Anti-SLC19A1 protein monoclonal antibody

Abstract

The invention discloses an anti-human SLC19A1 protein monoclonal antibody which comprises a first monoclonal antibody and a second monoclonal antibody, and the amino acid sequence of a heavy chain variable region of the first monoclonal antibody is shown as SEQ ID NO.7; the amino acid sequence of the light chain variable region of the first monoclonal antibody is as shown in SEQ ID NO.9; the amino acid sequence of the heavy chain variable region of the second monoclonal antibody is as shown in SEQ ID NO.11; and the amino acid sequence of the light chain variable region of the second monoclonal antibody is as shown in SEQ ID NO.13. The combination of the first monoclonal antibody and the second monoclonal antibody has a potential value of researching and intervening the combination of different structural domains of the human SLC19A1 protein and detecting the combination of different structural domains of the human SLC19A1 protein.

Description

Technical Field

[0001] The present invention belongs to the field of biological products and relates to an anti-SLC19A1 protein monoclonal antibody. Background Art

[0002] Solute carrier family (SLC) is the second largest membrane protein family after G protein-coupled receptors. Its main function is to promote the transport of various substrates across biological membranes and plays an important role in a variety of physiological and pharmacological processes.

[0003] SLC19A1 is a member of this family, and its ability to transport folic acid has attracted the attention of scholars at home and abroad. Folic acid is a general term for the water-soluble components of B vitamins and is one of the nutrients necessary for normal tissue growth and development in mammalian cells. Due to its important role in one-carbon unit transfer reactions, folic acid plays an important role in basic cell metabolism such as the synthesis of thymidine, purine nucleotides, serine and methionine. Mammalian cells cannot synthesize folic acid from scratch, so they need to use transport proteins such as SLC19A1 to actively absorb folic acid to maintain basic cell life activities. In addition to transporting folic acid, SLC19A1 is also a key transporter for antifolate drugs such as methotrexate and pemetrexed. These antifolate drugs have a similar chemical structure to folic acid and can competitively inhibit the metabolic process of folic acid, playing an important role in the treatment of various diseases such as various cancers, rheumatoid arthritis and psoriasis.

[0004] In 2019, two independent studies identified a new function of SLC19A1, namely the transport of messenger molecules in the cGAS-STING pathway: cyclic dinucleotide (CDN). The cGAS-STING signaling pathway is a cytoplasmic DNA response pathway that is widely present in eukaryotic cells. After the innate immune receptor cyclic guanosine monophosphate-adenylate synthetase (cGAS) recognizes the abnormal presence of double-stranded DNA in the cytoplasm, it catalyzes the production of a special CDN: 2'3'-cGAMP. Subsequently, 2'3'-cGAMP acts as a second messenger to bind and activate the stimulator of interferon genes (STING) on the endoplasmic reticulum, further activating downstream signaling pathways and inducing a broad spectrum of immune responses. Studies have found that SLC19A1 can not only transport endogenous 2'3'-cGAMP in mammalian cells, but also transport a variety of CDNs secreted by bacteria and synthetic CDN drugs, thereby activating STING and regulating the immune response of cells. The discovery of SLC19A1's function in transporting CDN has greatly deepened our understanding of the cGAS-STING pathway, which also suggests the potential of SLC19A1 as a drug target in the treatment of tumor immunotherapy and autoimmune diseases.

[0005] Monoclonal antibodies against membrane proteins can be used to study the function of the membrane protein, provide more information to reveal the physiological and genetic characteristics of the membrane protein, and may also achieve industrial applications such as disease treatment by intervening in the membrane protein. The existing technology lacks monoclonal antibodies against SLC19A1, so it is necessary to prepare monoclonal antibodies against SLC19A1 in order to promote theoretical and applied research on the membrane protein. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the first aspect of the present invention provides a biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10;

[0007] P1: Monoclonal antibody

[0008] The monoclonal antibody is a first monoclonal antibody, a second monoclonal antibody, or a combination of the first monoclonal antibody and the second monoclonal antibody;

[0009] The first monoclonal antibody maintains specific binding activity to human SLC19A1 protein;

[0010] The first monoclonal antibody comprises a first monoclonal antibody heavy chain and a first monoclonal antibody light chain;

[0011] The first monoclonal antibody heavy chain includes a first heavy chain CDR1, a first heavy chain CDR2 and a first heavy chain CDR3;

[0012] The first monoclonal antibody light chain includes a first light chain CDR1, a first light chain CDR2 and a first light chain CDR3;

[0013] The amino acid sequence of the first heavy chain CDR1 protein is shown in SEQ ID NO.15;

[0014] The amino acid sequence of the first heavy chain CDR2 protein is shown in SEQ ID NO.17;

[0015] The amino acid sequence of the first heavy chain CDR3 protein is shown in SEQ ID NO.19;

[0016] The amino acid sequence of the first light chain CDR1 protein is shown in SEQ ID NO.21;

[0017] The amino acid sequence of the first light chain CDR2 protein is shown in SEQ ID NO.23;

[0018] The amino acid sequence of the first light chain CDR3 protein is shown in SEQ ID NO.25;

[0019] The second monoclonal antibody retains specific binding activity to human SLC19A1 protein;

[0020] The second monoclonal antibody comprises a second monoclonal antibody heavy chain and a second monoclonal antibody light chain;

[0021] The second monoclonal antibody heavy chain includes a second heavy chain CDR1, a second heavy chain CDR2, and a second heavy chain CDR3;

[0022] The second monoclonal antibody light chain includes a second light chain CDR1, a second light chain CDR2, and a second light chain CDR3;

[0023] The amino acid sequence of the second heavy chain CDR1 protein is shown in SEQ ID NO.27;

[0024] The amino acid sequence of the second heavy chain CDR2 protein is shown in SEQ ID NO.29;

[0025] The amino acid sequence of the second heavy chain CDR3 protein is shown in SEQ ID NO.31;

[0026] The amino acid sequence of the second light chain CDR1 protein is shown in SEQ ID NO.33;

[0027] The amino acid sequence of the second light chain CDR2 protein is shown in SEQ ID NO.35;

[0028] The amino acid sequence of the second light chain CDR3 protein is shown in SEQ ID NO.37;

[0029] P2: Genetically engineered monoclonal antibodies

[0030] The genetically engineered monoclonal antibody is the first genetically engineered monoclonal antibody, the second genetically engineered monoclonal antibody, or a combination of the first genetically engineered monoclonal antibody and the second genetically engineered monoclonal antibody;

[0031] The first genetically engineered monoclonal antibody maintains specific binding activity to human SLC19A1 protein;

[0032] The first genetically engineered monoclonal antibody comprises a first genetically engineered monoclonal antibody heavy chain and a first genetically engineered monoclonal antibody light chain;

[0033] The first genetically engineered monoclonal antibody heavy chain includes a first heavy chain CDR1, a first heavy chain CDR2, a first heavy chain CDR3 and a tag peptide;

[0034] The first genetically engineered monoclonal antibody light chain includes a first light chain CDR1, a first light chain CDR2, a first light chain CDR3 and a tag peptide;

[0035] The amino acid sequence of the first heavy chain CDR1 protein is shown in SEQ ID NO.15;

[0036] The amino acid sequence of the first heavy chain CDR2 protein is shown in SEQ ID NO.17;

[0037] The amino acid sequence of the first heavy chain CDR3 protein is shown in SEQ ID NO.19;

[0038] The amino acid sequence of the first light chain CDR1 protein is shown in SEQ ID NO.21;

[0039] The amino acid sequence of the first light chain CDR2 protein is shown in SEQ ID NO.23;

[0040] The amino acid sequence of the first light chain CDR3 protein is shown in SEQ ID NO.25;

[0041] The second genetically engineered monoclonal antibody maintains specific binding activity to human SLC19A1 protein;

[0042] The second genetically engineered monoclonal antibody comprises a second genetically engineered monoclonal antibody heavy chain and a second genetically engineered monoclonal antibody light chain;

[0043] The second genetically engineered monoclonal antibody heavy chain includes a second heavy chain CDR1, a second heavy chain CDR2, a second heavy chain CDR3 and a tag peptide;

[0044] The second genetically engineered monoclonal antibody light chain includes a second light chain CDR1, a second light chain CDR2, a second light chain CDR3 and a tag peptide;

[0045] The amino acid sequence of the second heavy chain CDR1 protein is shown in SEQ ID NO.27;

[0046] The amino acid sequence of the second heavy chain CDR2 protein is shown in SEQ ID NO.29;

[0047] The amino acid sequence of the second heavy chain CDR3 protein is shown in SEQ ID NO.31;

[0048] The amino acid sequence of the second light chain CDR1 protein is shown in SEQ ID NO.33;

[0049] The amino acid sequence of the second light chain CDR2 protein is shown in SEQ ID NO.35;

[0050] The amino acid sequence of the second light chain CDR3 protein is shown in SEQ ID NO.37;

[0051] The label peptide is selected from a label peptide used for separating and purifying proteins, a fluorescent protein peptide, a label peptide specifically binding to a receptor, and a label peptide specifically binding to a ligand;

[0052] P3: Antibody derivatives

[0053] The antibody derivative is a first antibody derivative, a second antibody derivative, or a combination of a first antibody derivative and a second antibody derivative;

[0054] The first antibody derivative retains specific binding activity to human SLC19A1 protein;

[0055] The protein sequence portion of the first antibody derivative contains a first heavy chain CDR1, a first heavy chain CDR2, a first heavy chain CDR3, a first light chain CDR1, a first light chain CDR2 and a first light chain CDR3;

[0056] The amino acid sequence of the first heavy chain CDR1 protein is shown in SEQ ID NO.15;

[0057] The amino acid sequence of the first heavy chain CDR2 protein is shown in SEQ ID NO.17;

[0058] The amino acid sequence of the first heavy chain CDR3 protein is shown in SEQ ID NO.19;

[0059] The amino acid sequence of the first light chain CDR1 protein is shown in SEQ ID NO.21;

[0060] The amino acid sequence of the first light chain CDR2 protein is shown in SEQ ID NO.13;

[0061] The amino acid sequence of the first light chain CDR3 protein is shown in SEQ ID NO.25;

[0062] The second antibody derivative retains specific binding activity to human SLC19A1 protein;

[0063] The protein sequence portion of the second antibody derivative contains a second heavy chain CDR1, a second heavy chain CDR2, a second heavy chain CDR3, a second light chain CDR1, a second light chain CDR2 and a second light chain CDR3;

[0064] The amino acid sequence of the second heavy chain CDR1 protein is shown in SEQ ID NO.27;

[0065] The amino acid sequence of the second heavy chain CDR2 protein is shown in SEQ ID NO.29;

[0066] The amino acid sequence of the second heavy chain CDR3 protein is shown in SEQ ID NO.31;

[0067] The amino acid sequence of the second light chain CDR1 protein is shown in SEQ ID NO.33;

[0068] The amino acid sequence of the second light chain CDR2 protein is shown in SEQ ID NO.35;

[0069] The amino acid sequence of the second light chain CDR3 protein is shown in SEQ ID NO.37;

[0070] The antibody derivative is in the form selected from the group consisting of: enzyme-labeled antibodies, fluorescently labeled antibodies, chemically modified antibodies, antibody Fab fragments, humanized antibodies, murine antibodies, camelized antibodies, single-chain antibodies, chimeric monoclonal antibodies, and modified monoclonal antibodies;

[0071] P4: RNA combination

[0072] The RNA combination is the first RNA combination, the second RNA combination, or a combination of the first RNA combination and the second RNA combination;

[0073] The first RNA combination includes a first monoclonal antibody heavy chain RNA and a first monoclonal antibody light chain RNA;

[0074] The first monoclonal antibody heavy chain RNA can be translated to obtain the first monoclonal antibody heavy chain described in P1 or the first genetically engineered monoclonal antibody heavy chain described in P2;

[0075] The first monoclonal antibody light chain RNA can be translated to obtain the first monoclonal antibody light chain described in P1 or the first genetically engineered monoclonal antibody light chain described in P2;

[0076] The second RNA combination includes a second monoclonal antibody heavy chain RNA and a second monoclonal antibody light chain RNA;

[0077] The second monoclonal antibody heavy chain RNA can be translated to obtain the second monoclonal antibody heavy chain described in P1 or the second genetically engineered monoclonal antibody heavy chain described in P2;

[0078] The second monoclonal antibody light chain RNA can be translated to obtain the second monoclonal antibody light chain described in P1 or the second genetically engineered monoclonal antibody light chain described in P2;

[0079] P5: Genetic combination

[0080] The gene combination is the first gene combination, the second gene combination, or a combination of the first gene combination and the second gene combination;

[0081] The coding sequence of the first gene combination can encode the first monoclonal antibody heavy chain described in P1 or the first genetically engineered monoclonal antibody heavy chain described in P2; and the first monoclonal antibody light chain described in P1 or the first genetically engineered monoclonal antibody light chain described in P2;

[0082] The coding sequence of the second gene combination can encode the second monoclonal antibody heavy chain described in P1 or the second genetically engineered monoclonal antibody heavy chain described in P2; and the second monoclonal antibody light chain described in P1 or the second genetically engineered monoclonal antibody light chain described in P2;

[0083] P6: Gene expression cassette combination

[0084] The gene expression cassette combination is a first gene expression cassette combination, a second gene expression cassette combination, or a combination of the first gene expression cassette combination and the second gene expression cassette combination;

[0085] The gene expression product in the first gene expression cassette combination is the first RNA combination described in P4;

[0086] The gene expression product in the second gene expression cassette combination is the second RNA combination described in P4;

[0087] P7: Genetic Engineering Vector

[0088] The genetic engineering vector is the first genetic engineering vector, the second genetic engineering vector, or a combination of the first genetic engineering vector and the second genetic engineering vector;

[0089] The first genetic engineering vector contains the first gene expression cassette described in P6;

[0090] The first monoclonal antibody heavy chain RNA and the first monoclonal antibody light chain RNA are encoded in one or two vectors;

[0091] The second genetic engineering vector contains the second gene expression cassette described in P6;

[0092] The second monoclonal antibody heavy chain RNA and the second monoclonal antibody light chain RNA are encoded in one or two vectors;

[0093] P8: Cells

[0094] The cell is a first cell, a second cell, or a combination of a first cell and a second cell;

[0095] The first cell contains the first genetic engineering vector described in P7;

[0096] The coding gene in the gene expression cassette of the first genetic engineering vector is constitutively expressed or artificially induced;

[0097] When the first monoclonal antibody heavy chain RNA and the first monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells;

[0098] The second cell contains the second genetic engineering vector described in P7;

[0099] The coding gene in the gene expression cassette of the second genetic engineering vector is constitutively expressed or artificially induced;

[0100] When the second monoclonal antibody heavy chain RNA and the second monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells;

[0101] P9: Composition

[0102] The composition contains the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetically engineered vector described in P7 or the cell described in P8; and

[0103] P10: Test kit

[0104] The kit contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8.

[0105] In some embodiments, the amino acid sequence of the human SLC19A1 protein is as shown in SEQ ID NO.1.

[0106] In some embodiments, in P7, the backbone vector of the genetic engineering vector is a pTT3 vector.

[0107] In some embodiments, in P8, the cells are 293F cells.

[0108] In some embodiments, the amino acid sequence of the heavy chain variable region of the first monoclonal antibody or the first genetically engineered monoclonal antibody is as shown in SEQ ID NO.7;

[0109] The amino acid sequence of the light chain variable region of the first monoclonal antibody or the first genetically engineered monoclonal antibody is shown in SEQ ID NO.9.

[0110] In some embodiments, the amino acid sequence of the heavy chain variable region of the second monoclonal antibody or the second genetically engineered monoclonal antibody is as shown in SEQ ID NO.11;

[0111] The amino acid sequence of the light chain variable region of the second monoclonal antibody or the second genetically engineered monoclonal antibody is shown in SEQ ID NO.13.

[0112] The second aspect of the present invention provides use of the biomaterial described in the first aspect of the present invention in preparing a preparation for identifying human SLC19A1 protein.

[0113] The third aspect of the present invention provides a method for detecting human SLC19A1 protein in a sample for non-diagnostic purposes, the method comprising the following steps:

[0114] T1: Fix the sample to be tested on the solid surface to obtain an immobilized sample;

[0115] T2: incubating the immobilized sample with a monoclonal antibody to obtain an immobilized sample of the first incubation;

[0116] The monoclonal antibody is the first monoclonal antibody or the first genetically engineered monoclonal antibody described in the first aspect of the present invention;

[0117] T4: adding the monoclonal antibody specific binding substance connected with the label to the immobilized sample of the first incubation for incubation to obtain the immobilized sample of the second incubation;

[0118] T5: Characterizing the label indirectly bound to the immobilized sample in the second incubation, and determining whether human SLC19A1 protein exists in the sample to be detected based on the presence or absence of the label.

[0119] In some embodiments, any one of the following situations B1, B2, B3 and B4 is selected:

[0120] B1: In step T1, the sample to be detected is human SLC19A1 protein, or the sample to be detected contains human SLC19A1 protein;

[0121] B2: In step T1, the solid surface is the inner surface of the well of the microplate;

[0122] B3: In step T3, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody labeled with a fluorescent molecule; in step T4, the fixed sample of the second incubation is photographed with a fluorescence microscope, and the presence of human SLC19A1 in the sample to be tested is determined based on the photographing result;

[0123] B4: In step T3, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody labeled with horseradish peroxidase; in step T4, the fixed cells of the second incubation are developed with TMB colorimetric solution, and whether human SLC19A1 exists in the sample to be tested is determined based on the color development result.

[0124] In some embodiments, the amino acid sequence of the human SLC19A1 protein is as shown in SEQ ID NO.1. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] Figure 1 This is a western-blot detection diagram of antibody binding to human SLC19A1.

[0126] Figure 2 The figure is a flow cytometry screening diagram of antibodies binding to the human SLC19A1 epitope. DETAILED DESCRIPTION

[0127] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0128] Example 1: Preparation of human SLC19A1 protein

[0129] This example constructs the immunogen (expressed by the vector Bacmid-SLC19A1) and the tag protein (expressed by the vector Bacmid-flag-SLC19A1) for enrichment of antigen-specific B cells used in the present invention.

[0130] 1. Vector Construction

[0131] The GenBank number of the human SLC19A1 (hSLC19A1) gene used in the present invention is NM_001352512.2.

[0132] The SLC19A1 protein sequence is as follows (SEQ ID NO. 1): MVPSSPAVEKQVPVEPGPD.

[0133] The preparation steps of Bacmid-SLC19A1 are as follows: Shanghai Shenggong Bioengineering Technology Service Co., Ltd. was commissioned to synthesize the DNA sequence expressing the human SLC19A1 protein shown in GenBank No. NM_001352512.2, and the codons of the insect expression system were optimized to obtain the codon-optimized SLC19A1 coding sequence fragment. The SLC19A1 coding sequence fragment was PCR amplified using the following primers F1 and F2 to obtain an amplified product. The gel-purified amplified product was linearized with BamH1 and Hind3 enzymes and pFastBac vector with C-terminal 8*His tag (purchased from Invitrogen Life Sciences Co., Ltd.) was homologously recombined according to the steps in the kit instructions to obtain a recombinant vector, and the recombinant vector was transformed into Escherichia coli Top10 strain. The plasmid was extracted and sequenced by Sanger method to verify that the recombination was correct, and the coding sequence inserted in the recombinant vector was the same as the protein sequence translated from the aforementioned SLC19A1 gene coding sequence. The verified recombinant vector was transformed into Escherichia coli DH10Bac strain for blue-white shift screening and then bacmid was extracted and named Bacmid-SLC19A1.

[0134] F1 sequence (SEQ ID NO. 2): 5'-ACCATCGGGCGCCACCATGGGATCCATGGTGCCTAGTAGCCCAGC.

[0135] R1 sequence (SEQ ID NO. 3): 5'-AGGTAGAGATTCTCGCTGCCAAGCTTCTGATTCACATTCTGCACT.

[0136] The preparation steps of Bacmid-flag-SLC19A1 are as follows:

[0137] The flag tag was integrated between the carbon terminus of hSLC19A1 and the HIS tag in the above-mentioned pFastBac-hSLC19A1 plasmid. The preparation steps of Bacmid were the same as those of Bacmid-SLC19A1 above, and it was named Bacmid-flag-SLC19A1.

[0138] Flag tag amino acid sequence (SEQ ID NO.4): DYKDDDDK.

[0139] Flag tag DNA sequence (SEQ ID NO. 5): GACTACAAGGACGACGATGA CAAG.

[0140] 2. Immunogen and Tag Protein Expression

[0141] The Sf9 cells were diluted to a concentration of 0.5 × 10 6 After 200 μg / mL, the cells were evenly plated on a 6-well plate, 2 mL per well, and cultured at 27°C for 30 minutes to allow it to adhere completely to the plate.

[0142] Take a sterilized EP tube for each of the two recombinant vectors, and dilute 10 μL of Bacmid-SLC19A1 (concentration 500 ng / uL after dilution) and Bacmid-flag-SLC19A1 (concentration 500 ng / uL after dilution) with 90 μL of Grace medium (Thermo Fisher Scientific). Take another sterilized EP tube for each of the two recombinant vectors, dilute 6 μL of transfection reagent cellfectin (Thermo Fisher Scientific) with 100 μL of Grace medium, and then mix the contents of the two EP tubes corresponding to each vector and incubate at room temperature for 30 minutes. Add 800 μL of Grace medium to the above mixed solutions to obtain two transfection solutions.

[0143] Remove the culture medium from the 6-well plate and add 2 mL of Grace medium to each well to rinse the cells; then add the two transfection solutions dropwise into the wells containing cells and incubate at 27°C for 5 hours; remove the transfection solution and add 2 mL of ESF921 insect cell culture medium (Expression Systems) containing 10% fetal bovine serum; after incubation at 27°C for 72 hours, collect the supernatant, which is the P1 virus. Since the virulence of the P1 virus is weak, the virus particles need to be further amplified for subsequent large-scale expression. Add 10 mL of 2×10 6 5% P1 virus was added to Sf9 insect cells with a concentration of 100 μg / mL, and the supernatant was collected after shaking culture at 27°C for 72 hours, which was the P2 virus. Similarly, the P4 virus was finally used as the expression virus. The expression virus formed by Bacmid-SLC19A1 was called P4 baculovirus 1, and the expression virus formed by Bacmid-flag-SLC19A1 was called P4 baculovirus 2.

[0144] 3. Protein Expression and Purification

[0145] According to the instructions of Bac-to-Bac baculovirus expression system (Invitrogen), human SLC19A1 alone or human SLC19A1 linked to a flag tag was expressed in Sf9 insect cells. Sf9 insect cells were cultured at a density of 2 to 2.5 × 10 62% (v / v) P4 baculovirus 1 and P4 baculovirus 2 were added at a concentration of 1 cell / mL for infection, and the cells were collected by centrifugation after shaking culture at 27°C for 72 hours. The cells were then resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM imidazole, 10% glycerol) supplemented with 1 mM PMSF and homogenized by ultrasonic treatment on ice.

[0146] The protein was placed in a 4°C freezer with a final concentration of 1% (w / v) n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace) and 0.1% (w / v) Cholesteryl Hemisuccinate Tris Salt (CHS, Anatrace) and gently shaken for 2 hours. After centrifugation at 4°C, 18,000rpm for 30 minutes to remove insoluble components, the supernatant was incubated with the equilibrated nickel column material and gently stirred for 1 hour. The resin was then washed with a lysis buffer supplemented with 0.06% glyco-diosgenin (GDN, Anatrace). The target protein was then eluted with an eluent (50mM Tris-HCl pH 7.5, 500mM NaCl, 500mM imidazole, 10% glycerol, 0.12% GDN) and concentrated to 750μL using a 50kDa cutoff concentrator. The protein was then further finely purified by gel filtration chromatography (SEC) on Superdex 200, and the peak of the target protein was collected. After SDS-PAGE verification, the target proteins were combined and concentrated to 5-10 mg / ml and stored at -80°C for later use. The protein expressed by P4 baculovirus 1 was called SLC19A1, and the protein expressed by P4 baculovirus 2 was called flag-SLC19A1.

[0147] Example 2: Preparation of monoclonal antibodies

[0148] Monoclonal antibodies against hSLC19A1 were generated in mice based on an antigen-specific B cell receptor sequencing method. The immunization, screening, and preparation procedures are described below.

[0149] 1. Immunization of Balb / C mice

[0150] The SLC19A1 protein obtained in Example 1 was used as an immunogen. 100 μg / mL SLC19A1 protein (solvent 20 mM Hepes pH 7.5, 150 mM NaCl, 0.01% GDN) was diluted with 150 μg / mL CpG1826 (Invitrogen) (solvent PBS) to a final concentration of 50 μg / mL of SLC19A1 protein, and mixed to obtain a mixed solution a, and then the mixed solution a and complete Freund's adjuvant were mixed at a volume ratio of 1:1 to form a suspension 1; the mixed solution a and incomplete Freund's adjuvant were mixed at a volume ratio of 1:1 to form a suspension 2.

[0151] Six-week-old Balb / c mice were immunized with suspension 1 by subcutaneous multi-point injection, with each immunization dose of 0.2 mL. 30 and 60 days after the first immunization, suspension 2 was used for two more immunizations, with each immunization dose of 0.2 mL. 90 days after the first immunization, 25 μg / mL SLC19A1 protein (solvent PBS) without adjuvant was used for booster immunization, with an immunization dose of 0.2 mL. Single-cell sequencing analysis of B cell receptor (BCR) was performed 7 days later.

[0152] 2. Single-cell BCR sequencing analysis

[0153] The immunized mice were killed, and the spleen and draining lymph node cells were removed for blocking treatment. The specific steps are:

[0154] The isolated mouse spleen cells and draining lymph node cells were incubated with blocking solution (PBS + 2mM EDTA + 10% mouse serum + TruStain FcX TM The cells were incubated with anti-mouse CD16 / 32 antibody (1:100, Biolegend) for 20 minutes, and then the blocked cells were incubated with human SLC19A1 protein carrying the flag tag (flag-SLC19A1 protein obtained in Example 1) for 30 minutes, washed once with FACS buffer (PBS+2mM EDTA+2% BSA), centrifuged, and stained with a secondary antibody, specifically APC-eFluor TM 780-labeled anti-mouse CD3e antibody (Invitrogen), PerCP-Cy5.5-labeled anti-mouse B220 antibody (BioLegend), PE-Cy7-labeled anti-mouse CD19 antibody (BioLegend), Alexa Fluor TM 488-labeled anti-mouse GL7 antibody (Invitrogen), BV421-labeled anti-mouse CD38 antibody (BioLegend), DYKDDDDY Tag (PE, BioLegend) and LIVE / DEAD TMThe cells were stained with Fixable Violet Dead Cell Stain Kit (Yellow, Invitrogen) for 30 minutes, washed once with FACS buffer, centrifuged and resuspended, and CD3 was sorted using BD ArialIII. - B220 + CD19 + CD38 - GL7 + Flag + B cells, and single cell BCR sequencing analysis (10x genomic). Sequencing data were analyzed by Cell RangerLoupe VDJ Browser 4.0.0 software to select target sequences. Among the numerous antibody sequences, the present invention selected 5# antibody and 6# antibody for subsequent research.

[0155] The 5# antibody heavy chain variable region nucleic acid sequence (SEQ ID NO.6) is as follows: GAGGTGCAGCT GGTGGAGTCTGGGGGAGACTTAGTAAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGAAGCTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTACTTACATTTACTATC GAGACAGTATAAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAACCCCCTGGGACTGGGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA.

[0156] The protein sequence of the heavy chain variable region of antibody #5 (SEQ ID NO.7) is as follows: EVQLVESGGDLV KPGGSLKLSCAASGFTFRSYGMSWVRQTPDKRLEWVATISSGGTYIYYRDSIKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCATPWDWGFAYWGQGTLVTVSA.

[0157] The nucleic acid sequence of the 5# antibody light chain variable region (SEQ ID NO.8) is as follows: GATGTTGTGATG ACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGGAGAGTGGAGGCTGAGGATTTGGGAGAGTATTATTGCTGGCAAGGAACACATTTTCCGGTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA。

[0158] The protein sequence of the 5# antibody light chain variable region (SEQ ID NO.9) is as follows: DVVMTQTPLTLS VTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKVDSGV PDRFTGSGSGTDFTLKIRRVEAEDLGEYYCWQGTHFPVTFGAGTKLELK。

[0159] The nucleic acid sequence of the 6# antibody heavy chain variable region (SEQ ID NO.10) is as follows: CAGGTGCAGCT GAAAGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACCATCTCAGGGTTCTCATTAACCAGCTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGTAGTGATATGGAATGATGGAAGCACAACCTATAATTCAGCTCTCAAATCCAGACTGAGCATCAGCAGGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTCCAAACTGCTGACACAGCCATGTACTACTGTGTCAGTTATTACTACGAGAGACTGGACTATGTTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。

[0160] The protein sequence of the heavy chain variable region of antibody #6 (SEQ ID NO.11) is as follows: QVQLKESGPGLVAPSQSLSITCTISGFSLTSYGVHWVRQPPGKGLEWLVVIWNDGSTTYNSALKSRLSISRDNSKSQVFLKMNSLQTADTAMYYCVSYYYERLDYVMDYWGQGTSVTVSS.

[0161] The nucleic acid sequence of the variable region of the 6# antibody light chain (SEQ ID NO.12) is as follows: GATGTTGTGTT GACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAAC CGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACATATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA.

[0162] The protein sequence of the light chain variable region of antibody #6 (SEQ ID NO.13) is as follows: DVVLTQTPLSLP VSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSG VPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTYVPLTFGAGTKLELK.

[0163] The nucleotide and amino acid sequences of each CDR in the variable region of antibody #5 are as follows.

[0164] Heavy chain CDR1 coding sequence (SEQ ID NO. 14): GGATTCACTTTCAGAAGCT AT.

[0165] Heavy chain CDR1 protein sequence (SEQ ID NO.15): GFTFRSY.

[0166] Heavy chain CDR2 coding sequence (SEQ ID NO.16): AGTAGTGGTGGTACTTAC.

[0167] Heavy chain CDR2 protein sequence (SEQ ID NO.17): SSGGTY.

[0168] Heavy chain CDR3 coding sequence (SEQ ID NO. 18): TGTGCAACCCCCTGGGACT GGGGGTTTGCTTACTGG.

[0169] Heavy chain CDR3 protein sequence (SEQ ID NO. 19): CATPWDWGFAYW.

[0170] Light chain CDR1 coding sequence (SEQ ID NO. 20): AAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAAT.

[0171] Light chain CDR1 protein sequence (SEQ ID NO.21): KSSQSLLDSDGKTYLN.

[0172] Light chain CDR2 coding sequence (SEQ ID NO. 22): CTGGTGTCTAAAGTGGACT CT.

[0173] Light chain CDR2 protein sequence (SEQ ID NO.23): LVSKVDS.

[0174] Light chain CDR3 coding sequence (SEQ ID NO. 24): TGCTGGCAAGGAACACATTTTCCGGTCACGTTC.

[0175] Light chain CDR3 protein sequence (SEQ ID NO.25): CWQGTHFPVTF.

[0176] The nucleotide and amino acid sequences of each CDR in the variable region of antibody #6 are as follows.

[0177] Heavy chain CDR1 coding sequence (SEQ ID NO. 26): GGGTTCTCATTAACCAGCT AT.

[0178] Heavy chain CDR1 protein sequence (SEQ ID NO.27): GFSLTSY.

[0179] Heavy chain CDR2 coding sequence (SEQ ID NO. 28): TGGAATGATGGAAGC.

[0180] Heavy chain CDR2 protein sequence (SEQ ID NO.29): WNDGS.

[0181] Heavy chain CDR3 coding sequence (SEQ ID NO. 30): TGTGTCAGTTATTACTACGAGAGACTGGACTATGTTATGGACTACTGG.

[0182] Heavy chain CDR3 protein sequence (SEQ ID NO.31): CVSYYYERLDYVMDYW.

[0183] Light chain CDR1 coding sequence (SEQ ID NO. 32): AGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACAT.

[0184] Light chain CDR1 protein sequence (SEQ ID NO.33): RSSQSLVHSNGNTYLH.

[0185] Light chain CDR2 coding sequence (SEQ ID NO. 34): AAAGTTTCCAACCGATTTT CT.

[0186] Light chain CDR2 protein sequence (SEQ ID NO.35): KVSNRFS.

[0187] Light chain CDR3 coding sequence (SEQ ID NO. 36): TGCTCTCAAAGTACATATGT TCCGCTCACGTTC.

[0188] Light chain CDR3 protein sequence (SEQ ID NO.37): CSQSTYVPLTF.

[0189] IV. Antibody Expression and Purification

[0190] The entire light and heavy chain variable regions of the target antibodies 5# and 6# selected in the previous step were subjected to codon optimization analysis respectively, and the light chain variable region of antibody 5# was codon optimized and connected to the pTT3 (Addgene) vector containing the mouse light chain κ chain constant region to obtain antibody expression vector 1, the heavy chain variable region of antibody 5# was codon optimized and connected to the pTT3 (Addgene) vector containing the mouse heavy chain IgG2a constant region to obtain antibody expression vector 2, the light chain variable region of antibody 6# was codon optimized and connected to the pTT3 (Addgene) vector containing the mouse light chain κ chain constant region to obtain antibody expression vector 3, and the heavy chain variable region of antibody 6# was codon optimized and connected to the pTT3 (Addgene) vector containing the mouse heavy chain IgG2a constant region to obtain antibody expression vector 4.

[0191] The mouse antibody constant region sequence (the aforementioned light chain κ chain constant region and mouse heavy chain IgG2a constant region) was derived from the in vitro hybridoma cell JL2 (Xuyuan Zhang et al. The binding of amonoclonal antibody to theapical region of SCARB2 blocks EV71 infection. PROTEIN&CELL, 2017.8, 8(8): 590-600.). The above antibody expression vectors were commissioned to be synthesized by Sangon Biotechnology.

[0192] The four synthesized antibody expression vectors (antibody expression vectors 1-4) were extracted with an endotoxin-free plasmid mini-extraction kit (purchased from Tiangen Bio), and then antibody expression vector 1 and antibody expression vector 2 were mixed at a molar ratio of 1:1, and antibody expression vector 3 and antibody expression vector 4 were mixed at a molar ratio of 1:1. The two mixed antibody expression vectors were mixed evenly with transfection reagent PEI and then allowed to stand. Then, they were added to 293F cells (purchased from ATCC), cultured at 37°C, 5% CO2 in a shaking incubator for 5 days, and the supernatant was centrifuged.

[0193] Protein A filler (CaptivaTM PriMAB, cat: CA-PRI-1000) was loaded into the column, and the antibody was adsorbed by the affinity chromatography column. The bound antibody was eluted from the affinity chromatography column with 0.1M glycine (pH2.7) eluent, and the antibody in the collection tube was measured for protein concentration. The liquid in the tube containing protein was combined, and the antibody was diluted with PBS (the solution dissolving the antibody was replaced with PBS), added to a 30KD ultrafiltration centrifuge tube, and centrifuged and concentrated at 3000rpm and 4°C. Recombinant monoclonal antibodies #5 and #6 were obtained.

[0194] 5. Western blot detection of antibodies

[0195] The insect cell-expressed recombinant protein hSLC19A1 purified in Example 1 was subjected to SDS-PAGE and then transferred to the NC membrane. The diluted 5# and 6# recombinant monoclonal antibodies (1:1000 volume dilution) were used as primary antibodies, and horseradish-enzyme-labeled goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) was used as secondary antibodies. The reactivity of 5# and 6# recombinant monoclonal antibodies to hSLC9A1 insect cell-expressed protein was detected by Western blot technology. In the negative control, the Sf9 insect cell lysate that did not express the recombinant protein hSLC19A1 was transferred to the NC membrane, and the serum of hSLC19A1-positive mice was used as the primary antibody; in the positive control, the Sf9 insect cell lysate that expressed the recombinant protein hSLC19A1 was transferred to the NC membrane, and the serum of hSLC19A1-positive mice was used as the primary antibody.

[0196] Results Figure 1 From left to right are negative control, positive control, 5# antibody treatment result, 6# antibody treatment result. It can be seen that the prepared 5# recombinant monoclonal antibody can be used to detect hSLC19A1 insect cell expressed protein by Western blot technology, while 6# recombinant monoclonal antibody cannot.

[0197] 6. Detection of Antibody Epitopes

[0198] The GenBank number of hSLC19A1 protein is NM_001352512.2, and the GenBank number of mouse SLC19A1 protein is NM_001199271.1.

[0199] In order to map the antigenic epitopes of recombinant mAbs #5 and #6, based on the full-length protein sequence of hSLC19A1, the present invention designed five truncated or chimeric hSLC19A1 modified fragments: hσN-16aa (deleting amino acids 1-16 of hSLC19A1), h N-16aa m (replacing amino acids 1-16 of hSLC19A1 with amino acids corresponding to positions 1-16 of mouse SLC19A1), h 1 / 2loop m (replacing amino acids 52-61 of hSLC19A1 with amino acids corresponding to positions 52-59 of mouse SLC19A1), h 6 / 7loop m (replacing amino acids 214-249 of hSLC19A1 with amino acids corresponding to positions 213-253 of mouse SLC19A1) and h 7 / 8loop m (replacing amino acids 216-249 of hSLC19A1 with amino acids corresponding to positions 213-253 of mouse SLC19A1). m (replacing amino acids 292-305 of hSLC19A1 with amino acids 296-302 of mouse SLC19A1), which contain the main extracellular region of hSLC19A1 protein. The full-length sequence of wild-type hSLC19A1 protein (codenamed hSLC19A1 WT) was used as a control.

[0200] The above five modified hSLC19A1 proteins and wild-type hSLC19A1 proteins were fused with the green fluorescent protein coding sequence and cloned into the pTT-3 vector. After extracting the plasmids, they were transfected into HEK-293T cells respectively. After culturing at 37°C for 12 hours, the cells were collected. Resuspend the cells with 1mL of PBS containing 2% PFA and let stand at 4°C for 20 minutes to fix the cells. Centrifuge at 1500rpm for 3 minutes, discard the supernatant, and resuspend the cells with 1mL of perforation buffer. Centrifuge at 1500rpm for 3 minutes, discard the supernatant, and evenly suspend the cells with 1mL of 5# and 6# antibody permeabilization buffer diluted at a volume ratio of 1:1000, and incubate for 30 minutes. Centrifuge at 1500rpm for 3 minutes, discard the supernatant, resuspend the cells with 1mL of PBS buffer, discard the supernatant after centrifugation, and wash off the excess antibodies. Resuspend the cells with a permeabilization buffer containing a goat anti-mouse IgG secondary antibody diluted at a volume ratio of 1:1000, and incubate for 30 minutes. After centrifugation at 1500 rpm for 3 minutes, discard the supernatant and wash off the excess secondary antibody. Resuspend the cells with 0.2 mL PBS buffer and use flow cytometry to detect the binding of antibodies to different hSLC19A1 proteins, using the pTT-3 vector as a control.

[0201] The results are as follows Figure 2 As shown. Figure 2 It can be seen that the five truncated or chimeric hSLC19A1-GFP proteins can be expressed smoothly, which has the basis for analyzing the binding characteristics of 5# and 6#. The results show that both 5# and 6# antibodies can bind to wild-type hSLC19A1 protein, indicating that the results of the flow cytometry experiment are highly reliable. 5# antibody cannot bind to hσN-16aa (delete amino acids 1-16) and hN-16aa m (replace amino acids 1-16 with the amino acid sequence corresponding to mouse SLC19A1) forms of hSLC19A1 protein, but can bind to other forms of hSLC19A1 protein, indicating that 5# antibody recognizes amino acid residues 1-16 of hSLC19A1. 6# antibody can bind to other forms of protein except h 6 / 7loop m (replace amino acids 214-249 with the amino acid sequence corresponding to mouse SLC19A1), so 6# antibody recognizes amino acid residues 214-249 of hSLC19A1.

[0202] It can be seen that 5# and 6# antibodies are specific to human SLC19A1 (they do not recognize the corresponding mouse epitope) and have unique value in studying human SLC19A1, especially the difference between human and mouse SLC19A1. 5# and 6# antibodies recognize different epitopes, so they can be used to jointly study the functions of different domains of human SLC19A1 (especially different transmembrane domains).

[0203] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A biomaterial, wherein the biomaterial is any one of the following P1, P2, P3, P4, P5, P6, P7, P8, P9 and P10; P1: Monoclonal antibody The monoclonal antibody is a first monoclonal antibody, a second monoclonal antibody, or a combination of the first monoclonal antibody and the second monoclonal antibody; The first monoclonal antibody maintains specific binding activity to human SLC19A1 protein; The first monoclonal antibody comprises a first monoclonal antibody heavy chain and a first monoclonal antibody light chain; The first monoclonal antibody heavy chain includes a first heavy chain CDR1, a first heavy chain CDR2 and a first heavy chain CDR3; The first monoclonal antibody light chain includes a first light chain CDR1, a first light chain CDR2 and a first light chain CDR3; The amino acid sequence of the first heavy chain CDR1 protein is shown in SEQ ID NO.15; The amino acid sequence of the first heavy chain CDR2 protein is shown in SEQ ID NO.17; The amino acid sequence of the first heavy chain CDR3 protein is shown in SEQ ID NO.19; The amino acid sequence of the first light chain CDR1 protein is shown in SEQ ID NO.21; The amino acid sequence of the first light chain CDR2 protein is shown in SEQ ID NO.23; The amino acid sequence of the first light chain CDR3 protein is shown in SEQ ID NO.25; The second monoclonal antibody retains specific binding activity to human SLC19A1 protein; The second monoclonal antibody comprises a second monoclonal antibody heavy chain and a second monoclonal antibody light chain; The second monoclonal antibody heavy chain includes a second heavy chain CDR1, a second heavy chain CDR2, and a second heavy chain CDR3; The second monoclonal antibody light chain includes a second light chain CDR1, a second light chain CDR2, and a second light chain CDR3; The amino acid sequence of the second heavy chain CDR1 protein is shown in SEQ ID NO.27; The amino acid sequence of the second heavy chain CDR2 protein is shown in SEQ ID NO.29; The amino acid sequence of the second heavy chain CDR3 protein is shown in SEQ ID NO.31; The amino acid sequence of the second light chain CDR1 protein is shown in SEQ ID NO.33; The amino acid sequence of the second light chain CDR2 protein is shown in SEQ ID NO.35; The amino acid sequence of the second light chain CDR3 protein is shown in SEQ ID NO.37; P2: Genetically engineered monoclonal antibodies The genetically engineered monoclonal antibody is the first genetically engineered monoclonal antibody, the second genetically engineered monoclonal antibody, or a combination of the first genetically engineered monoclonal antibody and the second genetically engineered monoclonal antibody; The first genetically engineered monoclonal antibody maintains specific binding activity to human SLC19A1 protein; The first genetically engineered monoclonal antibody comprises a first genetically engineered monoclonal antibody heavy chain and a first genetically engineered monoclonal antibody light chain; The first genetically engineered monoclonal antibody heavy chain includes a first heavy chain CDR1, a first heavy chain CDR2, a first heavy chain CDR3 and a tag peptide; The first genetically engineered monoclonal antibody light chain includes a first light chain CDR1, a first light chain CDR2, a first light chain CDR3 and a tag peptide; The amino acid sequence of the first heavy chain CDR1 protein is shown in SEQ ID NO.15; The amino acid sequence of the first heavy chain CDR2 protein is shown in SEQ ID NO.17; The amino acid sequence of the first heavy chain CDR3 protein is shown in SEQ ID NO.19; The amino acid sequence of the first light chain CDR1 protein is shown in SEQ ID NO.21; The amino acid sequence of the first light chain CDR2 protein is shown in SEQ ID NO.23; The amino acid sequence of the first light chain CDR3 protein is shown in SEQ ID NO.25; The second genetically engineered monoclonal antibody maintains specific binding activity to human SLC19A1 protein; The second genetically engineered monoclonal antibody comprises a second genetically engineered monoclonal antibody heavy chain and a second genetically engineered monoclonal antibody light chain; The second genetically engineered monoclonal antibody heavy chain includes a second heavy chain CDR1, a second heavy chain CDR2, a second heavy chain CDR3 and a tag peptide; The second genetically engineered monoclonal antibody light chain includes a second light chain CDR1, a second light chain CDR2, a second light chain CDR3 and a tag peptide; The amino acid sequence of the second heavy chain CDR1 protein is shown in SEQ ID NO.27; The amino acid sequence of the second heavy chain CDR2 protein is shown in SEQ ID NO.29; The amino acid sequence of the second heavy chain CDR3 protein is shown in SEQ ID NO.31; The amino acid sequence of the second light chain CDR1 protein is shown in SEQ ID NO.33; The amino acid sequence of the second light chain CDR2 protein is shown in SEQ ID NO.35; The amino acid sequence of the second light chain CDR3 protein is shown in SEQ ID NO.37; The label peptide is selected from a label peptide used for separating and purifying proteins, a fluorescent protein peptide, a label peptide specifically binding to a receptor, and a label peptide specifically binding to a ligand; P3: Antibody derivatives The antibody derivative is a first antibody derivative, a second antibody derivative, or a combination of a first antibody derivative and a second antibody derivative; The first antibody derivative retains specific binding activity to human SLC19A1 protein; The protein sequence portion of the first antibody derivative contains a first heavy chain CDR1, a first heavy chain CDR2, a first heavy chain CDR3, a first light chain CDR1, a first light chain CDR2 and a first light chain CDR3; The amino acid sequence of the first heavy chain CDR1 protein is shown in SEQ ID NO.15; The amino acid sequence of the first heavy chain CDR2 protein is shown in SEQ ID NO.17; The amino acid sequence of the first heavy chain CDR3 protein is shown in SEQ ID NO.19; The amino acid sequence of the first light chain CDR1 protein is shown in SEQ ID NO.21; The amino acid sequence of the first light chain CDR2 protein is shown in SEQ ID NO.23; The amino acid sequence of the first light chain CDR3 protein is shown in SEQ ID NO.25; The second antibody derivative retains specific binding activity to human SLC19A1 protein; The protein sequence portion of the second antibody derivative contains a second heavy chain CDR1, a second heavy chain CDR2, a second heavy chain CDR3, a second light chain CDR1, a second light chain CDR2 and a second light chain CDR3; The amino acid sequence of the second heavy chain CDR1 protein is shown in SEQ ID NO.27; The amino acid sequence of the second heavy chain CDR2 protein is shown in SEQ ID NO.29; The amino acid sequence of the second heavy chain CDR3 protein is shown in SEQ ID NO.31; The amino acid sequence of the second light chain CDR1 protein is shown in SEQ ID NO.33; The amino acid sequence of the second light chain CDR2 protein is shown in SEQ ID NO.35; The amino acid sequence of the second light chain CDR3 protein is shown in SEQ ID NO.37; The antibody derivative is in the form of: enzyme-labeled antibody, fluorescent-labeled antibody, chemically modified antibody, antibody Fab fragment, humanized antibody, murine antibody, camelized antibody, single-chain antibody, chimeric monoclonal antibody and modified monoclonal antibody; P4: RNA combination The RNA combination is the first RNA combination, the second RNA combination, or a combination of the first RNA combination and the second RNA combination; The first RNA combination includes a first monoclonal antibody heavy chain RNA and a first monoclonal antibody light chain RNA; The first monoclonal antibody heavy chain RNA can be translated to obtain the first monoclonal antibody heavy chain described in P1 or the first genetically engineered monoclonal antibody heavy chain described in P2; The first monoclonal antibody light chain RNA can be translated to obtain the first monoclonal antibody light chain described in P1 or the first genetically engineered monoclonal antibody light chain described in P2; The second RNA combination includes a second monoclonal antibody heavy chain RNA and a second monoclonal antibody light chain RNA; The second monoclonal antibody heavy chain RNA can be translated to obtain the second monoclonal antibody heavy chain described in P1 or the second genetically engineered monoclonal antibody heavy chain described in P2; The second monoclonal antibody light chain RNA can be translated to obtain the second monoclonal antibody light chain described in P1 or the second genetically engineered monoclonal antibody light chain described in P2; P5: Genetic combination The gene combination is the first gene combination, the second gene combination, or a combination of the first gene combination and the second gene combination; The coding sequence of the first gene combination can encode the first monoclonal antibody heavy chain described in P1 or the first genetically engineered monoclonal antibody heavy chain described in P2; and the first monoclonal antibody light chain described in P1 or the first genetically engineered monoclonal antibody light chain described in P2; The coding sequence of the second gene combination can encode the second monoclonal antibody heavy chain described in P1 or the second genetically engineered monoclonal antibody heavy chain described in P2; and the second monoclonal antibody light chain described in P1 or the second genetically engineered monoclonal antibody light chain described in P2; P6: Gene expression cassette combination The gene expression cassette combination is a first gene expression cassette combination, a second gene expression cassette combination, or a combination of the first gene expression cassette combination and the second gene expression cassette combination; The gene expression product in the first gene expression cassette combination is the first RNA combination described in P4; The gene expression product in the second gene expression cassette combination is the second RNA combination described in P4; P7: Genetic Engineering Vector The genetic engineering vector is the first genetic engineering vector, the second genetic engineering vector, or a combination of the first genetic engineering vector and the second genetic engineering vector; The first genetic engineering vector contains the first gene expression cassette described in P6; The first monoclonal antibody heavy chain RNA and the first monoclonal antibody light chain RNA are encoded in one or two vectors; The second genetic engineering vector contains the second gene expression cassette described in P6; The second monoclonal antibody heavy chain RNA and the second monoclonal antibody light chain RNA are encoded in one or two vectors; P8: Cells The cell is a first cell, a second cell, or a combination of a first cell and a second cell; The first cell contains the first genetic engineering vector described in P7; The coding gene in the gene expression cassette of the first genetic engineering vector is constitutively expressed or artificially induced; When the first monoclonal antibody heavy chain RNA and the first monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells; The second cell contains the second genetic engineering vector described in P7; The coding gene in the gene expression cassette of the second genetic engineering vector is constitutively expressed or artificially induced; When the second monoclonal antibody heavy chain RNA and the second monoclonal antibody light chain RNA are encoded in two vectors, the two vectors are in the same cell or in different cells; P9: Composition The composition contains the monoclonal antibody described in P1, the genetically engineered monoclonal antibody described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetically engineered vector described in P7 or the cell described in P8; as well as P10: Test kit The kit contains the monoclonal antibody described in P1, the combination of the monoclonal antibody heavy chain and the monoclonal antibody light chain described in P2, the antibody derivative described in P3, the RNA combination described in P4, the genetic engineering vector described in P7 or the cell described in P8.

2. The biomaterial according to claim 1, characterized in that The amino acid sequence of the human SLC19A1 protein is shown in SEQ ID NO.

1.

3. The biomaterial according to claim 1 or 2, characterized in that In P7, the backbone vector of the genetic engineering vector is a pTT3 vector.

4. The biomaterial according to claim 1 or 2, characterized in that In P8, the cells were 293F cells.

5. The biomaterial according to any one of claims 1 to 4, characterized in that The amino acid sequence of the heavy chain variable region of the first monoclonal antibody or the first genetically engineered monoclonal antibody is shown in SEQ ID NO.7; The amino acid sequence of the light chain variable region of the first monoclonal antibody or the first genetically engineered monoclonal antibody is shown in SEQ ID NO.

9.

6. The biomaterial according to any one of claims 1 to 4, characterized in that The amino acid sequence of the heavy chain variable region of the second monoclonal antibody or the second genetically engineered monoclonal antibody is shown in SEQ ID NO.11; The amino acid sequence of the light chain variable region of the second monoclonal antibody or the second genetically engineered monoclonal antibody is shown in SEQ ID NO.

13.

7. Use of the biomaterial according to any one of claims 1 to 6 in preparing a preparation for recognizing human SLC19A1 protein.

8. A method for detecting human SLC19A1 protein in a sample for non-diagnostic purposes, the method comprising the following steps: T1: Fix the sample to be tested on the solid surface to obtain an immobilized sample; T2: incubating the immobilized sample with a monoclonal antibody to obtain an immobilized sample of the first incubation; The monoclonal antibody is the first monoclonal antibody or the first genetically engineered monoclonal antibody described in any one of claims 1 to 6; T4: adding the monoclonal antibody specific binding substance connected with the label to the immobilized sample of the first incubation for incubation to obtain the immobilized sample of the second incubation; T5: Characterizing the label indirectly bound to the immobilized sample in the second incubation, and determining whether human SLC19A1 protein exists in the sample to be detected based on the presence or absence of the label.

9. The method according to claim 8, characterized in that Select any one of the following cases B1, B2, B3 and B4: B1: In step T1, the sample to be detected is human SLC19A1 protein, or the sample to be detected contains human SLC19A1 protein; B2: In step T1, the solid surface is the inner surface of the well of the microplate; B3: In step T3, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody labeled with a fluorescent molecule; in step T4, the fixed sample of the second incubation is photographed with a fluorescence microscope, and the presence of human SLC19A1 in the sample to be tested is determined based on the photographing result; B4: In step T3, the monoclonal antibody specific binding substance connected with the label is a secondary antibody of the monoclonal antibody labeled with horseradish peroxidase; in step T4, the fixed cells of the second incubation are developed with TMB colorimetric solution, and whether human SLC19A1 exists in the sample to be tested is determined based on the color development result.

10. The method according to claim 8 or 9, characterized in that The amino acid sequence of the human SLC19A1 protein is shown in SEQ ID NO.1.

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