Cell strains causing two pathogenic missense mutations of composite heterozygous female HA patient, construction method and application

Abstract

The invention relates to the technical field of cell engineering, in particular to a cell strain causing two pathogenic missense mutations of a compound hybrid female HA patient, the cell strain comprises an FVIII-Met721Arg mutant cell strain and an FVIII-Pro283Thr mutant cell strain, the base sequence of the FVIII-Met721Arg mutant cell strain is shown as SEQ ID NO: 1, and the base sequence of the FVIII-Pro283Thr mutant cell strain is shown as SEQ ID NO: 3. The invention further provides a construction method and application of the cell strains causing the two pathogenic missense mutations of the composite heterozygous female HA patient. The discovery of the two new mutations not only enriches an F8 gene mutation database, but also clarifies the pathogenesis of the female hemophilia by combining the molecular pathogenic mechanism of the two new mutations and the random X chromosome inactivation result, thereby providing genetic counseling for family planning of hemophilia patients and providing a new strategy for precise treatment of the female hemophilia in future.

Description

Technical Field

[0001] The present invention relates to the technical field of cell engineering, and in particular to a cell line, a construction method and an application of two pathogenic missense mutations leading to compound heterozygous female HA patients. Background Art

[0002] Hemophilia A (HA) is one of the most common single gene inherited diseases caused by a defect in the F8 (Factor 8) gene, which can lead to a synthesis disorder or abnormal molecular structure of the F8 factor in the body, resulting in a reduction or complete absence of coagulation factor F8. Due to the X-linked recessive inheritance of hemophilia, women rarely show symptoms of hemophilia. The most common pathogenesis of female hemophilia is a combination of F8 gene mutation and X chromosome skewed inactivation. Even more rarely, homozygous or compound heterozygous mutations of the F8 gene and abnormalities in chromosome number or structure can also lead to female hemophilia.

[0003] The most common abnormality associated with HA is the inversion of intron 1 or 22. Other HA cases are caused by various point mutations distributed throughout the gene, including missense, nonsense, splice site, frameshift mutations, and severe deletions, of which missense mutations are the most common cause of HA. The human F8 gene is located on the long arm of the Xq28 region of the X chromosome and consists of 26 exons and 25 introns. The F8 protein product has no enzymatic activity. The mature FVIII protein consists of 2332 amino acids. Analysis of the human F8 gene revealed that the protein has a distinct domain structure expressed as A1-A2-B-A3-C1-C2. FVIII undergoes proteolysis at multiple sites to produce a 200 kDa heavy chain and an 80 kDa light chain to form a heterodimer, which is the main secreted form of FVIII. After being released into the circulating blood, the hFVIII heterodimer is hydrolyzed into 50kda, 43kda, and 73kda heterotrimers, which are the activated forms of FVIII.

[0004] Therefore, targeted genetic research on missense mutations in the F8 gene in female hemophilia patients can identify the pathogenic genes and their pathogenic mechanisms, which has potential clinical significance for genetic counseling and individualized treatment of hemophilia patients. Summary of the invention

[0005] The purpose of the present invention is to provide a cell line, construction method and application of two pathogenic missense mutations that cause compound heterozygous female HA patients, thereby enriching the F8 gene mutation database and providing a new strategy for the precise treatment of HA in the future.

[0006] The present invention provides cell lines with two pathogenic missense mutations that lead to compound heterozygous female HA patients, including a FVIII-Met721Arg mutant cell line and a FVIII-Pro283Thr mutant cell line.

[0007] Preferably, the base sequence of the FVIII-Met721Arg mutant cell strain is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the base sequence of the FVIII-Pro283Thr mutant cell strain is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

[0008] Preferably, the method for constructing the FVIII-Met721Arg / FVIII-Pro283Thr mutant cell line is specifically as follows: S11. Site-directed mutagenesis was performed on the BDD-FVIII wild-type plasmid to construct a plasmid expressing the FVIII-Met721Arg / FVIII-Pro283Thr cDNA sequence, wherein the mutation site of the FVIII- Met721Arg mutant cell line is c.2162T>G (p.Met721Arg) on ​​exon 14 of the F8 gene, and the mutation site of the FVIII- Pro283Thr mutant cell line is c.847C>A (p.Pro283Thr) on exon 7 of the F8 gene; S12. The FVIII-Met721Arg / FVIII-Pro283Thr plasmid containing the cDNA sequence was transfected into the cells using liposomes, and the FVIII-Met721Arg / FVIII-Pro283Thr mutant cell strain was obtained through drug screening.

[0009] Preferably, the base sequence of the BDD-FVIII wild-type plasmid is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6.

[0010] Preferably, the FVIII-Met721Arg plasmid containing the cDNA sequence is obtained by subjecting the BDD-FVIII wild type to site-directed mutagenesis based on the PCR method, and the FVIII-Pro283Thr plasmid containing the cDNA sequence is obtained by subjecting the BDD-FVIII wild type to site-directed mutagenesis based on the seamless cloning technology.

[0011] Preferably, the FVIII-Met721Arg plasmid / FVIII-Pro283Thr plasmid containing the cDNA sequence is transfected into human embryonic kidney cells HEK293T by liposome transfection.

[0012] Preferably, the drug screening is antibiotic screening, wherein the resistance drug is puromycin.

[0013] Preferably, when constructing the FVIII-Met721Arg plasmid containing the cDNA sequence, the primers used for site-directed mutagenesis are: upstream primer 5'-tttcggaacagaggcaGgaccgccttac-3', downstream primer 5'-Ctgcctctgttccgaaagtctgagttgt-3'; When constructing the FVIII-Pro283Thr plasmid containing the cDNA sequence, the primers used for site-directed mutagenesis were: upstream primer 5'-tctgattggatgccacagga-3', downstream primer 5'-gcgatggttcctcacaagaa-3'.

[0014] The present invention also provides the use of the cell strain with two pathogenic missense mutations that cause compound heterozygous female HA patients or the cell strain with two pathogenic missense mutations obtained according to the construction method of the cell strain with two pathogenic missense mutations that cause compound heterozygous female HA patients in the prevention, early diagnosis or treatment of female hemophilia.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention not only provides a research direction and a new theoretical basis for the early diagnosis and effective treatment of female hemophilia, but also provides a new molecular target and cell model for the development of specific drugs for the treatment of hemophilia in practice.

[0016] 2. The discovery of two new mutations in the present invention not only enriches the F8 gene mutation database, but also clarifies the cause of female hemophilia by combining the molecular pathogenic mechanism of the two new mutations and the results of random X chromosome inactivation, providing genetic counseling for family planning of hemophilia patients and new strategies for precise treatment of female hemophilia in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a wild-type plasmid map of BDD-FVIII in the examples of the present invention; Figure 2 This is a mutation region map of the FVIII-Met721Arg mutant cell line in the embodiment of the present invention; Figure 3 This is a mutation region diagram of the FVIII-Pro283Thr mutant cell line in the embodiment of the present invention; Figure 4 is an in vitro experimental structure diagram of the wild type group and the variant group in the embodiment of the present invention, wherein Figure 4 -A is a graph of FVIII mRNA expression levels analyzed by qRT-PCR, Figure 4 -B is a graph of FVIII:Ag levels in cell culture medium and cell lysate, Figure 4 -C is the expression level of FVIII:C in cell culture medium, Figure 4 -D is a Western blot analysis of recombinant FVIII protein; Figure 5 is an X chromosome inactivation analysis diagram of the HUMARA locus of the subject in the embodiment of the present invention, wherein Figure 5 -A is the PCR amplification diagram of the androgen receptor polymorphism (CAG) repeat sequence without HpaII digestion, Figure 5 -B is PCR amplification of the androgen receptor polymorphic (CAG) repeat sequence after HpaII digestion; Figure 6 This is a diagram of the DNA Sanger sequencing results of the F8 gene of the subject in the embodiment of the present invention; Figure 7 It is a conservative analysis diagram of Unipro UGENE software in an embodiment of the present invention; Figure 8 is the amino acid structure diagram of the mutant cell line before and after mutation in the embodiment of the present invention, wherein Figure 8 -A is the amino acid structure of FVIII before Met721Arg mutation, Figure 8 -B is the amino acid structure diagram of FVIII- Met721Arg after mutation, Figure 8 -C is the amino acid structure of FVIII-Pro283Thr before mutation, Figure 8 -D is the amino acid structure diagram of FVIII-Pro283Thr after mutation. DETAILED DESCRIPTION

[0019] 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.

[0020] The present invention provides cell lines with two pathogenic missense mutations that lead to compound heterozygous female HA patients, including a FVIII-Met721Arg mutant cell line and a FVIII-Pro283Thr mutant cell line.

[0021] like Figure 2 Provided FVIII-Met721Arg mutant cell line mutation region map, Figure 2 The first row is the normal base sequence, and the second row is the base sequence with successful mutation. The shaded area in the figure is the mutation area. The mutation site of the FVIII-Met721Arg mutant cell line is c.2162T>G (p.Met721Arg) on ​​exon 14 of the F8 gene.

[0022] like Figure 3 The mutation region map of FVIII-Pro283Thr mutant cell line provided, Figure 3 The first row is the normal base sequence, and the second row is the base sequence after successful mutation. The shaded area in the figure is the mutation area. The mutation site of the FVIII-Pro283Thr mutant cell line is c.847C>A (p.Pro283Thr) on exon 7 of the F8 gene.

[0023] The base sequence of the FVIII-Met721Arg mutant cell line is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0024] The base sequence of the FVIII-Pro283Thr mutant cell line is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4.

[0025] In this application, the main reagents, instruments and equipment used are shown in Table 1 and Table 2.

[0026]

[0027]

[0028] The BDD-FVIII wild-type plasmid used in the present invention is shown in the document “Targeted genome engineering based on CRISPR / Cas9 system to enhance FVIII expression in vitro”.

[0029] The BDD-FVIII used in the present invention is a wild-type plasmid with a full length of about 170 kDa. The protein is secreted in the form of a heterodimer formed by a 90 kDa heavy chain and an 80 kDa light chain, and exists in an activated form formed by 50 kDa, 43 kDa and 73 kDa heterotrimers.

[0030] The base sequence of the BDD-FVIII wild-type plasmid is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6.

[0031] Example 1: Construction of mutant plasmid containing cDNA sequence

[0032] like Figure 1 As shown, in the present application, the BDD-FVIII wild-type plasmid was subjected to site-directed mutagenesis based on the PCR method to construct a plasmid expressing the FVIII-Met721Arg cDNA sequence, and the specific steps are as follows: (1) Design mutation primers. The mutation primers are as follows: Upstream primer: 5′-tttcggaacagaggcaGgaccgccttac-3′; Downstream primer: 5′-Ctgcctctgttccgaaagtctgagttgt-3′.

[0033] (2) Amplify the BDD-FVIII wild-type plasmid using mutant primers. The amplification conditions are as follows: PCR system: The total reaction volume is 50 µL, including 8 ng of FVIII wild-type plasmid, 1 µL of upstream and downstream primers, 25 µL of 2×TransStart Fastpfu Fly PCR SuperMix, and deionized water to 50 µL; PCR conditions: 95°C pre-denaturation for 3 min; 95°C denaturation for 20 s, 59°C annealing for 20 s, 72°C extension for 3 min, a total of 25 cycles, and a final extension at 72°C for 5 min; Digestion of PCR products: Add 1 µL of DMT enzyme to the PCR product, mix well, and incubate in a 37°C water bath for 1 hour.

[0034] (3) Conversion ① Add 5 µL of DMT enzyme digestion product to 50 µL of competent cells, flick to mix, and place on ice for 30 minutes; ② Heat shock in a 42℃ water bath for 90 seconds, then immediately place on ice for 2 minutes; ③ Add 250 µL LB medium, incubate at 200 rpm and 37°C for 1 hour; ④ Take 150 µL of bacterial solution and spread it evenly on an agar plate with ampicillin resistance and culture it at 37℃ overnight.

[0035] (4) Plasmid amplification: Add 5 µL of ampicillin to 5 ml of LB medium, pick a single clone on the plate and add it to the above medium, and culture in a shaking incubator at 250 rpm and 37°C for 16 hours.

[0036] (5) Use the Omega plasmid extraction kit to extract the FVIII mutant plasmid. The specific steps are as follows: ① Take 5 ml of bacterial solution and centrifuge at 10000xg for 1 minute at room temperature; ② Pour off the culture medium, add 250 µL of Solution I / RNaseA mixture, and vortex to completely suspend the cells; ③ Add 250 µL Solution II and gently invert for 4-6 times to mix. The mixing time should not exceed 5 minutes. ④ Add 350 µL Solution III, gently invert several times until a white flocculent precipitate is formed, and centrifuge at 13,000 x g for 10 minutes at room temperature; ⑤ Transfer the supernatant to a HiBind Miniprep DNA binding column with a 2 ml collection tube, centrifuge at 14,000 x g for 10 minutes at room temperature, discard the filtrate, and replace the collection tube; ⑥ Add 500 µL HBC buffer, centrifuge at 14000xg for 1 minute at room temperature, discard the filtrate, and replace the collection tube; ⑦ Add 700 µL DNA washing solution, centrifuge at 14000xg for 1 minute at room temperature, discard the filtrate, and replace the collection tube; ⑧Repeat step 7 once; ⑨ Centrifuge the empty column at 14000xg for 2 minutes to dry the column matrix, place the column in a sterile 1.5ml centrifuge tube, add 30 µL of elution buffer, let stand at room temperature for 1 minute, centrifuge at 13000xg for 1 minute to elute the DNA, and store at -20℃.

[0037] (6) Confirmation of successful plasmid construction: The extracted plasmid is sent to BGI for sequencing to confirm the presence of the mutation.

[0038] In this application, site-directed mutagenesis was performed on the BDD-FVIII wild type based on seamless cloning technology to construct a plasmid expressing the FVIII-Pro283Thr cDNA sequence. The specific steps are as follows: (1) Reverse PCR amplification of BDD-FVIII plasmid using mutant primers, wherein the mutant primers are as follows: Upstream primer: 5′-ttcttgtgaggaaccatcgc-3′, downstream primer 5′-tcctgtggcatccaatcaga-3′; Amplification conditions were as follows: PCR system: The total reaction volume is 50 µL, including 300 ng of BDD-FVIII wild-type plasmid, 1 µL of upstream and downstream primers, 25 µL of 2×TransStart Fastpfu Fly PCR SuperMix, and deionized water to 50 µL; PCR conditions: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s, annealing at 57°C for 5 s, extension at 72°C for 100 s, for a total of 30 cycles, and a final extension at 72°C for 1 min.

[0039] (2) Recover PCR products by agarose gel electrophoresis.

[0040] (3) Using the patient's genomic DNA as a template, PCR amplify the target mutant gene fragment using mutation primers, where the mutation primers are as follows: Upstream primer: 5′-tctgattggatgccacagga-3′, downstream primer 5′-gcgatggttcctcacaagaa-3′; Amplification conditions were as follows: PCR system: The total reaction volume is 50 µL, including 300 ng of patient genome, 1 µL of upstream and downstream primers, 25 µL of 2×TransStart Fastpfu Fly PCR SuperMix, and deionized water to 50 µL; PCR conditions: pre-denaturation at 98°C for 1 min; denaturation at 98°C for 10 s, annealing at 57°C for 5 s, extension at 72°C for 80 s, for a total of 30 cycles, and a final extension at 72°C for 1 min.

[0041] It should be noted that the patient genomic DNA is the subject DNA extracted in Example 4.

[0042] (4) Recombination reaction: The total reaction volume is 10 µL, including 25 µL of MonCloneTM Hi-Fusion Cloning Mix V. The optimal amount of vector and target fragment is calculated according to the instructions, and deionized water is added to make up to 10 µL. The reaction system is placed at 50°C for 15 min.

[0043] (5) Conversion ① Add 5 µL of DMT enzyme digestion product to 50 µL of competent cells, flick to mix, and place on ice for 30 minutes; ② Heat shock in a 42℃ water bath for 90 seconds, then immediately place on ice for 2 minutes; ③ Add 250 µL LB medium, incubate at 200 rpm and 37°C for 1 hour; ④ Take 150 µL of bacterial solution and spread it evenly on an agar plate with ampicillin resistance and culture it at 37℃ overnight.

[0044] (6) Plasmid amplification: Add 5 µL of ampicillin to 5 ml of LB medium, pick a single clone on the plate and add it to the above medium, and culture in a shaking incubator at 250 rpm and 37°C for 16 hours.

[0045] (7) Use the Omega plasmid extraction kit to extract the FVIII mutant plasmid. The specific steps are as follows: ① Take 5 ml of bacterial solution and centrifuge at 10000xg for 1 minute at room temperature; ② Pour off the culture medium, add 250 µL of Solution I / RNaseA mixture, and vortex to completely suspend the cells; ③ Add 250 µL Solution II and gently invert for 4-6 times to mix. The mixing time should not exceed 5 minutes. ④ Add 350 µL Solution III, gently invert several times until a white flocculent precipitate is formed, and centrifuge at 13,000 x g for 10 minutes at room temperature; ⑤ Transfer the supernatant to a HiBind Miniprep DNA binding column with a 2 ml collection tube, centrifuge at 14,000 x g for 10 minutes at room temperature, discard the filtrate, and replace the collection tube; ⑥ Add 500 µL HBC buffer, centrifuge at 14000xg for 1 minute at room temperature, discard the filtrate, and replace the collection tube; ⑦ Add 700 µL DNA washing solution, centrifuge at 14000xg for 1 minute at room temperature, discard the filtrate, and replace the collection tube; ⑧Repeat step 7 once; ⑨ Centrifuge the empty column at 14000xg for 2 minutes to dry the column matrix, place the column in a sterile 1.5ml centrifuge tube, add 30 µL of elution buffer, let stand at room temperature for 1 minute, centrifuge at 13000xg for 1 minute to elute the DNA, and store at -20℃.

[0046] (8) Confirmation of successful plasmid construction: The extracted plasmid is sent to BGI for sequencing to confirm the presence of the mutation.

[0047] Implementation 2: Transfect cells and obtain stable mutant cell lines after drug screening

[0048] The empty vector, BDD-FVIII wild-type plasmid, and FVIII-Met721Arg plasmid / FVIII-Pro283Thr plasmid containing cDNA sequence were transfected into human embryonic kidney cells (HEK293T) using liposomes. The specific steps are as follows: (1) Cell recovery ① Take the HEK293T cells out of the -80℃ refrigerator and immediately put them into a 37℃ water bath for rapid thawing; ② After the cell mixture is completely thawed, add 1 ml of DMEM medium, then transfer to a centrifuge tube containing 5 ml of DMEM medium, centrifuge at 1000 rpm for 5 minutes, and remove the supernatant; ③ Add 1 ml of culture medium to the remaining cell pellet and mix gently using a pipette; ④ Add the above mixture to 25cm 2 Then, 4 ml of DMEM medium containing 10% fetal bovine serum (FBS) was added to the cell culture flask and cultured at 37°C with 5% CO2.

[0049] (2) Cell passaging: When about 80% of the cells are confluent, they are digested with trypsin to prepare a single cell suspension, which is then cultured in a six-well plate at 37°C and 5% CO2 for 24 hours.

[0050] (3) Transfection ① Transfection was performed when the cell confluence reached 80%, and the culture medium was replaced with fresh DMEM medium (containing 10% FBS) before transfection; ② Preparation of Solution A: Take 4 1.5 ml centrifuge tubes, add 100 µL DMEM medium (without FBS) to each tube, then add 10 µL Lipofectamine 2000 transfection reagent to each tube, vortex to mix and centrifuge briefly; ③ Preparation of Solution B: Take 4 1.5ml centrifuge tubes and add 100 µL DMEM medium (without FBS) to each tube. Add 2.5ug BDD-FVIII wild-type plasmid (FVIII-WT), FVIII mutant plasmid (FVIII-Pro283Thr and FVIII-Met721Arg) and empty vector to each tube, vortex to mix and centrifuge briefly; ④ Slowly add solution A to solution B, vortex to mix and centrifuge briefly, and incubate at room temperature for 5 minutes; ⑤ Add the above mixed solution into a six-well plate, gently shake the six-well plate to ensure that the mixed solution is evenly distributed, and culture at 37°C, 5% CO2 for 48 hours.

[0051] (4) Drug screening of stable point mutation cell lines ① Green fluorescence can be seen 48 hours after transfection. Digest the cells with trypsin containing 0.25% EDTA, dilute them at a ratio of 1:10 and inoculate them into a new six-well plate. After the cells are completely attached to the plate, add puromycin (the concentration of puromycin is 3.5µg / ml after screening) to screen the cells. A large number of cells die in the first 3 days, and the cell culture medium containing puromycin is replaced every day, and then every 3 days.

[0052] ②After 2 weeks, white monoclonal cell clusters can be seen with the naked eye in the six-well plate where the plasmid has been transferred. Select the monoclonal cells and continue to culture them in 24-well plates. When the cell density is >80%, digest them with trypsin containing 0.25% EDTA and then re-inoculate them in a new six-well plate at a ratio of 1:2 and continue to culture.

[0053] ③ Collect cells, extract genomic RNA, and detect by RT-PCR. For cell lines with positive RT-PCR results, use the limiting dilution method to plate the correctly sequenced cells in a 96-well plate for cell monoclonal cloning. When the monoclonal cells grow to a confluence of 80%, they are passaged at a 1:2 ratio. RNA is extracted from the cells in one well and identified by RT-PCR. Positive cells are expanded to obtain monoclonal cell lines and stored in liquid nitrogen at -80°C.

[0054] Example 3: Taking some stable mutant cell lines to test the expression levels of FVIII:mRNA, FVIII:Ag, FVIII:C, and FVIII protein

[0055] 1. Take some stable mutant cell lines and prepare samples (1) Use a 15 ml centrifuge tube to collect the culture medium in the six-well plate, centrifuge at 3000 rpm and 4°C for 10 minutes, aspirate the supernatant, and store it at -20°C for later use.

[0056] (2) RNA extraction ① Add 1 ml PBS to each well for washing, then add 1.2 ml RNAiso Plus lysis buffer and shake gently to evenly distribute it on the cell surface; ② Transfer the lysate containing cells to a 1.5 ml centrifuge tube, pipette repeatedly until there is no obvious precipitation in the lysate, and let it stand at room temperature for 5 minutes; ③ Add 240 µL of chloroform, vortex to mix, and let stand at room temperature for minutes; ④ Centrifuge at 12000xg, 4℃ for 15 minutes. Carefully remove the centrifuge tube from the centrifuge. The liquid is now divided into three layers. Transfer the upper clear liquid to a new 1.5ml centrifuge tube (do not aspirate the white middle layer); ⑤ Add an equal volume of isopropanol to the supernatant, mix by inverting, and let stand at room temperature for 10 minutes; ⑥ Centrifuge at 12000xg, 4℃ for 10 minutes, remove the supernatant. Add 1.2ml 75% ethanol to the remaining precipitate, wash the tube wall by inverting it, and centrifuge at 12000xg, 4℃ for 5 minutes; ⑦ After discarding the supernatant, dry the precipitate at room temperature for 3 minutes, add 15 µL RNase-free water to dissolve the precipitate, and store it at -20℃ for later use.

[0057] (3) Extraction of total protein ① Add 1 ml PBS to each well for washing, then add 100 µL mammalian active protein extraction reagent (containing 1 mmol / L PMSF) and incubate on ice for 30 minutes; ② Use a pipette to suspend the cell extraction product and transfer it to a 1.5 ml centrifuge tube, and centrifuge at 14000xg, 4ºC for 10 minutes; ③ Transfer the supernatant containing soluble protein to a new 1.5 ml centrifuge tube and store it at -20℃ for later use.

[0058] 2. Detection of FVIII mRNA expression level using real-time fluorescence quantitative PCR (qRT-PCR) qRT PCR was used to detect the FVIII: mRNA expression levels in the wild-type group and the mutant group. The specific detection steps are as follows: (1) Reverse transcription: Total RNA was reverse transcribed into cDNA using Primescript RT Master Mix reagent. The reaction conditions were 37°C for 15 min and 85°C for 5 s. The reaction system is shown in Table 3.

[0059]

[0060] (2) qRT-PCR was performed using TB Green Premix Ex Taq Ⅱ reagent, with β-actin as the internal reference, and three replicate wells were set for each sample. Reaction conditions: pre-denaturation at 95°C for 30 seconds; denaturation at 95°C for 5 seconds, annealing at 60°C for 34 seconds, for a total of 40 cycles. The primers used for qRT-PCR are shown in Table 4, and the reaction system is shown in Table 5.

[0061]

[0062]

[0063] 3. Detection of FVIII antigen (FVIII:Ag) levels in cell culture medium and cell lysate using ELISA kit The FVIII:Ag levels in the cell culture medium and cell lysate of the wild-type group and mutant group were detected using an enzyme-linked immunosorbent assay (ELISA) kit. The specific detection steps are as follows: (1) Before use, equilibrate the ELISA kit to room temperature and dilute the plasma to be tested at a ratio of 1:200 using diluent; (2) Add 50 µL of the sample to be tested and FVIII standard to a 96-well plate pre-coated with antibodies, then add 50 µL of Antibody Cocktail to each well, seal the 96-well plate, and incubate on a shaker at room temperature for 1 hour with the speed set to 400 rpm; (3) Add 350 µL of wash solution to each well and wash three times. After the last wash, turn the plate upside down and absorb the excess liquid with a clean paper towel. (4) Add 100 µL of developer to each well and incubate on a shaker set at 400 rpm in the dark for 10 min. (5) Add 100 µL of stop solution to each well, shake on a shaker for 1 minute to mix, and immediately record the OD value of each well at 450 nm using a microplate reader; (6) Draw an OD value-FVIII:Ag standard curve and calculate the FVIII:Ag content of the sample to be tested.

[0064] 4. Detection of Factor VIII Activity (FVIII:C) Levels Using the Phase I Method The FVIII:C levels in the wild-type group, mutant group, and control group were detected using a one-phase method. The specific steps are as follows: ① Add 1 ml of sterile water for injection to the factor VIII-deficient plasma, let it stand at room temperature for 20 minutes, then shake it well to completely dissolve it; ② Take 100 µL of CaCl2 and APTT reagent and put them into the automatic coagulation instrument; ③ Take 100 µL of the plasma to be tested and the plasma lacking factor VIII, and put them into the automatic coagulation analyzer; ④ Read the FVIII:C value on the coagulometer.

[0065] 5. Detection of FVIII protein expression levels in cell culture medium and cell lysate using Western Blotting assay Western Blotting was used to detect the FVIII protein expression levels in the cell culture medium and cell lysate in the wild-type group, mutant group, and control group. The specific steps are as follows: (1) Determine protein concentration, including: ①Prepare protein standard: add 0.8 ml of protein standard solution to a tube of protein standard (20 mg BSA), fully dissolve to prepare a 25 mg / ml protein standard solution, and then dilute to 0.5 mg / ml protein standard; ②Prepare BCA working solution: According to the number of samples, prepare BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1) and mix thoroughly; ③ Add 0.5mg / ml protein standard to a 96-well plate at 0, 1, 2, 4, 8, 12, 16, and 20 µL, and then add standard diluent to make up to 20 µL, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5mg / ml, respectively. Set up 3 replicate wells for each sample; ④ Add 20 µL of the sample to be tested into a 96-well plate, with 3 replicate wells for each sample; ⑤ Add 200 µL BCA working solution to each well and incubate at 37°C for 30 minutes; ⑥ Use an ELISA reader to measure the OD value of each well at 562 nm, draw a standard curve, and calculate the protein concentration of the sample to be tested.

[0066] (2) Protein denaturation: Mix the protein sample with 5× Loading Buffer in a volume ratio of 4:1, heat in a metal bath at 100°C for 5 min, cool to room temperature, and place in a -20°C refrigerator for later use.

[0067] (3) Detecting the FVIII protein expression levels in the cell culture medium and cell lysate in the wild-type group, mutant group, and control group, specifically including: ① Prepare PAGE gel: Use the Easy PAGE® Color Rapid Gel Preparation Kit (10%) of SEVEN to prepare PAGE gel. For the specific formula, refer to the instruction manual.

[0068] ② Electrophoresis: Place the prepared gel in the electrophoresis device and inject an appropriate amount of electrophoresis buffer solution into it. The protein loading amount is about 20μg and the Western Marker loading amount is 5μL. In the initial stage, perform electrophoresis at a constant voltage of 80V until the Western Marker indicator band completely enters the separation gel. Adjust the voltage to 120V and continue electrophoresis until the Loading Buffer migrates to the bottom of the gel.

[0069] ③ Transfer: Carefully remove the gel, cut the gel near the molecular weight of the target protein, and cut out a PVDF membrane of the same size as the gel, and place it in methanol for activation for 5 minutes. Place the sponge pad, three-layer transfer filter paper, PAGE gel, PVDF membrane, three-layer transfer filter paper and sponge pad from the black side to the white side of the transfer clamp, clamp it and place it in the electrotransfer tank, constant current 350mA, transfer for 50 minutes.

[0070] ④ Blocking: After the protein transfer step is completed, transfer the PVDF membrane to the protein incubation box, add an appropriate amount of TBST solution to wash for 10 min to remove the residual electrotransfer buffer, discard the TBST, then add 5% skim milk and incubate on a shaker at room temperature for 2 hours.

[0071] ⑤ Antibody incubation: After blocking, discard the rapid blocking solution, wash with TBST solution for 10 min, discard the TBST solution, add the primary antibody human anti-mouse F8 antibody and human anti-rabbit β-actin antibody diluted according to the recommended ratio in the instructions, and incubate overnight in a shaker at 4°C. After recovering the primary antibody, add TBST solution to wash 3 times, 10 min each time, then add the secondary antibody goat anti-mouse and goat anti-rabbit IgG antibodies diluted at a ratio of 1:10000, and incubate in a shaker at room temperature for one and a half hours.

[0072] ⑥ Luminescence color development: After the secondary antibody incubation is completed, the membrane is recovered and washed with TBST solution for 3 times, 10 min each time. According to the instructions, mix the A solution and B solution in the ECL detection kit to prepare the luminescence reagent. Soak the membrane evenly in this luminescence working solution, incubate it at room temperature in the dark for 5 minutes, and place the membrane in the ChemiDoc imaging system for exposure and imaging.

[0073] In order to study the molecular pathogenic mechanism of female hemophilia, in this application, plasmids containing wild-type or mutant FVIII cDNA were transiently transfected into HEK293T cells, and stable mutant cell lines were obtained after drug screening. Some stable mutant cell lines were taken to test the expression levels of FVIII: mRNA, FVIII: Ag, FVIII: C and FVIII protein. The test results are as follows: Figure 4 In this application, the FVIII: mRNA, FVIII: Ag and FVIII: C levels of the wild group are set to 100%, and Figure 4 WT represents the wild type group, MT1 represents the mutant FVIII-Met721Arg, MT2 represents the mutant FVIII-Pro283Thr, and Control represents the control group.

[0074] Depend on Figure 4 -A's qRT PCR test results showed that the FVIII: mRNA expression levels of the mutant FVIII-Met721Arg and mutant FVIII-Pro283Thr were no different from those of the wild-type group.

[0075] Depend on Figure 4 -B ELISA results showed that the levels of FVIII:Ag in the cell culture medium and lysate of mutant FVIII-Met721Arg and mutant FVIII-Pro283Thr were no different from those of the wild-type group.

[0076] Depend on Figure 4-C shows that the cell culture medium factor activity of the mutant FVIII-Met721Arg was significantly different from that of the wild-type group (P<0.0001), and the enzyme activity of the mutant protein was significantly reduced, which was no different from that of the control group; the cell culture medium factor activity of the mutant FVIII-Pro283Thr was slightly different from that of the wild-type group (P<0.05), and the enzyme activity of the mutant protein was slightly reduced. It should be noted that Figure 4 In -C, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

[0077] Depend on Figure 4 -D showed that the protein content of mutant FVIII-Met721Arg and mutant FVIII-Pro283Thr in cells was not significantly different from that in wild-type group; however, Figure 4 -D The results of western blotting of the cell culture medium showed that the A2 domain of the activated heterotrimer was active, and the extracellular active protein content (43 kDa) of the mutant FVIII-Met721Arg was significantly different from that of the wild type, but no different from that of the control group; the extracellular active protein content (43 kDa) of the mutant FVIII-Pro283Thr was slightly different from that of the wild type, which was consistent with the results of FVIII:C activity in the cell culture medium.

[0078] In summary, according to the test results of FVIII: mRNA, FVIII: Ag, FVIII: C, and FVIII protein expression levels of mutant FVIII-Met721Arg, mutant FVIII-Pro283Thr, and wild-type groups, mutant FVIII-Met721Arg and mutant FVIII-Pro283Thr did not affect F8 gene transcription, protein synthesis, and secretion, but may cause HA by destroying the spatial conformation of the FVIII protein, that is, the protein may eventually be inactivated to varying degrees due to changes in protein conformation, thereby causing the protein to lose activity after hydrolysis or affecting the production of active protein, thereby causing HA.

[0079] Example 4: Clinical manifestations of female HA patients

[0080] The proband is a 14-year-old girl whose parents are non-consanguineous. She was diagnosed with mild HA (FVIII:C7%) at the age of 6. Her first bleeding site was knee bleeding, and there was no family history. She is currently using an on-demand treatment regimen. In this study, we conducted gene mutation analysis on this female patient. In order to explore the molecular pathogenic mechanism of the new variant, we performed in vitro experiments and bioinformatics analysis.

[0081] The subjects signed the informed consent, and this study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University.

[0082] 1. Sample collection The peripheral blood of the subjects was collected using a sodium citrate vacuum blood collection tube and centrifuged at 3000 rpm for 15 minutes at room temperature. The plasma in the upper layer was aspirated for coagulation detection, and the blood cells in the lower layer were used for DNA extraction.

[0083] 2. Coagulation Test (1) Coagulation factor activity (FVIII:C) detection (2) Detection of von Willebrand factor antigen (VWF:Ag) content (3) Inhibitor detection Coagulation tests showed that the subject's FVIII:C was 7%, von Willebrand factor antigen (VWF:Ag) was 97%, and inhibitors were negative.

[0084] It should be noted that the FVIII:C detection steps in Example 4 are consistent with the FVIII:C detection steps in Example 3.

[0085] 3. Extraction of DNA from subjects The DNA was extracted using a blood genomic DNA extraction kit. The sample was the subject's peripheral blood. The specific steps are as follows: (1) Add 20 µL of protease to a 1.5 ml sterile centrifuge tube, add 300 µL of sample, add 200 µL of AL buffer, and vortex to mix; (2) Incubate at 56°C for 10 minutes, centrifuge briefly, and remove the liquid on the lid; (3) Add 200 µL of anhydrous ethanol and vortex to mix thoroughly; (4) Transfer the mixture to a QIAamp Mini spin column and centrifuge at 12,000 rpm for 1 min. Discard the filtrate and replace the collection tube. (5) Add 500 µL AW1 buffer, centrifuge at 12,000 rpm for 1 minute, discard the filtrate, and replace the collection tube; (6) Add 500 µL AW2 buffer, centrifuge at 12,000 rpm for 3 minutes, discard the filtrate, and replace the collection tube; (7) Place the QIAamp Mini spin column in a new 2 ml collection tube and centrifuge at 14,000 rpm for 1 min. (8) Place the QIAamp Mini spin column in a new 2 ml sterile centrifuge tube, add 100 µL AE buffer, incubate at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min to elute the DNA, collect the filtrate, and store at -20°C.

[0086] 4. X chromosome inactivation analysis (1) Enzyme digestion: DNA extracted from peripheral blood was digested with restriction endonuclease FastDigest HpaII (ThermoScientific) at 37°C for 1-3 h.

[0087] (2) Detection of random patterns of X chromosomes j The CAG repeat region of exon 1 of the HUMARA gene was amplified using the genomic DNA before and after restriction digestion as the reaction template, and the 5' end of the forward primer was labeled with FAM fluorescence.

[0088] kThe DNA template was PCR amplified using TranStart FastPfu Fly DNA Polymerase (Transgene), where the primers are as follows: AR-F (5'-TCCAGAATCTGTTCCAGAGCGTGC-3'), AR-R (5'-GCTGTGAAGGTTGCTGTTCCTCAT-3'); Amplification conditions were as follows: PCR process: pre-denaturation at 95°C for 15 min, 35 cycles (95°C for 20 s, 67°C for 20 s, 72°C for 25 s), 72°C for 7 min.

[0089] lPCR products were electrophoresed in an automatic capillary sequencer (3700xL), and the size of the site and the peak area were analyzed by data analysis software (GeneMapper4.2), where the degree of X chromosome inactivation was calculated as follows: , Wherein, d1 and d2 represent the peak values ​​of the 2nd band of PCR product of the digested sample, and u1 and u2 represent the peak values ​​of the 2nd band of PCR product of the undigested sample.

[0090] In the present application, the genomic DNA before and after restriction endonuclease HpaII digestion is used as a reaction template to amplify the CAG repeat region of exon 1 of the HUMARA gene, thereby detecting the X chromosome inactivation pattern of the subject. Figure 5 X-chromosome inactivation analysis of the HUMARA locus of the subject shown, wherein Figure 5 -A is the PCR amplification diagram of the androgen receptor polymorphism (CAG) repeat sequence without HpaII digestion, Figure 5 -B is the PCR amplification of the androgen receptor polymorphic (CAG) repeat sequence after HpaII digestion, Figure 5 -A and Figure 5-B shows that the subject is heterozygous for the F8 gene, inherited from both her parents. Figure 5 -A and Figure 5 -B test results show that the subject's X chromosome inactivation test is =55%.

[0091] 5. Gene mutation analysis Whole blood samples were used for whole exome sequencing of the subjects, which was completed by Shanghai Paisono Biotechnology Co., Ltd. using the Illumina NovaSeq platform. BGI purified and sequenced the PCR products, and the reactions were performed in both forward and reverse directions to confirm the presence of the mutation. The sequencing results were compared with the coding sequence (F8:NM_000132) and genomic sequence (NG_007994.1) in the NCBI database. The FVIII gene mutation database and the human gene mutation database were queried to confirm whether the mutation found was a new mutation.

[0092] Whole exome sequencing revealed two missense mutations in exons 7 and 14 of the subject's F8 gene, namely c.2162T>G, p.Met721Arg and c.847C>A, p.Pro283Thr. Subsequently, Sanger sequencing was performed on the DNA of the subject's F8 gene, as shown in Figure 2. Figure 6 The double peak graph corresponding to the mutant bases in the Sanger sequencing graph further confirmed the two missense mutations. In addition, no information about c.2162T>G, p.Met721Arg and c.847C>A, p.Pro283Thr was found in the FVIII gene mutation database and the human gene mutation database. These two missense mutations are new mutations. Therefore, the present application considers that the subject is a rare compound heterozygote causing female onset.

[0093] It should be noted that the website of the FVIII gene mutation database is https: / / f8-db.eahad.org / , and the website of the human gene mutation database is http: / / www.hgmd.cf.ac.uk / ac / index.php.

[0094] 6. Pathogenicity prediction PolyPhen2, SIFT, Mutation Taste and PROVEAN were used to predict the pathogenicity of FVIII-Met721Arg and FVIII-Pro283Thr. The results are shown in the following table:

[0095] It should be noted that the score range of PROVEAN is -14 to 14, and the threshold is -2.5. -14 to -2.5 is deleterious, and -2.5 to 14 is neutral. The smaller the score, the more harmful it is. The score range of SIFT is 0 to 1, and the threshold is 0.05. 0 to 0.05 is damaging, and 0.05 to 1 is harmless. The smaller the score, the more harmful it is. The prediction results of Mutation taster are divided into: harmful (Disease causing automatic), possibly harmful (Disease causing), possibly harmless (Polymorphism) and harmless (Polymorphism automatic). The integral range of Polyphen2 is 0-1. The closer the integral is to 1, the more it indicates that the mutation is likely to be harmful.

[0096] Therefore, it can be seen from the above table that FVIII-Met721Arg and FVIII-Pro283Thr are both pathogenic mutations.

[0097] It should be noted that the website of PolyPhen2 is http: / / genetics.bwh.harvard.edu / pph2 / , the website of SIFT is https: / / sift.bii.a-star.edu.sg / , and the website of Mutation Taster is https: / / www.mutationtaster.org / .

[0098] 7. Conservatism Analysis Unipro UGENE software was used to align homologous sequences from different species to assess the conservation of amino acid residues at different positions of FVIII protein. These species included chimpanzee, rhesus monkey, dog, house mouse, Norway rat, chicken, zebrafish and tropical clawed frog.

[0099] Analysis by Unipro UGENE software showed that the methionine at position 721 and the proline at position 283 of the FVIII protein are highly conserved in multiple species. For detailed results, see Figure 7 .

[0100] 8. Model Analysis First, the wild-type FVIII protein was modeled using the Swiss-model online tool, and then the amino acids were site-directed mutated using PyMOL software to display the amino acid structure before and after mutation and the surrounding hydrogen bonds and disulfide bonds. The amino acid structure of the mutant cell line before and after mutation is shown in the figure. Figure 8 As shown, Figure 8-A is the amino acid structure of FVIII- Met721Arg before mutation. The red part in the figure is methionine (M) at the 721st amino acid mutation site, and the yellow dotted line is the hydrogen bond; Figure 8 -B is the amino acid structure diagram of FVIII- Met721Arg after mutation. The red part in the figure is the arginine (R) at the 721st amino acid mutation site, and the yellow dotted line is the hydrogen bond; Figure 8 -C is the amino acid structure of FVIII- Pro283Thr before mutation. The red part in the figure is the proline (P) at the 283rd amino acid mutation site; Figure 8 -D is the amino acid structure diagram of FVIII-Pro283Thr after mutation. The red part in the figure is the threonine (T) at the 283rd amino acid mutation site.

[0101] Depend on Figure 8 -A and 8-B show that the mutation FVIII-Met721Arg changes the hydrophilicity of the region, that is, the hydrophobic amino acid (methionine, Met) is replaced by a polar positively charged hydrophilic amino acid (arginine, Arg). The surrounding hydrogen bonds have changed significantly, affecting the β-sheet structure of the FVIII protein and subsequently changing its spatial configuration. β-folding plays a role in protein folding, quaternary protein structure, protein interaction and protein aggregation. Figure 8 -C, 8-D show that the mutation FVIII-Pro283Thr changes the hydrophilicity of the region, that is, the hydrophobic amino acid (proline, Pro) is replaced by a polar, uncharged hydrophilic amino acid (threonine, Thr). The presence of proline in the peptide chain prevents helical extension and forms a helical structure of a proline knot. The mutation of FVIII-Pro283Thr changes the secondary structure of the protein FVIII. At the same time, in the study of the present application, both mutations showed reduced FVIII:C levels while maintaining normal FVIII:Ag levels. Thus, these findings suggest that mutations may destroy the spatial conformation of the FVIII protein, resulting in reduced FVIII activity.

[0102] In addition, the mature FVIII protein is composed of 2332 amino acids. Analysis of the human F8 gene revealed that the protein has a distinct domain structure, expressed as A1-A2-B-A3-C1-C2. Since the A region of the FVIII protein plays a vital role in several processes, such as promoting the binding of FVIII to vascular hematologic factor (vWF), stabilizing FVIII, promoting the inactivation of FVIII, mediating the binding of FVIII to factor IXa (FIXa), and participating in the circulation clearance of FVIII, etc. Therefore, the exon region where the two mutant cell strains are located obtained in the present application according to the Sanger sequencing map, that is, the mutant FVIII-Met721Arg is located at the 721 amino acid, and the mutant FVIII-Pro283Thr is located at the 283 amino acid. It can be seen that the change of the mutant FVIII-Met721Arg affects the domain A2, and the change of the mutant FVIII-Pro283Thr affects the domain A1.

[0103] It should be noted that exons are DNA sequences in genes that can encode proteins, and these sequences will be translated into proteins after being transcribed into mRNA. Domains are independently folded three-dimensional spatial structural units in protein molecules, and each of them performs a specific function. The formation of domains is based on the secondary structure or super secondary structure of proteins, and is a local folding region of a polypeptide chain at the tertiary structural level. Since exons encode proteins, mutations in exons may affect the structure and function of proteins. If a mutation occurs in the exon region of a coding domain, then this mutation may change the three-dimensional structure of the domain, thereby affecting its function, so in this application, the domain affected can be known based on the exon region where the two mutant cell strains are located.

[0104] This application is based on the subject, an extremely unusual female hemophilia patient, whose FVIII:C=7%<40%, and judged that she was a mild manifestation of hemophilia A. The subject was then subjected to whole exome sequencing to detect whether the subject's F8 gene had mutated. According to the whole exome sequencing results, two possible pathogenic gene mutations were found, namely mutation FVIII-Met721Arg and mutation FVIII-Pro283Thr. Therefore, it is considered that the subject is a rare compound heterozygote that causes female disease. Furthermore, due to the X-linked recessive inheritance of hemophilia, women rarely show symptoms of hemophilia. Therefore, in order to explore the cause of the subject's HA, the subject was tested for X chromosome inactivation in this application. According to the test results, the subject showed a 55% random X inactivation pattern (XCI). In addition, this application studied the pathogenic mechanism of these two mutations and found that both mutations did not affect transcription and secretion, but the mutation FVIII-Met721Arg seriously affected protein activity, and the mutation FVIII-Pro283Thr slightly affected protein activity. Considering that the variation of these two mutations in the A region domain may affect its physiological function by changing the spatial conformation of the FVIII protein, thereby having a differential effect on the protein activity, the cause of the mild phenotype (activity 7%) of female hemophilia can be known by combining the random X chromosome inactivation results and the pathogenic mechanism of the two mutations.

[0105] In summary, the discovery of two new mutations in this application not only enriches the F8 gene mutation database, but also clarifies the cause of female hemophilia by combining the molecular pathogenic mechanism of the two new mutations and the results of random X chromosome inactivation, provides genetic counseling for family planning of female hemophilia patients, and provides new strategies for the precise treatment of female hemophilia in the future.

[0106] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A cell line with two pathogenic missense mutations causing compound heterozygous female HA patients, characterized in that Including FVIII-Met721Arg mutant cell line and FVIII-Pro283Thr mutant cell line.

2. The cell line of claim 1 that causes two pathogenic missense mutations in compound heterozygous female HA patients, characterized in that: The base sequence of the FVIII-Met721Arg mutant cell line is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2; the base sequence of the FVIII-Pro283Thr mutant cell line is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO:

4.

3. A method for constructing a cell line with two pathogenic missense mutations that cause compound heterozygous female HA patients, characterized in that: The method for constructing the FVIII-Met721Arg / FVIII-Pro283Thr mutant cell line is specifically as follows: S11. Site-directed mutagenesis was performed on the BDD-FVIII wild-type plasmid to construct a plasmid expressing the FVIII-Met721Arg / FVIII-Pro283Thr cDNA sequence, wherein the mutation site of the FVIII- Met721Arg mutant cell line is c.2162T>G (p.Met721Arg) on ​​exon 14 of the F8 gene, and the mutation site of the FVIII- Pro283Thr mutant cell line is c.847C>A (p.Pro283Thr) on exon 7 of the F8 gene; S12. The FVIII-Met721Arg / FVIII-Pro283Thr plasmid containing the cDNA sequence was transfected into cells using liposomes, and the FVIII-Met721Arg / FVIII-Pro283Thr mutant cell line was obtained through drug screening.

4. The method for constructing a cell line with two pathogenic missense mutations that cause compound heterozygous female HA patients according to claim 3, characterized in that: The base sequence of the BDD-FVIII wild-type plasmid is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:

6.

5. The method for constructing a cell line with two pathogenic missense mutations that cause compound heterozygous female HA patients according to claim 4, characterized in that: The FVIII-Met721Arg plasmid containing the cDNA sequence was obtained by site-directed mutagenesis of the BDD-FVIII wild type based on the PCR method, and the FVIII-Pro283Thr plasmid containing the cDNA sequence was obtained by site-directed mutagenesis of the BDD-FVIII wild type based on the seamless cloning technology.

6. The method for constructing a cell line with two pathogenic missense mutations that cause compound heterozygous female HA patients according to claim 5, characterized in that: The FVIII-Met721Arg plasmid / FVIII-Pro283Thr plasmid containing the cDNA sequence is transfected into human embryonic kidney cells HEK293T by liposome transfection.

7. The method for constructing a cell line with two pathogenic missense mutations that cause compound heterozygous female HA patients according to claim 6, characterized in that: The drug screening is antibiotic screening, wherein the resistance drug is puromycin.

8. The method for constructing a cell line with two pathogenic missense mutations that cause compound heterozygous female HA patients according to claim 7, characterized in that: When constructing the FVIII-Met721Arg plasmid containing the cDNA sequence, the primers used for site-directed mutagenesis were: upstream primer 5′-tttcggaacagaggcaGgaccgccttac-3′, downstream primer 5′-Ctgcctctgttccgaaagtctgagttgt-3′; When constructing the FVIII-Pro283Thr plasmid containing the cDNA sequence, the primers used for site-directed mutagenesis were: upstream primer 5'-tctgattggatgccacagga-3', downstream primer 5'-gcgatggttcctcacaagaa-3'.

9. Use of a cell line with two pathogenic missense mutations resulting in compound heterozygous female HA patients according to any one of claims 1-2 or a cell line with two pathogenic missense mutations resulting in compound heterozygous female HA patients according to any one of claims 3-8 in the prevention, early diagnosis or treatment of female hemophilia.

Images