RyR2 gene mutation, detection kit, and method for constructing animal model thereof

Abstract

The present application discloses a RyR2 gene mutation, a detection kit, and a method for constructing an animal model thereof. The RyR2 gene mutation is a point mutation G4825R, which is associated with CPVT. Based on this, the present invention provides a kit specifically for detecting the point mutation, which can detect the point mutation based on nucleic acid detection technology or based on polypeptide detection technology. In addition, the present invention also provides a method for constructing a disease animal model with the point mutation and a corresponding disease animal model, so as to conduct medical research to deeply explore the pathogenesis of hereditary CPVT caused by gene mutation, which is of great significance for further exploring drug treatment targets that can intervene in CPVT.

Description

Technical field:

[0001] This invention patent application involves biotechnology, specifically the research findings on the RyR2 gene point mutation associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), which can be used to screen for diseases and to construct point mutation animal models for medical research. Background technology:

[0002] CPVT is a fatal ion channel disease that is common in children and adolescents. RyR2 gene mutations are the most common in CPVT patients. Current treatments have limited effects on CPVT patients. Therefore, it is necessary to study the potential molecular mechanism of CPVT caused by gene mutations in order to develop new targeted therapeutic drugs.

[0003] The RyR2 gene (NCBI number NG_008799.3) is the most common pathogenic gene of CPVT. The cardiac ion channel protein ryanodine receptor type 2 (RyR2) encoded by it is a homotetrameric, high-conductance, cation-selective channel that mediates the release of calcium ions in the sarcoplasmic reticulum (SR). Understanding the potential mechanism of RyR2 mutation is of great significance for understanding the occurrence and development of CPVT.

[0004] Unfortunately, due to the limitations of medical ethics and the incomplete penetrance of patients carrying mutations, there is still a lack of research on CPVT caused by RyR2 gene mutations at home and abroad. Therefore, the establishment of an effective animal model is an important prerequisite for in-depth exploration of the pathogenesis of CPVT caused by gene mutations, and is of great significance for further discovering drug treatment targets that can intervene in CPVT. Summary of the invention:

[0005] The present invention discovered a new RyR2 gene mutation site (p.G4825R), which causes CPVT. Compared with the normal RyR2 gene, the mutation changes the glycine at position 4825 to arginine.

[0006] In a first aspect, the present invention provides an isolated RyR2 mutant polypeptide or a fragment thereof, wherein the sequence of the RyR2 mutant polypeptide has a G4825R mutation compared to a normal RyR2 polypeptide.

[0007] In a second aspect, the present invention provides an isolated nucleic acid molecule or a fragment thereof encoding a mutant, wherein the nucleic acid molecule or the fragment thereof is a RyR2 gene or a fragment thereof, and compared with the normal RyR2 gene recorded in NCBI No. NG_008799.3, it has the following mutation: c.14473G>A mutation.

[0008] In a third aspect, the present invention provides a kit for screening a subject suffering from or suspected of suffering from CPVT, comprising a reagent suitable for detecting the p.G4825R mutant of the RyR2 gene.

[0009] In one embodiment, the reagent suitable for detecting the p.G4825R mutant of the RyR2 gene is a reagent for detecting the encoding gene based on nucleic acid detection technology, for example, selected from DNA polymerases, primers, probes, reporter markers such as fluorescent markers, etc. involved in nucleic acid sequencing.

[0010] In another embodiment, the reagent suitable for detecting the p.G4825R mutant of the RyR2 gene is a reagent for detecting the target polypeptide based on polypeptide detection technology, such as a specific antibody.

[0011] In a fourth aspect, the present invention provides a method for diagnosing a subject suspected of having CPVT, comprising obtaining a biological sample from the subject, detecting the presence of a p.G4825R mutant of the RyR2 gene, and confirming that the subject has or is at risk of having CPVT if the mutant is present.

[0012] In a fifth aspect, the present invention provides a method for detecting the p.G4825R mutant of the RyR2 gene in a subject suffering from or suspected of suffering from CPVT, comprising obtaining a biological sample from the subject, and detecting the p.G4825R mutant of the RyR2 gene in the biological sample based on nucleic acid detection technology or polypeptide detection technology.

[0013] In a sixth aspect, the present invention provides a method for diagnosing and treating a subject with CPVT, comprising obtaining a biological sample from the subject, detecting the presence of the p.G4825R mutant of the RyR2 gene, and confirming that the subject has or is at risk of having CPVT if the mutant is present; and administering a therapeutic agent to the confirmed subject.

[0014] Preferably the therapeutic agent is selected from a beta-blocker or a non-dihydropyridine calcium channel blocker.

[0015] Preferably, the beta-blocker is selected from propranolol, metoprolol and the like.

[0016] Preferably, the non-dihydropyridine calcium channel blocker is selected from verapamil or diltiazem wait.

[0017] According to the fourth to sixth aspects of the present invention, in one embodiment, the RyR2 gene p.G4825R mutant is detected based on nucleic acid detection technology;

[0018] In another embodiment, the RyR2 gene p.G4825R mutant is detected based on polypeptide detection technology.

[0019] In the seventh aspect, the present invention provides a targeting plasmid, comprising a nucleic acid molecule or a fragment thereof encoding a mutant of the RyR2 gene of a target animal, which is used to replace the corresponding endogenous gene sequence in the genome of the target animal, wherein the nucleic acid molecule or a fragment thereof encoding a mutant of the RyR2 gene of the target animal has c.14473G>A compared with the RyR2 gene of the wild-type target animal. Specifically, the targeting plasmid also comprises homologous sequences upstream and downstream of the target animal gene target site and the plasmid's own components, and preferably also comprises components such as positive and negative selection markers.

[0020] Those skilled in the art know that the corresponding tissue protein sequences of different animals are highly homologous. The gene point mutations found in the human body can be used to construct targeting plasmids, and the point mutations can be integrated into different animals to construct corresponding mutant animal models for medical research.

[0021] like Figure 1 As shown, the present invention also uses the NCBI database to compare the homology of RyR2 genes in humans, mice, rabbits, dogs, and cattle. Their sequences are consistent near G4825, and constructing their G4825R mutant animal models is also of research significance.

[0022] In an eighth aspect, the present invention provides a method for constructing an animal model of a RyR2 gene mutation, comprising using gene editing technology to mutate the 4825th amino acid residue of the RyR2 polypeptide of an animal corresponding to the human RyR2 polypeptide recorded in NCBI No. NG_008799.3 of the animal model from glycine to arginine.

[0023] Preferably, the gene editing technology is selected from one of the following: gene targeting technology based on homologous recombination, zinc finger nuclease technology, TALENs technology, CRISPR / Cas9 and CRISPR / Cas9 derivative technology.

[0024] The above are all mature gene editing technologies. No matter which gene editing technology is used, the purpose is to cause a point mutation in the coding gene of the target animal so that it can encode the RyR2 polypeptide with the G4825R mutation.

[0025] Specifically, the gene editing technology used in this article is a gene targeting technology based on homologous recombination. At this time, the steps of constructing the RyR2 gene mutation animal model include:

[0026] CRISPR / Cas9 technology was used to introduce the target point mutation by homologous recombination repair. The process is as follows: Cds9 mRNA and guide RNA were obtained by in vitro transcription, oligo donor DNA was obtained by synthesis, Cas9 mRNA, gRNA and donor DNA were microinjected into the fertilized eggs of animals, and F0 generation animals with the G4825R mutation of the RyR2 gene were obtained; F0 generation animals were mated with wild-type animals to obtain positive F1 generation animals.

[0027] In a ninth aspect, the present invention provides a RyR2 gene mutation animal model, in which the amino acid residue 4825 of the RyR2 polypeptide of the animal corresponding to the human RyR2 polypeptide recorded in NCBI No. NG_008799.3 is mutated from glycine to arginine.

[0028] According to the eighth or ninth aspect of the present invention, preferably, the animal model is selected from mammals, more preferably, selected from rats, mice, rabbits, dogs and cows.

[0029] Herein, the type of "nucleic acid" is not particularly limited, and may be any polymer containing deoxyribonucleotides and / or ribonucleotides corresponding to the coding gene of the mutant, including but not limited to DNA, RNA or cDNA. The nucleic acid, as will be understood by those skilled in the art, actually includes any one or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is given in most cases, in fact, the other strand complementary thereto is also disclosed. Those skilled in the art may also understand that one strand may be used to detect the other strand, and vice versa.

[0030] Herein, the type of "biological sample" is not particularly limited, as long as a nucleic acid sample reflecting whether a mutation exists in the biological sample can be extracted from the biological sample. According to an embodiment of the present invention, the biological sample can be at least one selected from human blood, skin, and subcutaneous tissue, preferably peripheral blood. Thus, sampling and detection can be conveniently performed, thereby further improving the efficiency of screening biological samples susceptible to CPVT. According to an embodiment of the present invention, the term "nucleic acid sample" used herein should be understood in a broad sense, and it can be any sample that can reflect whether a mutation exists in the biological sample, for example, it can be whole genome DNA directly extracted from the biological sample, it can also be a part of the whole genome containing the coding sequence of the pathogenic gene, it can be total RNA extracted from the biological sample, and it can also be mRNA extracted from the biological sample.

[0031] Beneficial effects:

[0032] The present invention has discovered a new RyR2 gene mutant G4825R associated with CPVT, and correspondingly provided a means for detecting the RyR2 gene mutant, so that disease screening and therapeutic intervention can be carried out in a timely manner; further, based on the RyR2 gene mutant, a targeting plasmid for constructing a corresponding disease animal model and a disease animal model construction method are provided, and the obtained disease animal model better simulates the pathogenesis of human CPVT in vivo, more objectively reflects the pathological characteristics of CPVT caused by RyR2 gene mutation, and deeply explores the pathogenesis of hereditary CPVT caused by gene mutation, which is of great significance for further discovering drug treatment targets that can intervene in CPVT. Description of the drawings:

[0033] Hereinafter, the specific contents of the present invention will be described in detail with reference to the accompanying drawings:

[0034] Figure 1 A comparison of the homology of the RyR2 genes of humans, mice, rabbits, dogs, and cattle is shown;

[0035] Figure 2 It shows the identification of the mutated gene in the proband of a clinical CPVT family;

[0036] Figure 3 The design strategy of site-directed knock-in of RyR2 gene mutation is shown;

[0037] Figure 4 The results of restriction enzyme digestion identification of the targeting plasmid are shown;

[0038] Figure 5 Mouse genome identification results are shown;

[0039] Figure 6 The results of changes in the expression level of RyR2 gene in model mice are shown;

[0040] Figure 7 This is the electrocardiogram of the model mouse in a resting state;

[0041] Figure 8 The electrocardiogram of the model mice under stress state is shown;

[0042] Fig. 9 A brief process of subsequent mouse breeding is shown; Specific implementation method:

[0043] The present invention is further described below in conjunction with specific embodiments, and the following embodiments are only exemplary and do not constitute any limitation to the scope of application of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0044] The equipment and materials used in the present invention are all commercially available. The sources of some equipment and reagents are listed below:

[0045] Required equipment and instruments

[0046]

[0047] Main reagents required

[0048] (1) Methanol (Shantou Xilong Scientific Co., Ltd., China);

[0049] (2) polyvinylidene fluoride membrane (Milipore, USA);

[0050] (3) Ammonium persulfate (Tianjin Damao Chemical Reagent Factory, China);

[0051] (4) Phosphate buffer (Beijing Zhongshan Jinqiao Co., Ltd., China);

[0052] (5) Isoflurane (Shenzhen Reward Life Science Technology Co., Ltd., China);

[0053] (6) Skim milk powder for blocking, protein immunoblotting membrane regeneration solution (Beijing Pulilai Co., Ltd., China);

[0054] (7) RIPA cell lysis buffer (strong), protein loading buffer, and BCA protein concentration assay kit (Shanghai Bio-Tech Co., Ltd., China);

[0055] (8) Protease / phosphatase inhibitors, gel markers, and fluorescent color development kit (Thermo Fisher Scientific, USA);

[0056] (9) Tetramethylethylenediamine, phenylmethylsulfonyl fluoride, Tris-base powder, SDS powder, Tween-20, glycine (Beijing Solebow Co., Ltd., China);

[0057] (10) Blood / cell / tissue genomic DNA extraction kit, RNAsimple total RNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd.);

[0058] (11)PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, Japan);

[0059] (12) PCR reverse transcription kit, TB Premix Ex TaqTM (Takara, Japan);

[0060] (13) Nuclear Extraction Reagent (NER) (Thermo Fisher Scientific, USA); (14) Primary antibody RyR2 (ab2868, abcam, USA);

[0061] (15) Primary Antibody Tubulin (ab18207, abcam, USA)

[0062] (21) mouse / rabbit secondary antibody (Beijing Zhongshan Golden Bridge Co., Ltd., China);

[0063] The inventors conducted high-throughput DNA sequencing of peripheral blood samples from a CPVT patient with a family history of sudden death and found that the RyR2 gene had a G4825R mutation, clarifying the association between the mutation and the onset of CPVT. For disease screening, discovering the associated gene point mutation is the key. The inventors provided information on the association between the RyR2 gene p.G4825R mutation and CPVT disease. Based on this information and combined with conventional technical means in the field, those skilled in the art can develop corresponding diagnostic reagents, diagnostic methods and systems in terms of nucleic acid detection technology and polypeptide detection technology, which will not be repeated here. Based on the discovery of this mutant, a corresponding disease animal model can also be constructed to facilitate medical research. The following will describe in detail the process of discovering the association between the RyR2 gene p.G4825R mutation and CPVT disease, as well as the process of constructing a disease animal model.

[0064] Example 1: Screening of mutated genes in clinical CPVT pedigree patients

[0065] The previous clinical data and auxiliary examination results of the proband were queried, and the clinical information of all family members of the proband was collected, including basic information, general condition, current medical history, past medical history, family history and changes in cardiac structure, etc. Then, a pedigree chart was drawn based on the basic conditions of the patient and his family members. The proband and his family members were fully informed of the purpose of the operation and agreed to it. After signing the informed consent form, 5 mL of peripheral venous blood was collected from all family members at the blood drawing department of the Second Affiliated Hospital of Nanchang University.

[0066] The present invention includes a female patient with a family history of sudden death. The proband had a normal electrocardiogram before the exercise treadmill test, but had frequent premature beats during exercise and occasional ventricular premature beats (VPs) after exercise. Her echocardiogram showed no abnormalities, and the main indicators of blood and urine tests were within normal ranges. The proband's daughter had similar symptoms of syncope and unfortunately died suddenly after school activities. Based on clinical manifestations, auxiliary examinations and family history, the proband was preliminarily judged to be a CPVT patient.

[0067] High-throughput sequencing was performed on DNA extracted from the proband's peripheral venous blood. The high-throughput sequencing results were sequenced in the HGMD database, and all genes with clear pathogenic relationships in the OMIM database were analyzed one by one. The function of the gene was analyzed in combination with the GeneCards database, and the candidate mutant gene was determined to be RyR2. Secondly, PCR amplification was performed on the RyR2 gene mutation region in the genomic DNA of family members, and the mutation site was verified by Sanger sequencing. After screening and verifying the mutated gene site, it was found that the RyR2 p.G4825R mutation was the first gene mutation discovered ( Figure 2 ). Since there were no previous reports on this mutation, this study used mutation annotation tools wANNOVAR, SIFT, Polyphen2, MutationAssessor, FATHMM, PROVEAN, and M-CAP to predict the pathogenicity of the selected sites and found that the RyR2 p.G4825R mutation may be a highly pathogenic mutation (see the table below).

[0068]

[0069] Example 2: Construction of RyR2 gene G4825R mutation animal model

[0070] CRISPR / CaS9 technology was used to introduce the target point mutation by homologous recombination repair. The process is as follows: Cds9 mRNA and guide RNA were obtained by in vitro transcription, oligo donor DNA was obtained by synthesis, and Cas9 mRNA, gRNA and donor DNA were microinjected into the fertilized eggs of C57BL / 6J mice to obtain F0 generation mice. After PCR product sequencing, F0 generation mice with target gene point mutations were obtained; F0 generation mice were mated with C57BL / 6J mice to obtain positive F1 generation mice ( Figure 3 , Figure 4 ).

[0071] Example 3: Extraction and identification of mouse DNA

[0072] 3.1 Extraction of mouse tail genomic DNA:

[0073] (1) Cut the tip of the mouse tail (length < 0.3 cm) and place it in a 1.5 mL nuclease-free centrifuge tube;

[0074] (2) Add 200 μL of buffer GA and 20 μL of proteinase K solution, mix the solution thoroughly and ensure that the mouse tail is submerged in the solution;

[0075] (3) Place the centrifuge tube in a 56°C constant temperature water bath and digest the rat tail overnight;

[0076] (4) Add 200 μL of buffer GB to the centrifuge tube, invert and mix thoroughly, and place at 70°C for 10 min. The solution will become clear at this time. Centrifuge briefly to remove water droplets on the inner wall of the tube;

[0077] (5) Add 200 μL of anhydrous ethanol and vortex thoroughly for 15 seconds. Flocculent precipitation may appear. Centrifuge briefly to remove water droplets on the inner wall.

[0078] (6) Add the resulting solution and flocculent precipitate to the adsorption column CB3 (the adsorption column is placed in the collection tube), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and place the adsorption column CB3 back into the collection tube;

[0079] (7) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and return the adsorption column to the collection tube;

[0080] (8) Add 600 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and put the adsorption column CB3 back into the collection tube;

[0081] (9) Repeat step 8;

[0082] (10) Centrifuge at 12000 rpm for 2 min and discard the waste liquid. Place the adsorption column CB3 at room temperature for 10 min to completely dry the residual rinse liquid PW;

[0083] (11) Transfer the adsorption column CB3 into a clean centrifuge tube, add 80 μL of elution buffer TE to the center, leave at room temperature for 5 min, centrifuge at 12000 rpm for 2 min, and collect the resulting liquid into a centrifuge tube;

[0084] 3.2 Identification of mouse RyR2 genotype:

[0085] In the subsequent mouse mating and breeding process, the mouse RyR2 genotype can be identified by short-fragment PCR. The mouse tail DNA is amplified by PCR and the obtained product is directly sequenced to determine the genotype (DNA identification results are shown in Figure 5 ). The primers and conditions required for mouse genotype identification in the subsequent mouse breeding steps are as follows.

[0086] (1) Primer design:

[0087] The PrimerSelect program in DNASTAR software was used for PCR amplification and sequencing primer design to ensure that there were no complementary structures and hairpin structures between the 5' and 3' primers and between themselves, and that sequencing could cover the mutation site region. The forward primer SEQ ID NO.1 was amplified, the reverse primer SEQ ID NO.2 was amplified, and the sequencing primer SEQ ID NO.3 was designed.

[0088] (2) PCR reaction system configuration table:

[0089]

[0090] Example 4: Determination of changes in expression of mutant genes in model mice

[0091] Western blot and Real time PCR techniques were used to detect the expression of RyR2 gene in the myocardium of WT and KI group mice.

[0092] 4.1 Total protein extraction from tissues:

[0093] (1) preparing RIPA lysis buffer (containing 1% phenylmethylsulfonyl fluoride and 10% each of pre-prepared protease and phosphatase inhibitors) according to the ratio;

[0094] (2) Heparinized mice (5 IU / g body weight of heparin sodium solution was injected intraperitoneally) were anesthetized with isoflurane gas and fixed on a workbench in a supine position;

[0095] (3) Rapidly open the chest cavity to obtain the mouse heart, place the obtained tissue in a pre-cooled PBS solution, and perfuse and flush the residual blood in the heart;

[0096] (4) Take 50 mg of tissue, dry it with absorbent paper, put it into a pre-cooled 1.5 mL centrifuge tube, and add 500 μL of RIPA lysis buffer to each tube;

[0097] (5) Cut the tissue into small pieces and homogenize it with a homogenizer until it is fully lysed;

[0098] (6) Precool a low-temperature refrigerated high-speed centrifuge to 4°C, place the centrifuge tube in the centrifuge and balance it, set it to 12,000 rpm and centrifuge for 15 min. After the centrifugation is completed, carefully remove the centrifuge tube (avoid oscillation and precipitation), fully separate the supernatant and add it to the precooled centrifuge tube, and place it in a -80°C refrigerator for freezing.

[0099] 4.2 Total RNA extraction from tissues:

[0100] (1) Add the specified amount of anhydrous ethanol to the deproteinizing solution RD and the rinse solution RW. The amount added is marked on the bottle.

[0101] (2) Grind the obtained tissue in liquid nitrogen, add 1 mL of lysis solution RZ to every 50-100 mg of tissue, and pulverize it with a homogenizer (the sample volume should be less than 1 / 10 of the volume of lysis solution RZ);

[0102] (3) The homogenized sample was placed at room temperature for 5 min to completely separate the nucleic acid and protein in the sample, and then centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was carefully transferred into a pre-cooled nuclease-free centrifuge tube;

[0103] (4) Add 200 μL of chloroform to the supernatant, cover the tube tightly, shake vigorously for 15 seconds, and leave at room temperature for 5 minutes;

[0104] (5) After centrifugation at 12,000 rpm for 10 min at 4°C, the sample can be separated into three layers: a yellow organic phase, an intermediate layer, and a colorless aqueous phase. The RNA-rich aqueous phase is transferred into a pre-cooled nuclease-free tube for the next step.

[0105] (6) Add 1 / 2 volume of anhydrous ethanol of the colorless aqueous phase to the tube, mix thoroughly and transfer all of it to the adsorption column CR3. Centrifuge at 12000 rpm for 30 s at 4°C and discard the waste liquid in the collection tube.

[0106] (7) Add 500 μL of pre-treated deproteinized solution RD to the adsorption column CR3, centrifuge at 12,000 rpm for 30 s at 4 °C, discard the waste liquid, and return CR3 to the collection tube;

[0107] (8) Add 500 μL of the previously treated rinse solution RW to the adsorption column CR3, let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 30 s at 4 °C, and discard the waste liquid;

[0108] (9) Repeat the previous step;

[0109] (10) Place the adsorption column CR3 back into the collection tube and centrifuge at 12,000 rpm for 2 min at 4 °C to completely remove the residual waste liquid;

[0110] (11) Place CR3 in a clean RNase-free collection tube, add 30-100 μL RNase-Free ddH2O, place at room temperature for 3 min, centrifuge at 12000 rpm for 2 min at 4°C, fully recover the obtained liquid, measure the RNA concentration, and store in a -80°C refrigerator for future use to prevent degradation;

[0111] 4.3 Western Blotting:

[0112] (1) Place the prepared gel pad and gel rack on a horizontal surface, clean the thick and thin glass plates and dry them thoroughly, fix the glass plates tightly and assemble them, and prepare appropriate amounts of concentrated gel and separation gel according to the preparation table in the instruction manual;

[0113] (2) Mix the separation gel prepared in the previous step and quickly pour it into the fixed thick and thin glass plates, and reserve a 2.5 cm space on the top for the concentrated gel to be poured. At this time, gently add anhydrous ethanol on the top of the separation gel to remove bubbles and seal the gel. Let it stand for at least 30 minutes. After the gel is fully solidified, discard the anhydrous ethanol and use filter paper to absorb the residual anhydrous ethanol. After it is fully removed, proceed to the next step;

[0114] (3) After the prepared concentrated gel is fully mixed, it is quickly and evenly poured onto the solidified separation gel, and floating bubbles are removed. A tooth comb is carefully inserted and allowed to stand for at least 30 minutes. After the gel is fully solidified, proceed to the next step;

[0115] (4) Remove the unsolidified gel attached to the glass plate, fix the glass plate stably in the electrophoresis tank and add the pre-prepared electrophoresis solution until the electrophoresis solution completely covers and submerges the gel, gently and vertically pull out the toothed comb, and use a 200 μL micropipette tip to carefully blow away impurities and bubbles in the sample well;

[0116] (5) Use a 10 μL micropipette to add the treated protein sample (add 1 / 4 volume of the sample to 5× Loading Buffer and treat in a boiling water bath for 10 min) into the designed loading well. The loading volume is 20 μL-40 μL (determined by the protein concentration), and add an appropriate amount of marker on both sides of the sample well (determined by the concentration of the separation gel);

[0117] (6) After loading, cover the lid and set the starting electrophoresis voltage to 80V. Increase the voltage appropriately as needed. Finally, determine the end time of electrophoresis based on the molecular weight of the target protein. After electrophoresis is complete, carefully remove the gel, discard the electrophoresis solution, and clean the electrophoresis tank for later use.

[0118] (7) After taking out the gel plate, carefully rinse it with ddH2O to remove the residual electrophoresis solution. Use a seesaw to carefully separate the thick and thin glass plates at the edges, and cut the gel appropriately according to the required molecular weight. Then, peel the remaining gel off the glass plate and gently transfer it to the filter paper soaked in the transfer solution and equilibrate for 5 minutes.

[0119] (8) Cut a polyvinylidene fluoride (PVDF) membrane of appropriate size and pre-activate it in methanol. Gently and evenly cover the PVDF membrane on the gel, remove all bubbles between the gel and the membrane, and place them in the order of sponge pad, filter paper, membrane, gel, filter paper, and sponge pad from top to bottom. Fix them with transfer clips and place them in the transfer tank in the correct direction of positive and negative electrodes. Pour the pre-prepared 1× SDS-PAGE transfer solution until the transfer clip is completely covered. Place ice packs to cool down. Place the transfer tank in a 4°C chromatography refrigerator. Set the constant current to 300A for transfer (the constant current value can be adjusted according to needs). Set the transfer time according to the required molecular weight.

[0120] (9) After the transfer is completed, turn off the power, discard the gel, carefully remove the PVDF membrane, and gently rinse it in 1× TBST for 5 min to wash away the excess transfer solution;

[0121] (10) Add the rinsed PVDF membrane in the previous step to the pre-prepared 5% skim milk for blocking and block at room temperature for at least 1 h (during blocking, place it on a horizontal shaker and shake gently to ensure full contact between the membrane and the milk);

[0122] (11) After blocking is completed, gently remove the PVDF membrane and rinse it in 1× TBST to remove residual skim milk;

[0123] (12) Cut the PVDF membrane according to the molecular weight required by the experiment, seal the cut PVDF membrane with plastic film, add the pre-prepared primary antibody diluent (prepare the antibody diluent of appropriate concentration according to the instructions), quickly seal after removing all bubbles, and place in a 4°C chromatography refrigerator for incubation overnight (at least 12 hours);

[0124] (13) After the incubation is completed the next day, the primary antibody dilution solution is recovered and stored in a 4°C chromatography refrigerator. The membrane is soaked in 1× TBST buffer and rinsed three times (10 min / time) on a horizontal shaker at room temperature.

[0125] (14) Prepare the corresponding secondary antibody dilution (dilution ratio is 1:4000 to 1:8000), seal the washed PVDF membrane with plastic film, add an appropriate amount of secondary antibody dilution and quickly seal it, place it on a horizontal shaker and shake gently, incubate at room temperature for at least 1 hour, after incubation is completed, remove the PVDF membrane and soak it in 1×TBST, place it on a horizontal shaker and rinse it 3 times at room temperature (10 min / time);

[0126] (15) After the membrane is washed, prepare an appropriate amount of color reagent (luminescent reagent A and B are mixed in a 1:1 ratio and used immediately after preparation), and use a pipette to gently and evenly blow the prepared color reagent to cover the surface of the PVDF membrane. The PVDF membrane that has fully reacted with the color reagent is exposed using a gel imaging instrument, and image acquisition is performed after setting appropriate exposure parameters;

[0127] (16) After image acquisition, the band data were analyzed using ImageLab 4.0.1 software and saved for statistical analysis.

[0128] 4.4 Real-time fluorescence quantitative PCR:

[0129] (1) Use the Takara reverse transcription kit to reverse transcribe the extracted RNA into cDNA. Prepare the reaction mixture on ice according to the ingredients in the table below. To ensure sufficient reaction solution, prepare the reaction mixture according to the number of reactions + 2, and then divide it into each reaction tube. Finally, add an appropriate amount of RNA sample and let it stand at room temperature for 30 minutes.

[0130]

[0131] (2) Prepare the reverse transcription reaction solution on ice according to the table below. To ensure sufficient reaction solution, prepare the amount of the reaction number + 2, and then dispense it into each reaction tube. After gently mixing, immediately start the reverse transcription reaction. The reverse transcription PCR reaction conditions are: 37°C × 15 min → 85°C × 5 s → maintain 4°C;

[0132]

[0133] (3) Determine the concentration of the synthesized cDNA for subsequent use. If long-term storage is required, store it at -20°C or lower.

[0134] (4) Using TB Premix Ex TaqTM kit was used for real-time fluorescence quantitative PCR reaction. The reaction system was as follows:

[0135]

[0136]

[0137] Add 20 μL of the prepared sample to a 96-well plate, with 3 wells for each type of sample. The reaction conditions are as follows: 95°C × 30 s pre-denaturation → 95°C × 5 s and 60°C × 30 s for a total of 40 cycles → maintain at 4°C;

[0138] (5) After the reaction, the cycle threshold (Ct) obtained by the system was obtained, and the relative expression level of the mRNA of the RyR2 gene was calculated using the 2-ΔΔCt method. The forward primer was SEQ ID NO.4, the reverse primer was SEQ ID NO.5, and the housekeeping gene GAPDH (Glyceraldehyde 3-phosohate dehydrogenase) was used as an internal reference. The forward primer was SEQ ID NO.6, and the reverse primer was SEQ ID NO.7. Both primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0139] The results are as follows Figure 6 As shown in the figure, the results of PCR and Western blot showed that compared with the WT group, the expression of RyR2 in the left ventricular tissue of KI mice did not change significantly at the mRNA level (P>0.05), and at the protein level, the expression of RyR2 in the heart of KI mice was lower than that of WT mice (P<0.05). The results showed that the reduced expression of RyR2 in cardiomyocytes may be the molecular basis of its pathogenicity.

[0140] Example 5: Echocardiographic detection of model mice

[0141] WT (n=7) and KI (n=10) mice of similar growth and development were selected at 9 weeks of age and were rapidly anesthetized using inhalation anesthesia isoflurane. Two-dimensional echocardiographic data of the experimental mice were obtained using a small animal ultrasound device. The two-dimensional echocardiographic data showed that there were no significant differences in cardiac structure and cardiac function between the WT and KI groups of mice, and both groups of data were within normal values. The results are shown in the following table:

[0142]

[0143] Example 6: Model mouse DSI implantable physiological signal telemetry system monitors electrocardiogram

[0144] 6.1 Implantation Surgery Procedure

[0145] (1) Preoperative preparation: Sterilize and disinfect surgical instruments and related supplies under high pressure; clean the operating table; sterilize under ultraviolet light in the operating room for 12 hours; debug the TA10-ETA-F10 implant to ensure that it can work properly, and soak the implant catheter in saline for more than 15 minutes.

[0146] (2) The mice were anesthetized with 1% isoflurane at a flow rate of 1.0 L / min. After anesthesia, the mice were fixed on the operating table in a supine position. The chest and abdomen were prepared and disinfected with a depilatory cream, and the mice were covered with a sterilized surgical drape.

[0147] (3) Specific intraoperative process: Use scissors to bluntly separate the implant pocket from the subcutaneous tissue. Use a 2 ml syringe to inject preheated saline into the pocket to clean the implant pocket. Place the implant body into the implant pocket and correctly expose the ends of the positive and negative wires of the implant. Make a small incision on the right pectoral muscle and the left upper abdomen respectively, lead out and bury the positive and negative wires, and simulate the position of the electrocardiogram II lead to install the positive and negative wires of the implant. Connect the American DSI (Data Sciences International) implantable physiological signal monitoring system and set the relevant parameters. Turn on the monitoring system switch and record the electrocardiogram for several minutes to check whether the electrode is successfully installed. Carefully clean the wound surface and wound again with saline, and then suture the wound with simple interrupted sutures.

[0148] (4) Postoperative care: Keep the animals individually in a sterilized, clean cage in a suitable environment (temperature: 25°C, humidity: 40-70%). Observe the wound every day. It takes about 1 week for the wound to fully recover.

[0149] 6.2 Real-time monitoring of the electrocardiogram of mice

[0150] Place the cage containing mice on the signal receiving panel of the American DSI ECG telemetry system, use a magnet to turn on the implant switch, and use the Dataquest ART 4.0 system to monitor the ECG of mice in real time when they are awake. Under the basic state, the ECG of mice is continuously monitored for at least 3 days, and each time it is recorded for 30-60 minutes. Under the acute stress state (2mg / kg epinephrine, intraperitoneal injection), the ECG of mice is continuously recorded for at least 1 hour, and the mice are prevented from falling asleep as much as possible, otherwise the measured heart rate will be lower than the actual situation. Under the basic and stress states, the occurrence of arrhythmias in mice is mainly observed.

[0151] The results are as follows Figure 7 , Figure 8 As shown in the figure, the electrocardiogram data obtained from WT and KI mice under basal conditions showed that no obvious spontaneous arrhythmias were found in the electrocardiograms of both groups of mice. However, the susceptibility of KI mice to ventricular arrhythmias such as ventricular premature beats, trigeminy, non-sustained or sustained ventricular tachycardia was significantly increased under stress. Sequence Listing <110> The Second Affiliated Hospital of Nanchang University <120> RyR2 gene mutation, detection kit, and method for constructing animal model thereof <141> 2022-06-20 <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <400> 1 ccaagcggca cagaaggt 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <400> 2 ccaagcggca cagaaggt 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <400> 3 ccaagcggca cagaaggt 18 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 gccttctacc cactcctg 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <400> 5 cctcctcggt gtctttca 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <400> 6 tgtttcctcg tcccgtag 18 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 caatctccac tttgccact 19

Claims

1. An isolated RyR2 mutant polypeptide, characterized in that The amino acid sequence of the mutant polypeptide is the normal RyR2 polypeptide amino acid sequence recorded in NCBI No. NG_008799.3, wherein the glycine at position 4825 is mutated to arginine.

2. An isolated nucleic acid molecule encoding a mutant, characterized in that The nucleotide sequence of the nucleic acid molecule is the nucleotide sequence of the normal RyR2 gene transcript coding region recorded in NCBI No. NG_008799.3, in which the guanine at position 14473 is mutated to adenine.

3. A kit for screening subjects suffering from or at risk of catecholamine-sensitive polymorphic ventricular tachycardia, characterized in that It contains a reagent suitable for detecting a RyR2 gene p.G4825R mutant in a biological sample from a subject, wherein the coding gene sequence of the RyR2 gene p.G4825R mutant is a mutation of guanine at position 14473 in the coding region of the transcript of the normal RyR2 gene recorded in NCBI No. NG_008799.3 to adenine; Among them, the reagent suitable for detecting the RyR2 gene p.G4825R mutant in a biological sample from a subject is a reagent for detecting the encoding gene based on nucleic acid detection technology, and the reagent for detecting the encoding gene based on nucleic acid detection technology includes DNA polymerase, primers, probes, and reporter markers involved in nucleic acid sequencing.

4. A method for constructing a RyR2 gene mutation mouse model, characterized in that: The method comprises using gene editing technology to mutate the glycine at position 4825 of the human RyR2 polypeptide recorded in NCBI No. NG_008799.3 on the RyR2 polypeptide of the mouse model into arginine.

5. The method for constructing a RyR2 gene mutation mouse model according to claim 4, characterized in that: The gene editing technology is selected from the following: gene targeting technology based on homologous recombination, zinc finger nuclease technology, TALENs technology and CRISPR / Cas9 technology.

6. A method for constructing a RyR2 gene mutation mouse model, characterized in that: Using CRISPR / Cas9 technology and homologous recombination repair, we introduce target point mutations. The process is as follows: Cds9 mRNA and guide RNA are obtained by in vitro transcription, oligodonor DNA is obtained by synthesis, Cas9 mRNA, gRNA and donor DNA are microinjected into fertilized eggs of mice to obtain F0 generation mice carrying the target point mutation; F0 generation mice are mated with wild-type mice to obtain positive F1 generation mice; The target point mutation is that the glycine corresponding to the glycine at position 4825 of the mouse RyR2 polypeptide and the human RyR2 polypeptide recorded in NCBI No. NG_008799.3 is mutated to arginine.

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