Amphiphilic polypeptide for liposome capture and transmembrane protein affinity activity analysis

Abstract

The invention discloses an amphiphilic polypeptide for liposome capture and transmembrane protein affinity activity analysis, and relates to the technical field of biology. The sequence of the amphiphilic polypeptide is GIGAVLKVLTTGLPALISWIKRLLNKLLKKLKLLKC, and the sequence of the amphiphilic polypeptide is GIGAVLKVLTTGLPALISWIKRLLNKLLKLLLKC. The amphiphilic polypeptide provided by the invention can be used for capturing and analyzing lipidosome, and compared with traditional hydrophobic alkyl capture, the amphiphilic polypeptide has stronger specificity and can effectively avoid non-specific binding. The lower non-specific binding enables the affinity analysis between the protein on the liposome and the ligand thereof to be possible.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an amphipathic polypeptide used for liposome capture and transmembrane protein affinity activity analysis. Background Art

[0002] In recent years, with the rapid progress of biotechnology, remarkable development achievements have been made in the field of biological drugs, especially antibody drugs. With its high specificity and low side effects, this type of drug has shown great potential in multiple therapeutic fields such as cancer treatment and autoimmune diseases. However, the realization of the effect of antibody drugs is inseparable from its precise recognition and binding with specific target proteins on the cell surface or inside. Among these targets, membrane proteins occupy an extremely important position. As a component of the cell membrane, membrane proteins are not only involved in key life processes such as substance transport and signal transduction, but are also core regulators of many disease-related pathways. Therefore, the development of drugs targeting membrane proteins, especially antibody drugs, has become a hot spot in current biopharmaceutical research.

[0003] However, the biological characteristics of membrane proteins have brought many challenges to the development of antibody drugs. First, the cell membrane acts as a natural barrier, limiting the ability of biological drugs to directly penetrate into the cell and bind to the target protein. This means that for membrane proteins located on the inner side of the cell membrane or in transmembrane structures, antibody drugs must overcome the barrier effect of the cell membrane to achieve effective treatment. Secondly, the biological activity of membrane proteins is highly dependent on their complex spatial configuration, especially those with multiple transmembrane structures. The transmembrane regions of these proteins not only maintain the overall conformation of the protein, but also determine its binding site and affinity with antibody drugs. Therefore, in the process of antibody drug development, how to maintain the natural conformation of membrane proteins has become an urgent problem to be solved.

[0004] In the current practice of antibody drug development, the activity analysis of membrane proteins with multiple transmembrane structures mainly relies on cell experiments. Although this method can simulate the drug's action environment in the body more realistically, it also has many limitations. On the one hand, cell experiments require cumbersome cell recovery, passaging and state adjustment processes, which are not only time-consuming, but also difficult to ensure that the state of each batch of cells is completely consistent, thus affecting the stability and repeatability of the experimental results. On the other hand, cell experiments have high requirements for experimental conditions, such as temperature, humidity, pH value, etc. These factors may affect the conformation and activity of membrane proteins, thereby interfering with the accuracy of drug screening.

[0005] In order to overcome the limitations of cell experiments, researchers began to explore new methods for in vitro activity detection. Among them, liposome technology has attracted much attention because it can simulate the cell membrane structure. Liposomes are closed vesicles with a bilayer structure formed by the self-assembly of phospholipid molecules, which can encapsulate biological macromolecules such as drugs and proteins. The polar phosphate head group and hydrophobic lipid tail of the phospholipid molecules enable liposomes to exist stably in aqueous solutions and simulate the hydrophobic and hydrophilic environment of the cell membrane. Using liposome technology, membrane proteins with multiple transmembrane structures can be stabilized on the surface of liposomes, thereby maintaining their natural conformation and realizing in vitro activity detection.

[0006] However, despite the excellent performance of liposome technology in simulating cell membrane structure, it also faces some challenges in practical application. In particular, when using surface plasmon resonance (SPR) technology for activity detection, there is often strong nonspecific adsorption between the detection chip modified with hydrophobic alkyl groups and the ligand protein (such as antibody). This nonspecific adsorption will not only reduce the sensitivity of the detection chip, but also may interfere with the results of protein activity detection, resulting in false positives or false negatives. The root cause of nonspecific adsorption lies in the interaction between the hydrophobic alkyl group and the hydrophobic structure of the protein.

[0007] Therefore, in the process of using liposome technology to conduct multiple transmembrane protein in vitro activity tests, how to effectively reduce or eliminate nonspecific adsorption and improve the sensitivity and specificity of the test has become a key issue in current research. Solving this problem will not only help accelerate the development of antibody drugs, but also provide a more accurate and effective drug screening platform for the treatment of membrane protein-related diseases. Summary of the invention

[0008] Based on the deficiencies in the prior art, the present invention provides an amphipathic polypeptide for liposome capture and transmembrane protein affinity activity analysis, which has extremely low nonspecific adsorption, can effectively capture liposomes, and at the same time, capture multiple transmembrane proteins embedded on the liposome membrane to the surface of the surface plasmon resonance detection chip. After capture, the affinity analysis of the multiple transmembrane proteins embedded on the liposome and their ligands can be performed.

[0009] Compared with cellular experiments, liposomes are easy to store and have better experimental repeatability. It is more convenient and reliable to use liposomes to conduct multiple affinity analyses of transmembrane proteins and their ligands.

[0010] Specifically, the present invention provides an amphipathic polypeptide, the sequence of the amphipathic polypeptide is GIGAVLKVLTTGLPALISWIKRLLNKLLKKLKKLLKC.

[0011] The present invention also provides a detection product, characterized in that the detection product is a surface plasmon resonance detection chip, the polypeptide is modified on the surface of the surface plasmon resonance detection chip, and the polypeptide modified on the surface of the surface plasmon resonance detection chip is the amphiphilic polypeptide.

[0012] The first method, the method of modifying the polypeptide on the surface of the surface plasmon resonance detection chip comprises:

[0013] The carboxyl groups on the surface of the surface plasmon resonance detection chip are activated by N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride reagents, and the activated carboxyl groups are condensed with the amino groups of the amphiphilic polypeptides to covalently link the amphiphilic polypeptides to the surface of the surface plasmon resonance detection chip.

[0014] The second method, the method of modifying the polypeptide on the surface of the surface plasmon resonance detection chip comprises:

[0015] N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride reagents were used to activate the carboxyl groups on the surface of the surface plasmon resonance detection chip;

[0016] fixing 2-(2-pyridyldithio)ethylamine hydrochloride onto the activated surface plasmon resonance detection chip;

[0017] The thiol group on the amphiphilic polypeptide undergoes a condensation reaction with 2-(2-pyridyldithio)ethylamine hydrochloride, thereby covalently connecting the amphiphilic polypeptide to the surface of a surface plasmon resonance detection chip.

[0018] The peptides obtained by the two methods are modified on the surface of the surface plasmon resonance detection chip. When the liposome sample flows through the modified detection chip surface, it is captured by the amphiphilic peptide on the chip surface. The chimeric protein on the liposome is also captured on the chip surface, so as to further analyze the affinity between the protein and its ligand.

[0019] The present invention also provides a method for detecting the affinity activity between a transmembrane protein and its ligand, using the above-mentioned detection product for detection.

[0020] Specifically, the method for detecting the affinity activity of a transmembrane protein and its ligand comprises the following steps:

[0021] (1) preparing transmembrane protein-liposome complexes;

[0022] (2) capturing the prepared transmembrane protein-liposome complex on the detection product;

[0023] (3) The ligand of the transmembrane protein is captured by the detection product having the transmembrane protein-liposome complex in step (2), and the specific binding and dissociation process between the transmembrane protein and its ligand is detected and recorded by surface plasmon resonance technology.

[0024] Specifically, in step (1), in the transmembrane protein-liposome complex, the molar ratio of the transmembrane protein to the liposome lipid is 1:1000.

[0025] 1× PBS was used as the running buffer.

[0026] In step (2), the transmembrane protein-liposome complex is diluted with running buffer and the test product is introduced at a flow rate of 10 μL / min for 7 min;

[0027] In step (3), the ligand of the transmembrane protein is diluted with running buffer, the flow rate is 10 μL / min, the binding time is 2 min, and the dissociation time is 5 min.

[0028] Beneficial effects of the present invention:

[0029] The amphiphilic polypeptide provided by the present invention can be used for the capture and analysis of liposomes. Compared with the traditional hydrophobic alkyl capture, the amphiphilic polypeptide has stronger specificity and can effectively avoid non-specific binding. The lower non-specific binding makes the affinity analysis between the protein on the liposome and its ligand more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The detection chip modified with hydrophobic alkyl has strong non-specific binding with proteins.

[0031] Figure 2 The detection chip modified with the amino group of the amphiphilic polypeptide of the present invention captures the binding process (A) and signal changes (B) of different types of liposomes. ①~⑨ represent the binding process and signal changes of the liposomes prepared by the formulas ①~⑨ in Table 3 and the detection chip.

[0032] Figure 3 It is the non-specific binding between the detection chip modified with the amino group of the amphiphilic polypeptide of the present invention and the protein.

[0033] Figure 4 The binding process (A) and signal changes (B) of different types of liposomes captured by the amphiphilic polypeptide thiol-modified detection chip of the present invention. ①~⑨ represent the binding process and signal changes of the liposomes prepared by formulas ①~⑨ in Table 3 and the detection chip.

[0034] Figure 5 It is the non-specific binding between the detection chip modified with thiol groups of the amphiphilic polypeptide of the present invention and the protein.

[0035] Figure 6 This is the affinity activity test of CD47 and Magrolimab Fab (amino connection); wherein, from top to bottom, the lines of different colors represent the binding and dissociation process of the anti-CD47 antibody Magrolimab Fab at concentrations of 138 nm, 69 nm, 34.5 nm, 17.25 nm, and 8.625 nm, respectively.

[0036] Figure 7 This is the affinity activity test of CD47 and Magrolimab Fab (thiol connection); wherein, from top to bottom, the lines of different colors represent the binding and dissociation process of the anti-CD47 antibody Magrolimab Fab at concentrations of 276nm, 138nm, 69nm, 34.5nm, 17.25nm, and 8.625nm, respectively. DETAILED DESCRIPTION

[0037] Example 1: Chips modified with hydrophobic alkyl groups have strong nonspecific binding

[0038] (1) Antibody preparation

[0039] Different commercially available antibodies or proteins were diluted at a ratio of 1:100 using PBS buffer and filtered using a 0.22 μm PES filter for later use.

[0040] (2) Surface plasmon resonance detection

[0041] Surface plasmon resonance detection was performed using a Biacore T200 molecular interaction analyzer. Before detection, a Series S L1 sensor chip was loaded. The surface of the chip was modified with hydrophobic long-chain alkanes and can be used for the analysis of lipids, liposomes, and exosomes.

[0042] The running buffer was PBS buffer; the sample loading flow rate was 30 μL / min, and the time was 2 min; the regeneration solution was 50 mM NaOH:isopropanol (volume ratio 3:2), the regeneration flow rate was 30 μL / min, and the regeneration time was 30 s.

[0043] (3) Experimental results

[0044] Binding of different antibodies to Series S L1 sensor chip Figure 1 As shown in the figure, there is strong non-specific binding between the antibody and the Series SL1 sensor chip, which may affect the detection of the protein.

[0045] Example 2: Screening of amphiphilic peptides

[0046] (1) Peptide selection and synthesis

[0047] The target polypeptide was artificially synthesized by solid phase synthesis, and the polypeptide sequence is shown in Table 1 below.

[0048] Table 1 Amphiphilic polypeptide sequences

[0049]

[0050]

[0051] (2) Detection chip surface modification

[0052] The amphiphilic peptides were coupled to the carboxyl groups of the Series S CM5 sensor chip by amino modification, thereby preparing a surface plasmon resonance detection chip modified with amphiphilic peptides. The specific process is as follows:

[0053] 1) Weigh 1 mg of peptide into a sample bottle, dissolve it completely with 100 μL of dimethyl sulfoxide, and then dilute it with 900 μL of ultrapure water;

[0054] 2) diluting the above polypeptide solution 50 times with 10 mM sodium acetate buffer at pH 4.0;

[0055] 3) Place the sealed bag containing the CM5 chip at room temperature for 15-30 minutes to equilibrate to prevent condensation on the chip surface; place the chip into the chip chamber and use 1×PBS (0.22 μm membrane filtered) as the running buffer;

[0056] 4) 0.1 mol / L N-hydroxysuccinimide (NHS) and 0.4 mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were mixed at a volume ratio of 1:1;

[0057] 5) Pass the mixture of NHS and EDC at a flow rate of 10 μL / min for 7 min;

[0058] 6) Passing the polypeptide solution described in step 2) at a flow rate of 10 μL / min for 14 minutes;

[0059] 7) 1 mol / L ethanolamine solution (pH 8.5) was introduced at a flow rate of 10 μL / min for 7 min to block excess carboxyl groups.

[0060] (3) Liposome preparation

[0061] In this experiment, liposomes were prepared by thin film hydration. The raw materials were dissolved in 5 mL of ethanol in proportion, and the final concentration of the mixed lipids was 1 mg / mL. After the lipids were fully dissolved, they were rotary evaporated under reduced pressure at 40°C to completely remove the organic solvent and a uniform lipid film could be observed on the bottle wall. After obtaining the film, 1 mL of 1×PBS was added to the bottle and oscillated to mix the film to make it fall off. After that, it was hydrated in a 45°C water bath for 30 minutes. After hydration, liquid nitrogen was used for 3 freeze-thaw processes, and then ultrasonicated for 10 minutes. After the ultrasonication was completed, the extruder was used to extrude repeatedly 11 times under a 100 nm polycarbonate membrane to obtain a finished liposome with uniform dispersion and consistent particle size.

[0062] The raw material formula for preparing liposomes is: 50% distearoylphosphatidylcholine (DSPC), 30% cholesterol, 10% dioleoylphosphatidylcholine (DOPC), and 10% phosphatidylserine (POPS).

[0063] (4) Liposome capture

[0064] The liposomes were respectively introduced into the detection chips with different peptides modified on the surface, with a flow rate of 10 μL / min and a time of 7 min. The changes in the detection signals before and after the introduction of the liposomes are shown in Table 2. The results show that the peptide of Seq1 can effectively capture the liposomes to the surface of the detection chip.

[0065] Table 2 Characteristics of amphiphilic peptides and liposome capture efficiency (signal response changes)

[0066] SeqID Hydrophobicity Hydrophobic polarity Charge number Signal response changes Seq_1 0.713 0.578 +9 2315.03 Seq_2 0.634 0.447 +1 95.34 Seq_3 0.394 0.466 +3 213.94 Seq_4 0.101 0.460 +4 -15.37 Seq_5 0.300 0.480 -1 4.28 Seq_6 0.631 0.442 +3 3.06 Seq_7 0.440 0.526 +1 395.12 Seq_8 0.546 0.530 0 1.54 Seq_9 0.516 0.479 +2 12.34 Seq_10 0.283 0.485 +2 3.58 Seq_11 0.759 0.456 +4 415.92 Seq_12 0.331 0.840 +13 587.12 Seq_13 0.518 0.446 -1 6.23 Seq_14 0.764 0.344 0 -1.21 Seq_15 0.338 0.511 +2 0.86 Seq_16 0.026 0.421 +5 1.01

[0067] Example 3: Signal response of different types of liposomes captured by amphiphilic peptides (amino group-linked)

[0068] (1) Preparation of peptide solution

[0069] The polypeptide was synthesized by solid phase synthesis, and the polypeptide sequence was shown in Seq 1. 1 mg of the polypeptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, diluted with 900 μL of ultrapure water, and stored at -80°C for later use.

[0070] (2) Detection chip surface modification

[0071] 1) dilute the above polypeptide solution 50 times with 10 mM sodium acetate buffer at pH 4.0;

[0072] 2) Place the sealed bag containing the CM5 chip at room temperature for 15-30 minutes to equilibrate to prevent condensation on the chip surface; place the chip into the chip chamber and use 1×PBS (0.22 μm membrane filtered) as the running buffer;

[0073] 3) Mix 0.1 mol / L NHS and 0.4 mol / L EDC in a 1:1 volume ratio;

[0074] 4) Pass the mixture of NHS and EDC at a flow rate of 10 μL / min for 7 min;

[0075] 5) Passing the polypeptide solution described in step 1) at a flow rate of 10 μL / min for 14 min;

[0076] 6) 1 mol / L ethanolamine solution (pH 8.5) was introduced at a flow rate of 10 μL / min for 7 min to block the excess carboxyl groups.

[0077] (3) Liposome preparation

[0078] The steps for preparing liposomes were the same as those described in Example 2. The liposome preparation formula is shown in Table 3 below.

[0079] Table 3 Liposome preparation formula

[0080] serial number formula ① 30% cholesterol, 70% DSPC ② 30% cholesterol, 60% DSPC, 10% DOPC ③ 30% cholesterol, 50% DSPC, 20% DOPC ④ 30% cholesterol, 65% DSPC, 5% POPS ⑤ 30% cholesterol, 55% DSPC, 10% DOPC, 5% POPS ⑥ 30% cholesterol, 45% DSPC, 20% DOPC, 5% POPS ⑦ 30% cholesterol, 60% DSPC, 10% POPS ⑧ 30% cholesterol, 50% DSPC, 10% DOPC, 10% POPS ⑨ 30% cholesterol, 40% DSPC, 20% DOPC, 10% POPS

[0081] (4) Liposome capture

[0082] The prepared liposomes were appropriately diluted with 1× PBS buffer, and then the liposomes with different formulations were passed into the detection chip with Seq1 peptide modified on the surface at a flow rate of 10 μL / min for 7 min. Figure 2 As shown, the amphiphilic peptide Seq1 has a good capture efficiency for various liposomes.

[0083] (5) Protein nonspecific adsorption detection

[0084] After diluting the commercially available antibody or protein at a ratio of 1:100 using PBS buffer, filter it with a 0.22μm PES filter membrane for later use. Non-specific adsorption detection of the detection chip with Seq1 peptide modified on the surface, the running buffer is PBS buffer; the sample loading flow rate is 30μL / min, the time is 2min; the regeneration liquid is 0.04mol / Ln-octyl β-D-glucopyranoside, the regeneration flow rate is 30μL / min, and the regeneration time is 30s. The test results are as follows Figure 3 As shown, the nonspecific adsorption was significantly lower than that of the detection chip modified with hydrophobic alkyl.

[0085] Example 4: Signal Response of Different Types of Liposomes Captured by Amphiphilic Peptides (Thydroxylinkage)

[0086] (1) Preparation of peptide solution

[0087] The peptide was synthesized by solid phase synthesis, and the peptide sequence is shown in Seq 1. 1 mg of the peptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, diluted with 900 μL of ultrapure water, and frozen at -80 degrees Celsius for later use.

[0088] (2) Detection chip surface modification

[0089] 1) dilute the above polypeptide solution 50 times with 10 mM sodium acetate buffer at pH 4.0;

[0090] 2) Place the sealed bag containing the CM5 chip at room temperature for 15-30 minutes to equilibrate to prevent condensation on the chip surface. Place the chip in the chip chamber and use 1×PBS (0.22μm membrane filtered) as the running buffer;

[0091] 3) Mix 0.1 mol / L NHS and 0.4 mol / L EDC in a ratio of 1:1 and use immediately after preparation;

[0092] 4) Mix 0.12 mol / L 2-(2-pyridinyldithio)ethaneamine hydrochloride (PDEA) and 0.15 mol / L sodium borate buffer (pH 8.5) at a ratio of 2:1 and use immediately after preparation;

[0093] 5) Pass the mixture of NHS and EDC at a flow rate of 10 μL / min for 7 min;

[0094] 6) Pass PDEA working solution at a flow rate of 10 μL / min for 4 min;

[0095] 7) Passing the polypeptide solution described in step 1) at a flow rate of 10 μL / min for 14 minutes;

[0096] 8) 0.05 mol / L L-cysteine, 1 mol / L sodium chloride, and 0.1 mol / L sodium acetate buffer (pH 4.0) were introduced at a flow rate of 10 μL / min for 4 min to block excess thiol groups.

[0097] (3) Liposome preparation

[0098] The liposome preparation steps were the same as those described in Example 3.

[0099] (4) Liposome capture

[0100] The prepared liposomes were appropriately diluted with 1×PBS buffer, and then the liposomes with different formulations in Table 3 of Example 3 were passed into the detection chip with Seq1 polypeptide modified on the surface at a flow rate of 10 μL / min for 7 min. Figure 4 As shown, the amphiphilic peptide Seq1 has a good capture efficiency for various liposomes.

[0101] (5) Protein nonspecific adsorption detection

[0102] Antibodies or proteins purchased from different companies were diluted in a ratio of 1:100 using PBS buffer and filtered with a 0.22μm PES filter for later use. Non-specific adsorption detection of the detection chip with Seq1 peptide modified on the surface, the running buffer was PBS buffer; the sample loading flow rate was 30μL / min, the time was 2min; the regeneration liquid was 0.04mol / L n-octyl β-D-glucopyranoside, the regeneration flow rate was 30μL / min, and the regeneration time was 30s. The test results are as follows Figure 5 As shown, the nonspecific adsorption was significantly lower than that of the detection chip modified with hydrophobic alkyl.

[0103] Example 5: Activity analysis after liposome capture (carboxyl linkage)

[0104] (1) Preparation of peptide solution

[0105] The peptide was synthesized by solid phase synthesis, and the peptide sequence was shown in Seq 1. 1 mg of the peptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, and then diluted with 900 μL of ultrapure water, and frozen at -80 degrees Celsius for later use.

[0106] (2) Detection chip surface modification

[0107] 1) dilute the above polypeptide solution 50 times with 10 mM sodium acetate buffer at pH 4.0;

[0108] 2) Place the sealed bag containing the CM5 chip at room temperature for 15-30 minutes to equilibrate to prevent condensation on the chip surface. Place the chip in the chip chamber and use 1×PBS (0.22μm membrane filtered) as the running buffer;

[0109] 3) Mix 0.1 mol / L NHS and 0.4 mol / L EDC in a ratio of 1:1;

[0110] 4) Pass the mixture of NHS and EDC at a flow rate of 10 μL / min for 7 min;

[0111] 5) Passing the polypeptide solution described in step 1) at a flow rate of 10 μL / min for 14 min;

[0112] 6) 1 mol / L ethanolamine solution (pH 8.5) was introduced at a flow rate of 10 μL / min for 7 min to block the excess carboxyl groups.

[0113] (3) Liposome preparation

[0114] The steps for preparing liposomes were the same as those described in Example 3. The liposome formula was No. ⑤.

[0115] (4) Protein chimera

[0116] 1) Take 200 μL of the prepared blank liposomes, add n-octylβ-D-glucopyranoside at a mass ratio of liposomes: OG = 1:5, mix and incubate at 4°C for 30 min;

[0117] 2) CD47 protein stored in 0.03% DDM buffer was added to the above solution at a molar ratio of protein: lipid = 1:1000, mixed and incubated at 4°C for 2 h;

[0118] 3) Add Bio-beads SM-2 at a mass ratio of detergent to polystyrene beads = 1:10, incubate for 1 hour, and collect the supernatant. Repeat five times, and add double the amount of polystyrene beads for the last time. After the detergent is adsorbed, remove the polystyrene beads to obtain the constructed proteoliposomes.

[0119] 4) The obtained CD47 protein-liposome solution was purified using Monomix Core 1000 filler to remove unbound proteins.

[0120] (5) Liposome capture

[0121] The prepared liposomes were appropriately diluted with 1×PBS buffer, and then the liposomes were passed into the detection chip with Seq1 polypeptide modified on the surface at a flow rate of 10 μL / min for 7 min.

[0122] (6) Analysis of affinity activity between anti-CD47 antibody and CD47 protein

[0123] After the detection chip with Seq1 polypeptide modified on the surface captured the CD47 protein-liposome, the anti-CD47 antibody Magrolimab Fab was passed through for detection. Anti-CD47 antibody Magrolimab Fab solutions of different concentrations (8.625nM-138nM) were passed through the chip, and the dynamic process of specific binding and dissociation of CD47 protein-liposome and anti-CD47 antibody Magrolimab Fab during the flow was detected and recorded by SPR. Finally, 20mM CHAPS was used to dissociate the liposomes and regenerate the surface of the biosensor. 1×PBS was used as the running buffer for the entire experiment. The flow rate of the anti-CD47 antibody Magrolimab Fab solution flowing through the chip was controlled at 10μL / min, the binding time was 2min, the dissociation time was 5min, and the flow rate of the regeneration solution through the chip was 30μL / min. The activity detection results are shown in the following figure. Figure 6 As shown, the equilibrium dissociation constant of the anti-CD47 antibody Magrolimab Fab and CD47 protein was measured to be approximately 8nmol / L, which is very close to the cell half-inhibitory concentration of approximately 6nmol / L reported in previous literature, indicating that this method can effectively detect the affinity activity of Magrolimab Fab and CD47 protein.

[0124] (7) Stability monitoring

[0125] Three identical CD47 protein-liposome samples were prepared and stored in a -80℃ refrigerator for affinity detection of anti-CD47 antibody Magrolimab Fab. Each sample was repeated 3 times, and the test was repeated every 7 days, for a total of 4 times. The monitoring results showed that the method had good repeatability.

[0126] Example 6: Activity analysis after liposome capture (carboxyl linkage and sulfhydryl linkage)

[0127] (1) Preparation of peptide solution

[0128] The peptide was synthesized by solid phase synthesis, and the peptide sequence is shown in Seq 1. 1 mg of the peptide was weighed into a sample bottle, completely dissolved with 100 μL of dimethyl sulfoxide, diluted with 900 μL of ultrapure water, and frozen at -80 degrees Celsius for later use.

[0129] (2) Detection chip surface modification

[0130] 1) Dilute the above polypeptide solution 50 times with 10 mM sodium acetate buffer at pH 4.0 and prepare it for use immediately;

[0131] 2) Place the sealed bag containing the CM5 chip at room temperature for 15-30 minutes to equilibrate to prevent condensation on the chip surface. Place the chip in the chip chamber and use 1×PBS (0.22μm membrane filtered) as the running buffer;

[0132] 3) Mix 0.1 mol / L NHS and 0.4 mol / L EDC in a ratio of 1:1;

[0133] 4) Mix 0.12 mol / L PDEA and 0.15 mol / L sodium borate buffer (pH 8.5) in a ratio of 2:1 and use immediately after preparation;

[0134] 5) Pass the mixture of NHS and EDC at a flow rate of 10 μL / min for 7 min;

[0135] 6) Pass PDEA working solution at a flow rate of 10 μL / min for 4 min;

[0136] 7) Passing the polypeptide solution described in step 1) at a flow rate of 10 μL / min for 14 minutes;

[0137] 8) 0.05 mol / L L-cysteine, 1 mol / L sodium chloride, and 0.1 mol / L sodium acetate buffer (pH 4.0) were introduced at a flow rate of 10 μL / min for 4 min to block excess thiol groups.

[0138] (3) Liposome preparation

[0139] The steps for preparing liposomes were the same as those described in Example 3. The liposome formula was No. ⑤.

[0140] (4) Protein chimera

[0141] 1) Take 200 μL of the prepared blank liposomes, add n-octylβ-D-glucopyranoside at a mass ratio of liposomes: OG = 1:5, mix and incubate at 4°C for 30 min;

[0142] 2) CD47 protein stored in 0.03% DDM buffer was added to the above solution at a molar ratio of protein: lipid = 1:1000, mixed and incubated at 4°C for 2 h;

[0143] 3) Add Bio-beads SM-2 at a mass ratio of detergent to polystyrene beads = 1:10, incubate for 1 h, and collect the supernatant. Repeat five times, and add double the amount of polystyrene beads for the last time. After the detergent is adsorbed, remove the polystyrene beads to obtain the constructed proteoliposomes.

[0144] 4) The obtained CD47 protein-liposome solution was purified using Monomix Core 1000 filler to remove unbound proteins.

[0145] (5) Liposome capture

[0146] The prepared liposomes were appropriately diluted with 1× PBS buffer, and then the liposomes were passed into the detection chip with Seq1 polypeptide modified on the surface at a flow rate of 10 μL / min for 7 min.

[0147] (6) Analysis of affinity activity between anti-CD47 antibody and CD47 protein

[0148] After the detection chip modified with Seq1 polypeptide on the surface captured the CD47 protein-liposome, the anti-CD47 antibody Magrolimab Fab was passed through for detection. Anti-CD47 antibody Magrolimab Fab solutions of different concentrations (8.625nM-276nM) were passed through the chip, and the dynamic process of specific binding and dissociation of CD47 protein-liposome and anti-CD47 antibody Magrolimab Fab during the flow was detected and recorded by SPR. Finally, 20mM CHAPS was used to dissociate the liposomes and regenerate the surface of the biosensor. 1×PBS was used as the running buffer for the entire experiment. The flow rate of the anti-CD47 antibody Magrolimab Fab solution flowing through the chip was controlled at 10μL / min, the binding time was 2min, the dissociation time was 5min, and the flow rate of the regeneration solution through the chip was 30μL / min. The activity detection results are shown in the following figure. Figure 7 As shown, the equilibrium dissociation constant of the anti-CD47 antibody Magrolimab Fab and CD47 protein was measured to be approximately 8nmol / L, which is very close to the previously reported cell half-inhibitory concentration of approximately 6nmol / L, indicating that this method can effectively detect the affinity activity of Magrolimab Fab and CD47 protein.

Claims

1. An amphipathic polypeptide for liposome capture and transmembrane protein affinity activity analysis, characterized in that: The sequence of the amphipathic polypeptide is GIGAVLKVLTTGLPALISWIKRLLNKLLKKLKKLLKC.

2. A detection product, characterized in that: The detection product comprises a surface plasmon resonance detection chip, the polypeptide is modified on the surface of the surface plasmon resonance detection chip for capturing liposomes, and the polypeptide modified on the surface of the surface plasmon resonance detection chip is the amphiphilic polypeptide according to claim 1.

3. The detection product according to claim 2, characterized in that: The method for modifying the polypeptide on the surface of the surface plasmon resonance detection chip comprises: The carboxyl groups on the surface of the surface plasmon resonance detection chip are activated by N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride reagents, and the activated carboxyl groups are condensed with the amino groups of the amphiphilic polypeptides to covalently link the amphiphilic polypeptides to the surface of the surface plasmon resonance detection chip.

4. The detection product according to claim 2, characterized in that: The method for modifying the polypeptide on the surface of the surface plasmon resonance detection chip comprises: N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride reagents were used to activate the carboxyl groups on the surface of the surface plasmon resonance detection chip; fixing 2-(2-pyridyldithio)ethylamine hydrochloride onto the activated surface plasmon resonance detection chip; The thiol group on the amphiphilic polypeptide undergoes a condensation reaction with 2-(2-pyridyldithio)ethylamine hydrochloride, thereby covalently connecting the amphiphilic polypeptide to the surface of a surface plasmon resonance detection chip.

5. A method for detecting the affinity activity of a transmembrane protein and its ligand, characterized in that: The detection is performed using the detection product described in any one of claims 2 to 4.

6. The method for detecting the affinity activity of a transmembrane protein and its ligand according to claim 5, characterized in that: The following steps are involved: (1) preparing transmembrane protein-liposome complexes; (2) capturing the prepared transmembrane protein-liposome complex on the detection product; (3) The ligand of the transmembrane protein is captured by the detection product having the transmembrane protein-liposome complex in step (2), and the specific binding and dissociation process between the transmembrane protein and its ligand is detected and recorded by surface plasmon resonance technology.

7. The method for detecting the affinity activity of a transmembrane protein and its ligand according to claim 6, characterized in that: In step (1), in the transmembrane protein-liposome complex, the molar ratio of the transmembrane protein to the liposome lipid is 1:1000.

8. The method for detecting the affinity activity of a transmembrane protein and its ligand according to claim 6, characterized in that: 1× PBS was used as the running buffer. In step (2), the transmembrane protein-liposome complex is diluted with running buffer and the test product is introduced at a flow rate of 10 μL / min for 7 min; In step (3), the ligand of the transmembrane protein is diluted with running buffer, the flow rate is 10 μL / min, the binding time is 2 min, and the dissociation time is 5 min.

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