Claudin18.2 gene engineering antibody based on DNA immunization and preparation method and application thereof

Abstract

The present application belongs to the technical field of bioengineering, and particularly relates to a Claudin18.2 gene engineering antibody based on DNA immunization and a preparation method and application thereof. The Claudin18.2 gene engineering antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises any one of the amino acid sequences shown in SEQ ID NO. 15-16; and the light chain variable region comprises the amino acid sequences shown in SEQ ID NO. 19-20. The Claudin18.2 gene engineering antibody is obtained by immunizing Balb / c mice with full-length plasmid DNA and full-length plasmid DNA+extracellular segment polypeptide as immunogens respectively, obtaining plasma cells capable of secreting specific antibodies against Claudin18.2, and then preparing specific mouse-derived gene engineering antibodies through single-cell optical guidance technology and genetic engineering technology. The gene engineering antibodies have high titer and strong specificity, and have specific recognition ability for natural protein Claudin18.2.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a DNA-based Claudin18.2 genetically engineered antibody, its preparation method, and its application. Background Technology

[0002] Single-cell photodynamic technology enables the in vitro cloning and expression of antibody genes on individual antigen-specific B cells, preserving the natural pairing of light and heavy chain variable regions. It offers advantages such as simplicity, speed, low cell requirement, and high efficiency. The single-B cell platform, combined with microfluidics and various sorting techniques, directly isolates, analyzes, and screens B cells at the single-cell level, accurately and efficiently identifying B cells that secrete target antibody molecules. Combined with single-cell sequencing technology, the target antibody sequence can then be obtained. This technology, by directly amplifying the VH and VL encoding genes of single B cells, is a powerful technique for preparing monoclonal antibodies from humans and immunized animals.

[0003] DNA immunization is a technique that uses recombinant DNA molecules to trigger an immune response. It involves introducing a specific gene fragment (usually an antigen gene of a pathogen) into host cells, thereby inducing a specific immune response in the host. This technique involves cloning the target gene into an appropriate expression vector, typically plasmid DNA, and then introducing it into host cells (such as mice or other animals). Once inside the host cells, the introduced DNA expresses the antigen protein, activating the immune system to produce a specific immune response to that antigen, including antibody and T-cell responses. Compared to traditional vaccines, DNA immunization offers advantages such as simplicity of production, lower cost, and longer-lasting immune effects, and is therefore widely used in vaccine development, disease prevention, and antibody production.

[0004] Claudins are a family of proteins responsible for maintaining tight junctions between cells and controlling molecular flow between cells by establishing barriers. Claudins are expressed in different isoforms in different tissues, and their aberrant expression is associated with tumor development and progression. In particular, Claudin18.2 is an important therapeutic target for gastrointestinal malignancies and has been investigated. Although research targeting Claudin18.2 is increasing, there are no reports of obtaining highly specific and sensitive Claudin18.2 monoclonal antibodies using gene DNA immunotherapy. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a DNA-based Claudin18.2 genetically engineered antibody, its preparation method, and its applications. This Claudin18.2 genetically engineered antibody is prepared by first generating a full-length Claudin18.2 (pVAX1-CLDN18.2) DNA plasmid and an extracellular polypeptide of Claudin18.2 (CLDN18.2). Then, Balb / c mice are immunized with the full-length plasmid DNA and the full-length plasmid DNA plus the extracellular polypeptide, respectively, to induce the proliferation and differentiation of B cells into plasma cells. These plasma cells secrete specific antibodies against Claudin18.2. Specific mouse-derived genetically engineered antibodies are then prepared using single-cell photoconductive technology and genetic engineering techniques. Enzyme-linked immunosorbent assay (ELISA) verifies that the genetically engineered antibody has a high titer. Immunohistochemistry and immunofluorescence experiments verify that the genetically engineered antibody has the specific recognition ability of the natural protein Claudin18.2, laying the foundation for further development of highly specific and highly sensitive Claudin18.2 pathological diagnostic antibodies.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The first objective of this invention is to provide a DNA-based Claudin18.2 genetically engineered antibody, wherein the Claudin18.2 genetically engineered antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises any one of the amino acid sequences shown in SEQ ID NO. 15-16; and the light chain variable region comprises the amino acid sequences shown in SEQ ID NO. 19-20.

[0007] Further, the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO. 15, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO. 19; or, The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO. 16, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO. 20.

[0008] A second objective of this invention is to provide a nucleic acid that encodes the heavy chain variable region and the light chain variable region of the aforementioned Claudin18.2 genetically engineered antibody.

[0009] Furthermore, the nucleic acid includes one or more of the nucleotide sequences shown in SEQ ID NO. 13-14 and SEQ ID NO. 17-18.

[0010] Furthermore, the nucleotide sequence shown in SEQ ID NO. 13 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 15 in the Claudin18.2 genetically engineered antibody; The nucleotide sequence shown in SEQ ID NO. 14 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 16 in the Claudin18.2 genetically engineered antibody; The nucleotide sequence shown in SEQ ID NO. 17 is used to encode the light chain variable region of the amino acid sequence shown in SEQ ID NO. 19 in the Claudin18.2 genetically engineered antibody; The nucleotide sequence shown in SEQ ID NO. 18 is used to encode the light chain variable region of the amino acid sequence shown in SEQ ID NO. 20 in the Claudin18.2 genetically engineered antibody.

[0011] A third objective of this invention is to provide a recombinant expression vector comprising the nucleic acid described above.

[0012] A fourth objective of this invention is to provide a recombinant expression cell comprising the recombinant expression vector described above, or the nucleic acid described in the market.

[0013] The fifth objective of this invention is to provide a method for preparing a DNA-based Claudin18.2 genetically engineered antibody, comprising the following steps: (a) Immunogen preparation; (b) Animal immunization; (c) Claudin 18.2 antigen-specific single plasma cell screening; (d) Preparation of genetically engineered antibodies.

[0014] The specific steps are as follows: (1) Immunogen preparation: Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid DNA immunogen and Claudin18.2 (CLDN18.2) extracellular polypeptide immunogen were prepared; wherein, the amino acid sequence of the Claudin18.2 extracellular polypeptide immunogen is shown in SEQ ID NO.1; the amino acid sequence of the Claudin18.2 full-length plasmid DNA immunogen is shown in SEQ ID NO.2; FWMSTANMYTGMGGC (SEQ ID NO.1) MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCA IAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV (SEQ ID NO.2) (2) Animal immunization experiments were conducted. Balb / c mice were immunized with two methods: full-length immunogen plasmid pVAX1-CLDN18.2 and full-length plasmid PVAX1-CLDN18.2 + extracellular polypeptide. The mice produced sensitized B lymphocytes, which can secrete specific antibodies against Claudin18.2. (3) Spleen cell suspensions were prepared for two types of immunized mice, mouse spleen mononuclear cells were isolated, and mouse CD138 positive plasma cells were obtained by sorting with MACS buffer and CD138 antibody magnetic beads; Specifically, the sorting method was: CD138 positive plasma cells were specifically sorted by Ficoll density gradient centrifugation and magnetic bead sorting. (4) Lentiviral packaging and construction of full-length Claudin18.2 overexpressing cell lines transfected with 293T cells; specifically, including lentiviral vector construction and lentiviral packaging, lentiviral infection of HEK-293T cells, and screening for stable overexpressing cell lines with puromycin; (5) Two groups of mouse-specific single plasma cells were screened using single-cell photoconductive technology and microfluidic technology based on the Beacon platform; specifically, this included treating the 293T-CLDN18.2 overexpressing cell line as a screening medium, screening mouse-specific single plasma cells, and exporting single positive plasma cells. (6) Using single B-cell sequencing technology, single cell RNA is obtained and cDNA is prepared. Antibody light and heavy chain variable region gene amplification and DNA fragment recovery are performed to obtain the antibody heavy and light chain variable region sequence. Specifically, the method of extracting single plasma cell RNA and preparing cDNA includes using magnetic beads to extract single plasma cell RNA with high affinity for antigen binding, and using specific primers for reverse transcription to prepare cDNA. (7) Construct a monoclonal antibody gene expression vector, ligate the obtained heavy and light chain variable region sequence into the expression vector to construct a recombinant expression vector, and transfect the recombinant expression vector into eukaryotic cells for eukaryotic expression and purification to obtain a monoclonal antibody against Claudin18.2. Specifically, the expression method includes: using a specific ratio of heavy and light chain plasmids, adding a specific diluent, mixing a specific transfection reagent, transfecting into CHOS or 293F cells, purifying, and obtaining the corresponding genetically engineered monoclonal antibody.

[0015] Further, the expression vectors are pcDNA3.4(pFUSEss-CHIg-mG2b) and pcDNA3.4(pFUSE2ss-CLIg-mk); the eukaryotic cells are eukaryotic Expi CHOS or 293F. Specifically, the eukaryotic expression and purification method includes: diluting the plasmid of the recombinant expression vector and PEI with diluent and adding them to CHOS cell suspension for transfection culture to obtain cell suspension, feeding after 24 h of culture, centrifuging to collect cell supernatant after 120 h, filtering the supernatant with a filter membrane, passing the supernatant through a Protein A affinity chromatography column 3-4 times, and finally eluting with glycine solution, collecting the eluent to obtain the purified Claudin18.2 genetically engineered antibody.

[0016] In addition, it also includes ELISA potency testing, IHC validation, and ICC validation methods.

[0017] Specifically, the method for validating ELISA titers includes the following steps: coating, washing, blocking, washing, primary antibody incubation, washing, secondary antibody incubation, washing, and color development. Finally, the absorbance is detected using a microplate reader.

[0018] The ICC validation method includes the following steps: cell smearing, washing, fixing, permeabilization, blocking, primary antibody incubation, room temperature equilibration and washing, secondary antibody incubation, nuclear staining and mounting (DAPI staining), and finally observation and image acquisition under a laser confocal microscope.

[0019] The IHC validation method includes the following steps: tissue fixation, sectioning, inactivation, antigen retrieval, washing, blocking, secondary antibody incubation, staining, counterstaining, mounting, etc., and finally observation and photography under a microscope.

[0020] The sixth objective of this invention is to provide an application of the Claudin18.2 genetically engineered antibody prepared by the above-described Claudin18.2 genetic engineering antibody, or the above-described nucleic acid, or the above-described recombinant expression vector, or the above-described recombinant expression cell, or the above-described preparation method, in the preparation of Claudin18.2 pathological diagnostic antibodies or gastric cancer and breast cancer detection kits.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention first prepares the full-length Claudin18.2 (pVAX1-CLDN18.2) plasmid vector and the extracellular peptide of Claudin18.2 (CLDN18.2). Balb / c mice to be immunized are divided into two groups: the Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid immunogen group and the Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid combined with the Claudin18.2 (CLDN18.2) extracellular peptide immunogen group. Immunization is performed according to the immunization protocol to induce the proliferation and differentiation of B cells in Balb / c mice into plasma cells, which can secrete specific antibodies against Claudin18.2. Among them, the Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid immunogenome successfully screened 7 single plasma cells capable of secreting Claudin18.2-specific antibodies using single-cell photodynamic therapy. Furthermore, 12 genetically engineered antibodies were successfully prepared using genetic engineering and a eukaryotic expression system. Two of these genetically engineered antibodies showed high titers as detected by indirect ELISA, and one antibody was found to recognize Claudin18.2 protein in natural tissues by IHC analysis. Meanwhile, Claudin18.2 (pVAX1-CLDN18.2) An immunogenome was created by combining the full-length pVAX1-CLDN18.2 plasmid with the extracellular peptide of Claudin18.2 (CLDN18.2). Using single-cell photodynamic therapy, eight single plasma cells capable of secreting Claudin18.2-specific antibodies were successfully screened. Ten genetically engineered antibodies were then successfully prepared using genetic engineering and a eukaryotic expression system. One of these antibodies showed high titer as detected by indirect ELISA, and another antibody was found to recognize Claudin18.2 protein in natural tissues by IHC assay. This lays the foundation for further development of highly specific and sensitive Claudin18.2 pathological diagnostic antibodies. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is an agarose gel electrophoresis image of the pVAX1-CLDN18.2 plasmid in Example 1 of this invention; Figure 2 This is a microscopic image of the Puromycin screening results of 293T-CLDN18.2 overexpressing cells in Example 2 of this invention; Figure 3 This is a Texas Red fluorescence reaction diagram for screening single plasma cells using Beacon technology in Example 4 of this invention. Figure 4 Figure 4 shows agarose gel electrophoresis images of antibody light and heavy chain variable regions amplified in Example 4 of the present invention, where A is an amplification image of the antibody heavy chain variable region and B is an amplification image of the antibody light chain variable region. Figure 5 The images shown are colony identification diagrams of antibody light and heavy chain T vector construction in Example 4 of the present invention, where A is the antibody heavy chain colony identification diagram and B is the antibody light chain colony identification diagram. Figure 6 Figure 4 shows the colony identification diagrams of the full-length antibody light and heavy chains constructed in Example 4 of the present invention, where A is the antibody heavy chain colony identification diagram and B is the antibody light chain colony identification diagram. Figure 7 This is a gel electrophoresis image of the genetically engineered antibody polyacrylamide in Example 4 of the present invention; Figure 8 This is a curve showing the titer of the genetically engineered antibody in Example 5 of the present invention. Figure 9 This is an IHC detection result diagram of the genetically engineered antibody Mu-E15-D1-R1 in Example 5 of the present invention; Figure 10 This is an image showing the IHC detection results of the genetically engineered antibody Mu-E15-S154-H1-R4 in Example 5 of this invention; Figure 11 This is an ICC detection result diagram of the genetically engineered antibody Mu-E15-D1-R1 in Example 5 of the present invention; Figure 12 This is an ICC detection result diagram of the genetically engineered antibody Mu-E15-S154-H1-R4 in Example 5 of the present invention. Detailed Implementation

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the raw materials used in the following examples are all commercially available products and can be purchased from the market.

[0025] The above-described technical features of the present invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.

[0026] Example 1: Preparation of Claudin18.2 (CLDN18.2) full-length plasmid immunogen The preparation method of the full-length Claudin18.2 (CLDN18.2) plasmid vector includes the following steps: (1) Design and synthesis of Claudin18.2 (CLDN18.2) specific primers ① Go to the NCBI website (website: https: / / www.ncbi.nlm.nih.gov / ), select the Nucleotide database, enter Claudin18.2 (CLDN18.2) and the search term homo sapiens, find and click Homo sapiensclaudin 18 (CLDN18), transcript variant 2; ② Click CDS to obtain the Claudin18.2 (CLDN18.2) protein coding region sequence; ③ Click Pick Primer, enter the full-length CDS sequence in PCR Template, enter 15-30bp from the beginning and end of the CDS sequence in Primer Parameters, click Get Primers and select the best specific primers based on primer Tm value, GC content, Self-complementarity and Self 3'complementarity score; ④ The designed specific primers were sent to Qingke Biotechnology Co., Ltd. for synthesis. The primer sequences are shown in Table 1.

[0027] Table 1 Primer Sequences

[0028] (2) Amplification and recovery of the full-length target gene fragment of Claudin18.2 (CLDN18.2) ① Using a plasmid containing the Claudin18.2 (CLDN18.2) gene as an amplification template, the full-length target gene was amplified by PCR. The PCR reaction system and amplification program settings are shown in Table 2 and Table 3, respectively.

[0029] Table 2 PCR reaction system

[0030] Table 3 Amplification Procedure

[0031] ② The amplification products were recovered using the Tiangen agarose recovery kit.

[0032] First, add 500 μL of BL equilibration buffer to the CA2 adsorption column, centrifuge for 2 min at 12000 rpm, and discard the lower layer. Add Claudin18.2 (CLDN18.2) amplification product to the CA2 adsorption column, incubate at room temperature for 2 min, centrifuge for 2 min at 12000 rpm, and discard the lower layer. Add 600 μL of PW wash buffer containing anhydrous ethanol to the CA2 column, centrifuge for 2 min at 12000 rpm, discard the lower layer, and repeat this step once. Centrifuge the empty CA2 column again at 12000 rpm for 2 min. Place the CA2 column in a fume hood for 5 min to dry completely, then transfer it to a clean 1.5 mL centrifuge tube. Finally, add 50 μL of ddH2O to the center of the CA2 adsorption membrane, incubate at room temperature for 2 min, then centrifuge for 2 min at 12000 rpm to collect the DNA solution.

[0033] (3) pVAX1 vector digestion and recovery ① pVAX1 vector digestion: Add 2 µg of pVAX1 plasmid to a 1.5 mL centrifuge tube, along with 1 μL each of restriction endonucleases EcoRI and XhoI, and 5 μL of digestion buffer NEBuffer™r3.1. Finally, bring the digestion system to 50 μL with ddH2O. After thorough mixing, place the tube in a 37°C water bath and digest for 3 h to linearize the pVAX1 vector.

[0034] ② Linearized vector recovery: The linearized vector was recovered using the Tiangen agarose recovery kit. First, 500 μL of BL equilibration buffer was added to the CA2 adsorption column, centrifuged for 2 min at 12000 rpm, and the lower layer was discarded. Then, Claudin18.2 (CLDN18.2) amplification product was added to the CA2 column, incubated at room temperature for 2 min, centrifuged for 2 min at 12000 rpm, and the lower layer was discarded. Next, 600 μL of PW wash buffer containing anhydrous ethanol was added to the CA2 column, centrifuged for 2 min at 12000 rpm, and the lower layer was discarded. This step was repeated once. The empty CA2 column was centrifuged again at 12000 rpm for 2 min. The CA2 column was then placed in a fume hood for 5 min to allow it to dry completely before being transferred to a clean 1.5 mL centrifuge tube. Finally, 50 μL of ddH2O was added to the center of the CA2 adsorption membrane, and the mixture was left at room temperature for 2 min. Then, it was centrifuged for 2 min at 12000 rpm to collect the DNA solution.

[0035] (4) Construction of pVAX1-CLDN18.2 plasmid ① The full-length target gene fragment of Claudin18.2 (CLDN18.2) was recombinated with the linearized pVAX1 vector. The amounts of the target fragment and linearized vector were calculated according to the formulas: Cloning vector usage = [0.02 × number of base pairs of cloning vector] ng, insert fragment usage = [0.04 × number of base pairs of insert fragment] ng, and added to the PCR tube. Then, 1 μL of Exnase II and 2 μL of 5×CE II Buffer were added to the PCR tube, and the recombination system was brought to a final volume of 10 μL with pure water. After thorough mixing, the PCR system was placed in a PCR instrument and the reaction conditions were set to 37℃ for 30 min.

[0036] ② Recombinant transformation: Add 5 μL of recombinant product to 50 μL of competent DH5α cells, gently mix, and let stand on ice for 30 min. Heat shock in a 42℃ water bath for 90 s, and then immediately place on ice for 4 min. Add 700 μL of antibiotic-free LB liquid medium and incubate at 37℃ and 220 rpm for 1 h. Then centrifuge the bacterial culture at 5000 rpm for 5 min, discard 600 μL of supernatant, resuspend the bacterial cells in the remaining liquid, and spread the resuspended bacterial cells evenly on LB solid culture dishes containing kanamycin resistance. Incubate upside down in a 37℃ incubator overnight.

[0037] (5) Identification of pVAX1-CLDN18.2 plasmid ① Colony PCR: Pick a single white, round colony from a culture dish and transfer it to a centrifuge tube. Incubate at 37°C in a shaker for 4 h. Then, add 1 μL of the bacterial culture as a template to a PCR tube, along with 0.5 μL of Claudin18.2 (CLDN18.2) upstream primer, 0.5 μL of pVAX1 vector downstream primer, 5 μL of 2×Taq Master Mix, and 3 μL of ddH2O. Place the tube in a PCR instrument for PCR amplification, and perform agarose gel electrophoresis on the amplified products. Figure 1 As shown, plasmids were extracted from clones with correct PCR bands.

[0038] ② Plasmid extraction: Add 100 μL of bacterial culture from a clone with the correct colony PCR band to 5 mL of LB medium containing kanamycin resistance and incubate overnight in a shaker at 37°C. After the E. coli reaches the logarithmic growth phase, extract the plasmid using a plasmid miniprep kit. Add 500 μL of BL equilibration buffer to adsorption column CP3, centrifuge at 12000 rpm for 2 min, and discard the lower layer. Centrifuge the E. coli culture at 12000 rpm for 2 min. Add 250 μL of P1 solution containing RNase A to the bacterial cells and mix thoroughly by pipetting. Add 250 μL of P2 solution to a centrifuge tube, gently invert 6-8 times until the solution becomes clear and viscous, then add 350 μL of P3 solution, immediately gently invert 6-8 times, and centrifuge at 12000 rpm for 10 min. Aspirate the supernatant and transfer it to a clean centrifuge tube. Add the supernatant to adsorption column CP3 and centrifuge at 12000 rpm for 1 min, discarding the waste liquid in the centrifuge tube. Add 600 μL of PW wash buffer containing anhydrous ethanol to CP3 and centrifuge at 12000 rpm for 1 min. After 2 min, discard the liquid in the tube and repeat the step once; centrifuge the CP3 with the discarded waste liquid again at 12000 rpm for 2 min; place the CP3 in a fume hood for 5 min to dry completely, and then place it in a clean 1.5 mL centrifuge tube; add 50 μL of ddH2O to the center of the CP3 adsorption membrane, place it at room temperature for 2 min, and then centrifuge at 12000 rpm for 2 min to collect the plasmid solution.

[0039] ③ Take 50 μL of plasmid solution from the clone with the correct colony PCR band and sequence it (measured by Wuhan Qingke Biotechnology Co., Ltd.). Analyze the sequencing results using Snapgene, BioXM 2.7.1 software and NCBI-BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) website.

[0040] Example 2: Construction of 293T-CLDN18.2 overexpression cell line (1) Packaging of lentiviruses ①Culturing cells: Culture 293T cells in 6-well plates and conduct experiments when the cell density is above 90% and the cells are in good condition; ② Plasmid treatment: Take two 1.5 mL EP tubes. Add 200 μL of basal culture medium to one tube, and add the packaged plasmids PMD (0.5 μg / well), PSA (0.75 μg / well), and pLVX-AcGFP-N1-CLDN18.2 (2 μg / well) to the tube and mix. Add 6 μL of PEI (1 mg / mL) to the other tube. ③ Mix and let stand: Add the prepared PEI to the EP tube containing the mixed plasmid, mix and let stand for 15-20 minutes to ensure that the PEI and plasmid are fully mixed and homogeneous. ④ Transfection: Take the prepared 293T cells, add the plasmid into the well plate, shake well and place in the incubator; ⑤ Change the medium: Change the medium after 6 hours of incubation; ⑥ Collect the virus: Collect the virus once every 24h, 48h and 72h. Mix the collected virus supernatant and centrifuge at 12000rpm for 5min. Then, remove the supernatant and filter it through a 0.45μm filter membrane.

[0041] (2) Infection of 293T cells ①Culturing cells: Culture 293T cells in 6-well plates and conduct experiments when the cells reach 50% of the culture dish and are in good condition; ② Infecting cells: Take out the prepared cells, wash them once with 1×PBS, then add 2mL of virus suspension to each well, and then supplement with complete culture medium to 3mL, and incubate in a 37℃ incubator.

[0042] ③ Culture medium change: Change the culture medium after 6 hours, and digest and re-inoculate the cells after 24 hours.

[0043] (3) Screening of stable cell lines ①Cell culture: 24 hours after infection, cells were plated and an uninfected cell control group was set up at the same time; ② Puromycin selection: When cells reach 50-70% confluence in the culture dish, add 2-8 μg / mL of puromycin. Change the culture medium every 2-3 days until all cells in the control group die. Figure 2 As shown.

[0044] Example 3: Animal Immunization (1) Immunizing Balb / c mice with the immunogen Claudin18.2 ① Immunization with pVAX1-CLDN18.2 plasmid: First, prepare and set the electroporation program; anesthetize the mice with isoflurane, shave the mice to expose the skin; draw 50 μL of plasmid solution (concentration of 1 mg / mL) into the syringe; after disinfecting the skin with 75% ethanol, inject the plasmid solution into the tibialis anterior muscle of the mouse's hind leg; insert the electrode into the mouse's hind leg muscle so that the plasmid injection site is in the center, and perform electroporation pulses.

[0045] ② Immunotherapy with extracellular peptides of Claudin18.2 (CLDN18.2): First, prepare 50 μg of peptide and add sterile 1×PBS to a volume of 200 μL. Then add an equal volume of Freund's incomplete adjuvant (Thermo Fisher Scientific, USA). Set the tissue homogenizer to 60 Hz and run for 15 min to emulsify the solution in the tube until it becomes milky white and opaque. Draw the solution into a syringe. Hold the mouse still with your left hand and insert the syringe with the needle tip facing upwards, parallel to the mouse's head, into the skin to inject the sample.

[0046] ③Immunization process: The immunization pathways for the Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid immunogen group and the Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid combined with the Claudin18.2 (CLDN18.2) extracellular peptide combination immunogen group are shown in Table 4.

[0047] Table 4 Immunization Procedure

[0048] (2) Detection of serum titer in Balb / c mice ① Orbital blood collection: In the second week after the fourth immunization, orbital blood was collected from Balb / c mice using capillary blood collection tubes. After standing, the blood was centrifuged for 10 minutes in a high-speed refrigerated centrifuge pre-cooled to 4°C at 3000 rpm. After centrifugation, the mouse serum was separated into clean centrifuge tubes.

[0049] ② Coating and blocking overexpressing cells: Add 0.1% gelatin to a 96-well plate and incubate at 37°C for 2 hours, then remove the gelatin. Add 1×10⁻⁶ gelatin to each well. 5 100 μL of cells per well were incubated overnight at 37°C. The plate was washed three times with 200 μL of 1×PBST for 3 min each time. 4% paraformaldehyde was added for fixation at room temperature for 30 min. The plate was washed three times with 200 μL of 1×PBST for 3 min each time. 2% BSA blocking solution was added to 200 μL of each well and the plate was blocked at 37°C for 1 h.

[0050] ③ Serum titer assay: First well was filled with stock serum, followed by 3-fold serial dilutions (serum dilution factor 1-729). The diluted samples were then added sequentially to 96-well plates coated with 293T-CLDN18.2, with two parallel control groups and a blank control group. 100 μL of sample and 1×PBS were added to each well, and the plates were incubated at 37°C for 1 h. After primary antibody incubation, the plates were washed three times with 1×PBST, and then HRP-labeled goat anti-mouse antibody was added, followed by incubation at 37°C for 40 min. After secondary antibody incubation, the plates were washed five times with 1×PBST and patted dry. Then, 90 μL of TMB chromogenic buffer was added to each well, and the plates were incubated at 37°C for 10 min. Finally, 50 μL of stop solution was added to terminate the reaction. The absorbance was read using a microplate reader at 450 nm.

[0051] (3) Immune shock in Balb / c mice The Balb / c mouse with the highest serum titer was selected for immunization shock. The 293T-CLDN18.2 cell density was first adjusted to 5 × 10⁻⁶ cells using 1×PBS. 6 1 cell / mL, draw 0.5mL into the syringe; hold the mouse with its head down, insert the syringe into the abdominal cavity at a 45-degree angle, and inject the sample after feeling that the needle tip can move.

[0052] Example 4: Preparation of Genetically Engineered Antibodies (1) Isolation of mouse spleen mononuclear cells ① Fix the mouse with one hand and gently press the skin around the mouse's eyes to cause the eyeballs to become congested and protrude. Use tweezers to grasp the mouse's eyeballs and quickly remove them, while gently pressing on the mouse's heart to accelerate blood flow. Sacrifice the mouse by dislocating its neck and immerse it in 75% alcohol for 3-5 minutes.

[0053] ② Place the mouse in a sterile operating table and expose the skin on the left side of the mouse. Use ophthalmic scissors to cut open the skin to find the dark red spleen at the left rib. Remove the connective tissue attached to the spleen and use forceps to remove the spleen and place it in a petri dish.

[0054] ③ Add 3-5 mL of RPMI 1640 medium + 10% fetal bovine serum (FBS) + 1:1000 DNAClean up to a petri dish, and then place the spleen in the dish.

[0055] ④ Make a small incision on one side of the spleen, and press the spleen with the handle of a disposable syringe while adding 1640 culture medium + 10% fetal bovine serum (FBS) + 1:1000 DNA Clean up to the dish. Press until there are no large red clumps in the spleen, then filter the liquid in the dish through a 40 µm filter membrane.

[0056] ⑤ Add 5 mL of Ficoll density gradient centrifuge solution to each of four 15 mL sterile centrifuge tubes. Add 5 mL of the filtrate along the tube wall to each centrifuge tube containing Ficoll. Centrifuge at 800 g for 25 min at 20°C, with the centrifugation speed set to 9 for the acceleration and 0 for the deceleration.

[0057] ⑥ After centrifugation, carefully aspirate the cells from the cloud layer using a pipette and transfer them to a centrifuge tube containing 10 mL of RPMI 1640 medium + 10% FBS. Gently mix with a pipette, and take 20 μL of the cell suspension for cell counting. Place the centrifuge tube in a high-speed refrigerated centrifuge and centrifuge at 800 g for 10 min at 4°C, discarding the supernatant.

[0058] (2) Sorting of CD138-positive plasma cells in mice ① Resuspend mononuclear cells in 10 mL of Magnetic Activated Cell Sorting (MACS) buffer and centrifuge at 300 g for 10 min at 20 °C. Simultaneously, collect 20 μL of the cell suspension for cell counting.

[0059] ② Each 1×10 7 Each cell was resuspended in 40 μL of MACS buffer and 10 μL of magnetic beads coated with CD138 antibody was added. After thorough mixing, the mixture was incubated at 4°C in the dark for 15 min.

[0060] ③ Each 1×10 7 Resuspend each cell in 1 mL of MACS buffer, centrifuge for 10 min at 300 g and 20°C. Discard the supernatant after centrifugation. For every 1×10⁻⁶ cells... 8 Each cell was resuspended in 500 μL of MACS buffer and stored on ice.

[0061] ④ Install the LS sorting column onto the sorter and rinse the column with 5 mL of MACS buffer. Add the cell suspension to the sorting column, then rinse the column with 3 mL of MACS buffer, repeating twice.

[0062] ⑤ Remove the sorting column from the sorter and place it on a 15 mL centrifuge tube. Add 5 mL of MACS buffer to the sorting column, then push the plug into the column to quickly wash out the CD138-positive plasma cells labeled with magnetic beads. Centrifuge at 300 g for 5 min at 4°C. Simultaneously, collect 20 μL of the cell suspension for cell counting.

[0063] ⑥ Resuspend the cells in basal medium (Base Media) to a density of 6.25 × 10⁻⁶. 6 Cells / mL.

[0064] (3) Specific single plasma cell screening based on Beacon single-cell optical guidance technology ① Use 293T-CLDN18.2 overexpressing cell lines as selection media a. Remove the old culture medium from the culture dish and wash the cells with 1×PBS, then remove the 1×PBS. b. Digest the cells with trypLE at room temperature for 1 min, then remove the trypLE; c. Add basal culture medium to resuspend the cells in a 1.5 mL centrifuge tube, and take 20 μL of the cell suspension for cell counting; d. Centrifuge cells at 500 g for 5 min; e. Take 1 mL of Plasma B Cell Media culture medium into a 1.5 mL centrifuge tube, add 1.5 μL of Texas Red fluorescent secondary antibody, and mix well by pipetting. f. Add fluorescent secondary antibody to a 1.5 mL centrifuge tube containing 293T-CLDN18.2 cells and mix well to achieve a cell density of 5 × 10⁻⁶ cells. 7 Cells / mL, mix well and store in a 4°C refrigerator protected from light.

[0065] ② Mouse-specific single plasma cell screening a. Resuspended plasma cells are added to the cell loading wells of the Beacon chip, avoiding the formation of air bubbles. Individual plasma cells are then guided into the chip's chambers using photoelectric positioning and microfluidic technology, where they secrete antibodies. b. Add 10 μL of the prepared 293T overexpression cells to the corresponding sample well, avoiding the generation of air bubbles. The 293T overexpression cells will enter the chip channel along with the culture medium. c. Observe the fluorescence reaction under Texas Red fluorescence channel conditions, such as Figure 3 As shown, plasma cells exhibiting strong fluorescence were picked from the microarray and extracted, then collected into 96-well PCR plates. 5 μL of lysis buffer (2×TCL) and 10 μL of mineral oil were added to each well of the PCR plate beforehand. Seal the PCR plate with sealing film, centrifuge at 400 g for 1 min, and store in a -80℃ freezer.

[0066] (4) Extract RNA from single plasma cells and prepare cDNA ① Remove the PCR plate from the -80℃ freezer, let it stand at room temperature, and then centrifuge it for 1 minute at a speed of 200 g; ② Add 10 μL of RNA Clean XP magnetic beads, which have been brought to room temperature, to the PCR plate. Mix well by pipetting and incubate at room temperature for 1 min until the magnetic beads are evenly distributed in the wells. Seal the PCR plate with sealing film and incubate at room temperature for 20 min. ③ Place the PCR plate on the Mag Well magnetic separator and let it stand for 5 minutes. Remove the supernatant from the wells without disturbing the magnetic beads. ④ Add 80% ethanol to the PCR plate, 100 μL per well, discard the supernatant after 30 s, and repeat this step twice; ⑤ Remove the PCR plate from the Mag Well magnetic separator and let it sit at room temperature for 2 minutes to dry the magnetic beads; ⑥ Add Mix 1 reagent (as shown in Table 5) to the PCR plate, 5 μL per well. Place the PCR plate in a PCR instrument for one reaction, setting the program to 72℃ for 3 min. Immediately after the reaction, place the PCR plate on ice. ⑦ Add Mix2 reagent to the PCR plate, the composition of which is shown in Table 6, 4 μL per well. After mixing thoroughly, place the PCR plate into the PCR instrument for a second reaction (program settings are shown in Table 7); ⑧ After the reaction is complete, add Mix3 reagent to the PCR plate. The composition is shown in Table 8, and the amount is 21 μL per well. After mixing thoroughly, place the PCR plate into the PCR instrument for three reactions (program settings are shown in Table 9). ⑨ Add 30 μL of AMPure XP magnetic beads that have been brought to room temperature into the PCR plate, mix thoroughly, and incubate at room temperature for 5 min. ⑩ Place the PCR plate on the Mag Well magnetic separator, let it stand for 2 minutes, and then aspirate the upper liquid. ⑪ Add 200 μL of 80% ethanol to each PCR well, incubate at room temperature for 30 s, then discard the supernatant. Repeat this step twice. ⑫ Place the PCR plate at room temperature for 2 minutes to dry the magnetic beads, then remove the PCR plate from the magnetic separator; ⑬ Add 15 μL of enzyme-free water to each well of the PCR plate, mix thoroughly, and incubate at room temperature for 2 min. ⑭ Place the PCR plate on the magnetic separator, let it stand for 1 minute, then aspirate the solution from the wells and store it in a clean PCR plate.

[0067] Table 5. Components of Mixed Reagent Mix1

[0068] Table 6. Components of Mix 2 Reagent

[0069] Table 7 Secondary PCR reaction procedure

[0070] Table 8. Components of Mix 3 Reagent

[0071] Table 9. Three-stage PCR reaction procedure

[0072] (5) Amplification of antibody light and heavy chain variable region genes and recovery of DNA fragments Using cDNA from a single plasma cell as a template, the variable region gene sequences of the antibody heavy and light chains were amplified separately. A 50 μL amplification mixture was prepared in a PCR tube containing: 18 μL ddH2O, 25 μL 2×PhantaMax Buffer, 1 μL dNTP, 1 μL PhantaMax Super-Fidelity DNA Polymerase, 1 μL template, and 2 μL forward and reverse degenerate primers. After thorough mixing, the mixture was placed in a PCR instrument for fragment amplification. The amplification program for the antibody light and heavy chain variable regions is shown in Table 10. After amplification, 1 μL of the amplification product was subjected to DNA electrophoresis on a 1% agarose gel. Figure 4 As shown in the figure. Subsequently, an agarose gel recovery kit was used to recover the correct DNA fragments identified by electrophoresis, and the recovery concentration was detected using a UV spectrophotometer.

[0073] Table 10. Antibody light and heavy chain variable region amplification program

[0074] (6) Construction of the variable region T vector of mouse anti-human Claudin18.2 antibody ① Add 1 μL of the target fragment from the antibody heavy or light chain variable region to a PCR tube, followed by 1 μL of pEASY®-Blunt Cloning Vector and 3 μL of pure water. Mix well and incubate at 37°C for 10 min. Add the ligation product to 50 μL of T1 competent cells, gently pat to mix, and incubate on ice for 30 min. Then heat shock in a 42°C water bath for 90 s and immediately place on ice for 2 min. Add 250 μL of antibiotic-free LB medium to the tube and incubate at 37°C for 1 h at 200 rpm. Take 8 μL of 500 mmol·L⁻¹ -1 IPTG and 40 μL 20 mg·mL -1Mix X-gal and spread evenly on ampicillin-resistant LB agar plates. Incubate at 37°C for 30 minutes to allow IPTG and X-gal to be absorbed. Centrifuge the culture at 5000 rpm for 5 min and discard 200 μL of the supernatant. Resuspend the cells in the remaining medium and spread evenly on plates. Incubate overnight at 37°C.

[0075] ② Pick a single white colony and incubate it in ampicillin-resistant LB medium at 37°C in a shaker. Once the bacterial culture becomes noticeably turbid, add 1 μL of the culture as a template to a PCR tube. Add 5 μL of 2×Taq Master Mix, 3 μL of purified water, and 0.5 μL of primers to the tube. Mix well and perform colony PCR, using the program settings shown in Table 11. For the heavy chain, use an upstream degenerate primer and a specific downstream primer; for the light chain, use a specific upstream primer and a downstream degenerate primer. After the reaction, perform agarose gel electrophoresis on the products. Figure 5 As shown. 300 μL of bacterial culture from clones with correct PCR bands was sequenced (antibody light and heavy chain variable region sequences were determined by Shanghai Sangon Biotech). The function of the antibody light and heavy chain variable region sequences was analyzed using the IMGT website (https: / / imgt.org / IMGT_vquest), and VDJ family analysis was performed.

[0076] Table 11 Colony PCR Procedure

[0077] (7) Construction of mouse anti-human Claudin18.2 full-length antibody vector By constructing the variable region genes of the antibody's light and heavy chains into vectors pFUSEss-CHIg-mG2b and pFUSE2ss-CLIg-mk containing the constant regions of the antibody's light and heavy chains, respectively, a vector for constructing the full-length mouse anti-human Claudin18.2 antibody was achieved.

[0078] First, plasmids pFUSEss-CHIg-mG2b and pFUSE2ss-CLIg-mk were linearized by double enzyme digestion to facilitate subsequent recombination with the variable region genes of the antibody light and heavy chains. Gel electrophoresis results showed that the bands of plasmids pFUSEss-CHIg-mG2b and pFUSE2ss-CLIg-mk after enzyme digestion were between 3000 bp and 5000 bp, consistent with the fragment size of the linearized vector.

[0079] Then, the variable region gene of the heavy chain was constructed into pFUSEss-CHIg-mG2b and the variable region gene of the light chain was constructed into pFUSE2ss-CHIg-mk using homologous recombination. After overnight incubation, several white single colonies grew on culture dishes containing the corresponding antibiotics. Single colonies were picked for expansion culture and colony PCR was used to confirm the success of the construction. Gel electrophoresis results are as follows: Figure 6 As shown, the results indicate that the full-length vectors of the heavy and light chains were successfully constructed. Sequencing results were analyzed using Snapgene, BioXM.2.7.1 software, and the NCBI-BLAST website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the sequences showed 100% overlap with the original base sequences.

[0080] The PCR amplification primers were designed based on the upstream and downstream restriction sites of the vector, as shown in Table 12. Then, the heavy and light chain variable region genes that were confirmed to be correct and functional were used as templates by sequencing.

[0081] Table 12 Primers for Heavy and Light Chain PCR Amplification

[0082] The nucleotide sequence encoding the first heavy chain variable region (E15-D1-MuIg-VH1) is shown below: GAGGTGAAACTGCAGCAGTCTGGACCTGAACTGGTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTTCTGGATACTCATTCACTAGTTATGTTATACACTGGGTGAAGCAGAAGCCTGGACAGGGCCTTGAGTGGATTGGATATATTCATCCTTACAATGGTGGTACTAAGAACAATGAG AAGTTCAAAGGCAGGGCCACACTGACTTCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGACTCGCCTCTTATGGTAGATATGGTTTGGACTACTGGGGTCAAGGAACCTCTGTCACCGTCTCCTCA(SEQ ID NO.13); The nucleotide sequence encoding the second heavy chain variable region (E15-S154-H1-MuIg-VH1) is shown below: CAGGTCAAACTGCAGGAGTCTGGGGCTGAACTGGCAAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTCACACGTTTACTACCTACTGGATACACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATTCATTAATCCTAGCACTGGTTATA CTGATTACAATCAGAAATTCAGGGACCAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAATACCTGGACGGTTTGGTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO.14); According to the codon encoding rules, the amino acid sequence of the first heavy chain variable region (E15-D1-MuIg-VH1) is as follows: EVKLQQSGPELVKPGASVKMSCKASGYSFTSYVIHWVKQKPGQGLEWIGYIHPYNGGTKNNEKFKGRATLTSDKSSSTAYMELSSLTSEDSAVYYCARLASYGRYGLDYWGQGTSVTVSS (SEQIDNO.15); The amino acid sequence of the second heavy chain variable region (E15-S154-H1-MuIg-VH1) is as follows: QVKLQESGAELAKPGASVKMSCKASGYTFTTYWIHWVKQRPGQGLEWIGFINPSTGYTDYNQKFRDQATLTADKSSSTAYMQLSSLTSEDSAVYYCAIPGRFGYWGQGTTVTVSS (SEQIDNO.16); The nucleotide sequence encoding the first light chain variable region (E15-D1-MuIg-VK1) is shown below: AAATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTTATGGCAATAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAAACTCCTCATCTATCTTGCA TCCAACCTAGAATTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACTGTCAGCAAAATAATGAGGATCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO.17); The nucleotide sequence encoding the second light chain variable region (E15-S154-H1-MuIg-VK1) is shown below: GACATTCAGCTGACGCAGTCTCCAGCATCCCTGTCCATGGCTATAGGAGAAAAAGTCACCATCAGATGCATAACCAGCACTGATATTGATGATGATATGAACTGGTTCCAGCAGAAGTCAGGGGAACCTCCTAAGCTCCTTATTTCAGAAGGCAATACTC TTCGTGCTGGAGTCCCATCCCGATTCTCCAGCAGTGGCTATGGTACAGATTTTGTTTTTACAATTGAAAACATGCTCTCAGAAGATGTTGCAGATTACTACTGTTTGCAAAGTGATAACTTGCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAAATAAAA (SEQ ID NO.18); According to the codon encoding rules, the amino acid sequence of the first heavy chain variable region (E15-D1-MuIg-VK1) is as follows: NIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKTPHLSCIQPRIGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPPTFGGGTKLEIK (SEQIDNO.19); The amino acid sequence of the second light chain variable region (E15-S154-H1-MuIg-VK1) is as follows: DIQLTQSPASLSMAIGEKVTIRCITSTDIDDDMNWFQQKSGEPPKLLISEGNTLRAGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPYTFGGGTKLEIK (SEQIDNO.20).

[0083] (8) Eukaryotic expression and purification of mouse anti-human Claudin18.2 full-length antibody Genetically engineered antibodies were expressed in large quantities by co-transfection of CHO cells with light and heavy chain plasmids, and purified using a protein A affinity chromatography column. The results of protein gel electrophoresis are as follows: Figure 7 The results showed two target bands in the eluent, located at 55 kD and 25 kD respectively, which are consistent with the molecular weight of the antibody light and heavy chains.

[0084] Example 5: Characterization and assay of genetically engineered antibodies (1) Determination of antibody titer in genetic engineering The titer of genetically engineered antibodies was detected using an indirect ELISA method. Readings were taken at 450 nm using a microplate reader, and the absorbance values ​​were analyzed. Figure 8 As shown in the figure. The results indicate that the Mu-E15-D1R1 antibody titer reached 1:729000, and the Mu-E15-S154-H1R4 antibody titer reached 1:243000.

[0085] (2) IHC identification of genetically engineered antibodies To verify that the genetically engineered mouse anti-human Claudin18.2 antibody can recognize Claudin18.2 in natural tissue samples, immunohistochemistry (IHC) was used to detect whether the genetically engineered antibody had the ability to bind to natural Claudin18.2 in human breast cancer and gastric cancer tissue samples. Figure 9 and Figure 10 As shown in the figure. The results showed that the genetically engineered antibodies Mu-E15-D1R1 and Mu-E15-S154-H1R4 could recognize natural Claudin18.2 in human breast cancer and gastric cancer tissue samples.

[0086] (3) ICC identification of genetically engineered antibodies To verify that the genetically engineered mouse anti-human Claudin18.2 antibody can recognize Claudin18.2 in natural tissue samples, immunocytochemistry (ICC) experiments were used to detect whether the genetically engineered antibody had the ability to bind to natural Claudin18.2 in gastric 293T-CLDN18.2 cells. Figure 11 and Figure 12As shown in the figure. The results showed that the genetically engineered antibodies Mu-E15-D1R1 and Mu-E15-S154-H1R4 could recognize native Claudin18.2 in 293T-CLDN18.2 cells.

[0087] Finally, it should be emphasized that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A DNA-based Claudin18.2 genetically engineered antibody, characterized in that, The Claudin18.2 genetically engineered antibody includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.15, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.19; or, The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.16, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.

20.

2. A nucleic acid, characterized in that, The nucleic acid encodes the heavy chain variable region and the light chain variable region of the Claudin18.2 genetically engineered antibody according to claim 1.

3. The nucleic acid according to claim 2, characterized in that, The nucleotide sequences encoding the heavy chain variable region are shown in SEQ ID NO. 13 or 14, and the nucleotide sequences encoding the light chain variable region are shown in SEQ ID NO. 17 or 18.

4. The nucleic acid according to claim 3, characterized in that, The nucleotide sequence shown in SEQ ID NO.13 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.15 in the Claudin18.2 genetically engineered antibody; The nucleotide sequence shown in SEQ ID NO.14 is used to encode the heavy chain variable region of the amino acid sequence shown in SEQ ID NO.16 in the Claudin18.2 genetically engineered antibody; The nucleotide sequence shown in SEQ ID NO.17 is used to encode the light chain variable region of the amino acid sequence shown in SEQ ID NO.19 in the Claudin18.2 genetically engineered antibody; The nucleotide sequence shown in SEQ ID NO.18 is used to encode the light chain variable region of the amino acid sequence shown in SEQ ID NO.20 in the Claudin18.2 genetically engineered antibody.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid according to any one of claims 2-4.

6. A recombinant expression cell, characterized in that, The recombinant expression cell comprises the recombinant expression vector of claim 5, or the nucleic acid of any one of claims 2-4.

7. The use of the Claudin18.2 genetically engineered antibody as described in claim 1, or the nucleic acid as described in any one of claims 2-4, or the recombinant expression vector as described in claim 5, or the recombinant expression cell as described in claim 6, in the preparation of Claudin18.2 pathological diagnostic antibodies or gastric cancer and breast cancer detection kits.

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