Human ghost protein mutant and application thereof

Abstract

The invention relates to a human ghost protein mutant. The human ghost protein mutant is mainly characterized in that a beta subunit of the mutant has a mutation site named D470G compared with a wild type amino acid sequence. In addition, the invention also relates to a nucleic acid molecule of the human ghost protein mutant, a biological material and application thereof. The single base mutation of the ghost protein beta subunit gene provided by the invention can effectively inhibit complement activation mediated red blood cell hemolysis. The hematopoietic stem cells are extracted, single base mutation is performed on the ghost protein beta subunit gene of the hematopoietic stem cells, and then the hematopoietic stem cells are transplanted back to the body, so that the hematopoietic stem cells are expected to be used for treating red blood cell hemolysis mediated by complement activation and related diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a human spectrin mutant and an application thereof. Background Art

[0002] Red blood cells, as an important component of the blood, undertake the important function of transporting oxygen and carbon dioxide. At the same time, they are also closely connected to the immune system. Under certain pathological conditions, the immune system may mistakenly identify red blood cells as foreign substances, activate the complement system, and lead to complement-mediated hemolysis of red blood cells. This hemolysis is associated with a variety of diseases, such as paroxysmal nocturnal hemoglobinuria (PNH) and autoimmune hemolytic anemia, which can cause a series of serious symptoms such as anemia, jaundice, and organ damage.

[0003] Although the molecular mechanism of complement-mediated erythrocyte hemolysis has been better understood in recent years, current treatments for related diseases still face challenges. The complement system is composed of more than 30 proteins, and its abnormal activation may lead to damage and lysis of erythrocyte membranes. Although complement inhibitors such as Eculizumab and Ultomiris have been used in clinical treatment, they reduce hemolysis by inhibiting complement components C5 or C3 proteins, but these inhibitors are not as effective as expected in clinical settings. This is mainly because these inhibitors mainly act on the extracellular signal transduction of erythrocytes, rather than directly targeting the final step of hemolysis. In addition, the complexity and diversity of complement activation pathways also pose challenges to treatment.

[0004] Researchers currently believe that the membrane attack complex (MAC) formed after complement activation will form holes in the erythrocyte membrane, leading to the loss of cell membrane integrity and ultimately causing rupture and death of erythrocytes. However, the processes that occur within erythrocytes after complement activation remain largely unknown. The erythrocyte membrane is composed of a phospholipid bilayer and cytoskeletal proteins arranged on the inner surface of the cytoplasm. Spectrin and actin are the most important proteins in the erythrocyte skeleton, which regulate the stability of the erythrocyte cell membrane and maintain its biconcave shape. When the stability and integrity of the cytoskeleton are impaired, mature erythrocytes are susceptible to damage and hemolysis occurs. Although spectrin may play an important role in the process of erythrocyte hemolysis, there are currently no reports of using spectrin as a target to inhibit complement-mediated erythrocyte hemolysis to treat related diseases. Summary of the invention

[0005] In order to solve the above problems in the prior art, the present invention provides a human spectrin mutant and its application, targeting the spectrin β subunit to treat complement activation-mediated erythrocyte hemolysis and related diseases.

[0006] In order to achieve the above-mentioned object, the first aspect of the present invention provides a human spectrin mutant, which mainly comprises a β subunit, and compared with the amino acid sequence of the wild-type human spectrin β subunit, the human spectrin mutant has a mutation site: D470G, and the amino acid sequence of the wild-type human spectrin β subunit is shown in SEQ ID NO. 1. The 1409th adenine base (A) of the human spectrin β subunit gene is mutated to a guanine base (G), that is, the 470th amino acid is replaced by aspartic acid (D) to glycine (G), and the mutation can prevent the spectrin β subunit from being lysed after the red blood cells are stimulated by complement activation, thereby inhibiting the hemolysis of the red blood cells; the mutation site of the mouse spectrin β subunit can achieve the effect of inhibiting the hemolysis of the mouse red blood cells after being stimulated by complement activation.

[0007] The second aspect of the present invention provides a nucleic acid molecule encoding the human spectrin mutant.

[0008] The third aspect of the present invention provides a biological material containing the nucleic acid molecule, the main feature of which is that the biological material is a recombinant DNA, a recombinant plasmid, a recombinant bacterium, an expression cassette, a transposon or a viral vector.

[0009] The fourth aspect of the present invention provides the use of the human spectrin mutant, the nucleic acid molecule, or the biological material in the preparation of a product that resists hemolysis of red blood cells.

[0010] Preferably, the erythrocyte hemolysis is complement-mediated erythrocyte hemolysis.

[0011] The human spectrin mutant and its encoding gene and application of the present invention can effectively inhibit complement activation-mediated erythrocyte hemolysis by targeting a single base mutation in the spectrin β subunit. By extracting hematopoietic stem cells, subjecting them to base mutations in the spectrin β subunit, and transplanting them back into the body, it is expected to be applied to the treatment of complement activation-mediated erythrocyte hemolysis and related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1a The blood smear microscopic examination result of the red blood cells in Example 1; Figure 1b This is the detection result of the free hemoglobin concentration in the supernatant in Example 1.

[0013] Figure 2 This is a graph showing the WB detection results of spectrin α subunit and β subunit after complement activation-mediated erythrocyte hemolysis in Example 2.

[0014] Figure 3 These are 2D and 3D SIM images of spectrin β subunit and actin after complement activation-mediated erythrocyte hemolysis in Example 3.

[0015] Figure 4This is the map of the working plasmid HY100-ABE-P2A-mcherry in Example 4.

[0016] Figure 5a This is a graph showing the results of free hemoglobin concentration detection in the supernatant of red blood cells with spectrin β subunit mutation after complement stimulation in Example 6; Figure 5b This is a 2D SIM image of the spectrin β subunit and actin in the erythrocytes with spectrin β subunit mutation in Example 6 after complement stimulation.

[0017] Figure 6 This is a diagram showing the expression of cell surface CD45.1 and CD45.2 molecular markers detected by flow cytometry.

[0018] Figure 7a 7a is a graph showing the results of blood smear microscopy of red blood cells of mice transplanted with mutant HSPCs in Example 9 subjected to a complement activation-mediated hemolysis experiment; 7b is a graph showing the results of the corresponding supernatant free hemoglobin concentration test. DETAILED DESCRIPTION

[0020] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0021] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, materials, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. already available in the prior art.

[0022] Example 1

[0023] Induction of complement activation-mediated hemolysis of erythrocytes in vitro

[0024] Whole blood samples from healthy people with blood type A were collected, anticoagulants were added, leukocytes were removed by filtration, centrifuged at 1000×g, 4°C for 10 min, the supernatant was removed, and the corresponding red blood cells were washed 3 times with saline to obtain the corresponding red blood cells, which were resuspended in saline to a 25% red blood cell suspension. The red blood cell suspension was then mixed with 1 / 5 volume of anti-A antibody and 3 / 2 volume of serum complement, and a negative control group (Control) with the same volume of saline was set up and incubated at 37°C for 30 min.

[0025] The red blood cell pellets were collected at 1000 × g and centrifuged at 4 °C for 10 min, and the red blood cell suspensions were resuspended with physiological saline. 10 μl of the red blood cell suspension was taken to prepare blood smears. The red blood cell morphology was observed under a microscope after staining with Wright-Gissam stain. Figure 1a As shown, most of the red blood cells in the complement activated group were deformed or even destroyed.

[0026] Take the supernatant after centrifugation of each group and use the free hemoglobin detection kit to detect the concentration of free hemoglobin. Figure 1b As shown in the figure, the concentration of free hemoglobin in the supernatant of the complement activated group was significantly increased compared with the control group, indicating that activated complement mediated hemolysis of erythrocytes.

[0027] Example 2

[0028] Cleavage of spectrin β subunit during complement activation-mediated erythrocyte hemolysis

[0029] Proteomic analysis revealed that the expression of multiple proteins in erythrocytes was significantly upregulated or downregulated under complement activation, including important proteins involved in biological processes related to erythrocyte skeleton organization and actin fiber assembly. Given that spectrin is widely believed to play a vital role in providing mechanical support for mammalian erythrocyte lipid membranes, the present invention identified the cleavage of spectrin during complement activation-mediated erythrocyte hemolysis.

[0030] Preparation of total red blood cell protein

[0031] According to the method described in Example 1, a control group and a complement activation group were set up, and red blood cell hemolysis was induced. After that, red blood cells of each group were collected by centrifugation, and protein lysis buffer RIPA containing protease inhibitor PMSF was added, mixed and incubated on ice for 30 minutes. After centrifugation at 12000×g and 4℃ for 20 minutes, the supernatant was aspirated and the protein concentration was detected using a BCA protein quantification kit. The protein sample was mixed with 5× Loading Buffer, boiled in boiling water for 10 minutes, and immediately loaded or stored in a -20℃ refrigerator.

[0032] SDS-PAGE protein electrophoresis

[0033] Prepare SDS-PAGE gel with a separation gel concentration of 6%, put the gel into an electrophoresis tank, add electrophoresis buffer, add each group of protein samples and protein markers into the gel wells, and perform electrophoresis at a constant voltage of 100V for the concentrated gel and 180V for the separation gel.

[0034] Electroblotting

[0035] Cut a PVDF membrane of appropriate size and soak it in methanol for 5 minutes. At the same time, cut a filter paper of appropriate size and soak it in 1× electrotransfer buffer. Take out the gel after electrophoresis and balance it in 1× electrotransfer buffer. Then assemble the transfer device in the order of sponge-2 layers of filter paper-protein gel-PVDF membrane-2 layers of filter paper-sponge (negative electrode to positive electrode of splint) to ensure that there are no bubbles. Put the transfer device and ice pack in the electrotransfer tank, pour 1× electrotransfer buffer into the electrotransfer tank, and transfer the membrane in 200mA constant current mode for 3 hours.

[0036] Antibody incubation

[0037] After electrotransfer, the PVDF membrane was removed and placed in a blocking solution containing 5% bovine serum albumin and incubated at room temperature for 2 hours; the blocked PVDF membrane was transferred to the diluted primary antibody and incubated at 4°C overnight; the PVDF membrane was removed from the primary antibody solution, rinsed three times with PBST solution, 10 minutes each time, and then transferred to the diluted secondary antibody and incubated on a shaker at room temperature for 1 hour; the PVDF membrane was removed from the secondary antibody solution, rinsed three times with PBST solution, 10 minutes each time.

[0038] Imaging

[0039] Mix equal volumes of developer A and B, drop the color mixture onto the surface of the dried PVDF membrane, place it in a fully automatic chemiluminescence imaging system, collect images, and identify and analyze the expression of spectrin α and β subunits.

[0040] like Figure 2 As shown in the figure, the spectrin β subunit in the complement activation group showed obvious cleavage, while the spectrin α subunit did not change significantly. This indicates that the spectrin β subunit will be cleaved during the complement activation-mediated erythrocyte hemolysis, which may directly lead to damage to the erythrocyte skeleton and induce hemolysis.

[0041] Example 3

[0042] Distribution of spectrin β-subunit and actin during complement activation-mediated hemolysis of erythrocytes

[0043] According to the method described in Example 1, a control group and a complement activation group were set up, and an erythrocyte hemolysis induction experiment was performed. The erythrocytes of each group were collected, fixed with 0.05% glutaraldehyde for 15 minutes, washed with PBS, and then 0.1% Triton X-100 was added and permeabilized for 15 minutes. Wash with PBS 3 times, add a solution containing 5% goat serum and 1% bovine serum to block for 1 hour. Then, antibodies corresponding to the β subunit of spectrin or actin were added to the blocking solution and incubated at 4°C overnight. After centrifugation, the supernatant was discarded, a fluorescently coupled secondary antibody was added, and the mixture was placed on a shaker and incubated in the dark for 1 hour. The prepared cells were then observed and photographed using a confocal microscope and a high-resolution microscope, and a structured illumination microscope (SIM) reconstruction was created using Zeiss Elyra Zen software and automatic settings.

[0044] like Figure 3 As shown in the figure, in the control group, the distribution of spectrin β subunit and actin inside or on the surface of erythrocytes was uniform, while in the complement activated group, the distribution of spectrin β subunit and actin was obviously uneven and disordered. This shows that complement activation leads to the destruction of the erythrocyte membrane skeleton during hemolysis.

[0045] Example 4

[0046] Obtaining human hematopoietic stem cells and single base mutation

[0047] Isolation of CD34 from healthy human peripheral blood + Hematopoietic stem cells

[0048] Peripheral blood was collected from healthy individuals, red blood cell lysis buffer was added, mixed and incubated for 10 minutes; the unlysed cells were suspended in MACS buffer and digested with collagenase IV and DNase at 37°C for 40 minutes; the cells were then incubated with anti-CD34 antibody-coupled microbeads at 4°C for 30 minutes, and hematopoietic stem cells (HSPCs) were isolated on the AutoMacs device. The isolated HSPCs were cultured in Stemspan supplemented with CC100 cytokine mixture. TM The cells were expanded in vitro in SFEMII medium.

[0049] Gene editing of human HSPCs

[0050] The mRNA sequence of human erythrocyte spectrin beta subunit (SPTB) was downloaded from the NCBI website (accession number NM_001355436.2), and a single base mutation target was designed at the cleavage motif. The sgRNA target sequence and PAM sequence were: 5'-GACCGACACGGCTGCCTACG AGG-3'. The corresponding fitting primer sequences were designed according to the BsaI restriction endonuclease site of the pGL3-U6-sgRNA-EGFP plasmid, which were 5'-ACCGGACCGACACGGCTGCCTACG-3' and 5'-AAACCGTAGGCAGCCGTGTCGGTC-3', respectively. The designed primer sequence was sent to a biological company for synthesis.

[0051] The pGL3-U6-sgRNA-EGFP plasmid was digested with BsaI restriction endonuclease, and 1% agarose gel electrophoresis was performed after digestion, and the digestion product was obtained by gel recovery. Take the above-synthesized primers and anneal to obtain a double-stranded DNA molecule with sticky ends. Prepare a connection system for the digested plasmid and the annealed product, and incubate with T4 DNA ligase at 16°C for 1h to obtain the connection product. Take 10ul of the connection product to transform DH5α Escherichia coli competent cells, and obtain recombinant Escherichia coli containing the recombinant plasmid pGL3-U6-sgRNA (hSPTB)-EGFP after colony verification. The recombinant bacteria were propagated in LB medium containing ampicillin antibiotics, and the recombinant plasmid was extracted and stored at -20°C.

[0052] Take an appropriate amount of amplified HSPCs and transfer the pGL3-U6-sgRNA (hSPTB)-EGFP recombinant plasmid and the ABE base editor working plasmid HY100-ABE-P2A-mcherry (containing adenine deaminase and Cas9 (D10A) protein and carrying mcherry protein) into the culture medium. Figure 4 ) HSPCs were co-transfected by electroporation, and then the cell genome was extracted for PCR sequencing to verify that the adenine base (A) at the 6th position of the target sequence corresponding to the sgRNA was mutated to a guanine base (G), that is, a nucleotide mutation (A1409G) was inserted into the SPTB gene, resulting in an amino acid change (D470G). The remaining cells were sorted by flow cytometry, and single cells were seeded into well plates to obtain monoclones, cultured monoclones, and genomic DNA was extracted for PCR sequencing to obtain HSPCs with correct SPTB mutations.

[0053] Example 5

[0054] In vitro erythroid differentiation of HSPCs

[0055] The SPTB mutant HSPCs and wild-type HSPCs in Example 4 were respectively cultured for in vitro differentiation of erythrocytes: on days 0-7, the cells were cultured in serum-free expansion medium II, the medium was supplemented with 1uM dexamethasone, 0.5% penicillin / streptomycin, 100ng / ml stem cell factor, 5ng / ml interleukin 3, and 4U / ml erythropoietin; on days 7-12, dexamethasone and interleukin 3 were omitted from the above medium; on days 12-17, the cells were cultured at 10 6 The cells were cultured at a density of 100 cells / ml, dexamethasone, stem cell factor and interleukin 3 were omitted from the culture medium, and 1 mg / ml human transferrin, 20% fetal bovine serum and 10% human serum were supplemented. The cells were taken to prepare smears and the maturation of red blood cells was observed after staining.

[0056] Example 6

[0057] Hemolysis experiment of gene mutation erythrocytes differentiated in vitro

[0058] The gene mutant and wild-type erythrocytes differentiated in vitro in Example 5 were taken, and the erythrocyte hemolysis induction experiment was carried out according to Example 1. After centrifugation, the supernatant was taken and the concentration of free hemoglobin therein was detected using a free hemoglobin detection kit. Figure 5a As shown, compared with wild-type erythrocytes, the concentration of free hemoglobin in the supernatant of erythrocytes with mutant spectrin β subunit was significantly reduced after complement stimulation, approaching the concentration of free hemoglobin in the supernatant of the negative control group.

[0059] Take each group of red blood cells and observe the distribution of actin and spectrin β subunit in the red blood cells according to Example 3. Figure 5b As shown, after complement stimulation, the distribution structure of actin and spectrin β subunit inside and on the surface of wild-type erythrocytes was destroyed, and the cells deformed and ruptured. However, after complement stimulation, the distribution of actin and spectrin β subunit inside and on the surface of erythrocytes with spectrin β subunit mutations was well maintained, and the erythrocytes did not undergo obvious deformation.

[0060] This indicates that the mutation of the spectrin β subunit gene site selected by the present invention can effectively inhibit complement activation-mediated erythrocyte hemolysis.

[0061] Example 7

[0062] Obtaining hematopoietic stem cells from mouse bone marrow

[0063] CD45.1 C57BL / 6 mice (8 weeks old) were killed by dislocation, the lower limbs of the mice were cut, the muscles of the lower limbs were stripped, the tibia and femur were removed and placed in a culture dish containing 75% alcohol, and the bone marrow cavity was flushed with pre-cooled PBS using an injection needle to remove bone marrow cells; centrifuged at 4300rpm, 4℃ for 5min, the supernatant was discarded, red blood cell lysis solution was added, incubated for 10min, an appropriate amount of PBS was added, and the mixture was filtered through a cell sieve to obtain a mouse bone marrow cell suspension, and CD45.1 HSPCs were isolated by magnetic bead sorting technology. The isolated HSPCs were cultured in Stemspan supplemented with CC100 cytokine mixture. TM The cells were expanded in vitro in SFEMII medium.

[0064] Example 8

[0065] Gene editing of mouse CD45.1 HSPCs

[0066] The mRNA sequence of mouse erythrocyte spectrin β subunit (Sptb) was downloaded from the NCBI website (accession number NM_013675.3), and a single base mutation target was designed at the cleavage motif. The sgRNA target sequence and PAM sequence were: 5'-GACCGACACAGCTGCCTATG AGG-3'. The corresponding fitting primer sequences were designed according to the BsaI restriction endonuclease site of the pGL3-U6-sgRNA-EGFP plasmid, which were 5'-ACCGGACCGACACAGCTGCCTATG-3' and 5'-AAACCATAGGCAGCTGTGTCGGTC-3', respectively. The designed primer sequence was sent to a biological company for synthesis.

[0067] The pGL3-U6-sgRNA-EGFP plasmid was digested with BsaI restriction endonuclease, and 1% agarose gel electrophoresis was performed after digestion, and the digestion product was obtained by gel recovery. Take the above-synthesized primers and anneal to obtain a double-stranded DNA molecule with sticky ends. Prepare a connection system for the digested plasmid and the annealed product, and incubate with T4 DNA ligase at 16°C for 1h to obtain the connection product. Take 10ul of the connection product to transform DH5α Escherichia coli competent cells, and obtain recombinant Escherichia coli containing the recombinant plasmid pGL3-U6-sgRNA (mSptb) -EGFP after colony verification. The recombinant bacteria were cultured in LB medium containing ampicillin antibiotics, and the recombinant plasmid was extracted and stored at -20°C.

[0068] Take the mouse CD45.1 HSPCs after appropriate amplification in Example 7, co-transform the pGL3-U6-sgRNA (mSptb) -EGFP recombinant plasmid and the ABE base editor working plasmid HY100-ABE-P2A-mcherry into HSPCs cells by electroporation, and then extract the cell genome for PCR sequencing identification to verify that the adenine base (A) at the 6th position of the target sequence corresponding to the sgRNA is mutated to a guanine base (G). The remaining cells are sorted by flow cytometry, and single cells are planted in a well plate to obtain a monoclone, the monoclone is cultured, and the genomic DNA is extracted for PCR sequencing identification to obtain CD45.1 HSPCs with correct SPTB mutations.

[0069] Example 9

[0070] Transplantation of mutant CD45.1 HSPCs into recipient mice

[0071] Male CD45.2 C57BL / 6 mice were used as recipient mice. When they were 10-12 weeks old, they were given 7.0 Gy of 60 After Co γ-ray irradiation, mice were transplanted with CD45.1 HSPCs mutated in Example 8 by tail vein injection.

[0072] One month after transplantation, peripheral blood was collected from the tail vein of mice, erythrocytes were lysed with erythrocyte lysis buffer, labeled with fluorescent antibodies, and incubated at 4°C for 30 minutes. After antibody labeling, the cells were washed with PBS, centrifuged, and detected by flow cytometry.

[0073] like Figure 6 As shown, most of the original hematopoietic stem cells of the recipient mice were replaced by mutant CD45.1 cells.

[0074] Extraction of red blood cells from mice transplanted with mutant HSPCs for complement activation-mediated hemolysis experiments

[0075] One month after transplantation, peripheral blood was collected from mice transplanted with mutant HSPCs and wild-type mice, and a complement activation-mediated hemolysis experiment was performed according to Example 1. Red blood cells of each group were collected after centrifugation, blood smears were prepared, and the morphology of red blood cells was observed after staining.

[0076] like Figure 7a As shown in Figure 2, after complement stimulation, most wild-type mouse red blood cells ruptured and hemolyzed to form ghost red blood cells (RBCs ghosts), while a considerable number of mutant mouse red blood cells maintained cell integrity and did not undergo hemolysis. The supernatant after centrifugation was used to detect the concentration of free hemoglobin, such as Figure 7b As shown, after complement stimulation, the free hemoglobin concentration in the mutant mouse red blood cell group was significantly decreased compared with the free hemoglobin concentration in the wild-type mouse red blood cell group.

[0077] Therefore, the transplanted HSPCs with spectrin β subunit base mutation provided by the present invention can resist the red blood cell hemolysis process mediated by complement activation to a certain extent.

[0078] In summary, the single base mutation of the spectrin β subunit provided by the present invention can effectively inhibit complement activation-mediated erythrocyte hemolysis. By extracting hematopoietic stem cells, performing base mutation on the spectrin β subunit, and transplanting them back into the body, it is expected to be applied to the treatment of complement activation-mediated erythrocyte hemolysis and related diseases.

[0079] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the specification and drawings should be regarded as illustrative rather than restrictive.

Claims

1. A human spectrin mutant, characterized in that: It comprises a beta subunit. Compared with the amino acid sequence of the wild-type human spectrin beta subunit, the human spectrin mutant has a mutation site: D470G.

2. A nucleic acid molecule encoding the human spectrin mutant according to claim 1.

3. A biological material containing the nucleic acid molecule according to claim 2, characterized in that: The biological material is recombinant DNA, recombinant plasmid, recombinant bacteria, expression box, transposon or virus vector.

4. Use of the human spectrin mutant according to claim 1, the nucleic acid molecule according to claim 2, or the biomaterial according to claim 3 in the preparation of a product that resists hemolysis of red blood cells.

5. The use according to claim 4, characterized in that: The erythrocyte hemolysis is complement-mediated erythrocyte hemolysis.

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