A method for increasing the abundance of transmembrane proteins displayed by enveloped virus-like particles

Abstract

The present invention provides a method for increasing the abundance of transmembrane proteins displayed by enveloped virus-like particles, comprising the following steps: introducing a gene encoding a fusion protein of a target protein and a gp41 protein intracellular region, and a gene encoding a skeleton protein of enveloped virus-like particles into a host cell, and culturing the host cell to bud and form enveloped virus-like particles displaying transmembrane proteins or functional fragments thereof. This method increases the abundance of transmembrane proteins or functional fragments displayed by enveloped virus-like particles without affecting the conformation of transmembrane proteins.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for efficiently displaying transmembrane proteins in enveloped virus-like particles. Background Art

[0002] Transmembrane proteins are a class of proteins that are widely present in organisms and can cross the cell membrane once or multiple times. The development of innovative drugs targeting transmembrane proteins has always been a hot research topic in the medical field. Among them, ion channels, transport proteins, G protein-coupled receptors and kinases are the largest class of drug targets in drug development. However, the preparation of transmembrane proteins faces many bottlenecks. For example, transmembrane proteins are usually embedded in the lipid bilayer, so their solubility in solution is often low. In addition, the structure of transmembrane proteins is complex, containing multiple transmembrane regions, ring structures and glycosylation modifications, which increase the difficulty of preparing transmembrane proteins.

[0003] Virus-like particles (VLP) are derived from the outer capsid protein of the virus. They are nanostructured tiny particles that are automatically assembled by one or more capsid protein components. VLP does not contain the viral genome and is non-infectious. It also has a structure similar to that of natural viruses. This special spatial structure can cause a strong immune response, including the production of antibodies and cell-mediated immune responses. Such characteristics make VLP have a wide range of application value. VLP is mainly divided into enveloped virus-like particles (eVLP) and non-enveloped virus-like particles (VLP). When correctly folded transmembrane proteins are expressed on the cell membrane, the virus-like particles formed by the capsid protein of the enveloped virus are wrapped around the cell membrane carrying the transmembrane protein by budding to form eVLP, which displays the transmembrane protein on the surface of the eVLP, so that these complex transmembrane proteins are converted into soluble, high-concentration eVLP-form proteins for immunization and antibody screening.

[0004] However, the display of transmembrane proteins by eVLP budding is random, and compared with other types of proteins, transmembrane proteins are usually expressed at lower levels. Only transmembrane proteins that are correctly folded and embedded in the cell membrane can be displayed by eVLP. Therefore, eVLPs formed by budding usually contain eVLPs that do not display transmembrane proteins or display low abundance, and the subsequent eVLP enrichment process cannot remove this part of eVLPs. eVLPs with low abundance of transmembrane proteins produce a large amount of non-specific antibodies after immunizing animals, making the screening efficiency of specific antibodies low.

[0005] Therefore, there is currently a lack of eVLP technology that can increase the abundance of transmembrane proteins displayed. Increasing the abundance of transmembrane proteins on eVLP can enhance the effectiveness of experimental animals' immune response to transmembrane proteins and the efficiency of antibody screening, which will help the development of innovative drugs targeting transmembrane proteins. Summary of the invention

[0006] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide a method for increasing the abundance of transmembrane proteins or functional fragments thereof displayed by enveloped virus-like particles (eVLPs) to solve the problems in the prior art.

[0007] To achieve the above-mentioned purpose and other related purposes, the present invention provides a method for displaying a transmembrane protein or a functional fragment thereof on eVLPs, comprising the following steps: introducing a gene encoding a fusion protein of a target protein and an intracellular region of a gp41 protein, and a gene encoding a backbone protein of an enveloped virus-like particle into a host cell, and culturing the host cell to bud and form enveloped virus-like particles displaying the transmembrane protein or a functional fragment thereof.

[0008] The gene encoding the fusion protein of the target protein and the intracellular region of the gp41 protein is introduced into the host cell in the form of a nucleic acid construct, and / or the gene encoding the enveloped virus-like particle skeleton protein is introduced into the host cell in the form of a nucleic acid construct.

[0009] The present invention also provides a VLP vaccine or VLP preparation, which comprises the virus-like particles displaying transmembrane proteins obtained by the above method.

[0010] The present invention also provides use of the above VLP preparation in preparing antibodies or screening antibodies.

[0011] As described above, the method of the present invention for increasing the abundance of transmembrane proteins or their functional fragments displayed by enveloped virus-like particles has the following beneficial effects: based on the interaction between the eVLP skeleton protein and the intracellular region of the gp41 protein, the target transmembrane protein is fused with the intracellular region of the gp41 protein to improve the membrane localization of the target transmembrane protein, thereby improving the efficiency of enveloped virus-like particles displaying transmembrane proteins or their functional fragments; simple operation steps reduce the complexity and cost of the production process; enhance the effect of the immune response of transmembrane proteins and the efficiency of antibody screening, and promote the research and development of innovative drugs targeting transmembrane proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Shown are the dynamic light scattering detection results of the eVLPs enriched by the present invention.

[0013] Figure 2 The Western blot (WB) shows that there is no Claudin 6 transmembrane protein displayed in the eVLPs enriched by the present invention. Legend: Pre: pre-stained protein marker; Positive: positive control; Full: concentrated liquid replacement sample; LS: supernatant of ultracentrifuged cells; 20%: ultracentrifuged sucrose layer; R: eVLP reconstituted supernatant reduction lane; N: reconstituted supernatant non-reducing lane.

[0014] Figure 3 The WB image shows that the Claudin 6 transmembrane protein is displayed in the eVLPs enriched by the present invention. Legend: Q: concentrated and exchanged sample; LS: supernatant layer of supernatant cells; 20%: supernatant sucrose layer; P: reconstituted sample centrifugation sediment; Pre: pre-stained protein marker; Positive: positive control; R1: reconstituted supernatant reduction lane; N1: reconstituted supernatant non-reduction lane.

[0015] Figure 4 Shown is a schematic diagram of the binding activity of Claudin 6-gp41-eVLP and its specific antibody. DETAILED DESCRIPTION

[0016] The present invention provides a method for displaying a transmembrane protein or a functional fragment thereof on an enveloped virus-like particle, comprising the following steps: introducing a gene encoding a fusion protein of a target protein and a gp41 protein intracellular region, and a gene encoding an enveloped virus-like particle skeleton protein (gag protein) into a host cell, and culturing the host cell to bud and form enveloped virus-like particles displaying the transmembrane protein or a functional fragment thereof.

[0017] The functional fragment is a partial fragment of a transmembrane protein.

[0018] In certain embodiments of the present invention, the gene encoding the fusion protein of the target protein and the gp41 protein is introduced into the host cell in the form of a nucleic acid construct, and / or the gene encoding the enveloped virus-like particle skeleton protein is introduced into the host cell in the form of a nucleic acid construct.

[0019] In certain embodiments of the present invention, the target protein is the intracellular region of a transmembrane protein or the intracellular region of a functional fragment of a transmembrane protein.

[0020] In certain embodiments of the present invention, the transmembrane protein is any transmembrane protein that can be used as a drug target, for example, a tetraspanin, a hexaspanin, a heptaphan or an eleventh transmembrane protein. Specifically, the transmembrane protein is selected from one or more of tight junction protein 6 (Claudin 6), tight junction protein 9 (Claudin 9), G protein coupled receptors (GPCRs), B lymphocyte surface antigen B1 (CD20), six-segment transmembrane epithelial antigen of the prostate 1 (STEAP1), and sodium-dependent phosphate transporter type 2b (NaPi2b).

[0021] In a preferred embodiment of the present invention, the transmembrane protein is a tetraspanin, more preferably Claudin 6 protein.

[0022] In certain embodiments of the present invention, the amino acid sequence of the intracellular region of the Claudin 6 protein comprises the amino acid sequence shown in SEQ ID NO.5.

[0023] In certain embodiments of the present invention, the nucleotide sequence encoding the intracellular region of Claudin 6 protein comprises the nucleotide sequence shown in SEQ ID NO.6.

[0024] In certain embodiments of the present invention, the gp41 protein is of human origin.

[0025] In certain embodiments of the present invention, the gp41 protein also includes a gp41 protein mutant.

[0026] In certain embodiments of the present invention, the amino acid sequence of the intracellular region of the gp41 protein comprises the amino acid sequence shown in SEQ ID NO.1, or a sequence in which at least one amino acid is substituted, deleted or added by the above amino acid sequence, or has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher homology with the above amino acid sequence.

[0027] In certain embodiments of the present invention, the nucleotide sequence encoding the intracellular region of the gp41 protein comprises the nucleotide sequence shown in SEQ ID NO.3.

[0028] In certain embodiments of the present invention, the eVLP backbone protein is one or more of HIV-1 gag protein, MLV gag protein, SIV gag protein, FIV gag protein, HTLV-1 gag protein or HTLV-1 gag protein.

[0029] In certain embodiments of the present invention, the amino acid sequence of the gag protein comprises the amino acid sequence shown in SEQ ID NO.2.

[0030] In certain embodiments of the present invention, the present invention further comprises codon optimization of the gag encoding gene.

[0031] In certain embodiments of the present invention, the nucleotide sequence encoding the gag protein comprises the nucleotide sequence shown in SEQ ID NO.4.

[0032] In certain embodiments of the present invention, the process of culturing cells is a process of assembling enveloped virus-like particles in cells, and displaying transmembrane proteins on the outer surface of eVLPs through the interaction between the backbone protein and the fusion protein.

[0033] In certain embodiments of the present invention, the nucleic acid construct comprises a nucleotide sequence encoding a backbone protein of an enveloped virus-like particle, or comprises a nucleotide sequence encoding a fusion protein.

[0034] In certain embodiments of the present invention, the nucleic acid construct encoding the backbone protein of the enveloped virus-like particle is obtained by constructing a nucleotide sequence encoding the backbone protein of the enveloped virus-like particle into an expression vector.

[0035] In certain embodiments of the present invention, the nucleic acid construct encoding the fusion protein is obtained by constructing a nucleotide sequence encoding the fusion protein into an expression vector.

[0036] In certain embodiments of the present invention, the expression vector is a mammalian expression vector, which is selected from any one of the following: pTT5 vector, pCAG vector, pcDNA expression vector, and pCMV expression vector.

[0037] In a preferred embodiment of the present invention, the gene encoding the backbone protein of the enveloped virus-like particle is constructed into the pTT5 vector.

[0038] In a preferred embodiment of the present invention, the gene encoding the fusion protein is constructed into the pcDNA3.4 vector.

[0039] In certain embodiments of the present invention, the nucleotide sequence of the nucleic acid construct encoding the enveloped virus-like particle backbone protein is shown as SEQ ID NO.7.

[0040] In certain embodiments of the present invention, the nucleotide sequence of the nucleic acid construct encoding the fusion protein is shown as SEQ ID NO.8.

[0041] In certain embodiments of the present invention, it further comprises inserting a tag sequence into the gene of the fusion protein. Preferably, the tag sequence is a Flag sequence.

[0042] In certain embodiments of the present invention, the ratio of the nucleic acid construct encoding the envelope virus-like particle backbone protein to the nucleic acid construct encoding the fusion protein introduced into the host cell is 1: (1-3). The ratio of the introduction into the host cell is selected from any of the following ranges: 1: (1-1.5), 1: (1.5-2), 1: (2-2.5), 1: (2.5-3). In a preferred embodiment of the present invention, the ratio of the nucleic acid construct encoding the envelope virus-like particle backbone protein to the nucleic acid construct encoding the fusion protein is 1: 2.

[0043] In certain embodiments of the present invention, the host cell is selected from HEK293 cells, HEK293T cells, CHO cells, Vero cells, BHK-21 cells, EXPI293 cells or insect cells. In a preferred embodiment of the present invention, the host cell is EXPI293 cells.

[0044] In certain embodiments of the present invention, the host cells are cultured at a temperature of 30-42° C. The temperature is selected from any of the following ranges: 30-32° C., 32-34° C., 34-36° C., 36-38° C., 38-40° C., 40-42° C. In a preferred embodiment of the present invention, the culture temperature is 37° C.

[0045] In certain embodiments of the present invention, the CO2 concentration for culturing host cells is 5% to 10%. The CO2 concentration is selected from any of the following ranges: 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%. In a preferred embodiment of the present invention, the CO2 concentration is 8%.

[0046] In certain embodiments of the present invention, the host cells are cultured for 60 to 80 hours. The time is selected from any of the following ranges: 60 to 65 hours, 65 to 70 hours, 70 to 75 hours, 75 to 80 hours. In a preferred embodiment of the present invention, the cell culture time is 72 hours.

[0047] In certain embodiments of the present invention, the host cells are cultured at a rotation speed of 80-150 rpm. The rotation speed is selected from any of the following ranges: 80-90 rpm, 90-100 rpm, 100-110 rpm, 110-120 rpm, 120-130 rpm, 130-140 rpm, 140-150 rpm. In a preferred embodiment of the present invention, the rotation speed is 110 rpm.

[0048] In certain embodiments of the present invention, collecting the supernatant comprises collecting by centrifugation.

[0049] The separation and purification steps include removing cell debris, concentrating, removing small molecule impurities, centrifuging and then resuspending the collected supernatant.

[0050] In certain embodiments of the present invention, the concentration method is to concentrate the supernatant after the above treatment using a hollow fiber column.

[0051] In certain embodiments of the present invention, the method for removing small molecule impurities is to dilute and replace the solution. The multiple of the solution replacement is 200-500.

[0052] In certain embodiments of the present invention, the centrifugation step is to subject the concentrated liquid-exchange sample to sucrose gradient density centrifugation, and the supernatant to 20% sucrose density ultracentrifugation. The speed of the ultracentrifugation is 80000-120000 g / min, preferably 100000 g / min. The time of the ultracentrifugation is 80-160 min, preferably 120 min.

[0053] In certain embodiments of the present invention, the preparation method further comprises dialyzing the crude pure sample to obtain a high-purity sample.

[0054] The present invention also provides a VLP preparation, which comprises enveloped virus-like particles of transmembrane virus obtained by the above method. The VLP preparation is a VLP vaccine.

[0055] The present invention also provides a method for generating an immune response, the method comprising administering the above-mentioned VLP vaccine or VLP preparation to an animal.

[0056] The present invention also provides use of the above VLP preparation in preparing antibodies or screening antibodies.

[0057] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0058] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0059] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0060] The eVLP backbone used in this example is HIV-1 gag protein, and the Gag protein alone has the ability to self-assemble into eVLP nanoparticles.

[0061] First, a nucleic acid construct containing a gag coding gene, i.e., a recombinant plasmid 1 (SEQ ID NO.7), is constructed. The present application optimizes the mammalian expression codon of the gag coding gene, adds a restriction endonuclease EcoR I cleavage site and a Kozak sequence at the 5' end, and adds a stop codon TAA and a restriction endonuclease Hind III cleavage site at the 3' end. The target gene is constructed in a pTT5 vector, i.e., a recombinant plasmid 1. Then, the recombinant plasmid 1 is transformed into Escherichia coli E.coli DH5α, a single colony is picked after overnight culture at 37°C, and colony PCR is performed after shaking at 37°C, and positive clones are selected for plasmid extraction. The extracted plasmid is sent to a third party for sequencing, and the clone with correct sequencing is selected for plasmid amplification and extraction. The extracted plasmid is sterile filtered and stored at -20°C for standby use.

[0062] First, a nucleic acid construct containing a Claudin 6 coding gene, namely a recombinant plasmid II, and a nucleic acid construct containing a Claudin6-gp41 fusion protein coding gene, namely a recombinant plasmid III (SEQ ID NO.8), were constructed respectively. The sequence of the nucleic acid construct encoding the Claudin 6 protein is the nucleotide sequence of the nucleic acid construct encoding the Claudin 6-gp41 fusion protein minus the nucleotide sequence encoding the gp41 protein. The present application optimizes the mammalian expression codon of the Claudin 6 coding gene, adds a restriction endonuclease KpnI restriction site and a Kozak sequence at the 5' end, and adds a Flag sequence, a stop codon TAA, and a restriction endonuclease XhoI restriction site at the 3' end. The target gene is constructed in a pcDNA3.4 vector, namely a recombinant plasmid II. The mammalian expression codons of the Claudin 6-gp41 fusion protein coding gene were optimized, and the restriction endonuclease KpnI restriction site and Kozak sequence were added at the 5' end; the Claudin 6 and gp41 fusion protein coding genes were directly inserted into the Flag sequence; the termination codon TAA and the restriction endonuclease XhoI restriction site were added at the 3' end. The target gene was constructed in the pcDNA3.4 vector, i.e., recombinant plasmid three. Then, the recombinant plasmids two and three were transformed into E. coli DH5α, and single colonies were picked after overnight culture at 37°C. Colony PCR was performed after shaking at 37°C, and positive clones were selected for plasmid extraction. The extracted plasmids were sent to a third party for sequencing, and the clones with correct sequencing were selected for plasmid amplification and extraction. The extracted plasmids were sterile filtered and stored at -20°C for later use.

[0063] The constructed recombinant plasmids were transfected into EXPI293 cells using PEI transfection reagent at a ratio of plasmid 1: plasmid 2 = 1: 2 and plasmid 1: plasmid 3 = 1: 2 for eVLP expression and assembly. The day before transfection, EXPI293 cells were passaged at 1×10^6 cells / mL and cultured at 37°C. On the day of transfection, the cells were counted and the cell density was adjusted to 2×10^6 cells / mL with a viability of more than 95%. Add 335 μL of recombinant plasmid 1 with a concentration of 0.8 μg / mL and 340 μL of recombinant plasmid 2 with a concentration of 1.56 μg / mL (or 445 μL of recombinant plasmid 3 with a concentration of 1.2 μg / mL) to EXPI293 culture medium to 20 mL (liquid A), add 3.6 mg of PEI transfection reagent with a concentration of 1 mg / mL to EXPI293 culture medium to 20 mL (liquid B), mix them well, slowly add liquid B to liquid A, mix well and let stand at room temperature for 20 minutes, then slowly drop the transfection mixture into 800 mL EXPI293 cells. After culturing for 72 hours at 37°C, 8% CO2, and 110 rpm, centrifuge at 4°C and 4200 rpm for 30 minutes to collect the culture supernatants of plasmid 1 & plasmid 2 and plasmid 1 & plasmid 3, respectively.

[0064] The obtained eVLP cell supernatant was centrifuged at 4000rpm, 4℃ for 20min to remove cell debris and filtered with a 0.22µm filter membrane. The supernatant was concentrated with a 750KD hollow fiber column and diluted with PBS, pH7.4, and the solution was changed more than 200 times to remove small molecule impurities. The concentrated solution sample was subjected to sucrose density gradient centrifugation. The supernatant was subjected to 20% sucrose density ultracentrifugation (100000g / min, 4℃, 2h). The precipitate was resuspended with PBS and centrifuged at 12000rpm for 5min. The precipitate was removed to obtain a crude eVLP sample. The crude sample obtained by ultracentrifugation was dialyzed to remove residual sucrose and small molecule impurities to obtain a higher purity sample. The retained sample was subjected to dynamic light scattering detection of the uniformity of enriched eVLP particles ( Figure 1 ) and Western blot analysis of transmembrane protein display ( Figure 2 and Figure 3 ),Depend on Figure 1 It can be seen that the measured diameter of eVLP is 158.59 nm, the polydispersity is 8%, and the enriched eVLP has high uniformity.

[0065] In this example, protein immunoblotting (WB) was used to detect protein expression on Claudin 6 eVLP and Claudin 6-gp41-eVLP to prove that the Claudin 6 tetraspanin protein was successfully displayed on eVLP. The specific steps are as follows:

[0066] 1) Prepare gel: prepare 10% separation gel and 5% stacking gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) respectively;

[0067] 2) Electrophoresis: Add 20 μL of sample to each well, perform electrophoresis at 180 V for 50 min;

[0068] 3) Electrotransfer (wet transfer): Place in the following order: transparent well plate, black sponge, filter paper, PVDF membrane, protein gel (placed in a mirror-symmetrical manner), filter paper (after adding this layer of filter paper, use a pipette to remove the bubbles in it), black sponge, black well plate, 80V (current maintained at 0.18-0.2A) for 1 hour;

[0069] 4) Blocking: 5% skim milk, slowly block on a horizontal shaker for 1 hour;

[0070] 5) Anti-Flag primary antibody incubation: incubate overnight at 4°C;

[0071] 6) Wash the membrane: wash 3 times with 1×TBS, 5 min each time;

[0072] 7) HRP fluorescent secondary antibody incubation: incubate at room temperature for 1 hour;

[0073] 8) Wash the membrane: wash 6 times with 1×TBS, 5 min each time;

[0074] 9) Development: ECL substrate (mix equal amounts of A and B solutions), pipette an appropriate amount of the ECL mixed solution and drop it on the PVDF membrane on the stage of the imaging device, set the exposure time to collect images.

[0075] As shown in the figure, the Flag-tagged Claudin 6 protein in Claudin 6 eVLP and Claudin 6-gp41 eVLP was detected by western blotting. The results showed that the enriched Claudin 6 eVLP did not display the Claudin 6 transmembrane protein ( Figure 2 ); The enriched Claudin 6-gp41 eVLP showed Claudin 6 transmembrane protein with a molecular weight of about 45kDa. At the same time, a broken band was detected with a molecular weight of about 35kDa, which may be caused by gp41 being cleaved by the cell's own protease ( Figure 3 ).

[0076] In this example, an enzyme-linked immunosorbent assay was used to detect the binding activity of Claudin 6-gp41-eVLP and its specific antibody, thereby proving that the Claudin 6-gp41 tetraspanin protein was successfully displayed on the eVLP in the correct conformation. The specific steps are as follows:

[0077] 1) Coating: 5 μg / mL Claudin 6-gp41 eVLP was coated on a 96-well plate (Corning, USA, Cat. No. 42592) at 4°C overnight (or 16 hours). The coating buffer used for dilution of Claudin 6-gp41 eVLP was 15 mM Na2CO3, 35 mM NaHCO3, 7.7 mM NaN3, pH 9.6;

[0078] 2) Washing: Wash the wells 4 times with 300 μL of washing buffer (TBS, 0.05% Tween-20, pH 7.4) per well. After washing, remove the remaining solution by aspiration and ensure that it is completely dry;

[0079] 3) Blocking: Each well was blocked with 200 μL blocking buffer (TBS, 2% BSA, pH 7.4) for 1.5 hours;

[0080] 4) Washing: Repeat step 2;

[0081] 5) Add samples: add 100 μL of Claudin 6 antibody (Cat# NC202) to each well and incubate at 37°C for 1 hour;

[0082] 6) Washing: Repeat step 2;

[0083] 7) Add detection antibody: add 100 μL anti-human immunoglobulin G antibody to each well and incubate at 37°C for 1 hour;

[0084] 8) Washing: Repeat step 2;

[0085] 9) Add 100 μL / well TMB solution and incubate at 37°C for 20-30 minutes, avoiding direct exposure to strong light;

[0086] 10) Stop the reaction: add 100 μL of stop solution to each well;

[0087] 11) Read the absorbance of each well on a spectrophotometer at 450 nm.

[0088] ELISA test results Figure 4 As shown, the EC50 value of Claudin 6-gp41 eVLP binding to Claudin 6 antibody is 2.33 ng / mL, indicating that Claudin 6-gp41 eVLP has good binding activity with Claudin 6 antibody, thereby proving that the Claudin 6 tetraspanin is successfully displayed on the eVLP in the correct conformation.

[0089] The sequences involved in this application are as follows:

[0090] SEQ ID NO.1

[0091] NRVRQGYSPLSFQTHLPIPRGPDRPEGIEEEGGERDRDRSIRLVNGSLALIWDDLRSLCLFSYHRLRDLLLIVTRIVELLGRRGWEALKYWWNLLQYWSQELKNSAVSLLNATAIAVAEGTDRVIEVVQGAYRAIRHIPRRIRQGLERILL

[0092] SEQ ID NO.2

[0093] MGARASVLSGGELDRWEKIRLRPGGKKKYKLKHIVWASRELERFAVNPGLLETSEGCRQILGQLQPSLQTGSEELRSLYNTVATLYCVHQRIEIKDTKEALDKIEEEQNKSKKKAQQAAADTGHSNQVSQNYPIVQNIQGQMVHQAISPRTLNAWVKVVEEKAFSPEVIPMFSALSEGATPQDLNTMLNTVGGHQAAMQMLKETINEEAAEWDRVHPVHAGPIAPGQMREPRGSDIAGTTSTLQEQIGWMTHNPPIPVGEIYKRWIILGLNKIVRMYSPTSILDIRQGPKEPFRDYVDRFYKTLRAEQASQEVKNWMTETLLVQNANPDCKTILKALGPGATLEEMMTACQGVGGPGHKARVLAEAMSQVTNPATIMIQKGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWKCGKEGHQMKDCTERQANFLGKIWPSHKGRPGNFLQSRPEPTAPPEESFRFGEETTTPSQKQEPIDKELYPLASLRSLFGSDPSSQ

[0094] SEQ ID NO.3

[0095] AATAGAGTGCGGCAGGGCTACAGCCCCCTGTCTTTCCAGACCCACCTGCCTATCCCCAGAGGCCCTGATCGGCCTGAGGGAATCGAGGAAGAGGGCGGCGAGAGAGACCGGGACAGAAGCATCCGGCTGGTGAACGGCAGCCTCGCCCTGATCTGGGACGACCTGAGATCTCTGTGCCTGTTCAGCTACCACAGACTGAGAGATCTGCTGCTGATCGTGACCAGAATTGTGGAACTGCTGGGCAGACGGGGCTGGGAAGCCCTGAAGTACTGGTGGAACCTGCTTCAATACTGGAGCCAGGAGCTGAAGAACAGCGCCGTGTCCCTGCTGAACGCCACCGCCATCGCTGTGGCCGAGGGAACAGACCGCGTCATCGAGGTGGTGCAGGGCGCTTATAGAGCCATCCGGCACATCCCAAGAAGGATCAGACAGGGCCTGGAAAGAATCCTGCTG

[0096] SEQ ID NO.4

[0097]

[0098] SEQ ID NO.5

[0099] CCTCPSGGSQGPSHYMARYSTSAPAISRGPSEYPTKNYV

[0100] SEQ ID NO.6

[0101] TGCTGCACCTGTCCTTCCGGCGGATCTCAGGGCCCAAGCCACTATATGGCCAGATACAGCACCAGCGCCCCTGCTATCAGCCGGGGCCCTAGCGAGTACCCCACAAAGAACTACGTG

[0102] SEQ ID NO.7

[0103]

[0104] SEQ ID NO.8

[0105]

[0106] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A method for displaying a transmembrane protein or a functional fragment thereof on an enveloped virus-like particle, characterized in that: The method comprises the following steps: introducing a gene encoding a fusion protein of a target protein and a gp41 protein intracellular region, and a gene encoding an enveloped virus-like particle skeleton protein into a host cell, culturing the host cell to bud and form enveloped virus-like particles displaying a transmembrane protein or a functional fragment thereof, wherein the amino acid sequence of the gp41 protein intracellular region is the amino acid sequence shown in SEQ ID NO.1; the amino acid sequence of the enveloped virus-like particle skeleton protein is the amino acid sequence shown in SEQ ID NO.2; and the target protein is the intracellular region of a transmembrane protein or the intracellular region of a functional fragment of a transmembrane protein.

2. The method according to claim 1, characterized in that The transmembrane protein is a transmembrane protein used as a drug target.

3. The method according to claim 2, characterized in that The transmembrane protein used as the drug target is a four-transmembrane protein, a seven-transmembrane protein or an eleven-transmembrane protein.

4. The method according to claim 3, characterized in that The four-transmembrane protein is Claudin 6 protein or CD20, or the seven-transmembrane protein is GPCRs, or the eleven-transmembrane protein is NaPi2b.

5. The method according to claim 1, characterized in that The nucleotide sequence encoding the intracellular region of the gp41 protein is the nucleotide sequence shown in SEQ ID NO.

3.

6. The method according to claim 1, characterized in that The nucleotide sequence encoding the enveloped virus-like particle skeleton protein is the nucleotide sequence shown in SEQ ID NO.

4.

7. The method according to claim 1, characterized in that The gene encoding the fusion protein of the target protein and the intracellular region of the gp41 protein is introduced into the host cell in the form of a nucleic acid construct, and / or the gene encoding the enveloped virus-like particle skeleton protein is introduced into the host cell in the form of a nucleic acid construct.

8. The method according to claim 7, characterized in that The method comprises any one or more of the following conditions: 1) The ratio of the nucleic acid construct encoding the envelope virus-like particle backbone protein to the nucleic acid construct encoding the fusion protein introduced into the host cell is 1: (1-3); 2) The host cell is selected from HEK293 cells, HEK293T cells, CHO cells, Vero cells, BHK-21 cells, EXPI293 cells or insect cells; 3) The host cell culture temperature is 30~42℃; 4) The CO2 concentration for culturing host cells is 5%~10%; 5) The host cells are cultured for 60 to 80 hours; 6) The rotation speed of the culture cells is 80~150rpm.

9. A VLP preparation, characterized in that The VLP preparation comprises enveloped virus-like particles that display transmembrane proteins and are obtained according to the method of any one of claims 1 to 8.

10. Use of the VLP preparation according to claim 9 in preparing antibodies or screening antibodies.

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