Method for improving VWF (von willebrand disease) dimerizing and secretory disorder von willebrand disease
Through laboratory diagnosis and cell expression of recombinant VWF protein, the C2724Y mutation caused VWF synthesis and secretion disorders were verified, the role of disulfide bonds in the CK domain chain was explored, and the problem of VWF deletion in vasculophilia was solved. A new direction for the diagnosis and treatment of type 3 VWD.
Patent Information
- Application Number
- CN202410842194.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-06-27
- Publication Date
- 2024-12-27
AI Technical Summary
The deletion or abnormal function of VWF protein in vasculophilia (VWD) leads to severe bleeding, especially the almost complete lack of VWF in plasma in patients with type 3 VWD, and there is a lack of effective diagnosis and treatment methods.
Through blood collection, laboratory diagnosis, VWF multimerization analysis, mutation screening, plasmid construction and cell expression of recombinant VWF protein, we verify whether the C2724Y mutation leads to synthesis and secretion disorders of VWF, and explore the disulfide bonds in the CK domain chain for the CK domain The structural stability and function of VWF protein.
The molecular pathogenesis of type 3 VWD was studied, and the important role of disulfide bonds in the CK domain chain in maintaining the structure and function of VWF was verified, and a new diagnostic and treatment direction was provided, which was of great significance for clinical diagnosis, treatment and prognosis. .
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of gene editing technology, and specifically to a method for improving VWF dimerization and secretion disorder von Willebrand disease. Background Art
[0002] Von Willebrand disease (VWD) is an autosomal dominant genetic disease caused by a mutation in the von Willebrand factor (VWF) gene. A reduction in the amount of VWF, as well as structural or functional abnormalities, can lead to VWD. According to the pathogenesis of VWF and the severity of clinical manifestations, VWD is divided into type 1 VWD, type 2 VWD, and type 3 VWD. Type 3 is the most serious type of VWD, with an incidence of approximately 0.5 to 1 / 1 million. VWF is completely or almost completely absent from the plasma of its patients, and most of its structure and function are also abnormal, which can lead to severe bleeding, even spontaneous joint bleeding, muscle bleeding, and visceral bleeding, which can threaten the patient's life in severe cases. Studying the molecular pathogenesis of type 3 VWD helps to understand the relationship between the structure and function of the VWF protein, which is of great significance for clinical diagnosis, treatment, and prognosis.
[0003] In view of this, we proposed a method to improve VWF dimerization and secretion disorders in von Willebrand disease. Summary of the invention
[0004] The object of the present invention is to provide a method for improving VWF dimerization and secretion disorder in von Willebrand disease to solve the problems raised in the above-mentioned background technology.
[0005] To achieve the above object, the present invention provides the following technical solution: a method for improving VWF dimerization and secretion disorder von Willebrand disease, the method comprising the following steps: S1, blood sampling and laboratory diagnosis; S2, VWF multimerization analysis; S3, mutation screening; S4, plasmid construction; S5, cell expression of recombinant VWF protein; S6. Data detection and collation.
[0006] Optionally, the S1 further comprises: Venous blood was collected and anticoagulated with 3.2% sodium citrate at a ratio of 1:9; Whole blood was centrifuged at 4000 g for 10 min to obtain plasma; VWF: Ag was measured by ELISA; The wells were coated with 10 mg / L VWF monoclonal antibody and blocked with 3% BSA-PBS for 2 h; After washing the plate three times with PBS-Tween20, normal mixed plasma and test plasma diluted with 0.5% BSA-PBS were added; The plate was washed three times with PBS-Tween20, and horseradish peroxidase-labeled SZ-34 was added and incubated at 37°C for 2 h; The plate was washed five times, 1 mg / ml o-phenylenediamine and 0.03% hydrogen peroxide were added to the substrate buffer, 0.05 ml 3M sulfuric acid was added to terminate the reaction after 5 minutes at room temperature, and the absorbance at 492 nm was measured after 10 minutes at room temperature; The VWF:Ag content of 50 normal mixed plasmas was set at 1000 U / L, and this plasma was used as a standard curve to calculate the VWF:Ag content of each plasma to be tested.
[0007] Optionally, the S2 further comprises: Patient plasma was diluted with loading buffer at a ratio of 1:5 and heated at 60°C for 30 min; VWF multimers were separated by 1.5% SDS-agarose gel electrophoresis at 3 mA constant current for 10 h, then the agarose gel was washed, air-dried, and coated with 5% skim milk powder for 1 h, and finally VWF multimers were detected using rabbit anti-human VWF primary antibody and goat anti-rabbit fluorescent secondary antibody; Optionally, the S3 further includes: The proband's blood cells were subjected to next-generation sequencing for genes related to hereditary bleeding disorders; The two mutations of the VWF gene found by the second-generation sequencing were verified by the first-generation sequencing of the proband's peripheral blood cell DNA. The primers for exon 28 and exon 51 of the VWF gene were designed as follows: VWF-6059034-F: 5'-GGATAGGTATCCGAACACGGAG-3'; VWF-6,059,034-R: 5'- ACAAGAGGGTTGCTTAGCCAT-3'; VWF-6,128,770-F: 5'-CAGCTCTGACGGTCGCTTC-3'; VWF-6,128,770-R: 5'-TCTGTGGGAATATGGAAGTCATTG-3'.
[0008] PCR reaction system (25 μl): dNTP 2 μl, 10×Ex tap buffer 2.5 μl, ExTap enzyme 0.2 μl, Primer F 1 μl, Primer R 1 μl, DNA or cDNA 1 μl, ddH2O 17.5 μl; PCR amplification conditions: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55-60°C for 35 seconds, extension at 72°C for 40-50 seconds, and repair extension at 72°C for 5-8 minutes after 35 cycles; PCR products were purified and recovered for sequencing analysis.
[0009] Optionally, the S4 further includes: The expression vector pSVHVWF contains the full-length cDNA sequence of wild-type human VWF; Based on the plasmid, the G at position 8171 of VWF was mutated to A using PCR-based site-directed mutagenesis; The sequences of the PCR primers were: 5′-gtgaggagcctgagtacaacgacatcactgc-3′; PCR reaction system (25μl): 5×HF buffer 5μl, 10 mM dNTP 0.5 μl, 10 mM Primer 1μl, DNA 1μl (50 ng), Phusion DNA polymerase 0.3 μl, ddH2O 16.2 μl; PCR amplification conditions: 98°C pre-denaturation for 30 seconds; 98°C denaturation for 10 seconds, 65°C annealing for 30 seconds, 72°C extension for 10 minutes, and 72°C repair extension for 15 minutes after 22 cycles; The PCR product was digested with Dpn I at 37°C for 3 hours and then transformed into DH5α competent cells by heat shock method. The plasmid was extracted and sequenced.
[0010] Optionally, the S5 further includes: HEK-293T cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum; Cells were cultured in 6-well plates and transfected when they were 50-70% confluent; Empty plasmid, VWF WT plasmid, VWF C2724Y plasmid, and a mixture of the two plasmids were transformed into HEK-293T cells; 48 h after transfection, the cell supernatant was collected, and the cells were washed with PBS and then lysed using lysis buffer (100 mM Tris / HCl, pH 7.5, 1% Triton X-100, supplemented with PMSF and a protease inhibitor cocktail); The cell supernatant was concentrated to 1 / 10 of the original volume using a 30 KDa ultracentrifuge tube; The lysate and culture supernatant were subjected to reducing (loading buffer containing β-mercaptoethanol) and non-reducing (loading buffer not containing β-mercaptoethanol) SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane and incubated with anti-VWF antibody for Western blot and VWF multimerization analysis.
[0011] Optionally, the S6 further includes: S61, plasma VWD phenotype detection in the proband; S62, analysis of plasma VWF multimers in the proband and his parents; Determination of S63 and VWF gene mutations; Expression of S64 and VWF C2724Y proteins; Multimeric analysis of S65 and VWF C2724Y proteins.
[0012] Compared with the prior art, the present invention provides a method for improving VWF dimerization and secretion disorder in von Willebrand disease, which has the following beneficial effects: 1. The method for improving VWF dimerization and secretion disorders in von Willebrand disease uses HEK-293T cells to study the expression and secretion of VWF wild-type and mutant proteins to verify whether the C2724Y mutation leads to VWF synthesis and secretion disorders, and to explore the role of intrachain disulfide bonds in the CK domain, including the disulfide bond Cys2724-Cys2774, in VWF protein synthesis, secretion and structural stability. The study will report a new type 3 VWD pedigree and clarify its molecular mechanism of disease, while also verifying the important role of intrachain disulfide bonds in the CK domain in maintaining VWF structure and function.
[0013] 2. The method to improve VWF dimerization and secretion disorders in von Willebrand disease and study the molecular pathogenesis of type 3 VWD, especially type 3 VWD caused by VWF missense mutations, will help to understand the relationship between the structure and function of VWF protein, which is of great significance for clinical diagnosis, treatment and prognosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 Schematic diagram of plasma VWF multimer analysis of the proband and his parents of the present invention; Figure 2 Schematic diagram of identification of VWF gene mutations of the present invention; Figure 3 Schematic diagram of VWF C2724Y plasmid construction and protein expression of the present invention; Figure 4 Schematic diagram of multimer analysis of the VWF C2724Y protein of the present invention; Figure 5 Schematic diagram of the positions of intrachain and interchain disulfide bonds in the VWF CK domain of the present invention. DETAILED DESCRIPTION
[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0016] like Figure 1-Figure 5 As shown, the present invention provides a technical solution: a method for improving VWF dimerization and secretion disorder von Willebrand disease, the method comprising the following steps: S1. Blood sampling and laboratory diagnosis, including the following steps: Venous blood was collected and anticoagulated with 3.2% sodium citrate at a ratio of 1:9; The whole blood was centrifuged at 4000 g for 10 minutes to obtain plasma. Coagulation parameters such as APTT and FVIII:C were measured using an automatic coagulometer (ACLTOP700) within 2 hours after blood collection.
[0017] VWF: Ag was measured by ELISA; The plate was coated with 10 mg / L VWF monoclonal antibody (SZ-29) and blocked with 3% BSA-PBS for 2 hours. After washing the plate three times with PBS-Tween20, normal mixed plasma and test plasma diluted with 0.5% BSA-PBS were added, where the standard curve was obtained from normal mixed plasma diluted 1:25 to 1:1000, and the test plasma was diluted 1:50 and incubated at 37°C for 2 hours; The plate was washed three times with PBS-Tween20, and horseradish peroxidase-labeled SZ-34 (E-SZ-34) was added and incubated at 37°C for 2 h; Wash the plate five times, add substrate buffer containing 1 mg / ml o-phenylenediamine (OPD) and 0.03% hydrogen peroxide (H2O2), add 0.05 ml 3M sulfuric acid to terminate the reaction after 5 minutes at room temperature, and measure the absorbance at 492 nm after 10 minutes at room temperature; The VWF:Ag content of 50 normal mixed plasmas was set at 1000 U / L, and this plasma was used as a standard curve to calculate the VWF:Ag content of each plasma to be tested.
[0018] S2, VWF multimerization analysis; Patient plasma was diluted 1:5 with loading buffer, and cell lysate and culture supernatant were diluted 1:3 with loading buffer consisting of 0.5 M Tris-HCl pH 6.8, 0.5 M EDTA, 0.1% SDS, 9 M Urea, heated at 60°C for 30 min; VWF multimers were separated by 1.5% SDS-agarose gel electrophoresis at 3 mA constant current for 10 h, then the agarose gel was washed, air-dried, and coated with 5% skim milk powder for 1 h. Finally, rabbit anti-human VWF primary antibody (Dako) and goat anti-rabbit fluorescent secondary antibody were used to detect VWF multimers.
[0019] S3. Mutation screening: The proband's blood cells were sequenced for genes related to hereditary bleeding disorders. Then, the proband's peripheral blood cell DNA was verified by first-generation sequencing for the two mutations of the VWF gene found by the second-generation sequencing. The primers were designed for exon 28 and exon 51 of the VWF gene as follows: VWF-6059034-F: 5'-GGATAGGTATCCGAACACGGAG-3'; VWF-6,059,034-R: 5'- ACAAGAGGGTTGCTTAGCCAT-3'; VWF-6,128,770-F: 5'-CAGCTCTGACGGTCGCTTC-3'; VWF-6,128,770-R: 5'-TCTGTGGGAATATGGAAGTCATTG-3'.
[0020] PCR reaction system (25μl): dNTP 2μl, 10×Ex tap buffer 2.5μl, ExTap enzyme 0.2μl, Primer F 1μl, Primer R 1μl, DNA or cDNA 1μl, ddH2O 17.5μl. PCR amplification conditions: 95℃ pre-denaturation for 3 minutes; 94℃ denaturation for 30 seconds, 55-60℃ annealing for 35 seconds, 72℃ extension for 40-50 seconds, 72℃ repair extension for 5-8 minutes after 35 cycles. PCR products were purified and recovered for sequencing analysis.
[0021] S4. Plasmid construction: The expression vector pSVHVWF containing the full-length cDNA sequence of wild-type human VWF was kindly donated by Professor Evan Sadler of the School of Medicine at Washington University in St. Louis, USA.
[0022] Based on the above plasmid, the G at position 8171 of VWF was mutated to A using PCR-based site-directed mutagenesis. The PCR primer sequence is: 5'-gtgaggagcctgagtacaacgacatcactgc-3'. PCR reaction system (25μl): 5×HF buffer 5μl, 10 mM dNTP 0.5 μl, 10 mM Primer 1μl, DNA 1μl (50 ng), Phusion DNA polymerase0.3 μl, ddH2O 16.2 μl. PCR amplification conditions: 98℃ pre-denaturation for 30 seconds; 98℃ denaturation for 10 seconds, 65℃ annealing for 30 seconds, 72℃ extension for 10 minutes, and 72℃ repair extension for 15 minutes after 22 cycles. After the PCR product was digested with Dpn I enzyme at 37℃ for 3 hours, it was heat-shocked and transferred into DH5α competent cells. The plasmid was extracted according to the instructions and sequenced.
[0023] S5. Cellular expression of recombinant VWF protein; HEK-293T cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum. Cells were cultured in 6-well plates and transfected when 50-70% of the total area was confluent. Referring to the instructions of PolyJet transfection reagent, empty plasmid, VWF WT plasmid, VWF C2724Y plasmid and a mixture of the two plasmids were transferred into HEK-293T cells. 48 hours after transfection, the cell supernatant was collected, the cells were washed with PBS and then lysed with lysis buffer (100 mM Tris / HCl, pH 7.5, 1% Triton X-100, and PMSF and protease inhibitor cocktail were added). The cell supernatant was concentrated to 1 / 10 of the original volume using a 30 KDa ultracentrifuge tube. The lysate and culture supernatant were subjected to reducing (loading buffer containing β-mercaptoethanol) and non-reducing (loading buffer not containing β-mercaptoethanol) SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane and incubated with anti-VWF antibody for Western blot and VWF multimerization analysis.
[0024] S6. Data detection and collation.
[0025] S61. Plasma VWD phenotype test of the proband: The proband had no abnormal blood routine test, but the coagulation routine test showed that the APTT was significantly prolonged, which was 66.8 seconds, and the use of 1:1 plasma could completely correct it (28.1 seconds). VWF: Ag (2%) was significantly reduced, RIPA (1.2mg 0, 0.6mg 0), VWF: Rco (2%), FVIII: C (2%) and other indicators were also significantly lower than normal values, while FXI: C (102%), FIX: C (65%) and FXII: C (86%) and other indicators were normal. Based on these experimental results, the proband can be diagnosed with type 3 VWD.
[0026] S62. Analysis of VWF multimers in the plasma of the proband and his parents. In order to analyze the pathogenicity of type 3 VWD, VWF multimers were analyzed in the plasma of the proband and his parents. The results showed that the VWF factor in the proband's plasma was severely deficient, with almost no VWF factor present, while the content and distribution of VWF factor in his parents' plasma were no different from those in standard plasma. This indicates that the proband's plasma lacks VWF factors that can work normally, leading to coagulation abnormalities. This result is consistent with the proband's other laboratory diagnosis; Determination of S63 and VWF gene mutations; In order to clarify the VWD gene mutation sites of the proband and his family, we extracted the proband's blood cell genomic DNA for second-generation gene testing. Sequencing results showed that the proband had a compound heterozygous mutation, and one VWF (NM_000552) allele mutation came from the mother: exon28: c.3814delT: p.C1272fs. It is speculated that this mutation involves the deletion of a single nucleotide T at position 3814 in exon 28, resulting in a frameshift mutation involving the loss of cysteine at position 1272. The other VWF gene mutation came from his father: exon 51: c.8171G>A: p.C2724Y. It is speculated that this mutation involves the replacement of cysteine at position 2724 at the carboxyl terminal. This amino acid change affects the formation of an intrachain disulfide bond (Cys2724-Cys2774) in the VWF monomer. Based on the results of the second-generation sequencing, we designed primers to amplify the nucleotide sequence near the mutation site and performed first-generation sequencing verification. The results showed that the mutation sequence of the proband's VWF gene was correctly identified. Cys2724 is located in the CK domain; S64, VWF C2724Y protein expression; In order to study the mechanism by which the C2724Y mutation causes type 3 VWD, we used a site-directed mutagenesis method to construct a VWF C2714Y mutant plasmid based on the VWF WT plasmid, and sequencing results indicated that the construction was successful. Subsequently, we transfected VWF WT, VWF C2724Y, and a mixture of the two plasmids into HEK293T cells, and used Western blotting to detect the expression of VWF in cell lysate and cell culture supernatant. The results showed that VWF WT protein was expressed in both cell lysate and culture supernatant, and VWF C2724Y mutant protein could accumulate in the form of pro-VWF in HEK-293T cells and could not be secreted outside the cells. This result suggests that the C2724Y mutation may be one of the reasons for the significant decrease in VWF content in the proband's plasma; S65, multimer analysis of VWF C2724Y protein; To study the reason why VWF C2724Y cannot be secreted outside the cell, we transfected VWF wild-type and C2724Y mutant plasmids into HEK293T cells, and used to detect the distribution of VWF protein multimers in cell lysate and cell culture supernatant. The results showed that in HEK-293T cell lysate, both VWF wild-type protein and VWF mutant protein could be expressed, but VWF wild-type protein mainly existed in dimer form, while VWF C2724Y mutant protein mainly existed in pro-VWF form, with a small amount of mature VWF monomer, and no dimer form; in the cell culture supernatant, only VWF wild-type protein was present, and its multimer distribution was normal, but no VWF mutant protein was expressed. These results suggest that the Cys2724 mutation causes VWF to be unable to form dimers, and thus cannot form VWF multimers as dimers and secrete them outside the cell.
[0027] As an application of this embodiment: By using HEK-293T cells to study the expression and secretion of VWF wild-type and mutant proteins, we will verify whether the C2724Y mutation leads to VWF synthesis and secretion disorders, and explore the role of intrachain disulfide bonds in the CK domain, including the disulfide bond Cys2724-Cys2774, in the synthesis, secretion and structural stability of VWF protein. The study will report a new type 3 VWD pedigree and clarify its molecular mechanism of disease, while also verifying the important role of intrachain disulfide bonds in the CK domain in maintaining the structure and function of VWF.
[0028] The above generally describes the present invention in detail, but it is obvious to a person skilled in the art that some modifications or improvements can be made to the present invention. Therefore, modifications or improvements that do not depart from the spirit of the present invention are within the scope of protection of the present invention.
Claims
1. A method for improving VWF dimerization and secretion disorder von Willebrand disease, characterized in that: The method comprises the following steps: S1, blood sampling and laboratory diagnosis; S2, VWF multimerization analysis; S3, mutation screening; S4, plasmid construction; S5, cell expression of recombinant VWF protein; S6. Data detection and collation.
2. The method for improving VWF dimerization and secretion disorder von Willebrand disease according to claim 1, characterized in that: Said S1 further comprises: Venous blood was collected and anticoagulated with 3.2% sodium citrate at a ratio of 1:9; Whole blood was centrifuged at 4000 g for 10 min to obtain plasma; VWF: Ag was measured by ELISA; The wells were coated with 10 mg / L VWF monoclonal antibody and blocked with 3% BSA-PBS for 2 h; After washing the plate three times with PBS-Tween20, normal mixed plasma and test plasma diluted with 0.5% BSA-PBS were added; The plate was washed three times with PBS-Tween20, and horseradish peroxidase-labeled SZ-34 was added and incubated at 37°C for 2 h; The plate was washed five times, 1 mg / ml o-phenylenediamine and 0.03% hydrogen peroxide were added to the substrate buffer, 0.05 ml 3M sulfuric acid was added to terminate the reaction after 5 minutes at room temperature, and the absorbance at 492 nm was measured after 10 minutes at room temperature; The VWF:Ag content of 50 normal mixed plasmas was set at 1000 U / L, and this plasma was used as a standard curve to calculate the VWF:Ag content of each plasma to be tested.
3. The method for improving VWF dimerization and secretion disorder von Willebrand disease according to claim 1, characterized in that: The S2 further comprises: Patient plasma was diluted with loading buffer at a ratio of 1:5 and heated at 60°C for 30 min; VWF multimers were separated by 1.5% SDS-agarose gel electrophoresis at 3 mA constant current for 10 h, then the agarose gel was washed, air-dried, and coated with 5% skim milk powder for 1 h. Finally, rabbit anti-human VWF primary antibody and goat anti-rabbit fluorescent secondary antibody were used to detect VWF multimers.
4. The method for improving VWF dimerization and secretion disorder von Willebrand disease according to claim 1, characterized in that: The S3 further comprises: The proband's blood cells were subjected to next-generation sequencing for genes related to hereditary bleeding disorders; The two mutations of the VWF gene found by the second-generation sequencing were verified by the first-generation sequencing of the proband's peripheral blood cell DNA. The primers for exon 28 and exon 51 of the VWF gene were designed as follows: VWF-6059034-F: 5'-GGATAGGTATCCGAACACGGAG-3'; VWF-6,059,034-R: 5'- ACAAGAGGGTTGCTTAGCCAT-3'; VWF-6,128,770-F: 5'-CAGCTCTGACGGTCGCTTC-3'; VWF-6,128,770-R: 5'-TCTGTGGGAATATGGAAGTCATTG-3'; PCR reaction system (25 μl): dNTP 2 μl, 10×Ex tap buffer 2.5 μl, ExTap enzyme 0.2 μl, Primer F 1 μl, Primer R 1 μl, DNA or cDNA 1 μl, ddH2O 17.5 μl; PCR amplification conditions: pre-denaturation at 95°C for 3 minutes; denaturation at 94°C for 30 seconds, annealing at 55-60°C for 35 seconds, extension at 72°C for 40-50 seconds, and repair extension at 72°C for 5-8 minutes after 35 cycles; PCR products were purified and recovered for sequencing analysis.
5. The method for improving VWF dimerization and secretion disorder von Willebrand disease according to claim 1, characterized in that: The S4 further comprises: The expression vector pSVHVWF contains the full-length cDNA sequence of wild-type human VWF; Based on the plasmid, the G at position 8171 of VWF was mutated to A using PCR-based site-directed mutagenesis; The sequences of the PCR primers were: 5′-gtgaggagcctgagtacaacgacatcactgc-3′; PCR reaction system (25μl): 5×HF buffer 5μl, 10 mM dNTP 0.5 μl, 10 mM Primer 1μl, DNA 1μl (50 ng), Phusion DNA polymerase 0.3 μl, ddH2O 16.2 μl; PCR amplification conditions: 98°C pre-denaturation for 30 seconds; 98°C denaturation for 10 seconds, 65°C annealing for 30 seconds, 72°C extension for 10 minutes, and 72°C repair extension for 15 minutes after 22 cycles; The PCR product was digested with Dpn I at 37°C for 3 hours and then transformed into DH5α competent cells by heat shock method. The plasmid was extracted and sequenced.
6. The method for improving VWF dimerization and secretion disorder von Willebrand disease according to claim 1, characterized in that: The S5 further comprises: HEK-293T cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum; Cells were cultured in 6-well plates and transfected when they were 50-70% confluent; Empty plasmid, VWF WT plasmid, VWF C2724Y plasmid, and a mixture of the two plasmids were transformed into HEK-293T cells; 48 h after transfection, the cell supernatant was collected, and the cells were washed with PBS and then lysed using lysis buffer (100 mM Tris / HCl, pH7.5, 1% Triton X-100, supplemented with PMSF and protease inhibitor cocktail); The cell supernatant was concentrated to 1 / 10 of the original volume using a 30 KDa ultracentrifuge tube; The lysate and culture supernatant were subjected to reducing (loading buffer containing β-mercaptoethanol) and non-reducing (loading buffer not containing β-mercaptoethanol) SDS-polyacrylamide gel electrophoresis, transferred to PVDF membrane and incubated with anti-VWF antibody for Western blot and VWF multimerization analysis.
7. The method for improving VWF dimerization and secretion disorder von Willebrand disease according to claim 1, characterized in that: The S6 further comprises: S61, plasma VWD phenotype detection in the proband; S62, analysis of plasma VWF multimers in the proband and his parents; Determination of S63 and VWF gene mutations; Expression of S64 and VWF C2724Y proteins; Multimeric analysis of S65 and VWF C2724Y proteins.