Assembled nanodisk integrated microsphere for transmembrane protein display and application thereof

Abstract

The invention relates to an assembled nanodisk integrated microsphere for transmembrane protein display and application of the assembled nanodisk integrated microsphere. Specifically, the invention provides a nanoparticle compound which comprises a solid-phase carrier as an inner core and a connecting layer attached to or combined with the outer surface of the solid-phase carrier, the outer side of the connecting layer is connected with a nano disc layer; the nano-disk layer is formed by arranging n independent nano-disk compounds, and each nano-disk compound comprises a nano-disk and a target protein embedded in the nano-disk. The nanoparticle compound only displays target transmembrane protein and does not contain host membrane protein which is not needed in virus-like particles (VLP), so that background noise is greatly reduced.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to an assembled nanodisc integrated microsphere for displaying transmembrane proteins and applications thereof. Background Art

[0002] Due to the hydrophobic transmembrane domains of membrane proteins, they cannot exist stably in isolation in solution. Current methods for displaying membrane proteins include nanodiscs and virus-like particles (VLPs). Nanodiscs are synthetic, nanoscale, disc-shaped lipid bilayers that provide stable, soluble cell membrane mimics for membrane protein research. Structurally, nanodiscs consist of a phospholipid bilayer (typically 1-10 nm in diameter) stabilized by amphiphilic molecules (membrane scaffold proteins (MSPs) or synthetic copolymers). In classic MSP-based nanodiscs, two scaffold proteins are wrapped around the lipid bilayer like adhesive tape. Newer "polymer nanodiscs" use amphiphilic copolymers (such as styrene-maleic acid) to dissolve cell membranes in the absence of detergents, yielding nanodiscs with a more native conformation. In both approaches, water-soluble disc-shaped lipid bilayer particles are produced that remarkably preserve the membrane protein's state and activity on the cell membrane. Consequently, these nanodiscs are increasingly being used by researchers and companies for membrane protein research and applications. Compared with virus-like particles, nanodisc-format membrane proteins have higher purity, but their disadvantage is that the particles are too small to be used as immunogens or for flow cytometry detection.

[0003] Therefore, there is an urgent need in the art to develop a nanoparticle complex with high-density membrane protein display. Summary of the Invention

[0004] The object of the present invention is to provide a nanoparticle complex with high-density membrane protein display.

[0005] In a first aspect of the present invention, a nanoparticle composite is provided, comprising: a solid phase carrier as a core, and a connecting layer attached to or bound to the outer surface of the solid phase carrier; a nanodisk layer is connected to the outer side of the connecting layer;

[0006] The nanodisc layer comprises n independently arranged nanodisc complexes, wherein the nanodisc complexes include nanodiscs and target proteins embedded in the nanodiscs; n is a positive integer of 10-100,000, preferably a positive integer of 100-50,000, and more preferably a positive integer of 500-10,000 or 1,000-5,000.

[0007] In another preferred embodiment, the solid phase support is modified or unmodified.

[0008] In another preferred embodiment, the target protein exposes the extracellular domain on the outside of the nanodisc layer.

[0009] In another preferred embodiment, the target protein includes a natural protein or a recombinant protein.

[0010] In another preferred embodiment, the target protein is a membrane protein.

[0011] In another preferred embodiment, the membrane proteins are membrane proteins of the same or different species.

[0012] In another preferred embodiment, the membrane proteins include (but are not limited to): at least one or more of lipid-anchored membrane proteins, single-pass transmembrane proteins, peripheral membrane proteins, embedded membrane proteins, integral membrane proteins, receptor proteins and G-protein coupled receptors.

[0013] In another preferred embodiment, the connecting layer includes (but is not limited to) a connecting method selected from the following group:

[0014] Streptavidin / Biotin, Ni-NTA / His-Tag, Strep-Tactin / Strep-tag II, SpyCatcher / SpyTag, Glutathione / GST-Tag, or a combination thereof.

[0015] In another preferred embodiment, the solid phase carrier includes: magnetic beads, resin, or a combination thereof.

[0016] In another preferred embodiment, the surface of the solid support contains a capture element, and the capture element is connected to the nanodisc complex via a connection method selected from the following group:

[0017] Streptavidin / Biotin, Ni-NTA / His-Tag, Strep-Tactin / Strep-tag II, SpyCatcher / SpyTag, Glutathione / GST-Tag, or a combination thereof.

[0018] In another preferred embodiment, the surface of the solid support is modified with elements selected from the following group:

[0019] Streptavidin, His antibody, Strep-tag antibody, myc-tag antibody, Flag-tag antibody, anti-GFP antibody, or a combination thereof.

[0020] In another preferred embodiment, the nanodisc comprises: lipid binding protein and lipid molecules; wherein,

[0021] The lipid binding protein surrounds the lipid molecules; the lipid molecules form a lipid layer, and the target protein is embedded in the lipid layer.

[0022] In another preferred embodiment, the embedding method includes: embedding the target protein into the lipid layer through an amphiphilic copolymer, a scaffold protein or an amphiphilic protein.

[0023] In another preferred embodiment, the amphiphilic copolymer includes styrene-maleic anhydride copolymer, diisobutylene-maleic acid (DIBMA), AASTY Poly (acrylic acid-co-styrene), AMPHIPOLS).

[0024] In another preferred embodiment, the scaffold protein includes MSP.

[0025] In another preferred embodiment, the amphiphilic protein includes SaponsinA.

[0026] In another preferred embodiment, the C-terminus of the amino acid sequence of the target protein embedded in the nanodisc includes a modification selected from the following group:

[0027] Biotin tag, Avi tag, His tag, Strep-tag, Flag-tag, myc-tag, HA-tag, GFP, or a combination thereof.

[0028] In another preferred embodiment, the particle size of the nanoparticle complex is 10-1000 nm, preferably 20-200 nm, and more preferably 30-50 nm.

[0029] In another preferred embodiment, the nanoparticle complex is captured by biotinylation or other labeling of the C-terminus of the nanodisc, and the nanosphere ensures the correct orientation of the extracellular domain of the target protein, similar to its natural positioning in the cell membrane, which helps to maintain functional integrity in research and therapeutic applications.

[0030] In another preferred embodiment, the membrane proteins include (but are not limited to): at least one or more of lipid-anchored membrane proteins, single-pass transmembrane proteins, peripheral membrane proteins, embedded membrane proteins, integral membrane proteins, receptor proteins and G-protein coupled receptors.

[0031] In another preferred embodiment, the lipid-anchored membrane protein includes (but is not limited to): RPE65.

[0032] In another preferred embodiment, the single-pass transmembrane protein includes (but is not limited to): enzyme-linked receptors, cytokine receptor superfamily, immunoglobulin superfamily, tumor necrosis factor receptor superfamily, Toll-like receptors, integrin family, cadherin, Notch receptor family, low-density lipoprotein receptor, receptor-like protein tyrosine phosphatase, or a combination thereof.

[0033] In another preferred embodiment, the integral membrane proteins include (but are not limited to): seven-transmembrane protein MRGPRX2, four-transmembrane protein CLDN18.2, ion channel protein Nav1.7, receptor protein CD19, or a combination thereof.

[0034] In another preferred embodiment, the integral membrane protein includes: a seven-transmembrane protein MRGPRX2, and the amino acid sequence of the seven-transmembrane protein MRGPRX2 is shown in SEQ ID NO: 1.

[0035] In another preferred embodiment, the integral membrane protein includes: a four-transmembrane protein CLDN18.2, and the amino acid sequence of the four-transmembrane protein CLDN18.2 is shown in SEQ ID NO:2.

[0036] In another preferred embodiment, the final concentration of the nanodisc is 0.1 mg / mL-20 mg / mL, preferably 0.5 mg / mL-10 mg / mL, and more preferably 1 mg / mL-5 mg / mL.

[0037] In a second aspect of the present invention, a method for preparing the nanoparticle complex according to the first aspect of the present invention is provided, comprising the following steps:

[0038] The nanodisc complex is combined with a solid phase carrier by a combination method selected from the group consisting of:

[0039] Streptavidin / Biotin, Ni-NTA / His-Tag, Strep-Tactin / Strep-tag II, SpyCatcher / SpyTag, Glutathione / GST-Tag, or a combination thereof.

[0040] In another preferred embodiment, the solid phase carrier includes: magnetic beads, resin, or a combination thereof.

[0041] In another preferred embodiment, the nanodisc comprises: lipid binding protein and lipid molecules; wherein,

[0042] The lipid binding protein surrounds the lipid molecules; the lipid molecules form a lipid layer, and the membrane protein is embedded in the lipid layer.

[0043] In another preferred embodiment, the C-terminus of the amino acid sequence of the membrane protein embedded in the nanodisc comprises a modification selected from the following group:

[0044] Biotin tag, Avi tag, His tag, Strep-tag, Flag-tag, myc-tag, HA-tag, GFP, or a combination thereof.

[0045] In the third aspect of the present invention, an analysis or screening method is provided, which uses the nanoparticle complex described in the first aspect of the present invention as a binding element to bind to cells or proteins for analysis or screening.

[0046] In another preferred embodiment, the method is suitable for screening therapeutic antibodies and studying receptor-ligand interactions.

[0047] In a fourth aspect of the present invention, a detection product is provided, wherein the product contains the nanoparticle complex described in the first aspect of the present invention.

[0048] In another preferred embodiment, the detection products include but are not limited to products or kits for the following detections:

[0049] Flow cytometry, enzyme-linked immunosorbent assay (ELISA), Western Blot, dynamic light scattering (DLS), or a combination thereof.

[0050] In a fifth aspect of the present invention, a flow cell sorting method is provided, comprising the following steps:

[0051] (a) providing a cell population to be screened,

[0052] (b) using the nanoparticle complex described in the first aspect of the present invention as a cell sorting marker to perform flow cytometry sorting to obtain cells bound to the nanoparticle complex.

[0053] In another preferred embodiment, the cell population is isolated from PBMCs of the peripheral blood of patients with target membrane protein-related diseases.

[0054] In another preferred embodiment, the cells include B cells.

[0055] In another preferred embodiment, the B cells express antibodies targeting target membrane proteins.

[0056] In another preferred embodiment, the target membrane protein includes at least one of a peripheral membrane protein, an embedded membrane protein, an integral membrane protein, a receptor protein and a G-protein coupled receptor.

[0057] In another preferred embodiment, the integral membrane protein is selected from the following group: seven-transmembrane protein MRGPRX2, four-transmembrane protein CLDN18.2, ion channel protein Nav1.7, receptor protein CD19, or a combination thereof.

[0058] In a sixth aspect of the present invention, a method for screening antibodies targeting a target membrane protein is provided, comprising:

[0059] (1) providing a B cell population to be screened;

[0060] (2) using the nanoparticle complex described in the first aspect of the present invention to label B cells expressing the target membrane protein antibody in the B cell population to be screened, to obtain a ternary complex of B cell-target membrane protein antibody-antigen complex;

[0061] (3) obtaining the ternary complex of the B cell-target membrane protein antibody-antigen complex in (2) by flow cytometry sorting, thereby obtaining sorted B cells;

[0062] (4) Cloning the sorted B cells in vitro to obtain antibodies targeting target membrane proteins.

[0063] In another preferred embodiment, the in vitro cloning includes: in vitro reverse transcription, PCR, sequencing, and recombinant expression.

[0064] In another preferred embodiment, the antigen complex is a nanodisc fused with a target membrane protein tagged with a fluorescent tag, preferably a nanodisc fused with a target membrane protein tagged with GFP.

[0065] In another preferred embodiment, the step (3) includes collecting GFP-positive cells, i.e., the ternary complex of B cells, target membrane protein antibody, and nanodisc, by flow cytometry sorting.

[0066] In the seventh aspect of the present invention, a use of the nanoparticle complex as described in the first aspect of the present invention is provided, for preparing a detection reagent or detection kit for detecting autoantibodies against target protein-related diseases, or for preparing medical materials for treating target protein-related diseases, or for preparing antibodies, or for receptor-ligand binding research.

[0067] In another preferred embodiment, the patient suffering from the "anti-target protein-related disease" has autoantibodies against the target protein.

[0068] In another preferred embodiment, the anti-target protein-related disease is selected from the following group:

[0069] Allergic diseases treated with MRGPRX2, malignancies treated with Claudin 18.2 or PD-L1, or a combination thereof.

[0070] In another preferred embodiment, the tumor is selected from the group consisting of gastric cancer, pancreatic cancer, esophageal cancer, or a combination thereof.

[0071] In an eighth aspect of the present invention, a biological detection kit for detecting target protein antibodies is provided, wherein the kit comprises: the nanoparticle complex as described in the first aspect of the present invention.

[0072] In another preferred embodiment, the biological detection comprises a detection selected from the following group:

[0073] Flow cytometry, enzyme-linked immunosorbent assay (ELISA), Western Blot, dynamic light scattering (DLS), or a combination thereof.

[0074] In another preferred embodiment, the target membrane proteins are membrane proteins of the same or different species.

[0075] In another preferred embodiment, the enzyme-linked immunosorbent assay (ELISA) detection kit further comprises: a microplate, a negative control, a positive control, a color developing solution A, a color developing solution B and a washing solution.

[0076] In another preferred embodiment, the microplate solid phase of the enzyme-linked immunosorbent assay (ELISA) detection kit is coated with the nanoparticle complex, and when certain specific membrane protein Nanodiscs are randomly adsorbed on the solid phase surface of the microplate, they often cause extracellular domain epitope masking, resulting in reduced antibody or ligand binding efficiency;

[0077] The nanoparticle complex specifically anchors the extracellular domain of the membrane protein to the surface of the nanoparticle complex through a directional fixation technology (such as C-terminal biotinylation and streptavidin nanosphere coupling, or C-terminal His tag / Ni-NTA microsphere specific coupling). Random coating will not affect the exposure of the epitope recognized by its antibody or ligand, reducing the risk of false negatives caused by epitope inaccessibility, and improving the sensitivity and specificity of detection based on membrane protein interactions.

[0078] In a ninth aspect of the present invention, a method for screening antibodies is provided, comprising using the nanoparticle complex described in the first aspect of the present invention or the biological detection kit described in claim 6 to detect whether a sample contains target protein antibodies.

[0079] In another preferred embodiment, the sample is selected from the following group: a whole blood sample, a serum sample, a plasma sample, a cerebrospinal fluid sample, a tissue sample, or a combination thereof.

[0080] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0081] In another preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0082] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 Shown is a schematic diagram of the structure of a nanosphere assembled using biotinylated MRGPX2 nanodisc as an example in one embodiment of the present invention, wherein the multi-channel transmembrane protein can be selected from MRGPX2 or other membrane proteins, and the biotinylated C-terminal capture nanodisc is combined with streptavidin-modified magnetic beads or resin to assemble nanospheres that can ensure the correct orientation of the extracellular domain of the protein.

[0084] Figure 2 Shown is a graph showing the results of detecting the binding activity of MRGPX2 nanospheres and MRGPX2 antibodies using flow cytometry in one embodiment of the present invention.

[0085] Figure 3 Shown is a graph comparing the activity of Claudin8.2 nanospheres and Claudin8.2 nanodiscs detected by ELISA in one embodiment of the present invention.

[0086] Figure 4 Shown is a diagram showing the size results of assembled nanosphers of different sizes detected using a Biotek nanoparticle size potential analyzer in one embodiment of the present invention.

[0087] Figure 5 Shown is a graph showing the results of Western Blot (WB) detection of MRGPX2 nanospheres and VLPs loaded with nanodiscs in one embodiment of the present invention. DETAILED DESCRIPTION

[0088] After extensive research, the inventors discovered that nanospheres, prepared by capturing nanodiscs at their C-termini with biotinylation or other tags, ensure the proper orientation of the protein's extracellular domain, similar to its natural positioning in the cell membrane, helping to maintain functional integrity in research and therapeutic applications. This led to the present invention.

[0089] the term

[0090] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0091] As used herein, the term “including” or “comprising” encompasses “comprising,” “consisting mainly of,” “consisting essentially of,” and “consisting of;” “consisting mainly of,” “consisting essentially of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0092] As used herein, the terms "Nanospheres", "nanospheres" and "nanoparticle complexes" are used interchangeably to refer to the nanoparticle complexes described in the first aspect of the present invention.

[0093] Nanomaterials

[0094] Nanomaterials, primarily granular materials, such as Fe₃O₄ nanoparticles, have extensive applications in biomedical fields such as magnetic resonance imaging contrast agents, magnetically targeted drug carriers, cell and biomolecule separation, biosensing and detection, and magnetically induced tumor hyperthermia due to their rich magnetic properties and excellent biocompatibility. Magnetic nanoparticles possess excellent magnetic properties, dispersibility, and stability, and are widely used in nanoprobe construction, magnetic resonance imaging and molecular imaging, magnetic hyperthermia, drug delivery, and integrated targeted diagnosis and therapy research. This invention combines nanodiscs with nanoparticle materials to develop a high-density membrane protein display format.

[0095] MRGPRX2(Mas-related G-protein coupled receptor X2)

[0096] MRGPRX2 is a multipass transmembrane protein belonging to the G protein-coupled receptor (GPCR) family. It has seven transmembrane domains and plays a key role in mast cell-mediated anaphylactoid reactions. Its transmembrane properties make it an important target for drug development.

[0097] Claudin 18.2 (CLDN18.2)

[0098] Claudin 18.2 (CLDN18.2) is a transmembrane protein belonging to the Claudin protein family with a typical four-transmembrane structure. CLDN18.2 has four transmembrane regions (TM1-TM4), with its N-terminus and C-terminus both located on the inner side of the cell membrane, while two extracellular loops (ECL1 and ECL2) are exposed to the extracellular environment. As a member of the Claudin family, it is mainly located in the tight junctions (TJs) of epithelial cells and is involved in regulating paracellular permeability and cell polarity. In healthy tissues, Claudin 18.2 is only limitedly expressed in differentiated epithelial cells of the gastric mucosa, while its expression is significantly upregulated in a variety of malignant tumors (such as gastric cancer, pancreatic cancer, esophageal cancer, etc.). Due to its specific high expression in tumors, Claudin 18.2 has become a popular target for antibody drugs (such as Zolbetuximab / IMAB362), CAR-T therapy, and ADC drugs.

[0099] The technical solution of the present invention has the following main advantages:

[0100] (1) The nanospheres of the present invention capture nanodiscs via biotinylation or other tagging of the C-terminus. The nanospheres ensure that the extracellular domains of proteins are correctly oriented, similar to their natural positioning in the cell membrane, helping to maintain functional integrity in research and therapeutic applications.

[0101] (2) The nanospheres of the present invention have high purity: Nanospheres display only the target transmembrane protein and do not contain unnecessary host membrane proteins such as virus-like particles (VLPs), thereby significantly reducing background noise.

[0102] (3) The nanospheres of the present invention have high density: each Nanosphere is densely packed with membrane proteins, which can provide a significantly higher transmembrane protein concentration compared to VLPs.

[0103] (4) The nanospheres of the present invention overcome the limitations of cell-based systems. Unlike VLPs, toxic or difficult-to-express transmembrane proteins can be tightly integrated into Nanospheres.

[0104] (5) The nanospheres of the present invention can be prepared by connecting various types of nanodisks to different types of resins (magnetic or non-magnetic) using any type of conjugation method, ensuring wide applicability and flexibility, making the nanospheres of the present invention adaptable to various experimental and industrial uses.

[0105] (6) The intracellular region of a specific membrane protein is too large to be expressed on the enveloped VLP. The Nanosphere of the present invention can overcome this limitation.

[0106] Experimental Method: Preparation of Nanodisc

[0107] A method for preparing membrane proteins based on styrene-maleic anhydride copolymer (SMA) nanodiscs. The specific preparation process can be referred to: Sun C et al. Single-particle cryo-EM studies of transmembrane proteins in SMA copolymer nanodiscs. Chem Phys Lipids. 2019 Jul; 221: 114-119. doi: 10.1016 / j.chemphyslip.2019.03.007. Epub 2019 Mar 30. PMID: 30940443; PMCID: PMC6500755, comprising the following steps:

[0108] (1) Eukaryotic expression of membrane proteins: The gene encoding the target membrane protein is cloned into a eukaryotic expression vector and transfected into mammalian cells or insect cells. The target membrane protein is efficiently expressed on the host cell membrane by regulating the expression conditions (such as expression time, culture medium composition, feed composition, etc.). The cell membrane fraction containing the target membrane protein is then separated by cell lysis and centrifugation;

[0109] The target membrane protein mentioned above can be any membrane protein. The present invention takes MRGPX2 and CLDN18.2 as examples, wherein,

[0110] (1) The amino acid sequence of MRGPRX2 is shown in SEQ ID NO: 1 (Uniprot: Q96LB1):

[0111] MDPTTPAWGTESTTVNGNDQALLLLCGKETLIPVFLILFIALVGLVGNGFVLWLLGFRMRRNAFSVYVLSLAGADFLFLCFQIINCLVYLSNFFCSISINFPSFFTTVMTCAYLAGLSMLSTVSTERCLSVLWPIWYRCRRPRHLSAVVCVLLWALSLLLSILEGKF CGFLFSDGDSGWCQTFDFITAAWLIFLFMVLCGSSLALLVRILCGSRGLPLTRLYLTILLTVLVFLLCGLPFGIQWFLILWIWKDSDVLFCHIHPVSVVLSSLNSSANPIIYFFVGSFRKQWRLQQPILKLALQRALQDIAEVDHSEGCFRQGTPEMSRSSLV(SEQ ID NO:1).

[0112] (2) The amino acid sequence of CLDN18.2 is shown in SEQ ID NO: 2 (Uniprot: P56856-2):

[0113] MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCA IAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV(SEQ ID NO:2).

[0114] (2) mixing the cell membrane fraction containing the target membrane protein obtained in step (1) with SMA polymer (Cube biotech, product number 18210 or other similar products) in a buffer solution (different mixing conditions can be used for different membrane proteins) to encapsulate the membrane protein in the nanodisc structure formed by SMA-phospholipid;

[0115] (3) removing impurities or excess components by centrifugation to obtain a solution containing SMA nanodisks;

[0116] (4) Using affinity purification (such as Ni column) and size exclusion chromatography, the SMA nanodisc complex loaded with the target membrane protein is separated and purified.

[0117] The present invention can use various membrane proteins, including but not limited to lipid-anchored membrane proteins (such as RPE65), single-pass transmembrane proteins, peripheral membrane proteins, embedded membrane proteins, integral membrane proteins (such as MRGPX2), receptor proteins and G-protein coupled receptors (such as MRGPX2, CLDN18.2) in the form of nanodiscs, combined with various microspheres.

[0118] The binding mode can be various common combinations such as Streptavidin / Biotin, Ni-NTA / His-Tag, Strep-Tactin / Strep-tag II, SpyCatcher / SpyTag, Glutathione / GST-Tag, etc.

[0119] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents involved in the present invention can be obtained from commercial sources unless otherwise specified.

[0120] Example 1 Assembly of Nanosphere

[0121] Based on the above experimental method, the present invention can assemble Nanospheres of various membrane proteins. In this example, nanodiscs of two membrane proteins (such as MRGPX2 and laudin 18.2) were randomly selected and assembled into Nanospheres of various membrane proteins using different binding modes (such as Streptavidin / Biotin and His antibody / His tag).

[0122] 1.1. Taking the biotinylated MRGPX2 nanodisc as an example, the specific assembly process is as follows:

[0123] (a) Taking MRGPX2 membrane protein as an example, the C-terminus of the MRGPX2 membrane protein sequence contains an Avi-tag or biotin tag, which is biotinylated by in vivo or in vitro labeling to obtain a biotinylated MRGPX2 nanodisc.

[0124] (b) Using streptavidin-magnetic beads as an example, transfer 20 μl of magnetic beads (Sangon Biotechnology, Order No. D112005 or other similar products) to a 1.5 ml EP tube. Add 400 μl of PBS and mix thoroughly. Place the tube on a magnetic rack. After approximately 1 minute, when the beads are fully adsorbed, discard the supernatant and resuspend in 100 μl of PBS.

[0125] (c) Add the biotinylated MRGPX2 Nanodisc to the treated magnetic beads, ensuring an excess of Nanodisc is added (typically, no more than 20 μg of beads are bound to 20 μg of Nanodisc). Incubate the beads on a rotary mixer at room temperature for 30 minutes. After incubation, place the EP on a magnetic stand. Once the beads are fully adsorbed, discard the supernatant and resuspend in 400 μl of PBS. Place the beads on the magnetic stand again, discard the supernatant, and resuspend in 100 μl of PBS.

[0126] The schematic diagram of the obtained Nanosphere structure is shown in the figure below. Figure 1The nanospheres of the present invention capture nanodiscs via their biotinylated C-termini, ensuring that the extracellular domains of proteins are properly oriented, similar to their natural positioning in the cell membrane, helping to maintain functional integrity in research and therapeutic applications.

[0127] 1.2. Taking His-tagged Claudin 18.2 as an example, the specific assembly process is as follows:

[0128] (a) Taking Claudin18.2 membrane protein as an example, the C-terminus of the Claudin18.2 membrane protein sequence was tagged with a His tag, and the preparation method was the same as the above Naodisc preparation method; a Claudin 18.2 nanodisc with a His tag was obtained.

[0129] (b) Place 20 μl of magnetic beads containing His antibodies (Beyotime, P2135, BeyoMag" Anti-His Magnetic Beads, or other similar products) into a 1.5 ml EP tube. Add 400 μl of PBS and mix thoroughly. Place the tube on a magnetic rack. After approximately 1 minute, when the beads are fully adsorbed, discard the supernatant and resuspend in 100 μl of PBS.

[0130] (c) Add 20 μg of His-tagged Claudin18.2 Nanodisc to the treated magnetic beads and incubate on a rotary mixer at room temperature for 30 min. After incubation, place the EP on a magnetic rack. Once the beads are fully adsorbed, discard the supernatant and resuspend in 400 μl of PBS. Place the EP on the magnetic rack again, discard the supernatant, and resuspend in 100 μl of PBS.

[0131] In addition, nanodisks of multiple membrane proteins can be assembled simultaneously on the nanosphere of the present invention. By loading multiple membrane proteins on a solid phase carrier in different proportions, a nanosphere loaded with multiple membrane proteins can be obtained.

[0132] Example 2 Flow cytometer detection of nanospheres

[0133] As a cell-free alternative to traditional fluorescence-activated cell sorting (FACS) techniques, Nanosphere technology enables stable and uniform display of transmembrane proteins, eliminating interference from intrinsic cellular activity. This technology platform is particularly suitable for therapeutic antibody screening and receptor-ligand interaction research.

[0134] Take MRGPX2 nanosphere as an example

[0135] The last 100ul in 1.1 of Example 1 was resuspended, and 2.5ul of 0.2mg / ml PE fluorescently labeled MRGX2 antibody (Anti-MRFPRX2-Ab-PE, Biolegend) was added to the prepared biotinylated MRGPX2 nanospheres. Blank magnetic beads were used as a control and incubated at room temperature for 30min. After incubation, the cells were placed on a magnetic rack, the supernatant was discarded, 400ul PBS was added to resuspend the cells, and the cells were placed on a magnetic rack again. After discarding the supernatant, 300ul PBS was added to resuspend the cells and tested on an Agilent Novocyte 2000R flow cytometer.

[0136] The results are as follows Figure 2 As shown, blank magnetic beads (Beads only) did not bind to the MRGX2 antibody, and the magnetic beads after adding biotinylated MRGPX2 nanodisc had obvious signals (the signal value of Q2-4 increased from 1.45% to 99.98%).

[0137] Example 3 ELISA detection of nanospheres

[0138] In the ELISA microplate solid-phase coating system, when certain specific membrane protein Nanodiscs are randomly adsorbed on the solid-phase surface of the well plate, they often cause masking of the extracellular domain epitope, resulting in reduced antibody or ligand binding efficiency. In contrast, Nanosphere nanospheres use directional immobilization technology (such as C-terminal biotinylation and streptavidin nanosphere coupling, or C-terminal His tag / Ni-NTA microsphere specific coupling) to specifically anchor the extracellular domain of membrane proteins to the nanosphere surface. Random coating will not affect the exposure of epitopes recognized by antibodies or ligands, reducing the risk of false negatives caused by epitope inaccessibility and improving the sensitivity and specificity of detection based on membrane protein interactions.

[0139] Taking Claudin18.2 nanosphere as an example

[0140] 3.1 Nanosphere Coating: Resuspend the last 100 μl of Claudin18.2 nanospheres prepared in 1.2 of Example 1, dilute 50-fold with PBS, and add 100 μl to an ELISA plate. Centrifuge at 3000 rpm for 5 minutes. Add the coating solution as a blank control and incubate at 2-8°C overnight. Wash the plate once using a plate washer. After washing, pat dry any remaining wash solution on absorbent paper. Use 0.5 μg of Claudin18.2 nanodisc coating per well.

[0141] 3.2 Blocking: Place 300 μL / well of 3% BSA in a microplate shaker at 37°C for 1 hour. Wash the plate three times using a plate washer. After washing, pat dry any remaining wash solution on absorbent paper.

[0142] 3.3 Primary Antibody Dilution: Dilute the primary antibody Anti-Claudin 18.2 three-fold starting from 10 μg / mL (i.e., 1:3 dilution) to obtain 11 concentration points. Load the sample at 100 μL / well according to the antibody gradient. Simultaneously add a blank control (diluent). Place the plate in a microplate shaker and shake at 600 rpm at 37°C for 1 hour. Wash the plate three times with a plate washer. After washing, pat the remaining wash solution in the plate dry on absorbent paper.

[0143] 3.4 Secondary Antibody Dilution: Dilution of the Anti-hFc-HRP enzyme-linked antibody: 1:15,000. Add the enzyme-linked antibody diluent and place the plate in a microplate shaker at 600 rpm at 37°C for 1 hour. Wash the plate three times using a microplate washer. After washing, pat dry any remaining wash solution on absorbent paper.

[0144] 3.5 Color Development & Termination & Reading

[0145] Add 100 μL / well of colorimetric solution and incubate in the dark at room temperature for 20 minutes or at 37°C for 10 minutes. Terminate the reaction by adding 50 μL / well of stop solution. Measure absorbance at 450 nm using a microplate reader.

[0146] The results are as follows Figure 3 As shown in the figure, in the ELISA microplate solid phase coating system, when certain specific membrane protein Nanodiscs are randomly adsorbed on the solid phase surface of the well plate, it often causes masking of the extracellular domain epitope, resulting in reduced antibody or ligand binding efficiency. In contrast, Nanosphere nanospheres use directional immobilization technology (such as C-terminal biotinylation and streptavidin nanosphere coupling, or C-terminal His tag / Ni-NTA microsphere specific coupling) to specifically anchor the extracellular domain of membrane proteins to the nanosphere surface. Random coating will not affect the exposure of epitopes recognized by antibodies or ligands, reducing the risk of false negatives caused by epitope inaccessibility and improving the sensitivity and specificity of detection based on membrane protein interactions.

[0147] Example 4 Dynamic Light Scattering (DLS) Detection of Nanospheres

[0148] Take MRGPX2 nanosphere as an example

[0149] Resuspend 200ul of MRGPX2 nanospheres of different sizes and add them into a cuvette, which is then placed into the Biotek nanoparticle size potential analyzer.

[0150] The results are as follows Figure 4 As shown in the figure, the assembled nanospheres of different sizes have a uniform size, i.e., a single peak. This indicates that the Nanospheres of the present invention can be selected from a particle size range of 30-1000 nm. Therefore, Nanospheres of different particle sizes can be selected for different application scenarios.

[0151] Example 5 WB detection of nanospheres

[0152] This example is used to demonstrate that nanospheres have high-density membrane proteins: each Nanosphere is densely packed with membrane proteins, which can provide significantly higher transmembrane protein concentrations compared to VLPs. Taking MRGPX2 nanosphere as an example,

[0153] Equal amounts (5 μg) of MRGPX2 nanospheres and MRGPX2 VLPs were added to reducing loading buffer and boiled at 95°C for 10 minutes. 10 μl of sample was loaded onto an SDS-PAGE gel and electrophoresed at a constant voltage of 180 volts for 35 minutes. After electrophoresis, the protein gel and activated PVDF membrane were placed in a transfer chamber in that order, filled with sufficient transfer buffer to submerge them, and the outside of the transfer chamber was kept ice-cooled, making sure not to touch the gel with gloves. The transfer was completed at a constant current of 400 mA for 45 minutes. After transfer, the PVDF membrane was transferred to a clean box with forceps and blocked with 5% nonfat milk (PBST) at room temperature for 1 hour or overnight at 4°C. After transfer, the membrane was transferred to another clean box with forceps and incubated with anti-HIS-HRP at a 1:5000 ratio at room temperature for 1 hour. The membrane was then transferred to a clean box and washed three times with PBST. The membrane was then developed and exposed.

[0154] Figure 5 The results show that the abundance of membrane proteins on the Nanosphere of the present invention is about 30-600 times higher than that on VLP. Different membrane proteins may behave differently, but overall, the abundance of membrane proteins is greatly increased.

[0155] Example 6 Nanospheres can be used as highly effective immunogens

[0156] The Nanospheres of this invention can serve as highly effective immunogens, enabling the generation of highly specific antibodies against complex, multi-spanning membrane proteins (such as GPCRs and ion channels). By displaying transmembrane proteins at high density and purity while retaining their native conformation, they significantly enhance immunogenicity recognition efficiency—a characteristic that makes them an ideal technology platform for the development of monoclonal antibodies (such as therapeutic antibody drugs) and polyclonal antibodies (such as diagnostic reagent raw materials).

[0157] Example 7: Nanospheres for receptor-ligand binding studies

[0158] The Nanospheres of the present invention have controllable directionality and almost zero background interference, and can perform detailed analysis of receptor-ligand binding dynamics, and can support research on immune checkpoints, hormone receptors, and other aspects.

[0159] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A nanoparticle composite, characterized in that include: A solid phase carrier serves as a core, and a connecting layer is attached to or bound to the outer surface of the solid phase carrier; the outer side of the connecting layer is connected to a nanodisk layer; The nanodisc layer comprises n independently arranged nanodisc complexes, wherein the nanodisc complexes include nanodiscs and target proteins embedded in the nanodiscs; n is a positive integer of 10-100,000, preferably a positive integer of 100-50,000, and more preferably a positive integer of 500-10,000 or 1,000-5,000.

2. The nanoparticle composite according to claim 1, wherein The target protein includes a natural protein or a recombinant protein.

3. The nanoparticle composite according to claim 1, wherein The target protein is a membrane protein, which includes (but is not limited to): at least one or more of lipid-anchored membrane proteins, single-pass transmembrane proteins, peripheral membrane proteins, embedded membrane proteins, integral membrane proteins, receptor proteins and G-protein coupled receptors.

4. The nanoparticle composite according to claim 1, wherein The connection layer includes (but is not limited to) a connection method selected from the following group: Streptavidin / Biotin, Ni-NTA / His-Tag, Strep-Tactin / Strep-tag II, SpyCatcher / SpyTag, Glutathione / GST-Tag, or a combination thereof.

5. The nanoparticle composite according to claim 1, wherein The solid phase carrier includes: magnetic beads, resin, or a combination thereof.

6. The nanoparticle composite according to claim 1, wherein The nanodisc comprises: lipid binding protein and lipid molecules; wherein, The lipid binding protein surrounds the lipid molecules; the lipid molecules form a lipid layer, and the target protein is embedded in the lipid layer.

7. The nanoparticle composite according to claim 1, wherein The embedding method includes: embedding the target protein into the lipid layer through an amphiphilic copolymer, a scaffold protein or an amphiphilic protein.

8. The nanoparticle composite according to claim 1, wherein The C-terminus of the amino acid sequence of the target protein embedded in the nanodisc includes a modification selected from the following group: Biotin tag, Avi tag, His tag, Strep-tag, Flag-tag, myc-tag, HA-tag, GFP, or a combination thereof.

9. The nanoparticle composite according to claim 1, wherein The particle size of the nanoparticle complex is 10-10000 nm, preferably 20-2000 nm, and more preferably 30-1000 nm.

10. The method for preparing the nanoparticle composite according to claim 1, wherein: The steps include: The nanodisc complex is combined with a solid phase carrier by a combination method selected from the group consisting of: Streptavidin / Biotin, Ni-NTA / His-Tag, Strep-Tactin / Strep-tag II, SpyCatcher / SpyTag, Glutathione / GST-Tag, or a combination thereof.

11. An analysis or screening method, characterized in that The nanoparticle complex according to claim 1 is used as a binding element to bind to cells or proteins for analysis or screening.

12. The method according to claim 11, wherein The method is suitable for screening therapeutic antibodies and studying receptor-ligand interactions.

13. A detection product, characterized in that: The product contains the nanoparticle composite according to claim 1.

14. The detection product according to claim 13, wherein: The detection products include but are not limited to products or kits for the following detections: Flow cytometry, enzyme-linked immunosorbent assay (ELISA), Western Blot, dynamic light scattering (DLS), or a combination thereof.

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