Zebra fish model for screening arrhythmia drugs and application of zebra fish model

Abstract

The invention relates to a zebrafish model for screening arrhythmia drugs and application thereof, and belongs to the technical field of biological medicines. According to the transgenic zebrafish provided by the invention, an appropriate arrhythmia zebrafish model can be screened out through a fluorescence intensity IOD value, the fluorescence intensity is in positive correlation with the transgenic expression quantity, the transgenic expression level can be quickly judged according to the fluorescence intensity, and an appropriate individual can be selected for subsequent experiments. Isoprenaline is an anti-arrhythmia drug which is widely used clinically at present, an isoprenaline treatment experiment verifies the effectiveness of the model for screening the anti-arrhythmia drug, and the model is not only suitable for researching the pathogenesis of hereditary arrhythmia diseases, but also suitable for researching the pathogenesis of the hereditary arrhythmia diseases. Technical support is also provided for research and development of arrhythmia treatment medicines, and important theoretical significance and wide application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to a zebrafish model for screening arrhythmia drugs and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Arrhythmias are disorders characterized by abnormalities in the frequency, rhythm, site of origin, conduction velocity, or activation sequence of cardiac impulses. Malignant arrhythmias can rapidly impair the heart's pumping function, leading to hemodynamic impairment and a major cause of syncope and sudden death. Statistics show that sudden cardiac death accounts for 88% of all sudden cardiac deaths, with approximately 540,000 deaths in my country each year. This has made antiarrhythmic drugs a key focus and hotspot in current drug research and development. Current antiarrhythmic drugs can cause a drop in blood pressure, exacerbate existing arrhythmias, or induce new ones. There is an urgent need to identify new antiarrhythmic drugs with improved efficacy and minimal side effects. Currently available arrhythmia models include mouse and zebrafish models. The physiological functional requirements of the mouse heart differ from those of humans. The electrophysiological activity of its cardiomyocytes focuses on rapid excitation-contraction coupling to maintain its pumping function at high heart rates. Consequently, the repolarization process is relatively simple and rapid, lacking a plateau phase. Compared with other models, the zebrafish model has high reproducibility, transparent body, heart rate closer to human heart rate, average QT interval, resting membrane potential and action potential (AP potential) amplitude similar to humans. Therefore, zebrafish can be used as an ideal model organism for studying arrhythmia diseases and establishing a large-scale in vivo screening model for antiarrhythmic active compounds. In summary, the present invention constructs KCNQ1 A transgenic zebrafish model of arrhythmia with overexpression of pathogenic mutations was developed. Using an inverted fluorescence microscope and a Zeiss microscope, cardiac function indicators were efficiently collected. Combined with cardiac function analysis, a new technology for evaluating antiarrhythmic activity was established to screen antiarrhythmic active compounds, providing a theoretical basis for the research and development of new antiarrhythmic drugs in my country.

[0003] KCNQ1 The gene is a member of the potassium voltage-gated channel subfamily Q and is located on human chromosome 11p15.5-p15.4. It encodes a voltage-gated potassium channel required for the repolarization phase of the cardiac action potential. KCNQ1 The encoded Kv7.1 channel forms the IKs current with the minK subunit, which plays an important role in cardiac action potential repolarization. Loss-of-function mutants cause long QT syndrome type 1 (LQT1). The zebrafish KCNQ1 protein sequence is 85% identical to that of the human KCNQ1 protein. KCNQ1 V180L It was found in clinical patients KCNQ1 Gene mutation, the present invention specifically established cardiac tissue overexpression KCNQ1 V180LTransgenic zebrafish provide a model animal for the study of the pathogenesis of inherited arrhythmias and their drug targets, and optimize appropriate model parameters to obtain a model application method for evaluating arrhythmias that is fast in modeling, high in throughput, low in cost, and convenient in testing. Summary of the Invention

[0004] In view of the deficiencies of the existing technology, the present invention provides a zebrafish model for screening arrhythmia drugs and applications thereof.

[0005] The technical solutions of the present invention are as follows: A zebrafish model for arrhythmia drug screening, wherein the zebrafish model is KCNQ1 V180L The mutant gene is inserted between the promoter and the fluorescent protein gene to achieve KCNQ1 V180L By co-expressing the mutant gene with the fluorescent protein gene, arrhythmia transgenic zebrafish were constructed. Under the conditions of exposure time 3.06ms and gain effect 4×, the transgenic zebrafish with fluorescence intensity IOD value of 650,000~960,000 were used as the zebrafish model for arrhythmia drug screening.

[0006] Preferably, according to the present invention, the transgenic zebrafish achieves gene recombination and stable inheritance through the Tol2 transposon system.

[0007] The method for constructing the zebrafish model for arrhythmia drug screening comprises the following steps: (1) Modify the Tol2 vector. cmlc2 promoter, KCNQ1 V180L Mutated genes and mcherry The genes were sequentially inserted into it, and the P2A element was introduced at the same time to obtain the pTol2-cmlc2-KCNQ1 V180L-P2A-mcherry-SV40PA transgenic expression vector. After linearization and purification steps, the linearized expression vector was obtained; (2) The Tol2 transposase plasmid is linearized, transcribed in vitro, and purified to obtain linearized Tol2 transposase mRNA; (3) The linearized expression vector obtained in step (1) and the linearized Tol2 transposase mRNA obtained in step (2) were mixed and injected into zebrafish fertilized eggs, and cultured to 48-72 hpf to obtain arrhythmia transgenic zebrafish; (4) Under the conditions of exposure time 3.06ms and gain effect 4×, transgenic zebrafish with fluorescence intensity IOD values ​​of 650,000~960,000 were screened to obtain a zebrafish model for arrhythmia drug screening.

[0008] According to the present invention, preferably, in step (1) cmlc2 The nucleotide sequence of the promoter is shown in SEQ ID NO.1, mcherry The nucleotide sequence of the gene is shown in SEQ ID NO.2. KCNQ1 V180L The mutated gene has a Gene ID of 3784 on NCBI.

[0009] According to the preferred embodiment of the present invention, the final concentration of the linearized expression vector injected in step (3) is 55-65 ng / μL, and the final concentration of the linearized Tol2 transposase mRNA injected is 190-210 ng / μL.

[0010] According to the preferred embodiment of the present invention, the time of injection into the zebrafish fertilized eggs in step (3) is controlled within 20 minutes after ovulation.

[0011] According to the preferred embodiment of the present invention, the injection volume in step (3) is 0.9-1.1 nL per fertilized egg.

[0012] Application of the above zebrafish model in screening drugs for treating or preventing arrhythmias.

[0013] Beneficial effects: The transgenic zebrafish provided by this invention can be used to screen suitable zebrafish models for arrhythmias based on fluorescence intensity (IOD) values. Fluorescence intensity is positively correlated with transgene expression, allowing rapid assessment of transgene expression levels and selection of suitable individuals for subsequent experiments. Isoproterenol is a widely used antiarrhythmic drug in clinical practice. Experiments using isoproterenol treatment have demonstrated the effectiveness of this model for antiarrhythmic drug screening. This model is not only suitable for studying the pathogenesis of inherited arrhythmias but also provides technical support for the development of therapeutic drugs for arrhythmias, possessing significant theoretical significance and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the map of the pTol2-cmlc2-KCNQ1 V180L-P2A-mcherry-SV40PA transgenic plasmid.

[0015] Figure 2 This is a statistical histogram of cardiac function indicators in transgenic zebrafish; A is cardiac output, B is stroke volume, and C is ventricular minor axis shortening fraction; In the figure: Tg (cmlc2: EGFP) represents the control group, Tg (cmlc2: KCNQ1 V180L -mCherry)-L, Tg(cmlc2:KCNQ1 V180L -mCherry)-M, Tg (cmlc2:KCNQ1V180L -mCherry)-H indicates low, medium, and high fluorescence intensity groups, respectively; *** indicates P ≤ 0.001 compared with the control group.

[0016] Figure 3 is the gene expression level of transgenic zebrafish 2 -ΔΔCT Histogram; In the figure: Tg (cmlc2: KCNQ1 V180L -mCherry)-L, Tg (cmlc2:KCNQ1 V180L -mCherry)-M, Tg(cmlc2:KCNQ1 V180L -mCherry)-H indicates low, medium, and high fluorescence intensity groups, respectively; ** indicates P ≤ 0.01 compared with the low fluorescence intensity group; *** indicates P ≤ 0.001 compared with the low fluorescence intensity group.

[0017] Figure 4 The bar graph shows the effects of different drug doses on the heart rate and heart volume of transgenic zebrafish; In the figure: Tg (cmlc2: EGFP) represents the control group, Tg (cmlc2: KCNQ1 V180L -mCherry) indicates the transgenic zebrafish group, and 0.2, 0.5, 1, 10, 15, and 20 indicate the corresponding concentrations of isoproterenol-treated groups; ### indicates P ≤ 0.001 compared with the control group; *** indicates P ≤ 0.001 compared with the transgenic zebrafish group; ns indicates P > 0.05 compared with the transgenic zebrafish group.

[0018] Figure 5 The bar graph shows the effect of isoproterenol on the heart rate of transgenic zebrafish.

[0019] Figure 6 The bar graph shows the effect of isoproterenol on cardiac output in transgenic zebrafish.

[0020] Figure 7 The bar graph shows the effect of isoproterenol on the stroke volume of transgenic zebrafish.

[0021] Figure 8 The bar graph shows the effect of isoproterenol on the fractional shortening of the ventricle in transgenic zebrafish.

[0022] Figures 5 to 8 Middle: Tg (cmlc2: EGFP) represents the control group, Tg (cmlc2: KCNQ1 V180L -mCherry)-L, Tg(cmlc2:KCNQ1 V180L-mCherry)-M, Tg (cmlc2:KCNQ1 V180L -mCherry)-H represents low, medium, and high fluorescence intensity groups, respectively; iso-L, iso-M, and iso-H represent low, medium, and high fluorescence intensity isoproterenol-treated groups, respectively; ### indicates P ≤ 0.001 compared with the control group; ## indicates P ≤ 0.01 compared with the control group; # indicates P ≤ 0.05 compared with the control group; *** indicates P ≤ 0.001 compared with the low / medium / high fluorescence intensity groups in the corresponding figures; ** indicates P ≤ 0.01 compared with the low / medium / high fluorescence intensity groups in the corresponding figures; * indicates P ≤ 0.05 compared with the low / medium / high fluorescence intensity groups in the corresponding figures; ns indicates P > 0.05 compared with the low / medium / high fluorescence intensity groups in the corresponding figures. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be further described below in conjunction with the embodiments and the accompanying drawings, but the scope of protection of the present invention is not limited thereto.

[0024] In the examples, the contents without specific conditions were carried out under conventional conditions; the reagents or instruments used without specifying the manufacturer were all common commercial products.

[0025] Example 1 Construction of transgenic zebrafish 1. Construction of transgenic vector: The Tol2 vector was modified by molecular cloning to express the gene specifically and strongly in the zebrafish heart. cmlc2 Promoter (nucleotide sequence is shown in SEQ ID NO.1), KCNQ1 V180L sequence (Gene ID: 3784) and mcherry The genes (nucleotide sequence shown in SEQ ID NO.2) were inserted into it in sequence; at the same time, the P2A plasmid element was introduced, which can achieve the co-expression of multiple genes on a single vector, and finally the pTol2-cmlc2-KCNQ1 V180L-P2A-mcherry-SV40PA transgenic expression vector was obtained, which can achieve the co-expression of multiple genes on the same promoter. cmlc2 Driven by KCNQ1 V180L and mcherry , and its transgenic plasmid map is as follows Figure 1 shown.

[0026] The transgenic plasmid was linearized by cleavage with the Xho I restriction endonuclease and purified using the TaKaRa MiniBEST DNA Fragment Purification Kit Ver. 4.0 (TaKaRa). The Tol2 transposase plasmid, purchased from the National Zebrafish Resource Center, was linearized with the Not I restriction endonuclease and in vitro transcribed using the mMESSAGE mMACHINE™ SP6 (Thermo Fisher Scientific) transcription kit according to the manufacturer's instructions. Tol2 transposase mRNA was purified using the GeneJET RNA Purification and Concentration Mini Kit (Thermo Fisher Scientific).

[0027] 2. Culture of zebrafish fertilized eggs: (1) Healthy, sexually mature Tg (cmlc2: EGFP) zebrafish were placed in a mating tank at a ratio of 1:1 between male and female, with a partition placed in the middle. The tank was placed in a dark environment. The partition was removed before turning on the light the next day. The fish were stimulated to ovulate by light and the ovulation time was controlled within half an hour to reduce the difference in development time between embryos. After ovulation, the adult fish were removed and the fertilized eggs were immediately collected and disinfected and cleaned. (2) The fertilized eggs were transferred into clean zebrafish embryo culture water and 0.2 ppm methylene blue was added. The eggs were cultured in a 28°C constant temperature incubator under a 14 h light / 10 h dark cycle until 48 hpf, which served as the control group.

[0028] The composition of embryo culture water is as follows: CaCl20.4mM, NaCl 5mM, MgSO40.16mM, KCl 0.17mM, prepared in deionized water.

[0029] 3. Construction of transgenic zebrafish: (1) The fertilized eggs obtained by the method described in step 2 (1) were microinjected within 20 minutes after ovulation; the purified linearized transgenic plasmid obtained in step 1 was mixed with the purified Tol2 transposase mRNA at a final concentration of 60 ng / μL and microinjected into the zebrafish fertilized eggs at a final concentration of 200 ng / μL. The injection volume was 1 nL per fertilized egg.

[0030] (2) The fertilized eggs were transferred into clean zebrafish embryo culture water and 0.2 ppm methylene blue was added. The eggs were cultured in a 28°C constant temperature incubator under a 14 h light / 10 h dark cycle until 48 hpf. The fluorescence was observed under a fluorescence microscope. Red fluorescence was observed in the zebrafish heart, indicating mcherry The normal expression of genes indicates that the transgenic expression system can work normally.

[0031] Example 2 Detection of cardiac function indicators and transgenic expression levels in transgenic zebrafish The fertilized zebrafish eggs described in step 3 of Example 1 were cultured until 72 hpf and anesthetized in a 0.02% tricaine solution. Heart images showing red fluorescence were acquired using a fluorescence microscope (Olympus inverted microscope, model X51) with an exposure time of 3.06 ms and a gain of 4×, and the zebrafish heart rate was analyzed. Based on the acquired images, the transgenic zebrafish cardiac fluorescence intensity was measured using Image-Pro Plus 6.0 software. Data were processed using GraphPad Prism 8.0.2 software. The normal resting heart rate of zebrafish at a suitable temperature is approximately 160 ± 20 beats / min. Arrhythmias can reduce the zebrafish heart rate by 20%-40%. According to the heart rate range, the transgenic zebrafish were divided into three groups: normal heart rate group (160±20 beats / minute), lower than normal heart rate group with a decrease of less than 40% (96-140 beats / minute), lower than normal heart rate group with a decrease of more than 40% (<96 beats / minute). The IOD values ​​of the heart fluorescence intensity of the three groups were statistically analyzed.

[0032] The experimental results showed that the IOD value of the fluorescence intensity of the heart of transgenic zebrafish in the normal heart rate range of 160±20 beats / minute was lower than 650,000, the IOD value of the fluorescence intensity of the heart of transgenic zebrafish in the heart rate range of 96-140 beats / minute was higher than 650,000 and lower than 960,000, and the IOD value of the heart of transgenic zebrafish in the heart rate range below 96 beats / minute was higher than 960,000. The fluorescence intensity IOD below 650,000 was set as the low fluorescence intensity group, 650,000-960,000 as the medium fluorescence intensity group, and 960,000 and above as the high fluorescence intensity group. The cells were grouped according to the fluorescence intensity, and cardiac function indicators such as cardiac output, stroke volume and ventricular short axis shortening rate were statistically analyzed.

[0033] like Figure 2 As shown, cardiac output, stroke volume, and ventricular fractional shortening of the transgenic zebrafish were significantly reduced, and positively correlated with cardiac fluorescence intensity, with statistical significance, indicating that the transgenic zebrafish were successfully constructed. However, when the fluorescence intensity is below 650,000, i.e., when the heart rate is normal, it is not suitable as a model animal for subsequent experiments. When the fluorescence intensity IOD value is 650,000 or above, arrhythmias are obvious, making it suitable as a model animal for subsequent antiarrhythmic drug screening.

[0034] The three groups of transgenic zebrafish were homogenized and total RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme). The procedures were performed according to the relevant instructions. Reverse transcription was performed into a cDNA template using HiScript IV RT SuperMix for qPCR (+gDNA wiper) (Vazyme). The procedures were performed according to the relevant instructions. RT-qPCR was performed using the zebrafish gene β-actin as an internal reference and ChamQ Universal SYBR qPCR Master Mix (Vazyme). The operation method was conventional in the field. CT values ​​were collected and statistically analyzed to obtain 2 -ΔΔCT The relationship between fluorescence intensity and transgenic expression was verified.

[0035] RT-qPCR primer sequences: β-actin (forward primer): 5′-AGAGCTATGAGCTGCCTGACG-3′; β-actin (reverse primer): 5′-CCGCAAGATTCCATACCCA-3′; KCNQ1 V180L (Forward primer): 5′-AACACAACCAGAAGATATGAACGAG-3′; KCNQ1 V180L (Reverse primer): 5'-GATGAGACCTCGAAAACCCCA-3'.

[0036] The results are as follows Figure 3 As shown, transgenic zebrafish KCNQ1 V180L Gene expression level 2 -ΔΔCT The value is positively correlated with the fluorescence intensity, so the fluorescence intensity can be used to make a preliminary judgment KCNQ1 V180L Gene expression level. The above results show that appropriate transgenic zebrafish can be selected for subsequent experiments based on fluorescence intensity.

[0037] Example 3 Therapeutic effects of isoproterenol on cardiac arrhythmias in transgenic zebrafish 1. The transgenic zebrafish cultured to 48 hpf in step 3 (2) of Example 1 were placed in a 0.02% tricaine solution to anesthetize them. Heart images showing red fluorescence were acquired using a fluorescence microscope with an exposure time of 3.06 ms and a gain of 4×.

[0038] The zebrafish cultured to 48 hpf in step 2 (2) of Example 1 were used as the control group.

[0039] 2. Treatment with different concentrations of isoproterenol The transgenic zebrafish were treated with isoproterenol and divided into a control group, a transgenic zebrafish group, and groups treated with 0.2, 0.5, 1, 10, 15, or 20 mg / mL isoproterenol. Each group was divided into three replicate wells, with 10 zebrafish per well. The following treatment steps were performed: embryo culture water was removed from the 12-well plate, 2 mL of embryo culture water was added to the control and transgenic zebrafish groups, and 2 mL of embryo culture water containing 0.2, 0.5, 1, 10, 15, or 20 mg / mL isoproterenol was added to the isoproterenol-treated groups. The zebrafish were then incubated for 24 hours in a 28°C incubator under a 14-hour light / 10-hour dark cycle. Heart rate, ventricular diastolic volume, and ventricular systolic volume were measured in each group.

[0040] The experimental results are as follows Figure 4 As shown in the figure, compared with the control group, the heart rate of zebrafish in the 0.2, 0.5, and 1 mg / mL isoproterenol treatment groups did not recover significantly, while the heart rate of zebrafish in the 10, 15, and 20 mg / mL isoproterenol treatment groups increased significantly, indicating that low-concentration administration is not effective for the treatment of arrhythmias. The diastolic / systolic volume of the zebrafish heart in the 0.2, 0.5, 1, and 10 mg / mL isoproterenol treatment groups did not change significantly, but the diastolic / systolic volume of the zebrafish heart in the 15 and 20 mg / mL isoproterenol treatment groups increased significantly, indicating that excessively high concentration administration will cause pericardial edema and heart deformities in zebrafish. Combined with the above experimental results, 10 mg / mL isoproterenol was subsequently used as the optimal concentration for the treatment of zebrafish arrhythmias.

[0041] 3. Isoproterenol recovery treatment Based on the collected images, image-pro plus 6.0 software was used to collect zebrafish heart fluorescence intensity. The transgenic zebrafish were divided into three groups according to the fluorescence intensity IOD value: low, medium, and high. The grouping method was as in Example 2, with a fluorescence intensity IOD value below 650,000 as the low fluorescence intensity group, a fluorescence intensity IOD value of 650,000-960,000 as the medium fluorescence intensity group, and a fluorescence intensity IOD value above 960,000 as the high fluorescence intensity group. The transgenic zebrafish in each of the above groups were treated with isoproterenol and divided into a control group, low, medium, and high fluorescence intensity groups, and low, medium, and high fluorescence intensity isoproterenol-treated groups, with 3 parallel wells per group and 10 fish per well. The following steps were performed: (1) embryo culture water was removed from the 12-well plate, and 2 mL of embryo culture water was added to the control group and the low, medium, and high fluorescence intensity groups. 2 mL of embryo culture water containing 10 mg / mL isoproterenol was added to the low, medium, and high fluorescence intensity isoproterenol treatment groups, respectively. The cells were cultured in a 28°C constant temperature incubator under a 14 h light / 10 h dark cycle for 24 h. Zebrafish cardiac function indicators were measured.

[0042] The experimental results are as follows Figures 5 to 8 As shown in the figure, compared with the control group, the heart rate, stroke volume, cardiac output and ventricular short axis shortening rate of the zebrafish hearts in the low, medium and high fluorescence intensity groups were significantly reduced, and the results were statistically significant. This shows that the transgenic zebrafish showed symptoms of arrhythmia and the transgenic zebrafish model was successfully established.

[0043] like Figure 5 As shown in Figure 2, after treatment with isoproterenol, the heart rates of zebrafish in the low and medium fluorescence intensity groups increased significantly, while the heart rates of zebrafish in the high fluorescence intensity group improved, but there was no significant difference. Figure 6 As shown in Figure 2, after treatment with isoproterenol, the cardiac output of zebrafish in the low and medium fluorescence intensity groups increased significantly, while the cardiac output of zebrafish in the high fluorescence intensity group improved, but there was no significant difference. Figure 7 As shown in Figure 3, after treatment with isoproterenol, the stroke volume of zebrafish in the low and medium fluorescence intensity groups increased significantly, while the stroke volume of zebrafish in the high fluorescence intensity group improved, but there was no significant difference. Figure 8 As shown, compared with the transgenic zebrafish group, the zebrafish ventricular short axis shortening rate in the low and medium fluorescence intensity isoproterenol treatment groups was significantly increased, while there was no significant difference in the high fluorescence intensity isoproterenol treatment group.

[0044] In summary, isoproterenol can reverse the low and medium fluorescence intensity KCNQ1 Arrhythmias caused by pathogenic mutations cannot be reversed by high fluorescence intensity KCNQ1Arrhythmias caused by pathogenic mutations indicate that when the expression level of the pathogenic mutation gene is too high, the arrhythmia cannot be alleviated by drugs and is not suitable for use as an antiarrhythmic drug screening model. Arrhythmias are obvious when the fluorescence intensity IOD value is 650,000 and above. Therefore, when the fluorescence intensity IOD value is between 650,000 and 960,000, it is the best experimental subject for transgenic zebrafish models for antiarrhythmic drug screening.

[0045] The present invention establishes cardiac tissue overexpression KCNQ1 V180L Transgenic zebrafish provide a model animal for the study of the pathogenesis of hereditary arrhythmia and its drug targets, with fluorescence intensity IOD and gene expression level 2 -ΔΔCT Based on the reference indicators, appropriate model parameters are optimized to obtain a model for evaluating arrhythmias with fast modeling, high throughput, low cost and convenient detection. The present invention can provide model animals and application methods for drugs for treating arrhythmias, and has broad market prospects.

Claims

1. A zebrafish model for arrhythmia drug screening, characterized in that: The zebrafish model is KCNQ1 V180L The mutant gene is inserted between the promoter and the fluorescent protein gene to achieve KCNQ1 V180L By co-expressing the mutant gene with the fluorescent protein gene, arrhythmia transgenic zebrafish were constructed. Under the conditions of exposure time 3.06ms and gain effect 4×, the transgenic zebrafish with fluorescence intensity IOD value of 650,000~960,000 were used as the zebrafish model for arrhythmia drug screening.

2. The zebrafish model according to claim 1, wherein The transgenic zebrafish achieves gene recombination and stable inheritance through the Tol2 transposon system.

3. The method for constructing the zebrafish model according to claim 1, characterized in that: The steps include: (1) Modify the Tol2 vector. cmlc2 promoter, KCNQ1 V180L Mutated genes and mcherry The genes were sequentially inserted into it, and the P2A element was introduced at the same time to obtain the pTol2-cmlc2-KCNQ1 V180L-P2A-mcherry-SV40PA transgenic expression vector. After linearization and purification steps, the linearized expression vector was obtained; (2) The Tol2 transposase plasmid is linearized, transcribed in vitro, and purified to obtain linearized Tol2 transposase mRNA; (3) The linearized expression vector obtained in step (1) and the linearized Tol2 transposase mRNA obtained in step (2) were mixed and injected into zebrafish fertilized eggs, and cultured to 48-72 hpf to obtain arrhythmia transgenic zebrafish; (4) Under the conditions of exposure time 3.06ms and gain effect 4×, transgenic zebrafish with fluorescence intensity IOD values ​​of 650,000~960,000 were screened to obtain a zebrafish model for arrhythmia drug screening.

4. The construction method according to claim 3, wherein: As described in step (1) cmlc2 The nucleotide sequence of the promoter is shown in SEQ ID NO.1, mcherry The nucleotide sequence of the gene is shown in SEQ ID NO.

2. KCNQ1 V180L The mutated gene has a Gene ID of 3784 on NCBI.

5. The construction method according to claim 3, wherein: In step (3), the final concentration of the linearized expression vector injected was 55-65 ng / μL, and the final concentration of the linearized Tol2 transposase mRNA injected was 190-210 ng / μL.

6. The construction method according to claim 3, wherein: The injection time of step (3) into the zebrafish fertilized egg is controlled within 20 minutes after ovulation.

7. The construction method according to claim 3, wherein: The injection volume in step (3) is 0.9~1.1nL per fertilized egg.

8. Use of the zebrafish model according to claim 1 in screening drugs for treating or preventing arrhythmias.

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