A membrane-like system containing CD133 and a preparation method thereof

Abstract

The present invention discloses a membrane-like system containing CD133 and a preparation method thereof, and relates to the field of biotechnology. The membrane-like system comprises combining CD133 protein, membrane skeleton protein and phospholipid bilayer, and assembling these three components to form a small disc of a membrane-like structure, i.e., a nanodisc containing CD133 protein, wherein the transmembrane region of the CD133 protein is wrapped by the phospholipid bilayer and runs through the entire phospholipid bilayer, wherein the extracellular region is on one side of the phospholipid bilayer of the nanodisc, and the intracellular region is on the other side. The nanodisc has high stability, and this structure provides a cell-like environment for the CD133 protein to maintain its functional state, and since the extracellular region of CD133 is exposed to the outside, it does not affect its binding with anti-CD133 antibodies.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a membrane-like system containing CD133 and a preparation method thereof. Background Art

[0002] The main membrane-like environments at present are: bicelles, micelles, liposomes and nanodiscs. Among them, Nanodiscs are mainly composed of membrane scaffold proteins (MSPs) and phospholipids. Purified membrane proteins are incubated with Nanodiscs under appropriate conditions, which can enable the membrane proteins to be correctly embedded in the phospholipid bilayer of Nanodiscs, thereby maintaining the natural conformation and function of membrane proteins, providing an ideal platform for the study of membrane proteins.

[0003] CD133 is a 5-transmembrane protein with a total length of 865 amino acids and a molecular weight of about 120kD. CD133 contains two large glycosylated extracellular loops and two smaller intracellular loops, which are specifically localized in plasma membrane protrusions and microvilli. It is of great significance to identify and find new tumor stem cell-related targets and signaling pathways through CD133. CD133 is a tumor marker, and its main function is to induce the proliferation and metastasis of endothelial cells by regulating vascular-related proteins and paracrine methods. Studies have found that high expression of CD133 is strongly correlated with the occurrence and development of various cancers, including glioma, liver cancer, lung cancer, gastric cancer, breast cancer and colon cancer. This situation may be related to the role of the protein in cell differentiation, proliferation and apoptosis. In the tumor and immune microenvironment, CD133 regulates biological processes such as tumor blood vessels, hypoxia, EMT, and immune escape by activating multiple signaling pathways and mediating multiple transcriptional protein factors, thereby promoting the occurrence and development of tumors. Therefore, studying the signaling pathways, mechanisms of action and principles of CD133 is of great significance for related disease research and drug development.

[0004] In vitro studies on CD133 are still limited to the study of single domains of CD133, and the function of CD133 has not been studied at the overall structural level. This is because it is difficult to obtain CD133 with complete structure and correct folding in vitro. Therefore, if CD133 protein that can effectively mimic the natural structure can be constructed in vitro, it will have far-reaching significance for the true and accurate study of CD133 function, the preparation of CD133 antibodies, and the screening and modification of CD133 inhibitors.

[0005] Although there are some public studies on membrane-like systems, such as CN 107090030 A, which discloses a membrane-like system containing FGFR, a phospholipid bilayer and a membrane skeleton protein MSP and a preparation method thereof; however, different membrane proteins, the materials required for assembly, and the conditions during the assembly process such as temperature, time, and the concentration ratio of protein and Nanodisc all need to be finely adjusted to achieve successful assembly; and it is also unclear how to effectively improve the assembly efficiency.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a membrane-like system containing CD133 and a preparation method thereof.

[0008] The present invention is achieved in that:

[0009] In a first aspect, an embodiment of the present invention provides a method for preparing a membrane-like system, which comprises the following steps:

[0010] (1) mixing a CD133 protein solution, a membrane skeleton protein solution, a phospholipid mother solution and a detergent;

[0011] (2) Remove detergent;

[0012] (3) purifying to obtain a membrane-like system containing CD133;

[0013] The CD133 protein solution contains CD133 protein, the membrane skeleton protein solution contains membrane skeleton protein (MSP), and the phospholipid mother solution contains phospholipid; the molar ratio of the CD133 protein, the membrane skeleton protein, the phospholipid and the detergent is 1:5:50-100:100-200.

[0014] The CD133 protein solution, membrane skeleton protein solution, phospholipid mother solution and detergent are fully mixed to form a nanodisc containing CD133 protein. MSP is an amphiphilic protein that can wrap phospholipids to form a nanoscale disc-shaped phospholipid bilayer. After mixing with the CD133 protein and the phospholipid bilayer, the CD133 protein migrates into it to form a nanodisc embedded with CD133 protein (CD133-Nanodisc). The extracellular region of CD133 is on one side of the phospholipid bilayer of the nanodisc, and the intracellular region of CD133 is on the other side of the phospholipid bilayer of the nanodisc. The transmembrane region of CD133 is wrapped by a phosphate bilayer and runs through the entire phospholipid bilayer. This type of membrane system provides a cell-like environment for CD133, helping CD133 maintain an active conformation. Since the extracellular region of CD133 is exposed to the outside, it does not affect its binding with anti-CD133 antibodies.

[0015] In some embodiments, the molar ratio of the feed is any one of 1:5:50:100, 1:5:75:100, 1:5:100:100, 1:5:50:125, 1:5:75:125, 1:5:100:125, 1:5:50:150, 1:5:75:150, 1:5:100:150, 1:5:50:175, 1:5:75:175, 1:5:100:175, 1:5:50:200, 1:5:75:200, and 1:5:100:200, or a range between any two of them.

[0016] In some embodiments, the phospholipids include any one or more of DPPC, DOPE, DMPC, POPC, DOPC, DMPG, and DOPG.

[0017] In some embodiments, the phospholipid comprises DPPC.

[0018] In some embodiments, the phospholipids include a combination of DPPC and DOPE. Compared with a single phospholipid solution, the combination of DPPC and DOPE can significantly shorten the self-assembly time of the nanodisc and improve the assembly efficiency.

[0019] In some embodiments, in the combination of DPPC and DOPE, the molar ratio of DPPC to DOPE is 1:2 to 3. The molar ratio can be any one of 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, or a range between any two thereof.

[0020] In some embodiments, the amino acid sequence of the CD133 protein includes the sequence shown in SEQ ID NO: 1, or has at least 60% identity with the sequence shown in SEQ ID NO: 1 while retaining the transmembrane region sequence in the sequence shown in SEQ ID NO: 1.

[0021] The at least 60% identity includes any one of 60%, 70%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100% or a range between any two of them.

[0022] The term "identity" used herein refers to the fact that the nucleotide or amino acid residues in two sequences are exactly the same at the same position.

[0023] In some embodiments, the amino acid sequence of the CD133 protein has at least 80% identity with the sequence shown in SEQ ID NO: 1 while retaining the transmembrane region sequence in the sequence shown in SEQ ID NO: 1.

[0024] In some embodiments, the membrane skeleton protein comprises: a derivative of apolipoprotein A1 or a truncated form of apolipoprotein A1.

[0025] In some embodiments, the amino acid sequence of the membrane skeleton protein includes the sequence shown in SEQ ID NO:2 or has at least 80% identity with the sequence shown in SEQ ID NO:2.

[0026] The at least 80% identity includes any one of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100% or a range between any two of them.

[0027] In some embodiments, the detergent comprises sodium cholate solution.

[0028] In some embodiments, the mixing includes performing a first incubation, and the conditions of the first incubation include: 40-42° C., 1-3 h.

[0029] In some embodiments, the temperature of the first incubation can be any one of 40, 41, or 42° C., or a range between any two of them; the time of the first incubation can be any one of 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, or 3 h, or a range between any two of them.

[0030] In some embodiments, step (2) includes: adding a hydrophobic adsorbent to the mixed product for a second incubation. The hydrophobic adsorbent is used to remove excess detergent. During the incubation process, the detergent is adsorbed by the hydrophobic adsorbent, and then the hydrophobic adsorbent is removed by filtration, and the filtrate is recovered, thereby achieving separation efficiency. The residual detergent content does not need to be detected. If the detergent is not effectively removed, the assembly cannot be successful.

[0031] In some embodiments, the conditions of the second incubation include: an incubation temperature of 40-42°C and a time of ≥3-6 hours. In some embodiments, the temperature of the first incubation can be any one of 40, 41, 42°C or a range between any two thereof; the time of the first incubation can be any one of ≥3, 3.5, 4, 4.5, 5, 5.5, 6 hours or a range between any two thereof.

[0032] In some embodiments, the hydrophobic adsorbent comprises XAD-2.

[0033] In some embodiments, the step (3) comprises: filtering to remove the hydrophobic adsorbent, recovering the liquid for chromatography, and separating to obtain a membrane-like system containing a phospholipid bilayer of CD133.

[0034] In a second aspect, an embodiment of the present invention provides a membrane-like system containing CD133 prepared according to the preparation method described in any of the preceding embodiments.

[0035] In a third aspect, an embodiment of the present invention further provides a use of a membrane-like system containing CD133 according to any of the foregoing embodiments, comprising at least one of the following (I) to (IV):

[0036] (I) preparing antibodies against CD133;

[0037] (II) Screening of CD133 inhibitors;

[0038] (III) preparing a reagent or kit for detecting CD133 autoantibodies;

[0039] (IV) Detection of CD133 autoantibodies for non-therapeutic or diagnostic purposes.

[0040] The present invention has the following beneficial effects:

[0041] The embodiments of the present invention combine CD133 protein, membrane skeleton protein and phospholipid bilayer to form a small disc of membrane-like structure, i.e., a nanodisc containing CD133 protein, wherein the transmembrane region of CD133 protein is wrapped by the phospholipid bilayer and runs through the entire phospholipid bilayer, wherein the extracellular region is on one side of the phospholipid bilayer of the nanodisc and the intracellular region is on the other side. The nanodisc containing CD133 protein has high stability, and this structure can maintain the natural membrane environment of CD133 protein and its functional state, and since the extracellular region of CD133 is exposed to the outside, it does not affect its binding with anti-CD133 antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0043] Figure 1 This is the SDS-PAGE gel electrophoresis diagram of CD133 protein;

[0044] Figure 2 This is the SDS-PAGE gel electrophoresis diagram of MSP protein;

[0045] Figure 3 is the SDS-PAGE gel electrophoresis image of the CD133-nanodisc of Example 1;

[0046] Figure 4is the SDS-PAGE gel electrophoresis image of the CD133-nanodisc of Comparative Example 1;

[0047] Figure 5 This is a WB image of the CD133-nanodisc and antibody after binding in Example 1;

[0048] Figure 6 These are the test results of the products of Examples 7 and 8 and Comparative Example 3 assembled for 3 hours. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0050] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0051] Example 1

[0052] This embodiment provides a method for preparing CD133-nanodiscs, comprising the following steps.

[0053] 1. Expression and purification of CD133 protein

[0054] The pcDNA3.1 eukaryotic expression vector containing the full-length sequence of CD133 (SEQ ID NO.1) was synthesized.

[0055] Take 100ng of pcDNA3.1-CD133 synthesis product and add it to 100μL TOP10 competent cells, incubate on ice for 30min, heat shock at 42℃ for 60s, incubate on ice for 2min, add 200mL LB medium (1% peptone, 0.5% yeast powder, 1% NaCl2), and shake culture at 37℃ for 16h. The bacterial solution is used to extract plasmids.

[0056] 300 mL of a liquid with a density of 2×10 6Add 293F cells to a 1L culture flask. Take 300mL of 150mM sodium chloride pH=7.4 buffer, add 300μg of plasmid, mix well and let stand for 15min; then slowly add it dropwise into the culture flask, continue to culture for 72h, and collect the cells. Resuspend the collected 293F cells with lysis buffer, and add 10mL of cell lysis buffer (20mM Tris, 100mM NaCl, 0.5% Triton, 1% DDM, pH7.2) to 1g of cells (wet weight). After lysis for 1h, ultrasonically disrupt at 300W for 20min, centrifuge using an ultracentrifuge, centrifuge at 40000g for 1h, and collect the supernatant. The nickel column was equilibrated with equilibration buffer (20mM Tris, 100mM NaCl, 0.5% Triton, 0.1% DDM), the supernatant was incubated with the nickel filler for 2h, then passed through the column, and the impurities were eluted with elution buffer (20mM Tris, 100mM NaCl, 0.5% Triton, 50mM imidazole), and finally CD133 was eluted with elution buffer (20mMTris, 100mM NaCl, 0.5% Triton, 300mM imidazole) and identified by SDS-PAGE gel electrophoresis.

[0057] The results are as follows Figure 1 The molecular weight of the purified CD133 band in gel electrophoresis is about 120 kD, which is consistent with the theoretical molecular weight.

[0058] 2. Expression and purification of MSP1D1 protein

[0059] The pET-28a+ expression vector containing the full-length sequence of MSP1D1 (SEQ ID NO. 2) was synthesized.

[0060] Transformation was performed, and plasmids were extracted from the correct clones. The extracted pET-28a-MSP1D1 plasmid was transformed into Escherichia coli BL21 (DE3) to obtain positive single clones with kanamycin resistance, which were stored in a -80°C ultra-low temperature refrigerator as expression strains.

[0061] (1) Take out the MSP1D1 strain stored at -80°C and place it on ice. Take out 200 μL and inoculate it into a test tube containing 5 mL LB medium. Add kanamycin with a final concentration of 50 μg and culture at 37°C overnight.

[0062] (2) Transfer to 500 mL LB medium at a ratio of 1:100 (5 mL) and add kanamycin at a final concentration of 50 μg;

[0063] (3) Cultivate at 37°C until OD 600When the p value reaches between 0.6 and 0.8, add 500 μL IPTG (final concentration is 1 mM), induce expression at 25°C for 3.5-4 h, and collect the cells by centrifugation;

[0064] (4) Ultrasonic disruption: The cells were resuspended in 20 mM Tris, 100 mM NaCl, and 0.5% Triton buffer, placed in an ice bath, and the ultrasonic disruptor was set to work for 3 s, rest for 3 s, and last for 5 min, and repeated three times; the cell disruption solution was then centrifuged at 12,000 g for 30 min; the supernatant was removed and stored at 4°C;

[0065] (5) Ni-NTA affinity column purification: The supernatant after ultrasonic crushing and centrifugation was mixed with an appropriate amount of nickel filler and allowed to bind at 4°C for more than 2 h; after binding, the sample was slowly loaded and the effluent was collected; after loading, two column volumes were washed with equilibrium buffer to collect the impurity protein peaks that were not attached to the column; then, the gradient buffer with increasing imidazole concentration was used for elution, and each protein peak was collected; 20 μL was taken out from each elution peak receiving tube for SDS-PAGE detection. The results are shown in Figure 2 , and the rest were stored at 4°C;

[0066] (6) The purified MSP1D1 was mixed with TEV enzyme (40 μg TEV enzyme was added to 1 mg MSP1D1), incubated at room temperature for 2 h, and then purified by Ni-NTA affinity column. The flow-through was collected to obtain MSP1D1 without His tag.

[0067] (7) Concentration: According to the SDS-PAGE test results, the elution peak receiving tube was concentrated using an ultrafiltration concentrator tube and centrifuged at 3000 rpm at 4°C until it was concentrated to about 5 mL. A was measured by UV spectrophotometer. 280 The absorbance value can be calculated to obtain the protein concentration.

[0068] 3. Preparation of Phospholipid Stock Solution

[0069] The phospholipids purchased in this experiment are all powders, and the phospholipid mother solution is commonly used in the assembly (the phospholipids used must first be dissolved in sodium cholate solution to obtain the phospholipid mother solution). The preparation process of the phospholipid mother solution is as follows:

[0070] (1) Weigh the required phospholipid powder, dissolve it in chloroform solution, and blow dry it with nitrogen slowly to make it spread on the bottom of the container to form a transparent or white film. In this example, 30 mg of DPPC is weighed;

[0071] (2) The container is sealed with a sealing film with a few air holes left, and then placed in a freeze-drying bottle and freeze-dried in a vacuum freeze dryer for 3.5 hours to remove residual chloroform;

[0072] (3) Add an appropriate amount of pre-prepared sodium cholate solution (usually the molar concentration of sodium cholate is twice the concentration of phospholipids) to the freeze-dried phospholipid film, then mix at low speed on a rotary shaker overnight to obtain the phospholipid mother solution, and then add 1 mL of 80 mM sodium cholate solution (20 mM Tris, 100 mM NaCl, pH 7.2);

[0073] (4) The mixed phospholipid stock solution can be stored at -80℃ for a long time.

[0074] 4. Assembly of CD133 Nanodiscs

[0075] (1) The purified CD133 protein, MSP1D1 protein, phospholipid mother solution and detergent were mixed in a certain ratio and incubated at 42° C. for 2 h. In this embodiment, the detergent was sodium cholate solution, and the molar ratio of CD133 protein: MSP1D1 protein: phospholipid: sodium cholate was 1:5:100:200.

[0076] (2) After the first incubation, an appropriate amount of hydrophobic adsorbent (XAD-2) was added to remove the sodium cholate molecules. After the addition of the hydrophobic adsorbent, the second incubation was carried out at 42°C for 6 h to complete the assembly.

[0077] The amount of XAD-2 used is proportional to the content of sodium cholate in the assembly solution. In this embodiment, the hydrophobic adsorbent is added in an amount of 0.5 g of XAD-2 filler per 1 mL of assembly solution. The detergent will be adsorbed by the XAD-2 filler during the incubation process, and then the filler will be removed by filtration, and the liquid will be recovered to achieve the separation effect. The residual detergent content does not need to be detected. If the detergent is not effectively removed by the XAD-2 filler, the assembly cannot be successful, that is, the electrophoresis detection can only detect the CD133 band, but not the MSP1D1 band.

[0078] 5. Removal of Nanodiscs without CD133 Protein Inserted by Affinity Chromatography

[0079] A TEV cleavage site was added after the 6×His tag in the MSP1D1 protein, and the MSP1D1 after cleavage does not contain the His tag. This design ensures that affinity chromatography is performed after assembly to remove the nanodiscs that are not inserted with membrane proteins.

[0080] The assembled CD133-nanodisc filter membrane was used to remove the XAD-2 filler to obtain the filtrate containing CD133-nanodisc; the filtrate containing CD133-nanodisc was incubated with nickel filler for 2 hours. After the loading, 2 column volumes were washed with equilibrium buffer to collect the impurity protein peaks that were not attached to the column; then, the gradient buffer with increasing imidazole concentration was used for elution to collect each protein peak; 20 μL was taken out from each elution peak receiving tube for SDS-PAGE detection, and the rest was stored at 4°C.

[0081] Example 2

[0082] This embodiment provides a method for preparing CD133-nanodiscs, which is substantially the same as that of embodiment 1, except that the first incubation temperature is changed to 40°C; the second incubation temperature is changed to 40°C.

[0083] Example 3

[0084] This example provides a method for preparing CD133-nanodiscs, which is substantially the same as that of Example 1, except that the molar ratio of CD133 protein:MSP1D1 protein:phospholipid:sodium cholate is 1:5:50:100.

[0085] Example 4

[0086] This example provides a method for preparing CD133-nanodiscs, which is substantially the same as that of Example 1, except that the molar ratio of CD133 protein:MSP1D1 protein:phospholipid:sodium cholate is 1:5:70:150.

[0087] Example 5

[0088] This example provides a method for preparing CD133-nanodiscs, which is substantially the same as that of Example 1, except that the phospholipid DPPC is replaced with a combination of DOPE and DPPC, and DOPE and DPPC are mixed in a molar ratio of 1:3.

[0089] Example 6

[0090] This example provides a method for preparing CD133-nanodiscs, which is substantially the same as that of Example 5, except that DOPE and DPPC are mixed in a molar ratio of 1:2.

[0091] Example 7

[0092] The embodiment provides a method for preparing CD133-nanodiscs, which is substantially the same as that of embodiment 5, except that the second incubation time is 3 hours. After the sample is concentrated 10 times by ultrafiltration, CD133 is detected by Western Blot, and MSP1D1 is detected by Coomassie Brilliant Blue staining.

[0093] Example 8

[0094] The embodiment provides a method for preparing CD133-nanodiscs, which is substantially the same as that of embodiment 6, except that the second incubation time is 3 hours. After the sample is concentrated 10 times by ultrafiltration, CD133 is detected by Western Blot, and MSP1D1 is detected by Coomassie Brilliant Blue staining.

[0095] Comparative Example 1

[0096] This example provides a method for preparing CD133-nanodiscs, which is substantially the same as that of Example 1, except that the second incubation temperature is changed to 37°C.

[0097] Comparative Example 2

[0098] This example provides a method for preparing CD133-nanodiscs, which is substantially the same as that of Example 1, except that the amount of DPPC used is 120 μM.

[0099] Comparative Example 3

[0100] This example provides a method for preparing CD133-nanodiscs, which is substantially the same as that of Example 1, except that the incubation time in the second step is 3 hours. After the sample is concentrated 10 times by ultrafiltration, CD133 is detected by Western Blot, and MSP1D1 is detected by Coomassie Brilliant Blue staining.

[0101] Verification Example 1

[0102] The nanodiscs prepared in Examples 1 to 6 and Comparative Examples 1 to 2 were tested. After the assembled CD133-nanodiscs were purified by nickel affinity chromatography, there should be two obvious bands. The results are as follows.

[0103] Table 1 Comparison of Examples 1 to 6 and Comparative Examples 1 to 2

[0104]

[0105] like Figure 3 As shown, lane 3 is the gel running result of the product of Example 1 (lanes 1 and 2 are control results, wherein: lane 1 is pure MSP1D1 protein with a molecular weight of 24kD; lane 2 is only pure CD133 protein with a molecular weight of 120kD). The results show that there are two bands near 24kD and 120kD at the same time, indicating that the assembly is successful.

[0106] After the assembled CD133-nanodiscs in Examples 2 to 6 were purified by nickel affinity chromatography, the electrophoresis results also showed two bands at the same time, indicating that the assembly was successful.

[0107] like Figure 4The electrophoresis result of the product of Comparative Example 1 is as follows: Figure 4 The electrophoresis results showed that only a band near 120 kD (CD133 protein) could be seen, and no band near 24 kD (MSP1D1 protein) could be seen, indicating that the assembly was unsuccessful. The electrophoresis results of the product of Comparative Example 2 were the same as those of Comparative Example 1, indicating that the assembly was unsuccessful.

[0108] Verification Example 2

[0109] The recognition of CD133-nanodiscs by CD133 antibodies was verified.

[0110] 1 mL of the product prepared by the method of Example 1 was taken, 40 μL of filler coupled with protein A and 1 μg of commercial antibody against CD133 were added, incubated at 4°C for 2 h, and then centrifuged at 1000g at 4°C to collect the filler, and then Western Blot detection was performed. At the same time, a control group was set up, and the product prepared by the method of Example 1 was replaced with pure MSP1D protein and pure CD133 protein, respectively.

[0111] The results are as follows Figure 5 As shown, lanes 1 and 2 are the results of the control group (lane 1 is pure MSP1D protein, lane 2 is pure CD133 protein), and lane 3 is the result of Example 1.

[0112] Depend on Figure 5 It can be seen that there is a CD133 band at 120 kD, which proves that CD133 in CD133-nanodiscs can normally recognize CD133 antibodies.

[0113] Examples 2 to 6 were detected in the same manner, and a band near 120 kD was also observed.

[0114] Verification Example 3

[0115] The incubation time was further optimized, as shown in Examples 7 and 8 and Comparative Example 3 (see Table 2), and the second incubation time was shortened to 3 hours. After the product was concentrated 10 times by ultrafiltration, CD133 was detected by Western Blot, and MSP1D1 was detected by Coomassie Brilliant Blue staining.

[0116] Table 2 Comparison of Examples 7, 8 and Comparative Example 3

[0117]

[0118]

[0119] Among them, see Figure 6As shown, the assembly of comparative example 3 was unsuccessful, and only the band of CD133 (120 kD) was shown, while the assembly of examples 7 and 8 was successful, and both MSP1D1 (24 kD) and CD133 (120 kD) had bands on the left and right. It can be seen that the combination of DPPC and DOPE phospholipids can greatly improve the assembly efficiency of CD133-nanodiscs.

[0120] The sequence information involved in this application is as follows.

[0121]

[0122]

[0123] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a membrane-like system, characterized in that: It includes the following steps: (1) mixing a CD133 protein solution, a membrane skeleton protein solution, a phospholipid mother solution and a detergent; (2) Remove detergent; (3) purifying to obtain a membrane-like system containing CD133; The CD133 protein solution contains CD133 protein, the membrane skeleton protein solution contains membrane skeleton protein, and the phospholipid mother solution contains phospholipid; the molar ratio of the CD133 protein, the membrane skeleton protein, the phospholipid and the detergent is 1:5:50-100:100-200.

2. The preparation method according to claim 1, characterized in that: The phospholipids include any one or more of DPPC, DOPE, DMPC, POPC, DOPC, DMPG and DOPG; Optionally, the phospholipid comprises DPPC; Optionally, the phospholipids include a combination of DPPC and DOPE; Optionally, in the combination of DPPC and DOPE, the molar ratio of DOPE to DPPC is 1:2-3.

3. The preparation method according to claim 1, characterized in that: The amino acid sequence of the CD133 protein includes the sequence shown in SEQ ID NO: 1, or has at least 60% identity with the sequence shown in SEQ ID NO: 1 while retaining the transmembrane region sequence in the sequence shown in SEQ ID NO: 1; Optionally, the amino acid sequence of the CD133 protein has at least 80% identity with the sequence shown in SEQ ID NO: 1 while retaining the transmembrane region sequence in the sequence shown in SEQ ID NO:

1.

4. The preparation method according to claim 1, characterized in that: The membrane skeleton protein includes: a derivative of apolipoprotein A1 or a truncated form of apolipoprotein A1; Optionally, the amino acid sequence of the membrane skeleton protein includes the sequence shown in SEQ ID NO:2 or has at least 80% identity with the sequence shown in SEQ ID NO:

2.

5. The preparation method according to claim 1, characterized in that: The detergent includes sodium cholate solution.

6. The preparation method according to any one of claims 1 to 5, characterized in that: The mixing includes a first incubation, and the conditions of the first incubation include: 40-42° C., 1-3 h.

7. The preparation method according to any one of claims 1 to 5, characterized in that: The step (2) comprises: adding a hydrophobic adsorbent to the mixed product for a second incubation; Optionally, the conditions of the second incubation include: an incubation temperature of 40 to 42° C. and a time of ≥3 to 6 hours; Optionally, the hydrophobic adsorbent comprises XAD-2.

8. The preparation method according to claim 7, characterized in that: The step (3) comprises: filtering to remove the hydrophobic adsorbent, recovering the liquid for chromatography, and separating to obtain a membrane-like system containing a phospholipid bilayer of CD133.

9. A membrane-like system containing CD133 prepared according to the preparation method according to any one of claims 1 to 8.

10. The use of the membrane-like system containing CD133 according to claim 9, comprising at least one of the following (I) to (IV): (I) preparing antibodies against CD133; (II) Screening of CD133 inhibitors; (III) preparing a reagent or kit for detecting CD133 autoantibodies; (IV) Detection of CD133 autoantibodies for non-therapeutic or diagnostic purposes.

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