. MYBPC3 Application of mutant genes in non-human animal models.

[0054] Different animals MYBPC3 Gene coding sequences have high homology, which can identify genes found in the human body. MYBPC3 Mutations in the sequence bind to different animals MYBPC3 In the sequence, different animals MYBPC3 People corresponding in the sequence MYBPC3 Mutating sequence mutation sites allows for the construction of targeting vectors and corresponding mutant non-human animal models for medical research. For example, mice... MYBPC3 The transcript number of the gene is NM_009608. Sequence alignment revealed that in mice... MYBPC3 The 3641st base of the transcript sequence corresponds to the wild-type human. MYBPC3 The 3708th base of the transcript sequence was used in mice via gene editing technology. MYBPC3 Deleting the 3641st base of the sequence can produce a mouse model of hypertrophic cardiomyopathy.

[0055] This application provides a method for constructing a non-human animal model with hypertrophic cardiomyopathy or for constructing a non-human animal model with hypertrophic cardiomyopathy. MYBPC3 Methods for constructing non-human animal models of mutated genes include: constructing a non-human animal model with hypertrophic cardiomyopathy using the aforementioned targeting vector, or constructing a non-human animal model with... MYBPC3 Non-human animal models with mutated genes; or gene editing technology used to modify non-human animals. MYBPC3 The coding sequence is similar to that of the human wild type. MYBPC3Deleting the base corresponding to position 3653 of the gene's coding sequence yields a non-human animal model of hypertrophic cardiomyopathy or a model of hypertrophic cardiomyopathy. MYBPC3 Non-human animal models of mutated genes, human wild type MYBPC3 The coding sequence of the gene is shown in SEQ ID NO.3. The above-mentioned targeting vectors or gene editing technologies can be used to modify non-human animals... MYBPC3 The coding sequence corresponds to the human wild type. MYBPC3 The coding sequence of the gene was deleted at position 3653, thereby obtaining a non-human animal model with hypertrophic cardiomyopathy. MYBPC3 Non-human animal models of mutated genes are beneficial for medical research on hypertrophic cardiomyopathy.

[0056] In one embodiment of this application, the gene editing technology is selected from at least one of the following gene editing methods: homologous recombination-based gene targeting technology, zinc finger nuclease technology, transcription activator-like effector nuclease (TALENs) technology, zinc finger nuclease (ENs) technology, CRISPR / Cas9 technology, CRISPR / Cas12a technology, CRISPR / Cas12i technology, prime editing technology, base editing technology, dual-base editing technology, single-stranded oligonucleotide (ssODN) or double-stranded DNA template repair technology, and viral vector-mediated integration.

[0057] In one embodiment of this application, the non-human animal or non-human animal model can be a mammal, such as at least one of mice, rats, cattle, Xenopus laevis, chickens, rabbits, dogs, chimpanzees, zebrafish, and pigs.

[0058] This application provides a non-human animal model of hypertrophic cardiomyopathy or a model of hypertrophic cardiomyopathy. MYBPC3 Non-human animal models of mutated genes, wherein the non-human animal model has hypertrophic cardiomyopathy or has MYBPC3 In non-human animal models of mutated genes MYBPC3 The coding sequence of the gene is similar to that of the human wild type. MYBPC3 The 3653rd base of the gene coding sequence is missing.

[0059] According to international guidelines for cardiomyopathy, adult myocardial hypertrophy is defined as a ventricular wall thickness of ≥15 mm in any myocardial segment. In this application, the following characteristics of cardiomyocytes are considered to indicate a hypertrophic cardiomyopathy cell model: a significant increase in the surface area of ​​cardiomyocytes in the mutant group compared to the wild-type control group. The following characteristics of non-human animal models are considered to indicate hypertrophic cardiomyopathy: increased heart / body weight compared to wild-type controls in the same litter, increased diastolic left ventricular anterior wall thickness as indicated by echocardiography, increased myocardial fibrosis as indicated by Masson staining, and / or increased expression of myocardial hypertrophy markers such as atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and myosin heavy chain gene 7 (MYH7).

[0060] This application provides the aforementioned hypertrophic cardiomyopathy cell model, or a non-human animal model of hypertrophic cardiomyopathy, or a model of hypertrophic cardiomyopathy. MYBPC3 Application of non-human animal models of mutated genes in screening drugs for the prevention or treatment of hypertrophic cardiomyopathy or in preparing products for evaluating the efficacy of treatment for hypertrophic cardiomyopathy.

[0061] In other words, hypertrophic cardiomyopathy cell models, or non-human animal models with hypertrophic cardiomyopathy, or... MYBPC3 The application of non-human animal models with mutated genes in screening drugs for the prevention or treatment of hypertrophic cardiomyopathy, or hypertrophic cardiomyopathy cell models, or non-human animal models with hypertrophic cardiomyopathy, or... MYBPC3 Application of non-human animal models of mutated genes in the preparation of products for evaluating the efficacy of treatments for hypertrophic cardiomyopathy. These products may, but are not limited to, drugs.

[0062] The above-mentioned hypertrophic cardiomyopathy cell models, or non-human animal models with hypertrophic cardiomyopathy, or those with MYBPC3 Non-human animal models of mutated genes can simulate the pathogenesis of human hereditary hemorrhage in vitro, providing a more objective reflection of the disease. MYBPC3 The pathological characteristics of hereditary hemorrhage caused by gene mutations are helpful in exploring the pathogenesis of hereditary hemorrhage caused by gene mutations, and are of great significance for further discovering drug targets that can be intervened in hereditary hemorrhage.

[0063] This application provides a device for detecting hypertrophic cardiomyopathy, comprising: The input module is used to receive the nucleotide sequence of the subject. The processing module is used to determine if the subject's nucleotide sequence matches that of a human wild-type human. MYBPC3 Compared to the coding sequence of the gene, the 3653rd base is deleted, generating the first treatment result. This first treatment result is used to indicate that the subject has hypertrophic cardiomyopathy, human wild-type. MYBPC3 The coding sequence of the gene is shown in SEQ ID NO.3; The output module is used to output the first processing result.

[0064] In one embodiment of this application, the device further includes a pre-storage module for storing human wild-type... MYBPC3 The coding sequence of a gene.

[0065] In one embodiment of this application, the input module is further configured to receive input of human wild type. MYBPC3 The coding sequence of a gene.

[0066] In one embodiment of this application, the processing module is further configured to, if it is determined that the nucleotide sequence of the subject is similar to that of a human wild-type... MYBPC3 The 3653rd base of the gene's coding sequence is identical, generating a second processing result, which is used to indicate that the subject does not have hypertrophic cardiomyopathy; the output module is also used to output the second processing result.

[0067] The following specific examples further illustrate the effects of the technical solution in this application.

[0068] According to the human shown in SEQ ID NO.1 MYBPC3 Mutated genes, using CRISPR / Cas9 technology based on homologous recombination, will carry human... MYBPC3 The single-stranded oligonucleotide donor DNA of the mutated gene was introduced into human embryonic stem cells stably expressing the Cas9 protein along with specific guide RNA. Cardiomyocytes were then induced to differentiate into cardiomyocytes. Cardiomyocytes derived from unmutated human embryonic stem cells were designated as the WT group.

[0069] Figure 1 Based on gene sequencing results, compared with the WT group, the homozygous (HO) group and the heterozygous (HE) group... MYBPC3 The gene has mutated, and the coding sequence is shown in SEQ ID NO.1. Therefore, a gene carrying the above-described mutation was successfully constructed. MYBPC3 The homozygous (HO) and heterozygous (HE) cell lines of the mutant gene were successfully used to construct cardiomyocyte models. Understandably, the homozygous cell line obtained is the HO group, and the heterozygous cell line is the HE group.

[0070] The expression of cardiac-specific troponin T (cTnT) in cardiomyocytes of each group was detected and statistically analyzed using flow cytometry and FITC fluorescent dye to obtain the differentiation efficiency of cardiomyocytes in each group. Figure 2 The graph shows the differentiation efficiency of cardiomyocytes in each group. Figure 2 In the middle (a), the differentiation efficiency of undifferentiated human embryonic stem cells (hESC) is shown. Figure 2(b) represents the differentiation efficiency of the WT group. Figure 2 (c) represents the differentiation efficiency of the HE group. Figure 2 In the figure, (d) represents the differentiation efficiency of the HO group. The horizontal axis represents the relative intensity of the fluorescence signal, in arbitrary fluorescence units, which is a dimensionless relative value. The vertical axis represents the cell number, which is a normalized relative count. "FITC-A-" indicates the percentage of cells with negative fluorescence signals, and "FITC-A+" indicates the percentage of cells with positive fluorescence signals. The cardiomyocyte differentiation efficiency of the WT group was 85%, that of the HE group was 84.1%, and that of the HO group was 83.8%. It can be seen that the differentiation efficiency levels of cardiomyocytes in each group are similar. MYBPC3 The gene mutation did not affect the differentiation efficiency of cardiomyocytes.

[0071] The surface area of ​​cardiomyocytes in each group was measured using flow cytometry. Figure 3 This is a comparison chart of the surface area of ​​cardiomyocytes in each group, where... Figure 3 (a) shows the surface area detection results of cardiomyocytes in each group. Figure 3 (b) is a statistical graph showing the surface area detection results of cardiomyocytes in each group. "FSC-A::FSC-A" indicates a two-parameter scatter plot with FSC-A as the horizontal and vertical axes. The horizontal axis represents the original intensity value of the forward scattered light signal, and the unit is the relative light intensity detected by the instrument. n=3. This indicates that p < 0.0001. It can be seen that, compared to the WT group, the surface area of ​​cardiomyocytes in both the HE and HO groups was significantly increased.

[0072] Cardiomyocytes were stained with 4',6-diamidinyl-2-phenylindole (DAPI), cTnT was stained with a green fluorescent dye secondary antibody, and actin was stained with a red fluorescent dye. The staining results of the three methods were then combined to form a merged result. The result is shown below. Figure 4 As shown, Figure 4 The images show immunofluorescence staining of cardiomyocytes in each group, with a scale bar of 20 μm. It can be seen that no obvious abnormalities were observed in the sarcomere structure of cardiomyocytes in each group.

[0073] After digestion, cardiomyocytes were reseeded at an appropriate density into culture plates with a nanopatterned surface, achieving a cell confluence of over 90% after seeding. After approximately 10 days of continued culture, the cardiomyocytes aligned regularly along the pattern, and their contraction direction became more uniform. Cardiomyocytes maintained in culture for approximately 35 days (±5 days) were then placed under an inverted microscope equipped with a camera system to record the dynamic process of spontaneous contraction. The spontaneous contraction activity of cardiomyocytes in each group was quantitatively assessed using the MUSCLEMOTION analysis system. Figure 5 This is a comparison diagram of the contractility of cardiomyocytes in different groups, where... Figure 5 (a) shows the results of the contractility test of myocardial cells in each group. Figure 5 (b) is a statistical graph showing the results of the contraction amplitude detection of myocardial cells in each group, n=3. This indicates that p < 0.05. This indicates p < 0.001. It can be seen that the contractility of cardiomyocytes in both the HE and HO groups was significantly higher than that in the WT group. In this example, the MUSCLEMOTION analysis system was used to quantitatively assess the spontaneous contractile activity of cardiomyocytes. Besides this, many other experimental methods exist, such as observing cell contractility using traction microscopy, calculating cell contractility using a microcolumn array, measuring cell contractility using a cantilever beam sensor, or detecting cell contractility using atomic force microscopy. All of these methods can obtain information about cell contractile function.

[0074] After digesting cardiomyocytes cultured for 30 days, reseeded them at an appropriate density in gelatin-pretreated culture plates, ensuring single-cell distribution. Cultured for another 3-5 days to allow cells to recover. On the day of the experiment, discard the culture medium and gently wash the cells twice with pre-warmed 37°C phosphate-powdered PBS to remove residual solution. Add 1 mL of 5 μM Fluo-4 AM calcium ion fluorescent probe working solution to each well. Incubate the culture plate in a 37°C CO2-free incubator in the dark for 30 min. After incubation, discard the Fluo-4 AM working solution from the wells and gently wash the cells three times with pre-warmed 37°C calcium-free PBS. After removing the waste solution, add 1 mL of pre-warmed PBS to each well. Start a confocal fluorescence microscope in a dark room, and install the electrical stimulation device and detection chamber. Remove the coverslip with forceps, place it in the detection chamber, and add approximately 500 μL of PBS to cover the sample. Set the electrical stimulation frequency to 1 Hz and use a 63x oil immersion microscope to acquire calcium signals. Using a linear scanning microscope at 63x oil immersion, cardiomyocytes with fluorescence intensity synchronized with contraction were selected. A straight line was used to select the intracellular region to be recorded (avoiding the nucleus; the scan time was set to 8 seconds). Cells were electrically stimulated several times at a frequency of 1 Hz, with at least eight consecutive stimulations, and changes in calcium transient fluorescence intensity were recorded. ImageJ software was used to analyze the calcium transient images. The main parameters acquired included calcium transient amplitude (ΔF / F0), time to peak, decay time, and duration. Figure 6 This is a comparison graph of calcium transient amplitudes, in which... Figure 6 (a) shows the results of calcium transient amplitude detection. Figure 6 (b) is a statistical chart of the calcium transient amplitude detection results; Figure 7The result of peak time detection is shown in the figure; Figure 8 The graph shows the results of calcium transient decay time detection. Figure 9 This is a comparison chart of the incidence of calcium alternation, in which... Figure 9 (a) shows the results of calcium alternation incidence detection. Figure 9 (b) is a statistical chart showing the results of calcium alternation incidence detection; n=3. This indicates that p < 0.05. p < 0.01, ns indicates no significant difference. It can be seen that compared to the WT group, the amplitude of calcium transients in both the HE and HO groups decreased, while calcium release kinetics (activation and reuptake rates) remained largely unchanged. Simultaneously, cells in the HO group were more prone to calcium exchange than those in the HE and WT groups, indicating... MYBPC3 Gene mutations lead to disruptions in cellular calcium cycling.

[0075] The above experiments demonstrated that the myocardial cells in the HE and HO groups exhibited enhanced contractility, disordered calcium circulation, and increased cell surface area. The myocardial cells in the HE and HO groups were a model of hypertrophic cardiomyopathy.

[0076] Through the above MYBPC3 Mutant genes can be used to construct cell models of hypertrophic cardiomyopathy (HCM) and non-human animal models of HCM, which is beneficial for simulating the pathogenesis of HCM in vitro and for exploring the pathogenesis mechanism of hereditary HCM caused by gene mutations. This is of great significance for further discovering drug targets that can be intervened in hereditary HCM.

[0077] The above description represents the preferred embodiments of this application, but should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this application, and these improvements and modifications are also considered to be within the scope of protection of this application.

Claims

1. A type of human MYBPC3 Mutant genes, characterized by, The human MYBPC3 The nucleotide sequence of the mutated gene is shown in SEQ ID NO.

1.

2. Detection of the human body as described in claim 1 MYBPC3 Application of reagents containing mutated genes in the preparation of kits for detecting hypertrophic cardiomyopathy.

3. A reagent kit for detecting hypertrophic cardiomyopathy, characterized in that, Including the detection of the human as described in claim 1 MYBPC3 Reagents for mutated genes.

4. An sgRNA, characterized in that, The sequence of the sgRNA is shown in SEQ ID NO.

2.

5. The application of sgRNA as described in claim 4 in the preparation of hypertrophic cardiomyopathy cell models.

6. A method for preparing a hypertrophic cardiomyopathy cell model, characterized in that, include: A hypertrophic cardiomyopathy cell model was prepared using the sgRNA described in claim 4; or Human embryonic stem cells were edited and induced to differentiate into cardiomyocytes. MYBPC3 A hypertrophic cardiomyopathy cell model was created by deleting the 3653rd base of the gene's coding sequence.

7. A target carrier, characterized in that, Including encoding targets non-human animals MYBPC3 Mutant gene or fragment thereof, which encodes the target animal's MYBPC3 The mutated gene or its fragment is similar to the human wild type. MYBPC3 The 3653rd base of the gene's coding sequence is missing, which is the human wild-type. MYBPC3 The coding sequence of the gene is shown in SEQ ID NO.

3.

8. The targeting vector as described in claim 7, used in constructing non-human animal models of hypertrophic cardiomyopathy or constructing models of... MYBPC3 Application of mutant genes in non-human animal models.

9. A method for constructing a non-human animal model of hypertrophic cardiomyopathy or constructing a model of hypertrophic cardiomyopathy. MYBPC3 The method for using non-human animal models of mutated genes is characterized by, include: Using the targeting vector described in claim 7, construct a non-human animal model of hypertrophic cardiomyopathy or construct a model of... MYBPC3 Non-human animal models of mutated genes; or Using gene editing technology to modify non-human animals MYBPC3 The coding sequence is similar to that of the human wild type. MYBPC3 Deleting the base corresponding to position 3653 of the gene's coding sequence yields a non-human animal model of hypertrophic cardiomyopathy or a model of hypertrophic cardiomyopathy. MYBPC3 Non-human animal models of mutated genes, specifically the human wild-type. MYBPC3 The coding sequence of the gene is shown in SEQ ID NO.

3.

10. A hypertrophic cardiomyopathy cell model prepared by the method of claim 6, or a non-human animal model of hypertrophic cardiomyopathy prepared by the method of claim 9, or a model of hypertrophic cardiomyopathy. MYBPC3 Application of non-human animal models of mutated genes in screening drugs for the prevention or treatment of hypertrophic cardiomyopathy or in preparing products for evaluating the efficacy of treatment for hypertrophic cardiomyopathy.

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