Method for constructing a zebrafish model of glanzmann thrombasthenia and applications thereof

Abstract

The application discloses a method for constructing a zebrafish model of Glanzmann thrombasthenia and application thereof. The application belongs to the technical field of genetic engineering and experimental animal model, and a CRISPR / Cas9 gene editing target point is designed for a zebrafish itga2b gene. sgRNA and Cas9 mRNA are introduced into zebrafish single-cell period fertilized eggs through microinjection, and a homozygote zebrafish with an itga2b gene mutation, i.e., a Glanzmann thrombasthenia model, is obtained through screening. It is verified that the model has typical bleeding phenotypes such as an increased bleeding area after gill injury, a prolonged bleeding time after tail fin injury and a significantly decreased platelet aggregation function. The model constructed by the application has advantages such as embryo transparency, dynamic observation of the bleeding and thrombosis process in vivo, and suitability for high-throughput drug screening. The zebrafish model can become a new carrier for researching Glanzmann thrombasthenia (GT) and screening of new candidate therapeutic drugs for GT.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to the research on platelet development, specifically to the construction method and application of a zebrafish model of Granzmann platelet dysfunction. Background Technology

[0002] Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited bleeding disorder caused by the absence or dysfunction of platelet membrane glycoproteins GPIIb / IIIa (αIIbβ3 integrin, also known as the glycoprotein IIb / IIIa complex, including the genes ITGA2B (CD41) and ITGB3 (CD61)). This results in the inability of fibrinogen to bridge platelets, leading to severely impaired platelet aggregation. GT is usually inherited in an autosomal recessive manner. Patients may be homozygous (especially in the presence of consanguineous marriage) or compound heterozygous (Bellucci S, Caen J. Molecular basis of Glanzmann's Thrombasthenia and current strategies in treatment [J]. Blood reviews, 2002, 16(3): 193-202.; Fiore M, d'Oiron R, Pillois X, et al. Anti-α(IIb) β(3) immunization in Glanzmann thrombasthenia: review of literature and treatment recommendations [J]. British journal of haematology, 2018, 181(2): 173-182.). As of February 2021, the Glanzmann Thrombasthenia database (https: / / glanzmann.mcw.edu / ) had included 475 GT mutations caused by ITGA2B and ITBG3 mutations, the most common of which are nonsense mutations, missense mutations, and splice site variations (Mathews N, Rivard GE, Bonnefoy A. Glanzmann Thrombasthenia: Perspectives from Clinical Practice on Accurate Diagnosis and Optimal Treatment Strategies [J]. Journal of bloodmedicine, 2021, 12: 449-463.). The clinical phenotype of GT is diverse; some patients experience severe bleeding, while others only experience minor bleeding. Manifestations may include petechiae, purpura, minor bruising, recurrent epistaxis, gingival bleeding and menorrhagia, gastrointestinal bleeding, intracranial hemorrhage, or hematuria.Most patients may experience spontaneous remission with age, but some patients experience a worsening of their condition in adulthood (Farsinejad A, Farajollahi MM, Kazemi A, et al. Different biochemical expression pattern of platelet surface glycoproteins suggests molecular diversity of Glanzmann's thrombasthenia in Iran [J]. Bloodcoagulation & fibrinolysis : an international journal in haemostasis and thrombosis, 2013, 24(6): 613-618.;Nurden A T. Glanzmann thrombasthenia [J]. Orphanet journal of rare diseases, 2006, 1: 10.;Jacquelin B, Tuleja E, Kunicki TJ, et al. Analysis of platelet membrane glycoprotein polymorphisms in Glanzmann thrombasthenia showed the French gypsy mutation in the alphaIIb gene to be strongly linked to the HPA-1b polymorphism in beta3). [J]. Journal of thrombosis and haemostasis: JTH, 2003, 1(3): 573-575.). For the treatment of Granzman's platelet dysfunction, generally no special treatment is needed. The main approach is for patients to avoid trauma, maintain good oral hygiene, and avoid taking antiplatelet drugs, aspirin, and nonsteroidal anti-inflammatory drugs (NSAIDs).Treatment of minor bleeding relies primarily on local hemostasis and / or the use of antifibrinolytic drugs, while platelet transfusion remains the standard treatment when these initial measures fail to control bleeding or when invasive procedures are performed. Unfortunately, platelet transfusion therapy may trigger an immune response against the deficient αIIbβ3 complex and / or HLA class I system (Fiore M, d'Oiron R, Pillois X, et al. Anti-α(IIb)β(3)immunization in Glanzmann thrombasthenia: review of literature and treatment recommendations [J]. British journal of haematology, 2018, 181(2): 173-182.). These antiplatelet antibodies are present in approximately 20-30% of patients, which may render platelet transfusions ineffective, thus raising significant clinical concern (Fiore M, Giraudet JS, Alessi MC, et al. Emergency management of patients with Glanzmann thrombasthenia: consensus recommendations from the French reference center for inherited platelet disorders [J]. Orphanetjournal of rare diseases, 2023, 18(1): 171.). There are reports that recombinant coagulation factor VIIa is effective for Glanzmann thromboasthenia with platelet antibodies and / or no response to platelet transfusion (Poon MC, d'Oiron R, Zotz RB, et al. The international, prospective Glanzmann Thrombasthenia Registry: treatment and outcomes in surgical intervention [J]. Haematologica, 2015, 100(8): 1038-1044.). There are also reports that a small number of GT patients may experience persistent and recurrent life-threatening bleeding that is unresponsive to existing hemostatic treatment, especially those with platelet antibodies and refractory conditions.

[0003] Among existing model organisms, mouse models are suitable for mechanistic studies but not for large-scale drug screening. Relying solely on mouse models results in extremely low efficiency in GT drug development, failing to meet clinical needs. While zebrafish are used for GT disease research quickly and allow for large-scale screening of potential therapeutic drugs, current models only exhibit transient expression; a stable, phenotypic, and high-throughput drug screening zebrafish model for GT is lacking. This technological gap severely restricts the development of GT therapeutics. Therefore, constructing a stably inherited cd41 model is crucial. KO The zebrafish model has significant scientific and clinical value. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a method for constructing and applying a zebrafish model of Granzmann platelet dysfunction. The zebrafish model of Granzmann platelet dysfunction provided by this invention is a zebrafish model of thrombocytopenia caused by an itga2b gene mutation.

[0005] This invention is achieved through the following technical means: This invention provides a method for constructing a zebrafish model of Granzmann platelet dysfunction, utilizing CRISPR / Cas9 gene editing technology to target and knock out the zebrafish itga2b gene to obtain cd41. KO Mutant zebrafish are zebrafish models of Granzmann platelet dysfunction. cd41 KO This refers to loss-of-function mutations in the itga2b gene that result in low or no expression of the CD41 / GPIIb protein it encodes.

[0006] Furthermore, the specific steps include the following: (1) Design and synthesize sgRNA targeting the zebrafish itga2b gene; (2) Mix sgRNA with Cas9 mRNA to prepare a gene knockout injection system; (3) The gene knockout injection system was microinjected into zebrafish fertilized eggs to obtain F0 generation chimeric zebrafish; (4) Cross F0 generation chimeric zebrafish with wild-type zebrafish and screen to obtain F1 generation heterozygous knockout zebrafish; (5) Self-crossing was performed on the F1 generation heterozygous knockout zebrafish to obtain the F2 generation homozygous knockout zebrafish, namely cd41. KO Mutant zebrafish, namely the zebrafish model of Granzmann platelet dysfunction.

[0007] Furthermore, the target sequence for knockout of the zebrafish itga2b gene is shown in SEQ ID NO.1; the sgRNA sequence is shown in SEQ ID NO.2.

[0008] This invention also provides a method for constructing a zebrafish model of Granzman platelet dysfunction with a transgenic background, comprising the following steps: The above cd41 KO Mutant zebrafish are crossed with transgenic zebrafish lines, and the offspring are then self-crossed to obtain offspring with a transgenic background. cd41 KO Mutant zebrafish, a zebrafish model of Granzman platelet dysfunction with a transgenic background.

[0009] This invention also provides the application of the zebrafish model of Granzmann platelet dysfunction constructed by the above-described method in screening drugs effective against Granzmann platelet dysfunction.

[0010] Furthermore, the zebrafish model of Granzmann platelet dysfunction is caused by abnormal expression of the itga2b gene.

[0011] Furthermore, in the zebrafish model of Granzman platelet dysfunction, the itga2b gene was missing 10 bases, which caused premature termination of itga2b gene expression and truncated 3 immunoglobulin-like domains.

[0012] Furthermore, symptoms of Granzmann's thrombocytopenia include normal platelet counts but impaired or significantly reduced platelet aggregation function.

[0013] Furthermore, the zebrafish model of Granzmann platelet dysfunction exhibits a bleeding tendency, characterized by: significantly longer bleeding time after caudal fin amputation compared to wild-type zebrafish; and significantly larger bleeding area after gill injury compared to wild-type zebrafish.

[0014] Furthermore, the following steps are included: (1) The candidate drug was administered to the zebrafish model of the Glanzmann platelet dysfunction, and the group was designated as the administration group; (2) Use untreated models or wild-type zebrafish as the control group; (3) Detect phenotypic indicators reflecting platelet function; (4) If the treatment group shows improvement compared to the control group, the candidate drug is considered to have the potential to treat Granzman's platelet dysfunction.

[0015] Furthermore, this also includes using the Granzmann platelet dysfunction zebrafish model to construct a humanized mutation model and screen for personalized drugs for patients, specifically including the following steps: (1) Construct humanized mutant plasmids; (2) mRNA was transcribed using an in vitro transcription kit; (3) mRNA was injected into the embryos of a zebrafish model of Granzman platelet dysfunction via microinjection to obtain a humanized mutant model, which was then used for drug screening.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The zebrafish model of Granzmann platelet dysfunction provided by the present invention can be used to screen drugs effective against Granzmann platelet dysfunction. Effective drugs are screened by treating mutant zebrafish with drugs during the embryonic period, and the experimental cycle is short.

[0017] (2) The zebrafish model of Granzman platelet dysfunction in this invention can be used to track the pathogenesis of the disease over a long period of time, from the embryonic period to adulthood, due to its stable heritability.

[0018] (4) Humanized mutant models were constructed using the zebrafish model of Granzmann platelet dysfunction, such as missense mutations on ITGA2B such as c.480C>G, c.659A>G, c.857T>A, and c.1355T>G. After constructing humanized mutant plasmids, mRNA was transcribed using an in vitro transcription kit. The mRNA was then injected into the embryos of the zebrafish model of Granzmann platelet dysfunction via microinjection to obtain humanized mutant models. These models were then used for drug screening, upgrading the model from "simulating disease" to "simulating patient," thus enabling personalized drug screening in the era of precision medicine. Attached Figure Description

[0019] Figure 1 To obtain cd41 using CRISPR-Cas9 targeted gene knockout technology KO Schematic diagram of mutant zebrafish (A: wild-type zebrafish and cd41) KO Exon 3 of the itga2b gene targeting mutant zebrafish; B: wild-type zebrafish and cd41 KO The CD41 / GPIIb protein encoded in mutant zebrafish.

[0020] Figure 2 cd41 KOMultilineage phenotypic results of mutant zebrafish 6 dpf (t-test, P>0.05; mean ± standard error) (A: in situ hybridization results of mpl probe; B: statistical results of in situ hybridization of mpl probe; C: in situ hybridization results of cmyb probe; D: statistical results of in situ hybridization of cmyb probe; E: in situ hybridization results of eos5 probe; F: statistical results of in situ hybridization of eos5 probe; G: in situ hybridization results of mfap4 probe; H: statistical results of in situ hybridization of mfap4 probe; I: in situ hybridization results of βe1 probe; J: statistical results of in situ hybridization of βe1 probe; K: in situ hybridization results of rag1 probe; L: statistical results of in situ hybridization of rag1 probe).

[0021] Figure 3 cd41 KO Phenotypic diagrams of juvenile mutant zebrafish (t-test, P<0.001; mean ± standard error) (A: Result of venous needle puncture injury in juvenile fish at 6 dpf; B: Statistical diagram of hemostasis time; C: Result of arterial laser injury in juvenile fish at 6 dpf; D: Statistical diagram of the time for the first platelet to arrive at the wound; E: Statistical diagram of the number of thrombus cells aggregated at the wound within 120 s).

[0022] Figure 4 cd41 KO Phenotypic diagrams of adult mutant zebrafish (t-test, P<0.001; mean ± standard error) (A: Results of tail cutting injury in adult fish; B: Statistical diagram of hemostasis time; C: Results of gill injury in adult fish; D: Statistical diagram of bleeding area; E: Results of in vitro fibrinogen spreading experiment; F: Statistical diagram of thrombus cell spreading).

[0023] Figure 5 cd41 KO Plots of mutant zebrafish used for drug screening (LMM (Linear Mixed Model), P<0.01; mean ± standard error) (A: Schematic diagram of drug treatment; B: Comparison of mutants before and after laser damage after drug treatment; C: Statistical graph of the time for the first platelet to reach the wound; D: Statistical graph of the number of thrombus cells aggregated in the wound within 120s). Specific implementation methods The following examples further illustrate specific implementations of the present invention, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described below are those that can be implemented or understood by those skilled in the art by referring to existing technology. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0024] The terms "wild type" or "WT" used in this invention refer to wild-type zebrafish.

[0025] The term "dpf" as used in this invention refers to the number of days after fertilization.

[0026] Materials and methods: (1) Zebrafish farming Zebrafish farming is described in the literature (Westerfield M: The zebrafish: guide for the laboratory use of zebrafish (Brachdanio rerio). Edition by Eugene, OR, M. Westerfield, 1993).

[0027] (2) The following strains are used in the embodiments of the present invention: AB wild-type zebrafish (hereinafter referred to as wild-type zebrafish). cd41 KO mutant zebrafish Tg(cd41:GFP) Genetically modified zebrafish.

[0028] Example 1 Constructing a zebrafish model of Granzmann platelet dysfunction involves the following steps: Figure 1 This demonstrates the use of CRISPR-Cas9 targeted gene knockout technology to obtain cd41. KO Schematic diagram of mutant zebrafish, where A represents wild-type zebrafish and cd41. KO In mutant zebrafish, exon 3 of the itga2b gene is targeted, while B represents wild-type zebrafish and cd41. KO The CD41 / GPIIb protein encoded in mutant zebrafish.

[0029] (1) Design and synthesize sgRNA targeting the zebrafish itga2b gene; choose itga2b (cd41) The sequence following the ATG start codon in exon 3 of the gene is used as the target sequence for targeted knockout. Based on predictions from the website http: / / www.crisprscan.org / , the target sequence with high score, high target efficiency, and low off-target efficiency is shown in SEQ ID NO.1. The AGG following this sequence constitutes the PAM region. The sgRNA sequence is shown in SEQ ID NO.2.

[0030] Figure 1 Figure A shows exon 3 of the itga2b gene in wild-type zebrafish. The itga2b-deficient knockout scheme was generated from exon 3 of the itga2b gene, as shown in cd41 of the figure. KOExon 3 of the mutant zebrafish targeting the itga2b gene is shown.

[0031] (2) Mix the sgRNA with Cas9 mRNA to prepare a gene knockout injection system; (3) The gene knockout injection system was microinjected into zebrafish fertilized eggs to obtain F0 generation chimeric zebrafish (F0 mutant line containing itga2b (-10bp, +0)). (4) Cross F0 generation chimeric zebrafish with wild-type zebrafish and screen to obtain F1 generation heterozygous knockout zebrafish; (5) Self-crossing was performed on the F1 generation heterozygous knockout zebrafish to obtain the F2 generation homozygous knockout zebrafish, namely cd41. KO Mutant zebrafish, i.e., zebrafish model of Granzmann platelet dysfunction; The obtained CD41 KO In mutant zebrafish, exon 3 of the itga2b gene is missing 10 bases, resulting in a frameshift mutation that terminates prematurely, producing a truncated itga2b protein. Compared to the wild-type itga2b protein, it lacks three important immunoglobulin-like functional domains.

[0032] Figure 1 B in the figure represents the CD41 / GPIIb protein encoded in wild-type zebrafish, as well as cd41 KO The CD41 / GPIIb protein encoded in mutant zebrafish, cd41 KO The CD41 / GPIIb protein encoded in mutant zebrafish was predicted to be truncated, retaining only the first FG-GAP domain and lacking the remaining six FG-GAP repeat sequences, three immunoglobulin-like domains, transmembrane domain, and acidic structure.

[0033] Example 2 Various genetically modified zebrafish strains and cd41 KO Mutant zebrafish were hybridized and then self-crossed to obtain transgenic individuals. cd41 KO Mutant zebrafish; This embodiment uses Tg(cd41:GFP) Taking genetically modified zebrafish as an example, Tg(cd41:GFP) Transgenic zebrafish strains containing platelet-specific promoters cd41 A recombinant vector encoding the promoter, nitroreductase NTR, and fluorescent protein eGFP was introduced into zebrafish fertilized eggs, and stable transgenic lines that specifically expressed NTR in platelets were obtained through screening. Tg(cd41:GFP) Genetically modified zebrafish and cd41 KO After hybridization of mutant zebrafish, the offspring are self-crossed to obtain individuals with... cd41 Genetically modified background cd41 KO mutant zebrafish Tg(cd41:GFP) ; cd41 KO .

[0034] Example 3 Whole-mount in situ hybridization detection cd41 KO Expression of platelet-related genes in mutant zebrafish: Whole-cell in situ hybridization was used to stain 6dpf zebrafish to detect whether the expression of genes from different lineages in the mutant was abnormal.

[0035] The experimental procedure was performed according to the standard experimental procedure in the cited literature (Thisse C, Thisse B. (2008). High-resolution in situ hybridization to whole-mount zebrafish embryos. NATUREPROTOCOLS. VOL.3 NO.1).

[0036] Figure 2 It is shown as cd41 KO The multi-lineage phenotypic results of the mutant zebrafish 6 dpf are shown in the figure. A represents the in situ hybridization results of the mpl probe, B represents the statistical results of the in situ hybridization of the mpl probe, C represents the in situ hybridization results of the cmyb probe, D represents the statistical results of the in situ hybridization of the cmyb probe, E represents the in situ hybridization results of the eos5 probe, F represents the statistical results of the in situ hybridization of the eos5 probe, G represents the in situ hybridization results of the mfap4 probe, H represents the statistical results of the in situ hybridization of the mfap4 probe, I represents the in situ hybridization results of the βe1 probe, J represents the statistical results of the in situ hybridization of the βe1 probe, K represents the in situ hybridization results of the rag1 probe, and L represents the statistical results of the in situ hybridization of the rag1 probe.

[0037] Among them, mpl represents the megakaryocyte / platelet lineage marker; cmyb represents the hematopoietic stem / progenitor cell marker; eos5 and mfap4 represent the myeloid lineage marker; βe1 represents the erythroid lineage marker; and rag1 represents the lymphoid lineage marker.

[0038] Depend on Figure 2 It can be seen that the in situ hybridization statistical results of the six probes mpl, cmyb, eos5, mfap4, βe1, and rag1 are all ns, confirming that cd41 KOThe hematopoietic defect of the mutant is platelet lineage specific and does not have non-specific effects on other hematopoietic lineages.

[0039] Example 4 Coagulation experiments of juvenile and adult fish, and in vitro fibrinogen-induced phenotype of adult fish blood: (1) Use a needle to injure the veins of juvenile fish. Start timing after needle puncture and measure the time required for bleeding to coagulation in juvenile fish. (2) The arteries of juvenile fish were damaged by laser. From the start of laser damage, the time when the first thrombus cell reached the wound and the number of thrombus cells that accumulated in the wound within 120 seconds were recorded. Figure 3 cd41 is shown KO Phenotypic diagrams of juvenile mutant zebrafish, where A is the result of venous needle puncture injury in juvenile fish at 6 dpf, B is a statistical diagram of hemostasis time, C is the result of arterial laser injury in juvenile fish at 6 dpf, D is a statistical diagram of the time it takes for the first platelet to arrive at the wound, and E is a statistical diagram of the number of thrombus cells aggregated at the wound within 120s.

[0040] Depend on Figure 3 It can be known that cd41 KO Mutant zebrafish have a longer venous bleeding time than wild-type zebrafish, cd41 KO Mutant zebrafish showed fewer thrombus cells aggregated after arterial injury compared to wild-type. (3) Use a blade to cut adult wild-type and gp9 SMU15 The tail is cut off, and a timer is started after the cut to measure the time required for the adult fish to bleed and clot. (4) Damage to the gill mucosa of fish with sodium hydroxide and count the bleeding area from the start of the damage to the point where bleeding stops; (5) Take blood from adult fish and observe the spread of thrombus cells under the stimulation of fibrinogen.

[0041] Figure 4 cd41 is shown KO Phenotypic diagrams of adult mutant zebrafish, where A shows the results of tail cutting injury in adult fish, B shows the statistics of hemostasis time, C shows the results of gill injury in adult fish, D shows the statistics of bleeding area, E shows the results of in vitro fibrinogen spreading experiment, and F shows the statistics of thrombus cell spreading.

[0042] Depend on Figure 4 From AD, we can see that cd41 KO The mutant zebrafish experienced more severe bleeding in both duration and area compared to the wild type; Figure 4 From E and F, we know that cd41 KO Mutant zebrafish thrombi have a weaker ability to spread under the stimulation of fibrinogen compared to wild-type zebrafish.

[0043] like Figure 3 and Figure 4 As shown, whether it is a juvenile or an adult fish, cd41 KO The mutant has fewer thrombus cells than the wild type, cd41 KO The mutant hemorrhage time and hemorrhage area were both more severe than those of the wild type, cd41 KO The mutant cells spread less when stimulated by fibrinogen compared to wild-type thrombus cells.

[0044] Example 5 Using CD41 KO Drug screening using mutants Use wild type with CD41 KO The mutant embryos were treated with drugs (0, 10, 100 μM) at 1.5 dpf, and the caudal hematopoietic tissue (CHT) was subjected to arterial laser injury at 6 dpf to observe the aggregation of zebrafish thrombus cells. The drug used was tranexamic acid (TXA).

[0045] Figure 5 cd41 is shown KO The diagram shows mutant zebrafish used for drug screening. A is a schematic diagram of drug treatment, B is a comparison diagram of the mutant before and after laser damage after drug treatment, C is a statistical diagram of the time it takes for the first platelet to reach the wound, and D is a statistical diagram of the number of thrombus cells that aggregate in the wound within 120 seconds.

[0046] like Figure 5 As shown, tranexamic acid was used to treat CD41 KO After treating the mutant, it was found that tranexamic acid treatment resulted in cd41. KO The mutant thrombus cell clusters had a higher number of clusters than the untreated zebrafish, indicating that cd41... KO The mutant can be used for drug screening of GT disease, and the effect can be visualized. It can then be used to screen other drugs with potential for treating Granzman's platelet dysfunction.

[0047] Example 6 For the human mutation case mentioned in the literature reference (Siddiqi, Muhammad Younus Jamal et al. “Glanzmann Thrombasthenia in Pakistani Patients: Identification of 7 NovelPathogenic Variants in the Fibrinogen Receptor αIIbβ3.” Cells vol. 12,2 213.4 Jan. 2023), the mutation was detected by zebrafish cd41.KO Humanized mutant models were constructed by overexpressing mutant mRNA. The human ITG A2B sequence was downloaded from NCBI, and the PCS2-HCD41 plasmid was constructed. Then, the mutant plasmid was constructed using PCR point mutation. The mutation sites were missense mutations c.480C>G, c.659A>G, c.857T>A, and c.1355T>G. The point mutation PCR primers are as follows: c.480C>G: The top primer FP sequence is shown in SEQ ID NO.3; the bottom primer FP sequence is shown in SEQ ID NO.4; c.659A>G: The top primer FP sequence is shown in SEQ ID NO.5; the bottom primer FP sequence is shown in SEQ ID NO.6; c.857T>A: The top primer FP sequence is shown in SEQ ID NO.7; the bottom primer FP sequence is shown in SEQ ID NO.8; c.1355T>G: The top primer FP sequence is shown in SEQ ID NO.9; the bottom primer FP sequence is shown in SEQ ID NO.10; Subsequently, the mRNA was transcribed using the SP6 in vitro transcription kit, and then injected into cd41 via microinjection. KO Humanized mutation models were constructed in embryos.

[0048] This humanized mutation model was used for drug screening to obtain personalized drugs for patients.

[0049] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A method for constructing a zebrafish model of Granzmann platelet dysfunction, characterized in that, cd41 KO mutant zebrafish, which is a zebrafish model of glanzmann thrombasthenia; cd41 KO This refers to loss-of-function mutations in the itga2b gene that result in low or no expression of the CD41 / GPIIb protein it encodes.

2. The construction method according to claim 1, characterized in that, Specifically, the steps include the following: (1) Design and synthesize sgRNA targeting the zebrafish itga2b gene; (2) Mix sgRNA with Cas9 mRNA to prepare a gene knockout injection system; (3) The gene knockout injection system was microinjected into zebrafish fertilized eggs to obtain F0 generation chimeric zebrafish; (4) Cross F0 generation chimeric zebrafish with wild-type zebrafish and screen to obtain F1 generation heterozygous knockout zebrafish; (5) Self-crossing was performed on the F1 generation heterozygous knockout zebrafish to obtain the F2 generation homozygous knockout zebrafish, namely cd41. KO Mutant zebrafish, namely the zebrafish model of Granzmann platelet dysfunction.

3. The construction method according to claim 1, characterized in that, The target sequence for knockout of the zebrafish itga2b gene is shown in SEQ ID NO.1; the sgRNA sequence is shown in SEQ ID NO.

2.

4. A method for constructing a zebrafish model of Granzman's platelet dysfunction with a transgenic background, characterized in that, Includes the following steps: The claim 1 cd41 KO Mutant zebrafish are crossed with transgenic zebrafish lines, and the offspring are then self-crossed to obtain offspring with a transgenic background. cd41 KO Mutant zebrafish, a zebrafish model of Granzman platelet dysfunction with a transgenic background.

5. The application of the zebrafish model of Granzmann platelet dysfunction constructed by the construction method according to any one of claims 1-3 in screening drugs effective against Granzmann platelet dysfunction.

6. The application according to claim 5, characterized in that, The zebrafish model of Granzman platelet dysfunction was caused by abnormal expression of the itga2b gene, which was missing 10 bases, leading to premature termination of its expression and truncation of three immunoglobulin-like domains.

7. The application according to claim 5, characterized in that, The symptoms of Granzman's platelet dysfunction are a normal platelet count but a lack of or significantly reduced platelet aggregation function.

8. The application according to claim 5, characterized in that, The zebrafish model of Granzman platelet dysfunction described above has a bleeding tendency.

9. The application according to claim 5, characterized in that, Includes the following steps: (1) The candidate drug was administered to a zebrafish model of Granzmann platelet dysfunction and designated as the drug administration group; (2) Use untreated models or wild-type zebrafish as the control group; (3) Detect phenotypic indicators reflecting platelet function; (4) If the treatment group shows improvement compared to the control group, the candidate drug is considered to have the potential to treat Granzman's platelet dysfunction.

10. The application according to claim 5, characterized in that, This also includes using the Granzmann platelet dysfunction zebrafish model to construct a humanized mutation model and screen for personalized drugs for patients, specifically including the following steps: (1) Construct humanized mutant plasmids; (2) mRNA was transcribed using an in vitro transcription kit; (3) mRNA was injected into the embryos of a zebrafish model of Granzman platelet dysfunction via microinjection to obtain a humanized mutant model, which was then used for drug screening.

Images