Claudin18.2 genetically engineered antibody based on DNA immunity and preparation method and application of Claudin18.2 genetically engineered antibody

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a Claudin18.2 genetically engineered antibody based on DNA (deoxyribonucleic acid) immunity as well as a preparation method and application of the Claudin18.2 genetically engineered antibody. 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 amino acid sequences as shown in SEQ ID NO. 15-16; the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO. 19-20. According to the Claudin18.2 genetically engineered antibody, full-length plasmid DNA and full-length plasmid DNA + extracellular domain polypeptide serve as immunogens to immunize Balb / c mice, plasma cells capable of secreting specific antibodies aiming at Claudin18.2 are obtained, then the specific mouse-derived genetically engineered antibody is prepared through the single-cell photoconductive technology and the genetic engineering technology, and the genetically engineered antibody is high in titer, high in specificity, good in stability and good in stability. The specificity is high, and the specific recognition capability on the natural protein Claudin18.2 is realized.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and in particular relates to a Claudin18.2 genetic engineering antibody based on DNA immunization, and a preparation method and application thereof. Background Art

[0002] Single B cell photoconductive technology enables in vitro cloning and expression of antibody genes from single antigen-specific B cells, preserving the natural pairing of light and heavy chain variable regions. It offers advantages such as simplicity, rapidity, low cell count requirements, and high efficiency. The single B platform combines microfluidics with various sorting technologies to directly isolate, analyze, and screen B cells at the single-cell level, allowing for precise and efficient screening of B cells secreting target antibody molecules. Combined with single-cell sequencing technology, the target antibody sequence can 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 immune animals.

[0003] DNA immunization utilizes recombinant DNA molecules to stimulate an immune response. This technology involves introducing specific gene fragments (typically pathogen antigen genes) into host cells, thereby prompting the host to produce a specific immune response. This technology 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 specific immune responses to the antigen, including antibodies and T cells. Compared to traditional vaccines, DNA immunization offers advantages such as simple production, lower cost, and longer-lasting immune effects. Therefore, it is 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 the flow of molecules between cells by establishing barriers. Claudins are expressed as distinct isoforms in different tissues, and their abnormal expression has been linked to the development and progression of tumors. Claudin18.2, in particular, is an important target for the treatment of digestive system malignancies. While research on Claudin18.2 is increasing, there are no reports of highly specific and sensitive monoclonal antibodies against Claudin18.2 generated by genetic DNA immunization. Summary of the Invention

[0005] In response to the deficiencies in the prior art, the present invention provides a Claudin18.2 genetically engineered antibody based on DNA immunization, and a preparation method and application thereof. The Claudin18.2 genetically engineered antibody is prepared by first preparing a full-length DNA plasmid of Claudin18.2 (pVAX1-CLDN18.2) and an extracellular segment polypeptide of Claudin18.2 (CLDN18.2). Balb / c mice are then immunized with the full-length plasmid DNA and the full-length plasmid DNA + extracellular segment polypeptide as immunogens, respectively, so that B cells in the Balb / c mice proliferate and differentiate into plasma cells, which can secrete specific antibodies against Claudin18.2. Specific mouse-derived genetically engineered antibodies are then prepared through single-cell photoconductive technology and genetic engineering technology, and the titer of the genetically engineered antibodies is verified to be high by enzyme-linked immunosorbent assay. Immunohistochemistry and immunofluorescence experiments verify that the genetically engineered antibodies have specific recognition capabilities for the natural protein Claudin18.2, which lays the foundation for the further development of highly specific and sensitive Claudin18.2 pathological diagnostic antibodies.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: The first object of the present invention is to provide a Claudin18.2 genetically engineered antibody based on DNA immunization, 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 SEQIDNO.15-16; the light chain variable region comprises the amino acid sequence shown in SEQIDNO.19-20.

[0007] Furthermore, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.15, and the light chain variable region comprises 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] The second object of the present invention is to provide a nucleic acid encoding the heavy chain variable region and light chain variable region of the above-mentioned 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 Claudin18.2 genetically engineered antibody with the amino acid sequence shown in SEQ ID NO.16; The nucleotide sequence shown in SEQ ID NO.17 is used to encode the light chain variable region of the Claudin18.2 genetically engineered antibody with the amino acid sequence shown in SEQ ID NO.19; 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] The third object of the present invention is to provide a recombinant expression vector comprising the nucleic acid described above.

[0012] The fourth object of the present invention is to provide a recombinant expression cell, which comprises the recombinant expression vector described above, or the nucleic acid described on the market.

[0013] A fifth object of the present invention is to provide a method for preparing a Claudin18.2 genetically engineered antibody based on DNA immunization, comprising the steps of: (a) Preparation of immunogens; (b) animal immunization; (c) Claudin18.2 antigen-specific single plasma cell screening; (d) Preparation of genetically engineered antibodies.

[0014] The specific steps are: (1) Immunogen preparation: Prepare a full-length plasmid DNA immunogen of Claudin18.2 (pVAX1-CLDN18.2) and an extracellular segment polypeptide immunogen of Claudin18.2 (CLDN18.2); wherein the amino acid sequence of the extracellular segment polypeptide immunogen of Claudin18.2 is shown in SEQ ID NO.1; the amino acid sequence of the full-length plasmid DNA immunogen of Claudin18.2 is shown in SEQ ID NO.2; FWMSTANMYTGMGGC (SEQ ID NO.1) MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCA IAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV (SEQ ID NO.2) (2) Animal immunization experiments were conducted, using two methods to immunize Balb / c mice: the full-length plasmid pVAX1-CLDN18.2 and the full-length plasmid PVAX1-CLDN18.2 + extracellular segment peptide. The mice produced sensitized B lymphocytes that secreted specific antibodies against Claudin18.2. (3) Splenocyte suspensions were prepared from the two immunized mice, and mouse spleen mononuclear cells were isolated. Mouse CD138-positive plasma cells were then sorted using MACS buffer and CD138 antibody magnetic beads. Specifically, the sorting method was to specifically sort CD138-expressing positive plasma cells using Ficoll density gradient centrifugation and magnetic bead sorting. (4) Lentiviral packaging and 293T transfection of full-length Claudin18.2 overexpressing cell lines; specifically, including lentiviral vector construction and lentiviral packaging, lentiviral infection of HEK-293T cells, and puromycin selection of overexpressing stable cell lines; (5) Two groups of immune mouse-specific single plasma cells were screened using single-cell optical guidance technology and microfluidic technology based on the Beacon platform; specifically, the process included treating the 293T-CLDN18.2 overexpressing cell line as a screening medium, screening mouse-specific single plasma cells, and deriving single positive plasma cells; (6) Using single B cell sequencing technology, single cell RNA is obtained and cDNA is prepared, and antibody light and heavy chain variable region gene amplification and DNA fragment recovery are performed to obtain antibody heavy and light chain variable region sequences; specifically, the method for extracting single plasma cell RNA and preparing cDNA includes using a magnetic bead method to extract single plasma cell RNA with high antigen binding affinity, and using specific primers for reverse transcription to prepare cDNA; (7) Constructing a monoclonal antibody gene expression vector, connecting the obtained heavy and light chain variable region sequences into the expression vector to construct a recombinant expression vector, and transfecting 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, transferring into CHOS or 293F cells, and purifying to obtain the corresponding genetically engineered monoclonal antibody.

[0015] Furthermore, the expression vectors are pcDNA3.4 (pFUSEss-CHIg-mG2b) and pcDNA3.4 (pFUSE2ss-CLIg-mk); and the eukaryotic cells are eukaryotic Expi CHOS or 293F. Specifically, the eukaryotic expression and purification method includes: diluting the recombinant expression vector plasmid and PEI with a diluent, adding the diluted products to a CHOS cell suspension for transfection and culturing to obtain a cell suspension, culturing for 24 hours, feeding, collecting the cell supernatant after 120 hours by centrifugation, filtering the supernatant with a filter membrane, and then passing the supernatant through a Protein A affinity chromatography column 3-4 times. Finally, eluting with a glycine solution, collecting the eluate, and obtaining a purified Claudin18.2 genetically engineered antibody.

[0016] In addition, ELISA titer detection, IHC verification, and ICC verification methods are also included.

[0017] Specifically, the ELISA titer verification method includes: coating, washing, blocking, washing, primary antibody incubation, washing, secondary antibody incubation, washing, color development and other steps, and finally detecting the absorbance by a microplate reader.

[0018] The ICC verification method includes: cell climbing, washing, fixation, permeabilization, blocking, primary antibody incubation, room temperature equilibration and washing, secondary antibody incubation, nuclear staining and sealing (DAPI staining), and finally observation under a laser confocal microscope to collect images.

[0019] The IHC verification method includes: tissue fixation, sectioning, inactivation, antigen retrieval, washing, blocking, secondary antibody incubation, color development, counterstaining, sealing and other steps, and finally observation under a microscope and taking pictures.

[0020] The sixth object of the present invention is to provide a use of the Claudin18.2 genetically engineered antibody or the Claudin18.2 nucleic acid or the recombinant expression vector or the recombinant expression cell or the Claudin18.2 genetically engineered antibody prepared by the above-mentioned 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 present invention has the following beneficial effects: The present invention first prepares a full-length Claudin18.2 (pVAX1-CLDN18.2) plasmid vector and a Claudin18.2 (CLDN18.2) extracellular segment polypeptide, and divides the Balb / c mice to be immunized into a Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid immunogen group and a combined immunogen group of the Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid and the Claudin18.2 (CLDN18.2) extracellular segment polypeptide. Immunization is performed separately according to the immunization scheme, so that B cells in the Balb / c mice proliferate and differentiate into plasma cells, and the plasma cells can secrete specific antibodies against Claudin18.2. Among them, the Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid immunogen group successfully screened out 7 single plasma cells that can secrete antibodies specific to Claudin18.2 using single-cell photoconductive technology, and successfully prepared 12 genetically engineered antibodies using genetic engineering technology and eukaryotic expression system. Two genetically engineered antibodies had high titers as detected by indirect ELISA, and one antibody could recognize Claudin18.2 protein in natural tissues through IHC detection; and Claudin18.2 ( A combination immunogen panel, combining the full-length pVAX1-CLDN18.2 plasmid and the extracellular domain of Claudin18.2 (CLDN18.2) peptide, was developed. Using single-cell photoluminescence technology, eight plasma cells secreting antibodies specific for Claudin18.2 were successfully screened. Ten genetically engineered antibodies were then produced using genetic engineering techniques and a eukaryotic expression system. One of these genetically engineered antibodies demonstrated a high titer by indirect ELISA, and another antibody recognized Claudin18.2 protein in native tissues by IHC. This approach paves the way for the development of highly specific and sensitive antibodies for the diagnosis of Claudin18.2 in pathology. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in this application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is an agarose gel electrophoresis diagram of the pVAX1-CLDN18.2 plasmid in Example 1 of the present invention; Figure 2 This is a microscopic image of the Puromycin screening results of 293T-CLDN18.2 overexpressing cells in Example 2 of the present invention; Figure 3 This is a graph showing the Texas Red fluorescence reaction of Beacon single plasma cells screened in Example 4 of the present invention; Figure 4 Figure 4 is an agarose gel electrophoresis diagram of the amplified light and heavy chain variable regions of the antibody in Example 4 of the present invention, wherein Figure A is an amplified diagram of the antibody heavy chain variable region, and Figure B is an amplified diagram of the antibody light chain variable region; Figure 5 A colony identification map was constructed for the antibody light and heavy chain T vector in Example 4 of the present invention, wherein A is the antibody heavy chain colony identification map, and B is the antibody light chain colony identification map; Figure 6 A colony identification map was constructed for the full-length antibody light and heavy chains of the antibody in Example 4 of the present invention, wherein A is the colony identification map of the antibody heavy chain, and map B is the colony identification map of the antibody light chain; Figure 7 This is the polyacrylamide gel electrophoresis diagram of the genetically engineered antibody in Example 4 of the present invention; Figure 8 This is a titer curve of the genetically engineered antibody in Example 5 of the present invention; Figure 9 This is a diagram showing the IHC test results of the genetically engineered antibody Mu-E15-D1-R1 in Example 5 of the present invention; Figure 10 This is a diagram showing the IHC test results of the genetically engineered antibody Mu-E15-S154-H1-R4 in Example 5 of the present invention; Figure 11 This is a graph showing the ICC test results of the genetically engineered antibody Mu-E15-D1-R1 in Example 5 of the present invention; Figure 12 This is a graph showing the ICC test results of the genetically engineered antibody Mu-E15-S154-H1-R4 in Example 5 of the present invention. DETAILED DESCRIPTION

[0024] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials involved in the following examples are common commercial products and can be purchased from the market unless otherwise specified.

[0025] The above-mentioned technical features of the present invention and the technical features specifically described below (such as 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 A method for preparing a full-length Claudin18.2 (CLDN18.2) plasmid vector comprises the following steps: (1) Design and synthesis of Claudin18.2 (CLDN18.2)-specific primers ① Go to the NCBI website (https: / / www.ncbi.nlm.nih.gov / ), select the Nucleotide database, enter the search terms Claudin18.2 (CLDN18.2) and homo sapiens, and find and click Homo sapiensclaudin 18 (CLDN18), transcript variant 2; ② Click on CDS to obtain the Claudin18.2 (CLDN18.2) protein coding region sequence; ③ Click Pick Primer, enter the full-length CDS sequence in the PCR Template field, enter the first and last 15-30 bp of the CDS sequence in the Primer Parameters field, click Get Primers, and select the optimal specific primers based on the primer Tm value, GC content, self-complementarity, and self-3'complementarity scores. 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 the plasmid containing the Claudin18.2 (CLDN18.2) gene as the amplification template, the full-length target gene was amplified by PCR. The PCR reaction system and amplification program settings are shown in Tables 2 and 3, respectively.

[0029] Table 2 PCR reaction system

[0030] Table 3 Amplification procedures

[0031] ② Use Tiangen Agarose Recovery Kit to recover the amplified product.

[0032] First, add 500 μL of BL equilibration solution to the CA2 column and centrifuge for 2 minutes at 12,000 rpm. Discard the lower layer. Add the Claudin18.2 (CLDN18.2) amplified product to the CA2 column, let it sit at room temperature for 2 minutes, then centrifuge for 2 minutes at 12,000 rpm. Discard the lower layer. Add 600 μL of PW rinse solution containing anhydrous ethanol to the CA2 column and centrifuge for 2 minutes at 12,000 rpm. Discard the lower layer and repeat this step once. Centrifuge the empty CA2 column again at 12,000 rpm for 2 minutes. Place the CA2 column in a fume hood for 5 minutes. Once completely dry, place it in a clean 1.5 mL centrifuge tube. Finally, add 50 μL of ddH2O to the center of the CA2 membrane. Let it sit at room temperature for 2 minutes. Then, centrifuge for 2 minutes at 12,000 rpm to collect the DNA solution.

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

[0034] ② Linearized vector recovery: Use the Tiangen Agarose Recovery Kit to recover the linearized vector. First, add 500 μL of BL equilibration buffer to the CA2 column. Centrifuge for 2 minutes at 12,000 rpm, and discard the lower layer. Add the Claudin18.2 (CLDN18.2) amplified product to the CA2 column. Let it stand at room temperature for 2 minutes, then centrifuge for 2 minutes at 12,000 rpm. Discard the lower layer. Add 600 μL of PW rinse buffer containing anhydrous ethanol to the CA2 column. Centrifuge for 2 minutes at 12,000 rpm, and discard the lower layer. Repeat this step once. Centrifuge the empty CA2 column again at 12,000 rpm for 2 minutes. Place the CA2 column in a fume hood for 5 minutes. Once the CA2 column is completely dry, place it in a clean 1.5 mL centrifuge tube. Finally, 50 μL of ddH2O was added to the center of the CA2 adsorption film, and the mixture was allowed to stand at room temperature for 2 min. The mixture was then centrifuged at 12,000 rpm for 2 min to collect the DNA solution.

[0035] (4) Construction of pVAX1-CLDN18.2 plasmid ① Recombinate the full-length Claudin18.2 (CLDN18.2) target gene fragment with the linearized pVAX1 vector. Calculate the required amounts of target fragment and linearized vector using the formula: Cloning vector amount = [0.02 × number of base pairs in the cloning vector] ng, insert amount = [0.04 × number of base pairs in the insert] ng. Add these to a PCR tube. Add 1 μL of Exnase II and 2 μL of 5× CE II Buffer to the tube, and make up the recombinant system to 10 μL with purified water. Mix the recombinant system thoroughly and place it in a PCR instrument. Set the reaction conditions to 37°C for 30 minutes.

[0036] ② Recombinant transformation: Add 5 μL of the recombinant product to 50 μL of competent DH5α, flick to mix, and place on ice for 30 min. Heat shock in a 42°C water bath for 90 s and immediately place on ice for 4 min. Add 700 μL of LB liquid medium without resistance and culture in a constant temperature shaker at 37°C and 220 rpm for 1 h. Then centrifuge the bacterial solution at 5000 rpm for 5 min, discard 600 μL of the supernatant, use the remaining liquid to resuspend the bacteria, and evenly spread the resuspended bacteria on an LB solid culture dish containing kanamycin resistance. Incubate inverted in a constant temperature incubator at 37°C overnight.

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

[0038] ② Plasmid extraction: Add 100 μL of the bacterial suspension from the clone with the correct PCR band to 5 mL of LB medium containing kanamycin resistance and incubate overnight at 37°C in a shaker. After the E. coli reaches the logarithmic growth phase, extract the plasmid using a plasmid extraction kit. Add 500 μL of BL balance solution to the adsorption column CP3, centrifuge at 12000 rpm for 2 minutes, and discard the lower waste liquid; centrifuge the E. coli culture at 12000 rpm for 2 minutes; add 250 μL of P1 solution containing RNaseA to the bacteria, and use a pipette to mix thoroughly; add 250 μL of P2 solution to the centrifuge tube, gently turn up and down 6-8 times, and when the solution becomes clear and viscous, add 350 μL of P3 solution, immediately turn up and down 6-8 times, and then centrifuge at 12000 rpm for 10 minutes, aspirate the upper solution and transfer it to a clean centrifuge tube; add the supernatant to the adsorption column CP3 and centrifuge at 12000 rpm for 1 minute, and discard the waste liquid in the centrifuge tube; add 600 μL of PW rinsing solution containing anhydrous ethanol to CP3, and centrifuge at 12000 rpm for 1 minute. min, pour out the liquid in the tube and repeat this step once; centrifuge the CP3 with the waste liquid discarded again at 12000 rpm for 2 min; place the CP3 in a fume hood for 5 min, and after the CP3 is completely dry, place it in a clean 1.5 mL centrifuge tube; add 50 μL of ddH2O to the center of the adsorption film of CP3, let it stand at room temperature for 2 min, and then centrifuge it at 12000 rpm in a centrifuge 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 for sequencing (determined by Wuhan Qingke Biotechnology Co., Ltd.) and analyze the sequencing results using Snapgene and BioXM 2.7.1 software and NCBI-BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0040] Example 2: Construction of 293T-CLDN18.2 overexpressing cell line (1) Lentivirus packaging ①Cultivate cells: Culture 293T cells in a 6-well plate 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, take the packaging plasmids PMD (0.5 μg / well), PSA (0.75 μg / well), and pLVX-AcGFP-N1-CLDN18.2 (2 μg / well) and mix; take 6 μL of PEI (1 mg / mL) packaging reagent 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; ④ Transfection: Take the prepared 293T cells, add the plasmid into the well plate, shake well and place in the incubator; ⑤ Medium change: Change the culture medium after 6 hours of culture; ⑥ Virus collection: Collect the virus once every 24h, 48h, and 72h. Mix the collected virus supernatants and centrifuge them at 12000rpm for 5min. Aspirate the supernatant and filter it with a 0.45μm filter membrane.

[0041] (2) Infection of 293T cells ①Cultivate cells: Culture 293T cells in a 6-well plate. Experiments are performed when the cells have grown to 50% of the culture dish and are in good condition. ② Infect cells: Remove the prepared cells, wash once with 1× PBS, then add 2 mL of virus suspension to each well, then add complete culture medium to 3 mL, and culture in a 37°C incubator.

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

[0043] (3) Screening of stable cell lines ①Cultivate cells: 24 hours after infection, plate the cells and set up an uninfected cell control group; ②Puromycin screening: When the cells grow to 50-70% of the culture dish, add 2-8μg / mL of puromycin and replace the culture medium every 2-3 days until all the cells in the control group die. Figure 2 shown.

[0044] Example 3: Animal immunization (1) Immunization of Balb / c mice with the immunogen Claudin18.2 ①pVAX1-CLDN18.2 plasmid immunization: First, prepare and set up the electroporator program; anesthetize the mouse with isoflurane and shave the hair to expose the mouse skin; draw 50 μL of plasmid solution (concentration 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 with a syringe; insert the electrode into the mouse's hind leg muscle, aligning the plasmid injection site in the center, and then perform the electroporation pulse.

[0045] ② Claudin18.2 (CLDN18.2) extracellular peptide immunization: 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, USA). Set the tissue grinder power to 60 Hz and the working time to 15 minutes. Emulsify the solution in the tube to a milky white opaque state and draw it up with a syringe. Fix the mouse with your left hand and with your right hand, point the needle tip of the syringe with the liquid drawn up in advance with the flat surface facing up and pierce the skin parallel to the mouse's head to inject the sample.

[0046] ③Immunization process: The immunization processes of the Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid immunogen group and the Claudin18.2 (pVAX1-CLDN18.2) full-length plasmid and Claudin18.2 (CLDN18.2) extracellular segment polypeptide combination immunogen group are shown in Table 4.

[0047] Table 4 Immunization process

[0048] (2) Balb / c mouse serum titer detection ① Orbital blood collection: Two weeks after the fourth immunization, collect orbital blood from Balb / c mice using capillary blood collection tubes. After allowing the blood to rest, centrifuge it in a high-speed refrigerated centrifuge pre-cooled to 4°C for 10 minutes at 3000 rpm. After centrifugation, separate the mouse serum into a clean centrifuge tube.

[0049] ② Coating overexpressing cells and blocking: Add 0.1% gelatin to 96-well plates and place in a 37°C constant temperature incubator for 2 hours, then remove the gelatin and add 1×10 5 cells, 100 μL per well, cultured overnight in a 37°C constant temperature incubator; washed 3 times with 1×PBST, 200 μL per well each time, for 3 minutes; fixed with 4% paraformaldehyde at room temperature for 30 minutes; washed 3 times with 1×PBST, 200 μL per well each time, for 3 minutes; added 2% BSA blocking solution, 200 μL per well, and blocked at 37°C for 1 hour.

[0050] ③ Serum titer assay: Serum stock solution was added to the first well, followed by a three-fold serial dilution (serum dilution factor 1-729). The diluted samples were sequentially added to a 96-well plate coated with 293T-CLDN18.2. Two parallel columns of control and blank groups were set up. 100 μL of sample and 1× PBS were added to each well and incubated at 37°C for 1 hour. After primary antibody incubation, the plate was washed three times with 1× PBST, followed by the addition of HRP-conjugated goat anti-mouse antibody and incubation at 37°C for 40 minutes. After secondary antibody incubation, the plate was washed five times with 1× PBST and patted dry. 90 μL of TMB colorimetric solution was then added to each well, and the color was developed at 37°C for 10 minutes. Finally, 50 μL of stop solution was added to terminate the colorimetric reaction. The absorbance was read on a microplate reader at a wavelength of 450 nm.

[0051] (3) Immune shock in Balb / c mice A Balb / c mouse with the highest serum titer was selected for immune shock. First, the 293T-CLDN18.2 cell density was adjusted to 5×10 6 cells / mL, draw 0.5 mL into the syringe; grab the mouse with its head facing downward, insert the syringe into the abdominal cavity at a 45-degree angle, and inject the sample after you feel the needle tip can move.

[0052] Example 4: Preparation of genetically engineered antibodies (1) Isolation of mouse spleen mononuclear cells ① Secure the mouse with one hand and gently press the skin around the eye to cause the eyeball to bulge and become congested. Grasp the eyeball with forceps and quickly remove it, while gently pressing the heart to increase blood flow. Execute the mouse by cervical dislocation and soak in 75% alcohol for 3-5 minutes.

[0053] ② Place the mouse on a sterile operating table and expose the skin on the left side of the mouse. Use ophthalmic scissors to cut the skin open. A deep red spleen will be found near the left rib cage. Remove the connective tissue attached to the spleen and remove it with forceps, then 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 the dish, and then place the spleen in the dish.

[0055] ④ Make a small incision on one side of the spleen and press down on it with the handle of a disposable syringe. Simultaneously, add 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1:1000 DNA Clean-up to the dish. Press until no large red particles remain in the spleen. Filter the liquid in the dish through a 40 µm filter.

[0056] ⑤ Add 5 mL of Ficoll density gradient centrifuge medium to each of four 15 mL sterile centrifuge tubes. Pour the filtrate along the tube wall into the centrifuge tube containing Ficoll, 5 mL per tube. Centrifuge at 800 g for 25 min at 20°C, using an acceleration of 9°C and a deceleration of 0°C.

[0057] ⑥ After centrifugation, carefully remove the cells from the cloud layer with a pipette and transfer them to a centrifuge tube containing 10 mL of RPMI1640 medium + 10% FBS. Gently pipette to mix thoroughly. 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 minutes at 4°C. Discard the supernatant.

[0058] (2) Isolation of mouse CD138-positive plasma cells ① Resuspend the mononuclear cells in 10 mL of magnetic activated cell sorting (MACS) buffer and centrifuge at 300 g for 10 min at 20°C. Collect 20 μL of the cell suspension for cell counting.

[0059] ② Every 1×10 7 Resuspend the cells in 40 μL of MACS buffer and add 10 μL of magnetic beads coated with CD138 antibody. Mix thoroughly and incubate in a 4°C refrigerator in the dark for 15 min.

[0060] ③ Every 1×10 7 Resuspend the cells in 1 mL MACS buffer and centrifuge for 10 min at 300 g and 20°C. Discard the supernatant. 8 The cells were resuspended in 500 μL MACS buffer and stored on ice.

[0061] ④ Install the LS column on the sorter and rinse the column with 5 mL of MACS buffer. Add the cell suspension to the column and rinse with 3 mL of MACS buffer. Repeat twice.

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

[0063] ⑥ Resuspend the cells in basal medium to a density of 6.25×10 6 cells / mL.

[0064] (3) Specific single plasma cell screening based on Beacon single cell light-guided technology ① Treatment of 293T-CLDN18.2 overexpressing cell line as screening medium 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 by aspiration. 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 the cells at 500 g for 5 min; e. Take 1 mL of Plasma B Cell Media into a 1.5 mL centrifuge tube, add 1.5 μL of Texas Red fluorescent secondary antibody, and mix thoroughly by pipetting. f. Add fluorescent secondary antibody to the 1.5 mL centrifuge tube containing 293T-CLDN18.2 cells and mix well to make the cell density 5×10 7 cells / mL, mix well and store in a 4°C refrigerator away from light.

[0065] ② Screening of mouse-specific single plasma cells a. Add the resuspended plasma cells to the cell loading well of the Beacon, avoiding bubbles. Using photoelectric positioning and microfluidics technology, single plasma cells enter the chamber of the chip and secrete antibodies therein. b. Add 10 μL of treated 293T overexpressing cells to the corresponding loading wells, avoiding bubbles. The 293T overexpressing cells enter the chip channel along with the culture medium. c. Observe the fluorescence reaction under the conditions of Texas Red fluorescence channel, such as Figure 3 As shown, plasma cells with strong fluorescence were picked from the chambers and removed from the chip, then collected into a 96-well PCR plate. In the PCR plate, 5 μL of 2× TCL lysis buffer and 10 μL of mineral oil were added to each well in advance. Seal the PCR plate with sealing film, centrifuge at 400 g for 1 min, and store in a -80°C refrigerator.

[0066] (4) Extraction of single plasma cell RNA and preparation of cDNA ① Take the PCR plate out of the -80°C freezer, place it at room temperature, and centrifuge it for 1 min at 200 g. ② Add 10 μL of RNA Clean XP magnetic beads, which have returned to room temperature, to the PCR plate. Mix thoroughly by pipetting, then incubate at room temperature for 1 minute. Once the beads are evenly distributed throughout the wells, seal the PCR plate with sealing film and incubate at room temperature for 20 minutes. ③ Place the PCR plate on a Mag Well magnetic separator and let it sit 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 MagWell magnetic separator and place it at room temperature for 2 minutes to dry the magnetic beads; ⑥ Add Mix 1 to the PCR plate (components listed in Table 5), 5 μL per well. Place the PCR plate in a thermal cycler and perform a single reaction, setting the temperature to 72°C for 3 minutes. Immediately place the PCR plate on ice after the reaction is complete. ⑦ Add Mix 2 to the PCR plate (components listed in Table 6), 4 μL per well. Mix thoroughly and place the plate in a PCR instrument for a secondary reaction (program settings shown in Table 7). ⑧ After the reaction is complete, add Mix 3 to the PCR plate (components shown in Table 8), 21 μL per well. Mix thoroughly and place the PCR plate in a PCR instrument for three reactions (program settings shown in Table 9). ⑨ Add AMPure XP magnetic beads that have returned to room temperature to the PCR plate, 30 μL per well, mix thoroughly, and incubate at room temperature for 5 minutes; ⑩ Place the PCR plate on a Mag Well magnetic separator, let it stand for 2 minutes, and then aspirate the upper liquid; ⑪ Add 80% ethanol to the PCR wells, 200 μL per well, incubate at room temperature for 30 seconds, and 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 enzyme-free water to the PCR plate, 15 μL per well, mix thoroughly, and incubate at room temperature for 2 min; ⑭ Place the PCR plate on a magnetic separator, let it stand for 1 minute, then aspirate the solution in the wells and store it in a clean PCR plate.

[0067] Table 5 Mixed reagent Mix1 ingredients

[0068] Table 6 Mixed reagent Mix2 composition

[0069] Table 7 Secondary PCR reaction procedure

[0070] Table 8 Mixed reagent Mix3 ingredients

[0071] Table 9 Three-step PCR reaction program

[0072] (5) Antibody light and heavy chain variable region gene amplification and DNA fragment recovery Using the cDNA of a single plasma cell as an amplification template, the variable region gene sequences of the antibody heavy chain and light chain were amplified separately. Prepare 50 μL of amplification system in a PCR tube: 18 μL ddH2O, 25 μL 2×Phanta Max Buffer, 1 μL dNTP, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL template, and 2 μL upstream and downstream degenerate primers. After thoroughly mixing, place it in a PCR instrument for fragment amplification. The antibody light and heavy chain variable region amplification program settings are shown in Table 10. After the amplification is completed, take 1 μL of the amplified product and perform DNA electrophoresis on a 1% agarose gel, as shown in Figure 10. Figure 4 The correct DNA fragments were then recovered by electrophoresis using an agarose gel recovery kit and the recovered concentration was measured using a UV spectrophotometer.

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

[0074] (6) Construction of mouse anti-human Claudin18.2 antibody variable region T vector ① Take 1 μL of the target fragment of the antibody heavy chain or light chain variable region and add it to a PCR tube. Then add 1 μL of the vector pEASY®-Blunt Cloning Vector and 3 μL of pure water. After mixing, react at 37°C for 10 minutes. Add the ligation product to 50 μL of T1 competent cells, flick to mix, and let it stand on ice for 30 minutes. Then heat shock in a 42°C water bath for 90 seconds and immediately place on ice for 2 minutes. Add 250 μL of resistance-free LB medium to the tube and incubate at 37°C for 1 hour at 200 rpm. Take 8 μL of 500 mmol·L -1 IPTG and 40 μL 20 mg mL -1Mix with IPTG and X-gal and evenly spread on an ampicillin-resistant LB culture plate. Incubate at 37°C for 30 minutes to allow IPTG and X-gal to be absorbed. Centrifuge the culture at 5000 rpm for 5 minutes and discard 200 μL of the supernatant. Resuspend the cells in the remaining medium and spread evenly on a plate. Incubate in a 37°C incubator overnight.

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

[0076] Table 11 Colony PCR program

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

[0078] First, the 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 the plasmids pFUSEss-CHIg-mG2b and pFUSE2ss-CLIg-mk after enzyme digestion were located between 3000 bp and 5000 bp, which is consistent with the fragment size of the linearized vector.

[0079] Then, the variable region gene of the heavy chain was constructed on pFUSEss-CHIg-mG2b by homologous recombination, and the variable region gene of the light chain was constructed on pFUSE2ss-CHIg-mk. After overnight culture, several white single colonies grew on the culture dish containing the corresponding resistance. Single colonies were picked for expansion and colony PCR was used to verify whether the construction was successful. Gel electrophoresis was as follows Figure 6 As shown, the results showed that the full-length vectors of the heavy chain and light chain were successfully constructed, and the sequencing results were analyzed using Snapgene, BioXM.2.7.1 software and NCBI-BLAST website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), which were 100% identical to the original base sequence.

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

[0081] Table 12 Heavy and light chain PCR amplification primers

[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: EVKLQQSGPELVKPGASVKMSCKASGYSFTSYVIHWVKQKPGQGLEWIGYIHPYNGGTKNNEKFKGRATLTSDKSSSTAYMELSSLTSEDSAVYYCARLASYGRYGLDYWGQGTSVTVSS (SEQIDNO.15); The amino acid sequence of the second heavy chain variable region (E15-S154-H1-MuIg-VH1) is: 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: NIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKTPHLSCIQPRIGVPARFSGSGSRTDFTLTIDPVEADDAATYYCQQNNEDPPTFGGGTKLEIK (SEQIDNO.19); The amino acid sequence of the second light chain variable region (E15-S154-H1-MuIg-VK1) is: DIQLTQSPASLSMAIGEKVTIRCITSTDIDDDMNWFQQKSGEPPKLLISEGNTLRAGVPSRFSSSGYGTDFVFTIENMLSEDVADYYCLQSDNLPYTFGGGTKLEIK (SEQIDNO.20).

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

[0084] Example 5: Characterization of genetically engineered antibodies (1) Determination of the titer of genetically engineered antibodies The titer of genetically engineered antibodies was detected by indirect ELISA, and the absorbance was analyzed using a microplate reader at a wavelength of 450 nm. Figure 8 The results showed that the titer of Mu-E15-D1R1 antibody reached 1:729000, and the titer of Mu-E15-S154-H1R4 antibody reached 1:243000.

[0085] (2) IHC identification of genetically engineered antibodies In order to verify that the mouse anti-human Claudin18.2 genetically engineered antibody can recognize Claudin18.2 in natural tissue samples, immunohistochemistry (IHC) experiments were performed to test whether the genetically engineered antibody has the ability to bind to natural Claudin18.2 in human breast cancer and gastric cancer tissue samples. Figure 9 and Figure 10 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 In order to verify that the genetically engineered mouse anti-human Claudin18.2 antibody can recognize Claudin18.2 in natural tissue samples, immunocytochemistry (ICC) experiments were performed to detect whether the genetically engineered antibody has the ability to bind to natural Claudin18.2 in gastric 293T-CLDN18.2 cells, such as Figure 11 and Figure 12The 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 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 may have various changes and modifications. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Claudin18.2 genetically engineered antibody based on DNA immunization, 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 any one of the amino acid sequences shown in SEQ ID NO.15-16; the light chain variable region includes the amino acid sequence shown in SEQ ID NO.19-20.

2. The Claudin18.2 genetic engineering antibody based on DNA immunization according to claim 1, characterized in that: The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.15, and the light chain variable region comprises 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.

3. 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 or 2.

4. The nucleic acid according to claim 3, characterized in that The nucleic acid includes one or more of the nucleotide sequences shown in SEQ ID NO.13-14 and SEQ ID NO.17-18.

5. The nucleic acid according to claim 4, 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.

6. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to any one of claims 3 to 5.

7. A recombinant expression cell, characterized in that: The recombinant expression cell comprises the recombinant expression vector according to claim 6, or the nucleic acid according to any one of claims 3-5.

8. The method for preparing a Claudin18.2 genetically engineered antibody based on DNA immunization according to claim 1 or 2, characterized in that: The steps include: (a) Preparation of immunogens; (b) animal immunization; (c) Claudin18.2 antigen-specific single plasma cell screening; (d) Preparation of genetically engineered antibodies.

9. The preparation method according to claim 8, characterized in that: The immunogen is a Claudin18.2 full-length plasmid DNA or a combination of a Claudin18.2 full-length plasmid DNA and an extracellular segment polypeptide; the Claudin18.2 full-length DNA plasmid has an amino acid sequence shown in SEQ ID NO.2; and the extracellular segment polypeptide has an amino acid sequence shown in SEQ ID NO.

1.

10. Use of the Claudin18.2 genetically engineered antibody as described in claim 1 or 2, or the nucleic acid as described in claims 3-5, or the recombinant expression vector as described in claim 6, or the recombinant expression cell as described in claim 7, or the Claudin18.2 genetically engineered antibody prepared by the preparation method as described in claim 8 or 9 in the preparation of Claudin18.2 pathological diagnostic antibodies or gastric cancer and breast cancer detection kits.

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