TRPV1 nano-disc protein as well as preparation method and application thereof

Abstract

The invention discloses a TRPV1 nanodisk protein and a preparation method and application thereof, the TRPV1 nanodisk protein comprises TRPV1, MSP and lipid, and the ratio of the TRPV1 to the MSP to the lipid is 1: (2-4): 200. Compared with TRPV1 in a form of a detergent, the TRPV1 nano-disc protein disclosed by the invention has the advantages that the protein stability and expression quantity are improved, and the immune titer of macromolecular antigen immunization is improved.

Description

Technical Field

[0001] The present invention relates to the field of immunotherapy, and in particular to a TRPV1 nanodisc protein and a preparation method and application thereof. Background Art

[0002] TRPV1 channel is a representative member of the TRPV subfamily and is the most clearly characterized TRP channel. TRPV1 channels are mainly distributed in sensory neurons of the peripheral nervous system (PNS), such as small and medium-diameter neurons of dorsal root ganglion (DRG), trigeminal ganglion (TG) and sympathetic ganglion. TRPV1 channels are also distributed in peptidergic or non-peptidergic C fibers and some Aδ fibers.

[0003] TRPV1 has been widely studied as a highly effective pain target. Some proinflammatory mediators, such as prostaglandins and sympathetic amines, can sensitize TRPV1 nociceptors, thereby increasing pain or hyperalgesia. In bone cancer, TRPV1 is overexpressed in DRG neurons, leading to neuropathic pain. In a rat model of bone cancer, endogenous tissue-derived formaldehyde upregulates TRPV1 expression through mitogen-activated protein kinase (MAPK) and phosphoinositide kinase signaling, supporting the role of TRPV1 in cancer pain. In chronic pancreatitis, both the expression and function of TRPV1 in DRG neurons are upregulated, leading to hyperalgesia. Many TRPV1 antagonists have progressed to clinical trials, such as AMG 517, AZD 1386, ABT-102, MK-2295, ABT-102, MK-2295, but to date, the current research has stagnated due to extreme side effects, such as hyperthermia and loss of sensitivity to thermal pain, limiting their therapeutic use.

[0004] Compared with small molecules, large molecules have obvious advantages. Their limited central nervous system (CNS) permeability (when targeting peripheral therapy), low immunogenicity, high selectivity and favorable half-life make the development of TRPV1 antibodies an attractive alternative to biologics. However, due to the dynamic nature of its conformation and extremely small extracellular region, screening for functionally active antibodies remains a well-known challenge. As the first step in antibody screening, antigen preparation has become one of the key technologies and a prerequisite for the development of functional targeted antibodies. The current common method is to separate and purify membrane proteins with detergents, but the stability of the protein remains to be considered.

[0005] In order to screen functional TRPV1 antibodies, it is urgent to find a suitable method to prepare TRPV1 antigens that are functionally and structurally complete and can stably exist in the extracellular environment, thereby improving the immune titer and the probability of antibody screening. Summary of the invention

[0006] Since the prior art lacks a high titer TRPV1 antigen, the present invention provides a nanodisc-packaged TRPV1 antigen for immunization and screening.

[0007] The present invention solves the above technical problems through the following technical solutions:

[0008] The first aspect of the present invention provides a TRPV1 Nanodisc protein, wherein the TRPV1 Nanodisc protein comprises: TRPV1, MSP and lipid, and TRPV1:MSP:lipid=1:(2-4):200.

[0009] Preferably, the lipid is soybean lipid.

[0010] Preferably, the amino acid sequence of TRPV1 is as shown in SEQ ID NO:6.

[0011] Preferably, the TRPV1:MSP:lipid=1:3:200.

[0012] Preferably, the MSP is selected from MSP2N2, MSP1E3D1 and MSP1D1.

[0013] Preferably, the TRPV1 Nanodisc protein further comprises an N-terminal MBP tag, wherein the tag is selected from strerp, flag, GST and MBP tags, and preferably the amino acid sequence of the MBP tag is shown in SEQ ID NO: 1, and more preferably the base sequence of the MBP tag is shown in SEQ ID NO: 2.

[0014] Preferably, the amino acid sequence of MSP2N2 is as shown in SEQ ID NO: 3; preferably, the amino acid sequence of MSP2N2 further includes a tag and / or an RBS sequence.

[0015] The amino acid sequence of RBS is FCLTLRRRYTMG (SEQ ID NO: 4), which is located between the promoter and the target gene. Its main function is to enable the mRNA of the target gene to be translated into protein by binding to the ribosome after transcription. A suitable RBS sequence can increase the expression level of the target gene and make protein synthesis more efficient.

[0016] The second aspect of the present invention provides a method for preparing TRPV1 nanodisc protein, wherein the preparation method comprises: mixing the TRPV1, MSP and lipid in corresponding proportions.

[0017] In some preferred embodiments of the present invention, the mixing step is to add SEC buffer to the dried lipid, convert it into a transparent solution through ultrasound, add TRPV1, MSP and lipid in corresponding proportions, and turn it over at 4°C for 1 hour.

[0018] In some more preferred embodiments of the present invention, the method further comprises a purification step, wherein the purification comprises adding 100 mg / mL Bio-beads SM-2 and inverting at 4°C overnight; removing the Bio-beads SM-2 by gravity filtration, and performing gel filtration chromatography after concentration and centrifugation.

[0019] In some embodiments of the present invention, the TRPV1 and MSP are prepared by the following steps: preparing a recombinant vector comprising a base sequence encoding TRPV1 in the TRPV1 Nanodisc protein, the tag and MSP to produce the TRPV1 and MSP.

[0020] In some preferred embodiments of the present invention, the base sequence encoding the TRPV1 is shown as SEQ ID NO:7, the base sequence encoding the tag is shown as SEQ ID NO:2, and / or the base sequence encoding MSP is shown as SEQ ID NO:5.

[0021] In some embodiments of the present invention, the base sequence encoding the TRPV1 and the base sequence encoding the MSP protein are on the same vector or on separate vectors;

[0022] In some preferred embodiments of the present invention, the backbone of the recombinant vector is pEGBacMam plasmid and / or pET28a plasmid, and / or the recombinant vector is a recombinant baculovirus vector.

[0023] In some embodiments of the present invention, the recombinant vector is introduced into prokaryotic cells or eukaryotic cells to produce the TRPV1 and MSP. The eukaryotic cells are preferably insect cells such as sf9, or mammalian cells.

[0024] The third aspect of the present invention provides the use of the TRPV1 Nanodisc protein as described in the first aspect or the TRPV1 Nanodisc protein obtained by the preparation method described in the second aspect in macromolecular antigen immunity or biomolecule interaction.

[0025] The fourth aspect of the present invention provides the use of the TRPV1 Nanodisc protein as described in the first aspect or the TRPV1 Nanodisc protein obtained by the preparation method described in the second aspect in the preparation of a nucleic acid-encoded small molecule drug.

[0026] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0027] The reagents and raw materials used in the present invention are commercially available.

[0028] The positive and progressive effects of the present invention are:

[0029] The present invention designs an MBP tag sequence at the N-terminus of the protein, which is convenient for purifying heterologously expressed proteins on the one hand, and on the other hand, the cloning transformation also improves the stability and expression of the protein. Secondly, the present invention purified the MSP2N2 protein, explored the mixing ratio of protein, lipid and MSP protein, and successfully assembled the TRPV1 protein in the form of nanodiscs. The packaged protein can be applied to macromolecular antigen immunity, nucleic acid-encoded small molecule drug library, biomolecular interaction experiments, etc. In addition, the TRPV1 nanodisc protein provided by the present invention has improved protein stability and immune titer compared to the TRPV1 in the detergent form, and is more convenient for fixation and screening in ELISA experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The human TRPV1 sequence was cloned into the pEGBacMam vector using seamless cloning technology.

[0031] Figure 2 The human MSP2N2 sequence was cloned into the pET 28a vector using seamless cloning technology.

[0032] Figure 3 A is the SEC graph of the purification of human TRPV1 protein in detergent form. Figure 3 B is an SDS-PAGE image of the purified human TRPV1 protein in detergent form.

[0033] Figure 4 A is the SEC image of the purified human TRPV1 protein in the form of nanodiscs. Figure 4 B is an SDS-PAGE image of the purified human TRPV1 protein in the form of nanodiscs.

[0034] Figure 5 This is the result of serum titer after the third immunization.

[0035] Figure 6 This is the result of serum titer after the fourth immunization.

[0036] Figure 7 This is the result of serum titer after the fifth immunization. DETAILED DESCRIPTION

[0037] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0038] Table 1 Commercial information of reagents / materials

[0039]

[0040] Table 2 Commercial information of instruments

[0041] name supplier Part Number Incubator Shanghai Zhichu ZCZY-CS8ES Spectrophotometer Thermofish NanoDrop One Gel filtration columns cytiva Superrose 6Increase 10 / 300GL Gel electrophoresis apparatus Tianneng VE-180 electrophoresis tank ELISA reader Molecular Devices FlexStation 3

[0042] Example 1: Gene synthesis and plasmid construction

[0043] The inventors sent the wild-type TRPV1 gene, MBP gene, and modified clone sequence to Nanjing Qingke Biotechnology Co., Ltd. for synthesis, carrying EcoR1 and Not1 restriction sites, and ligated and recombined into the pEGBacMam plasmid. The pEGBacMam recombinant plasmid containing TRPV1 and MBP is as follows: Figure 1 As shown, the pET 28a recombinant plasmid containing MSP2N2 is as follows Figure 2 shown.

[0044] ① The 6×His tag sequence and the MBP sequence were connected by a GSS linker, and the MBP and the HRV 3C protease cleavage site were connected by a NSSSNNNNNNNNNNLG linker.

[0045] MBP amino acid sequence (SEQ ID NO: 1):

[0046] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT

[0047] MBP base sequence (SEQ ID NO:2):

[0048]

[0049] MSP2N2 amino acid sequence (SEQ ID NO: 3):

[0050] STFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEAL KENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQGTPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQE

[0051] RBS amino acid sequence (SEQ ID NO:4):

[0052] FCLTLRRRYTMG

[0053] MSP2N2 base sequence (SEQ ID NO: 5):

[0054] TCTACCTTCAGTAAACTTCGCGAACAACTGGGCCCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAGCCATATCTCGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAAAAGGTGGAACCGCTGCGTGCGGAACTGCAAGAGGGGGCACGCCAAAAACTCCATGAGCTCCAAGAGAAGCTCAGCCCATTAGGCGAAGAAATGCGCGATCGCGCCCGTGCACATGTTGATGCACTCCGGACTCATTTGGCGCCGTATTCGGATGAACTTCGCCAGCGTTTGGCCGCACGTCTCGAGGCGCTGAAAGAAAACGGGGGTGCCCGCTTGGCTGAGTACCACGCGAAAGCGACAGAACACCTGAGCACCTTGAGCGAAAAAGCGAAACCGGCGCTGGAAGATCTACGCCAGGGCTTATTGCCTGTTCTTGAGAGCTTTAAAGTCAGTTTTCTGTCAGCTCTGGAAGAATATACTAAAAAGCTGAATACCCAGGGTACCCCCGTGACGCAGGAATTCTGGGACAACCTGGAAAAAGAAACCGAGGGACTGCGTCAGGAAATGTCCAAAGATTTAGAAGAGGTGAAGGCCAAGGTTCAGCCATATCTCGATGACTTTCAGAAAAAATGGCAGGAAGAGATGGAATTATATCGTCAAAAGGTGGAACCGCTGCGTGCGGAACTGCAAGAG

[0055] Amino acid sequence of wild-type human TRPV1 (TRPV1-WT) (SEQ ID NO: 6):

[0056] MKKWSSTDLGAAADPLQKDTCPDPLDGDPNSRPPPAKPQLSTAKSRTRLFGKGDSEEAFPVDCPHEEGELDSCPTITVSPVITIQRPGDGPTGARLLSQDSVAASTEKTLRLYDRRSIFEAVAQNNCQDLESLLLFLQKSKKHLTDNEFKDPETGKTCLLKAMLNLHDGQNTTIPLLLEIARQTDSLKELVNASYTDSYYKGQTALHIAIERRNMALVTLLVENGADVQAAAHGDFFKKTKGRPGFYFGELPLSLAACTNQLGIVKFLLQNSWQTADISARDSVGNTVLHALVEVADNTADNTKFVTSMYNEILMLGAKLHPTLKLEELTNKKGMTPLALAAGTGKIGVLAYILQREIQEPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVLEVIAYSSSETPNRHDMLLVEPLNRLLQDKWDRFVKRIFYFNFLVYCLYMIIFTMAAYYRPVDGLPPFKMEKTGDYFRVTGEILSVLGGVYFFFRGIQYFLQRRPSMKTLFVDSYSEMLFFLQSLFMLATVVLYFSHLKEYVASMVFSLALGWTNMLYYTRGFQQMGIYAVMIEKMILRDLCRFMFVYIVFLFGFSTAVVTLIEDGKNDSLPSESTSHRWRGPACRPPDSSYNSLYSTCLELFKFTIGMGDLEFTENYDFKAVFIILLLAYVILTYILLLNMLIALMGETVNKIAQESKNIWKLQRAITILDTEKSFLKCMRKAFRSGKLLQVGYTPDGKDDYRWCFRVDEVNWTTWNTNVGIINEDPGNCEGVKRTLSFSLRSSRVSGRHWKNFALVPLLREASARDRQSAQPEEVYLRQFSGSLKPEDAEVFKSPAASGEK

[0057] Wild-type human TRPV1 (TRPV1-WT) base sequence (SEQ ID NO:7):

[0058]

[0059] Example 2: Preparation of recombinant baculovirus

[0060] (1) The recombinant pEGBacMam plasmid containing the target gene was introduced into Escherichia coli DH10Bac competent cells (Shanghai Weidi) through heat shock transformation, and cultured at 37°C for 48-72 hours in LB solid medium containing 50μg / mL kanamycin (aladdin), 7μg / mL gentamicin (aladdin), 10μg / mL tetracycline (aladdin), 100μg / mL Bluo-gal (Thermofish), and 40μg / mL IPTG (aladdin). Uniform white spots were selected and added to 5mL LB liquid medium containing three antibiotics (50μg / mL kanamycin, 7μg / mL gentamicin, and 10μg / mL tetracycline), and cultured at 37°C and 200rpm for 12-16 hours to extract the recombinant bacmid.

[0061] (2) Take a 6-well cell culture plate (Nest) and add 1×10 6 / 2mL sf9 insect cells were cultured in a constant temperature and humidity incubator at 27℃ for 30min, and 100μL Insect Medium (Sf-900 TM III SFM) was added with 10 μL of transfection reagent (Cellfectin), and 5 μg of recombinant baculovirus plasmid was added to another 100 μL Insect Medium. After mixing, the mixture was incubated at room temperature for 20 minutes. The transfection complex was evenly added to a 6-well plate, and the culture was continued at 27°C for 72 hours. The mixture was centrifuged at 6000 rpm at 4°C for 15 minutes, and the supernatant was added with 2% FBS. The mixture was stored at 4°C away from light to obtain the P1 generation recombinant baculovirus.

[0062] Take the P1 recombinant baculovirus and infect 2 mL of cells at a density of 5×10 5 / mL sf9 insect cells were cultured at 27°C for 72 hours, centrifuged at 4°C and 6000 rpm for 15 minutes, the supernatant was added with 2% FBS, and stored at 4°C in the dark to obtain the P2 generation recombinant baculovirus.

[0063] The separation density is 1×10 6 / mL of 40mL sf9 insect cells, cultured for 24 hours, took P2 generation recombinant baculovirus at a ratio of 1:100, cultured at 27°C, 120rpm for 96 hours, centrifuged at 4°C 6000rpm for 15 minutes, filtered the supernatant with a 0.22μm filter device, added 2% FBS, and stored in the dark at 4 degrees to obtain P3 generation recombinant baculovirus.

[0064] The separation density is 1×10 6 / mL of 200mL sf9 insect cells, after culturing for 24 hours, the P3 generation recombinant baculovirus was infected at a ratio of 1:100, and cultured at 27°C, 120rpm for 96 hours, centrifuged at 4°C, 6000rpm for 15 minutes, the supernatant was filtered with a 0.22μm filter device, 2% FBS was added, and stored in the dark at 4 degrees to obtain the P4 generation recombinant baculovirus.

[0065] Example 3: Protein purification and packaging

[0066] HEK-293S cells were passaged at a concentration of 1.5 × 10 6 / mL, 37°C, 8% CO2, 60% humidity, 120rpm conditions for 24h, P4 generation recombinant baculovirus was used to infect mammalian cells at a ratio of 1:10, and sodium butyrate was added for 12-18h on a shaking platform to a final concentration of 10mM.

[0067] Protein purification (detergent group): After 72 hours, centrifuge at 6000 rpm for 15 minutes at 4°C, collect the cells, resuspend the cell pellet in Lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% Protease Inhibitor Cocktail, EDTA-Free, 0.5% LMNG / 0.05% CHS, 1 mM TCEP), and invert and dissolve the membrane at 4°C for 3 hours. After the membrane was dissolved, the mixture was centrifuged at 4°C and 40,000 rpm for 45 minutes. The supernatant was flipped and combined with the MBP affinity chromatography filler at 4°C for 2.5 hours. The combined supernatant was passed through a gravity column, and the impurities were eluted with Wash buffer (50mM HEPES, pH7.4, 150mM NaCl, 1mMTCEP, 0.01% LMNG / 0.001% CHS). The target protein was eluted with Elute buffer (50mM HEPES, pH7.4, 150mM NaCl, 0.01% LMNG / 0.001% CHS, 1mM TCEP, 40mM Maltose). The target protein was concentrated with a 100KDa ultrafiltration tube to a volume of 500μL and subjected to gel filtration chromatography. The gel column model used was Superrose 6Increase 10 / 300GL (cytiva), and the buffer was SEC buffer (50mM HEPES, pH 7.4, 150 mM NaCl, 1 mM TCEP, 0.01% LMNG / 0.001% CHS), collect protein samples, test A280 concentration, and perform SDS-PAGE gel electrophoresis to detect the size and purity of the target protein.

[0068] Protein packaging (Nanodisc protein group): Take the purified TRPV1 protein sample at the SEC peak tip containing 50mM HEPES, pH7.4, 150mM NaCl, 1mM TCEP, 0.01% LMNG / 0.001% CHS components, and package it according to the molar ratio of TRPV1:MSP2N2:soybean lipid = 1:3:200. Calculate the required Soybean Polar Lipid Extract Polar (Avanti), draw the mother liquor (soybean mother liquor is 25mg / mL) with the protein loading needle, and beat the soybean mother liquor to the bottom of a clean and dry glass tube, and then use a nitrogen blower to blow dry the chloroform of the soybean mother liquor in the glass tube, which will form a little light yellow residue at the bottom of the glass tube. Then add SEC buffer and sonicate for 10 minutes. During this period, the liquid in the glass tube will first appear white turbidity and then gradually clarify and become a light yellow solution. Add TRPV1, MSP2N2 and soybean to the EP tube in proportion and flip at 4℃ for 1h. Then, 100 mg / mL Bio-beads SM-2 was added to remove the detergent in the solution, and the mixture was turned over at 4°C overnight. The Bio-beads SM-2 was removed by gravity filtration, and the filtered protein solution was concentrated to a volume of 500 μL, centrifuged at 13,000 rpm, 4°C for 10 min, and gel filtration chromatography was performed. The gel column model used was Superrose 6 Increase 10 / 300 GL (cytiva), and the buffer was SEC buffer (50 mM HEPES, pH 7.4, 150 mM NaCl). The protein samples were collected, the A280 concentration was tested, and the size and purity of the target protein were detected by SDS-PAGE gel electrophoresis.

[0069] The results of purification of human TRPV1 protein in detergent form are as follows Figure 3 A and Figure 3 As shown in B, the results of purification of human TRPV1 protein in nanodisc form are as follows Figure 4 A and Figure 4 As shown in B.

[0070] Example 4: Animal Immunization

[0071] Immunizing animals (usually mice) with antigens is the first and most important step in the preparation of monoclonal antibodies. Whether the animal's immune response to the antigen is good and whether it can produce antibodies with high titers and good specificity directly determines the difficulty of later screening of monoclonal hybridoma cell lines and the effectiveness of the antibodies obtained.

[0072] The antigen used for immunization was produced by two different protein preparation methods, nanodisc and detergent. The immunized animals were balb / c, 6-8 week old female mice, and the specific immunization scheme was as follows:

[0073] 1. Primary immunization: A certain dose of antigen (100 μg protein / animal) is mixed with Freund's complete adjuvant in a volume ratio of 1:1. The insufficient volume of antigen is replaced with the purified buffer of the corresponding protein. After emulsification, the animal is routinely immunized by subcutaneous injection at multiple points on the back.

[0074] 2. Second immunization: On the 14th day, half the amount of antigen used in the initial immunization (50 μg / animal) was mixed with Freund's incomplete adjuvant at a volume ratio of 1:1. The insufficient antigen volume was replaced with the purified buffer of the corresponding protein. After emulsification, the animals were routinely immunized by subcutaneous injection at multiple points on the back.

[0075] 3. The third immunization: On the 28th day, half the amount of antigen used in the initial immunization (50 μg / animal) was mixed with Freund's incomplete adjuvant at a volume ratio of 1:1. The insufficient volume of the antigen was replaced with the purified buffer of the corresponding protein. After emulsification, the animals were routinely immunized by subcutaneous injection at multiple points on the back.

[0076] 4. Triple-immune antiserum detection: On the 35th day, blood was collected from mice for titer detection by ELISA and coated with the protein prepared by the detergent method.

[0077] 5. Fourth immunization: On the 42nd day, half the amount of antigen used in the first immunization (50 μg / animal) was mixed with Freund's incomplete adjuvant at a volume ratio of 1:1. The insufficient amount of antigen was replaced with the corresponding protein purification buffer. After emulsification, the immunized animals were routinely injected subcutaneously at multiple points on the back.

[0078] 6. Tetra-immune antiserum detection: On the 49th day, blood was collected from mice for titer detection by ELISA and coated with the protein prepared by the detergent method.

[0079] 7. Fifth immunization: On the 56th day, half the amount of antigen used in the initial immunization (50 μg / animal) was mixed with Freund's incomplete adjuvant in a volume ratio of 1:1. The insufficient volume of antigen was replaced with the purified buffer of the corresponding protein. After emulsification, the animals were immunized by subcutaneous injection at multiple points on the back.

[0080] 8. Pentaimmune antiserum detection: On the 63rd day, blood was collected from mice for titer detection by ELISA and coated with the protein prepared by the detergent method.

[0081] 9. Generally, the fifth immunization and serum test are completed, and the immunization is terminated when the serum is diluted to 1:10000 and the test value is positive.

[0082] Example 5: Titer detection

[0083] (1) Antigen coating: Antigen coating concentration: 10 μg / mL, 100 μL per well, overnight coating at 4°C.

[0084] (2) Washing: Discard the coating solution and wash twice with PBST, 300 μL per well, oscillate for 30 seconds, and remove all water at the last wash.

[0085] (3) Blocking with blocking solution: Add 200 μL of 2% BSA (prepared immediately before use) to each well and block at 4°C overnight or at 37°C for 2 h.

[0086] (4) Washing: Discard the coating solution and wash twice with PBST, 300 μL per well, oscillate for 30 seconds, and remove all water at the last wash.

[0087] (5) Add detection solution: Add antibody to the corresponding protein (usually 10 μg / mL initial concentration, 2-fold gradient dilution), 100 μL per well, incubate at 37°C for 2 h.

[0088] (6) Washing: Discard the coating solution and wash three times with PBST, 300 μL per well, oscillate for 30 seconds, and remove all water at the last wash.

[0089] (7) Add secondary antibody: enzyme-labeled secondary antibody, diluent 2% BSA, the dilution ratio is generally (1:2500), 100 μL per well, incubate at 37°C for 1 h.

[0090] (8) Washing: Discard the coating solution and wash three times with PBST, 300 μL per well, oscillate for 30 seconds, and remove all water at the last wash.

[0091] (9) Color development: Add 100 μL of color development solution to each well and incubate at room temperature / 37°C in the dark for 10 min.

[0092] (10) Stopping: Add 100 μL of stop solution to each well and measure the absorbance at OD450 nm with a microplate reader within 10 min.

[0093] Table 3 Serum titer results after the third immunization

[0094]

[0095]

[0096] Table 4 Serum titer results after the fourth immunization

[0097]

[0098] Table 5 Serum titer results after the fifth immunization

[0099]

[0100] The serum titer results after the third immunization are shown in Table 3 and Figure 5 The serum titer results after the fourth immunization are shown in Table 4 and Figure 6 The fifth serum titer results are shown in Table 5 and Figure 7 shown.

Claims

1. A TRPV1 nanodisc protein, characterized in that The TRPV1 nanodisc protein comprises: TRPV1, MSP and lipid, and TRPV1:MSP:lipid=1:(2-4):

200.

2. The TRPV1 Nanodisc protein according to claim 1, characterized in that The lipid is soybean lipid; and / or, the amino acid sequence of TRPV1 is as shown in SEQ ID NO:6; and / or, the TRPV1:MSP:lipid=1:3:200; and / or, the MSP is selected from MSP2N2, MSP1E3D1 and MSP1D1.

3. The TRPV1 Nanodisc protein according to claim 1, characterized in that The TRPV1 Nanodisc protein further comprises an N-terminal tag, which is selected from, for example, strep, flag, GST and MBP tags. Preferably, the amino acid sequence of the MBP tag is as shown in SEQ ID NO: 1, and more preferably, the base sequence of the MBP tag is as shown in SEQ ID NO:

2.

4. The TRPV1 Nanodisc protein according to any one of claims 1 to 3, characterized in that The amino acid sequence of MSP2N2 is shown in SEQ ID NO: 3; preferably, the amino acid sequence of MSP2N2 also includes a tag and / or an RBS sequence.

5. A method for preparing the TRPV1 nanodisc protein according to any one of claims 1 to 4, characterized in that: The preparation method comprises: mixing the TRPV1, MSP and lipid in corresponding proportions; Preferably, the mixing step is to add SEC buffer to the dried lipid, to make it a transparent solution through ultrasound, to add TRPV1, MSP and lipid in corresponding proportions, and to turn at 4°C for 1 hour; More preferably, the method further comprises a purification step, wherein the purification comprises adding 100 mg / mL Bio-beads SM-2 and inverting at 4°C overnight; removing the Bio-beads SM-2 by gravity column filtration, and performing gel filtration chromatography after concentration and centrifugation.

6. The preparation method according to claim 5, characterized in that: The TRPV1 and MSP are prepared by the following steps: preparing a recombinant vector comprising a base sequence encoding TRPV1 in the TRPV1 nanodisc protein, the tag and MSP to produce the TRPV1 and MSP; Preferably, the base sequence encoding the TRPV1 is shown as SEQ ID NO:7, the base sequence encoding the tag is shown as SEQ ID NO:2, and / or the base sequence encoding MSP is shown as SEQ ID NO:

5.

7. The preparation method according to claim 6, characterized in that: The base sequence encoding the TRPV1 and the base sequence encoding the MSP protein are on the same vector or on separate vectors; Preferably, the backbone of the recombinant vector is pEGBacMam plasmid and / or pET28a plasmid, and / or the recombinant vector is a recombinant baculovirus vector.

8. The preparation method according to claim 7, characterized in that: The recombinant vector is introduced into prokaryotic cells or eukaryotic cells to produce the TRPV1 and MSP. The eukaryotic cells are preferably insect cells such as sf9, or mammalian cells.

9. Use of the TRPV1 Nanodisc protein according to any one of claims 1 to 4 or the TRPV1 Nanodisc protein obtained by the preparation method according to any one of claims 5 to 8 in macromolecular antigen immunization or biomolecular interaction, wherein the biomolecular interaction refers to affinity screening between proteins or between proteins and antibodies or small molecules, and the biomacromolecular antigen immunization refers to obtaining corresponding antibodies through animal immunization.

10. Use of the TRPV1 Nanodisc protein according to any one of claims 1 to 4 or the TRPV1 Nanodisc protein obtained by the preparation method according to any one of claims 5 to 8 in the preparation of nucleic acid-encoded small molecule drugs, preferably in a DNA-encoded compound library.

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