CN111705085B - Method for constructing animal model and application - Google Patents

Abstract

The invention provides a method for constructing an animal model, wherein the animal model introduces frame shift mutation by editing Cldn18 gene, and does not express Cldn18.2 protein. The animal model prepared by the method expresses an antibody capable of being combined with human Cldn18.2 protein after immunization, and the antibody is specifically combined with the Cldn18.2 protein.

Description

Method for constructing animal model and application

Technical Field

The application relates to a method for establishing a genetically modified animal model and application thereof, in particular to a construction method based on a Claudin (Cldn) 18.2 protein deletion animal model and application thereof in biological medicine research.

Background

Antibodies are immunoglobulins which are produced by the conversion of B lymphocytes into plasma cells following stimulation of an organism by an antigenic substance and which are capable of specific binding reactions with the corresponding antigen. Since antibody drugs exert therapeutic effects through immunological mechanisms with less toxicity, they have higher specificity, reliability and lower toxic and side effects than conventional drugs, and are considered as one of the most promising classes of human disease therapeutic drugs, and have become a hot spot in drug development worldwide nowadays.

The Tight Junction (TJs) structure between cells is a transmembrane protein complex, the stability of which requires the coordinated activities of several different proteins to be maintained, whereas the Cldn protein, which is a scaffold protein constituting the tight junction structure, is mainly distributed on epithelial cells and mesenchymal cells, and plays a barrier and barrier role in maintaining the organization and function of cell tissues. It has been shown that about 90% of malignant tumors originate from epithelial cells, and the tight junction between epithelial cells is closely related to the occurrence and development of malignant tumors, which are often accompanied by the reduction of tight junction function and the change of expression of tight junction protein.

To date, 27 members of the Cldn family have been found in mammals with molecular weights of 20-27 KDa, and a distribution with high tissue-organ specificity; the functions mainly include intercellular adhesion, cell polarity maintenance, cell bypass permeability regulation, cell proliferation and cell differentiation regulation, and the like. The Cldn family protein structure includes 4 transmembrane regions, two extracellular loops and one intracellular loop, with the N-and C-termini being cytoplasmic.

The Cldn18 gene belongs to the Cldn family and expresses two protein subtypes by alternative splicing: cldn18.1 and cldn18.2. Studies have shown that cldn18.2 is normally expressed only at low levels in differentiated parietal cells, undetectable in gastric stem cells and other normal human organs, but is significantly expressed in a variety of tumors, including 80% of gastrointestinal adenomas, 60% of pancreatic tumors, and some biliary, ovarian and pulmonary tumors. The function, structural characteristics (two extracellular loops) and specific expression (almost no expression on normal tissues and low risk of side effects) of Cldn18.2 make the molecule an ideal tumor treatment target.

Although there have been many studies that have shown that Cldn18.2 is a very good target for tumor therapy (Ugur Sahin et al. Clin Cancer Res.2008 Dec 1;14(23): 7624-34); tureci O et al Gene.2011 Aug 1;481(2):83-92, but to date, only 1 antibody drug targeting Cldn18.2 (Ganymed' IMAB 362) is currently in preparation for the third-phase clinic, with the major indications being gastric and pancreatic cancer. The published data show that IMAB362 can directly act on Cldn18.2 to stimulate immune responses such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and the like, can be used alone, and can also be combined with chemotherapeutic drugs (such as epirubicin, oxaliplatin and capecitabine) to play roles in recruiting T cells and changing the tumor microenvironment, so as to achieve the effect of killing Cldn18.2 positive tumors. In contrast, IMAB362 does not have a killing effect and fewer side effects for normal tissues that express little or no cldn18.2. Furthermore, Ganymed is also developing Cldn18.2 and CD3 bispecific antibodies, as well as antibody-conjugated drugs.

Gastric and pancreatic cancers have been the worst-prognosis malignancies and the most complex diseases to treat. Because of the strong heterogeneity and low efficacy of these two types of tumors, immunotherapy has been an area of concern. Since the research results of anti-cldn18.2 antibody IMAB362 greatly prove the feasibility of cldn18.2 as a drug target for tumor therapy, there is a need in the art to accelerate the development of various drugs targeting cldn18.2.

However, due to the high degree of conservation of the sequence of the Cldn18 gene in different species (Tureci O et al Gene 2011 Aug 1;481(2): 83-92), especially the high homology existing between mouse and human Cldn18.1 and Cldn18.2 molecules, it is difficult to obtain an antibody specifically targeting the Cldn18.2 protein by the conventional method for immunizing ordinary mice. In addition, the extracellular regions of the Cldn18.1 and Cldn18.2 proteins expressed by the Cldn18 gene are similar, and only the first extracellular domain has eight amino acid differences, so that the problem that cross reaction is easily caused to generate side effect needs to be specially considered in the development process of the antibody. In the course of research and development, Ganymed has undertaken various immunization strategies to stimulate the mouse immune system and overcome immune tolerance, using virus-like particles (VLPs), peptide conjugates, and different fragments encoding human cldn18.2 protein in addition to conventional reagents, and performed multiple immunizations and boosts on more than 100 mice in total. After obtaining the hybridoma, the obtained antibody is identified by using various methods including flow analysis and fluorescence microscopy, the cross-reactivity of the antibody with Cldn18.1 and Cldn18.2 is analyzed, and no more than 50 hybridoma cell lines secreting monoclonal antibodies are obtained by screening (see patent CN200680043664.X, CN 201380024247.0). For the development process of antibodies of other targets, such as the development of antibodies of PD-1 targets, the immunization process only uses PD-1 antigen and immunologic adjuvant to immunize mice for 2 times, and the boosting immunization is carried out for 1 time, namely thousands of hybridoma clones are obtained (see CN 105531288A). It can be seen that the prior art has the disadvantages of large workload, complex flow, low efficiency, and huge time, capital and manpower consumption in screening the cldn18.2-targeting antibody.

In view of the great application value of the Cldn18.2 protein in the fields of tumor and immunotherapy, more and more domestic and foreign pharmaceutical enterprises are expected to participate in the research and development of antibody drugs targeting the Cldn18.2 protein in the future. In order to improve the research and development efficiency and success rate, the invention provides a method for establishing a Cldn18.2 gene knockout animal model worldwide, and a Cldn18.2 gene knockout animal is obtained. Specifically, the present invention aims to prepare a non-human animal model in which no Cldn18.2 protein is expressed but Cldn18.1 protein is normally expressed by performing gene editing at a specific position of Cldn18 gene to generate frame shift mutation. The method has wide application prospect in the aspects of research and development, screening, validity verification and the like of the drug targeting the Cldnn 18.2 protein.

Disclosure of Invention

In a first aspect, the invention relates to a method for constructing an animal model comprising introducing a loss-of-function mutation in the Cldn18 gene in the animal model such that the Cldn18.2 protein is not expressed in the animal model.

Preferably, the animal model expresses cldn18.1 protein.

Preferably, the loss-of-function mutation is one or more.

Preferably, the loss-of-function mutation is a frameshift mutation.

Preferably, the loss-of-function mutation is introduced in exon 1 of the Cldn18 gene. Preferably, the method comprises constructing an animal model using sgRNA for Cldn18 gene. Further preferably, the sgRNA-targeted target site is selected from the sequence of all or part of the 1 st exon of Cldn18.

Still further preferably, the targeted target site of the sgRNA is as set forth in SEQ ID NO: 1-9.

In a specific embodiment of the invention, the sgRNA targets a target site as set forth in SEQ ID NO: shown at 7.

In a specific embodiment of the invention, the method comprises introducing a loss-of-function mutation in the animal model Cldn18 gene using sgRNA directed against Cldn18 gene such that the animal model does not express Cldn18.2 protein, wherein the targeted target site of the sgRNA is as set forth in SEQ ID NO: 1-9.

In one embodiment of the present invention, the method comprises the steps of:

the first step is as follows: preparing a polypeptide comprising SEQ ID NO: 1-9 of any one of sgRNA vectors;

the second step is that: mixing an in-vitro transcription product of the sgRNA vector and Cas9mRNA to obtain a mixed solution, injecting the mixed solution into cytoplasm or nucleus of mouse fertilized eggs, transferring the injected fertilized eggs into a culture solution for culture, and then transplanting the fertilized eggs into an oviduct of a receptor mother mouse for development to obtain an F0 generation mouse.

Further still include:

the third step: the F0 mouse is tested by using a PCR technology, and the successful mutation of the Cldn18 gene in the cell is verified to obtain a Cldn18 gene mutation positive mouse;

the fourth step: and (3) expanding the population quantity of the positive mice screened in the third step in a hybridization and selfing mode, and establishing stable Cldn18 gene mutation mice.

Preferably, the sequences of the PCR detection primer pair used in the third step are shown in SEQ ID NO: 13 and SEQ ID NO: as shown at 14.

Preferably, the animal model is a non-human animal. Preferably, the non-human animal is a rodent. More preferably, the rodent is a mouse.

In a second aspect of the present invention, there is provided a method for constructing an animal model, comprising introducing a frameshift mutation into exon 1 of Cldn18 gene of the animal model, such that the animal model does not express Cldn18.2 protein.

Preferably, the animal model expresses cldn18.1 protein.

Preferably, the frame shift mutation is an insertion or deletion of 1 or more pairs of bases, and the number of inserted or deleted bases is not a multiple of 3. Preferably 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19 or 20 base pairs are inserted or deleted.

Preferably, the cldn18.2 protein is not expressed in cells, tissues or organs of the animal model obtained.

In a third aspect of the present invention, there is provided a Cldn18 gene mutant cell, comprising introducing a loss-of-function mutation in the Cldn18 gene of the cell using a sgRNA directed against the Cldn18 gene such that the Cldn18.2 protein is not expressed by the cell, wherein a targeted target site of the sgRNA is as set forth in SEQ ID NO: 1-9.

Preferably, the cell expresses cldn18.1 protein.

Preferably, the loss-of-function mutation is one or more.

Preferably, the loss-of-function mutation is a frameshift mutation.

Preferably, the loss-of-function mutation is introduced in exon 1 of the Cldn18 gene.

Further preferably, the cell is constructed using a sgRNA directed against the Cldn18 gene, wherein the targeted target site of the sgRNA is as set forth in SEQ ID NO: 1-9. Preferably, the non-human animal from which the cell is derived is further preferably derived from a rodent, and further preferably derived from a mouse.

Preferably, the cells are from all cells that can originally express the cldn18.2 protein.

More preferably, the cell is derived from a somatic cell, a stem cell, a fertilized egg cell, or the like.

In one embodiment of the invention, the cells are from epithelial cells, mesenchymal cells, parietal cells, tumor cells, and the like. The tumor cells include but are not limited to gastric cancer cells, pancreatic cancer cells, and bile duct, ovarian or lung tumor cells.

Preferably, the cell may be any cell isolated from an animal model constructed by the above method.

In a fourth aspect of the present invention, the sgRNA targeting Cldn18 gene is provided, wherein the sgRNA targeting target site is selected from the exon sequence of Cldn18 coding region, and the coding region is exon region No. 1.

Preferably, the sgRNA-targeted target site is screened from the exon region of Cldn18 No. 1.

Further preferably, the sgRNA targets a target site as set forth in SEQ ID NO: 1-9.

Still further preferably, the sgRNA targets a target site such as SEQ ID NO: shown at 7.

Preferably, the DNA double-stranded sequences corresponding to the sgrnas are SEQ ID NOs: 18 and SEQ ID NO: 19.

in a fifth aspect of the present invention, a sgRNA vector is provided, which includes the sgRNA described above.

In a sixth aspect of the invention, a method of preparing a sgRNA vector includes the steps of:

(1) providing a polypeptide with a sequence shown in SEQ ID NO: 1-9 to obtain upstream and downstream sequences of the sgRNA, and preparing a forward oligonucleotide sequence and a reverse oligonucleotide sequence;

preferably, the sgRNA target sequence is SEQ ID NO: 7, and the upstream sequence of the obtained sgRNA is shown as SEQ ID NO: 18 and the downstream sequence is shown as SEQ ID NO: 19, adding TAGG to the 5 'end of the upstream sequence to obtain a forward oligonucleotide, and adding AAAC to the 5' end of the downstream sequence to obtain a reverse oligonucleotide, wherein the sequence of the obtained forward oligonucleotide is shown as SEQ ID NO: 10 is shown in the figure; the sequence of the reverse oligonucleotide is shown as SEQ ID NO: 11 is shown in the figure;

(2) synthesizing a fragment DNA containing a T7 promoter and sgRNA scaffold, wherein the fragment DNA containing the T7 promoter and the sgRNA scaffold is shown as SEQ ID NO: 12, sequentially carrying out enzyme digestion on the EcoRI and BamHI on the fragments to be connected to a skeleton vector pHSG299, and carrying out sequencing verification to obtain a pT7-sgRNAG2 vector;

(3) separately synthesizing the forward oligonucleotide and the reverse oligonucleotide described in step (1), preferably the oligonucleotide of SEQ ID NO: 10 and SEQ ID NO: 11, denaturing and annealing the synthesized sgRNA oligonucleotide to form a double strand which can be connected to the pT7-sgRNA 2 vector in the step (2);

(4) and (4) respectively connecting the double-stranded sgRNA oligonucleotides annealed in the step (3) with a pT7-sgRNA 2 vector, and screening to obtain a sgRNA vector.

The seventh aspect of the present invention relates to an application of the sgRNA and the sgRNA vector described above to Cldn18 gene editing.

Preferably, the use comprises mutating exon 1 of the Cldn18 gene of an animal model. So that the cldn18.2 protein is not expressed in cells, tissues or organs of the animal model.

In an eighth aspect of the present invention, the sgRNA or the sgRNA vector is used to mutate Cldn18 gene in exon 1 of Cldn18 gene.

The ninth aspect of the present invention relates to a method for preparing an animal model, comprising the steps of:

the first step is as follows: obtaining the sgRNA vector according to the steps (1) to (4) of preparing the sgRNA vector;

the second step is that: mixing an in-vitro transcription product of the sgRNA vector and Cas9mRNA to obtain a mixed solution, injecting the mixed solution into cytoplasm or nucleus of mouse fertilized eggs, transferring the injected fertilized eggs into a culture solution for culture, and then transplanting the fertilized eggs into an oviduct of a receptor mother mouse for development to obtain an F0 generation mouse;

the third step: f0 mouse is tested by PCR technology to verify Cldn18 gene modified animal in cell;

preferably, the sequences of the PCR detection primer pair used in the third step are shown in SEQ ID NO: 13 and SEQ ID NO: as shown at 14.

The tenth aspect of the present invention relates to a method for preparing an animal model with Cldn18 gene mutation, comprising the following steps:

the first step is as follows: obtaining the sgRNA vector according to the steps (1) to (4) of preparing the sgRNA vector;

the second step is that: mixing an in-vitro transcription product of the sgRNA vector and Cas9mRNA to obtain a mixed solution, injecting the mixed solution into cytoplasm or nucleus of mouse fertilized eggs, transferring the injected fertilized eggs into a culture solution for culture, and then transplanting the fertilized eggs into an oviduct of a receptor mother mouse for development to obtain an F0 generation mouse;

the third step: the F0 mouse is tested by using a PCR technology, and the successful mutation of the Cldn18 gene in the cell is verified to obtain a Cldn18 gene mutation positive mouse;

the fourth step: expanding the population quantity of the positive mice screened in the third step in a hybridization and selfing mode, and establishing stable Cldn18 gene mutation-/-mice;

preferably, the sequences of the PCR detection primer pair used in the third step are shown in SEQ ID NO: 13 and SEQ ID NO: as shown at 14.

In an eleventh aspect, the invention relates to a stably passable animal model, wherein the animal is prepared by the method described above. Preferably, the Cldn18 gene of the animal model has a mutation such that the encoded Cldn18.2 protein is not expressed in cells, tissues or organs of the animal model.

Preferably, the animal model is a non-human mammal. Preferably, the non-human mammal is a rodent. More preferably, the rodent is a mouse. Further preferably, the mouse is a C57BL/6, BALB/C mouse or progeny thereof.

The twelfth aspect of the invention relates to the application of the animal model obtained by the method in preparing anti-tumor drugs or drugs for treating immune-related diseases. Preferably, the anti-tumor drug or the drug for treating immune-related diseases is an antibody.

In a thirteenth aspect, the invention relates to the use of the animal model obtained by the above method in the preparation of a medicament for treating tumors.

Preferably, the tumor is a gastrointestinal tumor, a gastrointestinal adenoma, a pancreatic tumor, a bile duct tumor, an ovarian tumor or a lung tumor. Preferably, the tumor is malignant.

In one embodiment of the invention, the tumor is gastric cancer or pancreatic cancer.

In a fourteenth aspect, the present invention relates to a method for screening an antibody, comprising the steps of:

(1) immunizing animals with deletion of the Cldn18.2 protein;

(2) 2 weeks after immunization, boost immunization;

(3) after 4 days of boosting immunity, removing the spleen of the animal and separating spleen cells of the animal;

(4) hybridizing and fusing the animal spleen cells separated in the step (3) with myeloma cells to obtain hybridoma cells;

(5) and (4) screening the hybridoma cells obtained in the step (4) to obtain an antibody targeting the Cldnn 18.2 protein.

Preferably, the animal with the Cldn18.2 protein deletion is an animal model obtained by the method.

Preferably, the immunization in step (1) is divided into three times, and is performed on days 1, 12 and 30 respectively; in the step (1), the immunization adopts 60 mu g of full-length DNA segment for encoding human Cldnn 18.2 to carry out hind limb muscle injection or electric shock pulse; the step (2) in the boost immunity through the tail vein injection using transfected human Cldnn18.2 plasmid nucleic acid CHO cells.

In a fifteenth aspect, the invention relates to a hybridoma cell comprising a hybrid fusion of a lymphocyte isolated from the animal model described above and a myeloma cell.

Preferably, it is obtained by immunizing an animal model prepared by the method of the present invention and then subjecting spleen cells isolated from the animal model to hybrid fusion with myeloma cells.

In a sixteenth aspect, the invention relates to an antibody expressed by the hybridoma of the invention. The antibody can specifically bind to human cldn18.2 protein.

In a seventeenth aspect, the present invention relates to a method for treating a tumor, said method comprising administering to an individual an effective amount of an antibody produced by a hybridoma cell of the invention.

An eighteenth aspect of the invention relates to the use of an animal model obtained by the above method in product development requiring an immunological process involving cells, the manufacture of antibodies, or as a model system for pharmacological, immunological, microbiological and medical research.

Preferably, the cell is a mammalian cell; further preferably, the cell is a human cell.

Preferably, the antibody is a mammalian antibody; further preferably, the antibody is a human antibody.

In a nineteenth aspect, the invention relates to the use of an animal model obtained by the above method for producing and using animal experimental disease models for etiology studies and/or for developing diagnostic and therapeutic strategies.

The twentieth aspect of the invention relates to the use of the animal model obtained by the method in screening, verifying, evaluating or studying the function of the Cldn18 gene, the Cldn18 antibody, a drug or a drug effect directed at the Cldn18 target site, a drug for immune-related diseases and an anti-tumor drug.

In a twenty-first aspect, the invention relates to a method of producing an antibody, said method comprising constructing an animal model using the method described above.

The "loss-of-function mutation" in the present invention means that the mutation causes complete loss of activity of a gene, failure to express the gene completely or partially, or complete or partial loss of function of the expressed protein.

The "frame shift mutation" of the present invention refers to the insertion or deletion of 1, 2 or several pairs of bases (not a multiple of 3 or 3, i.e., addition or subtraction of bases does not correspond to 1 or more triplets) at a certain point or a certain segment in a DNA sequence, which results in a shift in the transcription of amino acid triplet codons, a complete change in the base sequence from the damaged point, and the translation into an abnormal amino acid.

The term "treating" (or "treatment") as used herein means slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily refer to the complete elimination of all disease-related signs, symptoms, conditions, or disorders. The term "treatment" or the like refers to a therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

In a particular embodiment, the non-human animal is a rodent, which is a mouse of strain C57BL selected from BALB/C, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57 BL/Ola.

The "tumor" according to the present invention is selected from the group consisting of: leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein said leukemia is selected from the group consisting of: acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; the lymphoma is selected from the group consisting of: hodgkin's lymphoma and non-hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and waldenstrom's macroglobulinemia; and said sarcoma is selected from the group consisting of: osteosarcoma, ewing's sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

The "immune-related diseases" described in the present invention include, but are not limited to, allergy, asthma, dermatitis, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorder, etc.

An "effective amount" as referred to herein, refers to an amount or dose of a product of the invention (e.g., an antibody) that provides the desired treatment after administration to a patient or organ in a single or multiple doses.

The "individual" according to the invention is selected from a human or non-human mammal. Such non-human mammals include, but are not limited to, rodents, canines, monkeys, and the like.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. These techniques are explained in detail in the following documents. For example: molecular Cloning A Laboratory Manual, 2nd Ed., ed. By Sambrook, FritschandManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (d.n. glovered., 1985); oligonucleotide Synthesis (m.j. gaited., 1984); mullisetal U.S. Pat. No.4, 683, 195; nucleic Acid Hybridization (B.D. Hames & S.J. Higgins.1984); transformation And transformation (B.D. Hames & S.J. Higgins.1984); culture Of Animal Cells (r.i. freshney, alanr.liss, inc., 1987); immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J.Abelson and M.Simon, eds., In-chief, Academic Press, Inc., New York), specific, volumes, 154 and 155 (Wuetal. eds.) and Vol.185, "Gene Expression Technology" (D.Goeddel, ed.); gene Transfer Vectors For Mammarian Cells (J.H.Miller and M.P.Caloseds, 1987, Cold Spring Harbor Laboratory); immunochemical Methods In Cell And Molecular Biology (Mayer And Walker, eds., Academic Press, London, 1987); handbook Of Experimental Immunology, Volumes V (d.m.weir and c.c.blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

The foregoing is merely a summary of aspects of the invention and is not, and should not be taken as, limiting the invention in any way.

All patents and publications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference. Those skilled in the art will recognize that certain changes may be made to the invention without departing from the spirit or scope of the invention. The following examples further illustrate the invention in detail and are not to be construed as limiting the scope of the invention or the particular methods described herein.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: the detection result of the sgRNA activity is shown, wherein Con is a negative control, and PC is a positive control;

FIG. 2: map schematic diagram of pT7-sgRNA G2 plasmid;

FIG. 3: sequencing results of F0 mouse numbered F0-7;

FIG. 4: sequencing results of F0 mouse numbered F0-25;

FIG. 5: f1-6 XF 1-14, and the mouse is sequenced to identify that 17bp of deletion on a target sequence generates DNA frame shift mutation;

FIG. 6: f1-19 XF 1-25, and the mouse is identified by sequencing to insert 2bp on a target sequence, so that DNA frame shift mutation is generated;

FIG. 7: after the second immunization, the results of mouse serum FACS tests, wherein the number 7 mouse is FL1-H, SSC-H subset 1.13%, FL1-H, SSC-H subset 48.4%, FL1-H, SSC-H subset 1.77%, FL1-H, SSC-H subset 32.1%, FL1-H, SSC-H subset 1.95%, FL1-H, SSC-H subset 45.8%, FL1-H, SSC-H subset 1.27%, FL1-H, SSC-H subset47.3%, FL1-H, SSC-H subset 1.22%, FL1-H, SSC-H subset 44.6%;

FIG. 8: FACS detection results of mAbs produced by hybridomas derived from mouse No. 7, in which NC (FL 1-H, SSC-H subset 3.55%) was a negative control, PC (FL 1-H, SSC-H subset 95.4%) was a positive control, numbered 4C3 (FL 1-H, SSC-H subset 98.5%), 5B10 (FL 1-H, SSC-H subset 99.6%), 5D9 (FL 1-H, SSC-H subset 98.1%), 5G11 (FL 1-H, SSC-H subset 98.9%), the clones with 9E6 (FL 1-H, SSC-H subset 99.7%) were 5 positive clones screened, and the clones with numbers of 9B12 (FL 1-H, SSC-H subset 36.1%), 9E7 (FL 1-H, SSC-H subset 40.6%) were clones with relatively weak signals, poor specificity or less secreted antibody;

FIG. 9: FACS detection results of mAbs produced by hybridomas derived from mouse # 36, in which NC (FL 1-H, SSC-H subset 1.833%) was a negative control, PC (FL 1-H, SSC-H subset 73.8%) was a positive control, clones numbered 1B6 (FL 1-H, SSC-H subset 74.9%), 1E1 (FL 1-H, SSC-H subset 71.4%), 1E4 (FL 1-H, SSC-H subset 73.4%), 3E7 (FL 1-set H, SSC-H subset 76.1%), 7H7 (FL 1-H, SSC-H subset 74.7%), 8D11 (FL 1-H, SSC-H subset 71.5%) were 6 positive clones numbered 7C5 (FL 1-H, SSC 53-H subset 8.63%), and FL 588H subset 8-H3H 2-H subset 8%) (FL 73763-H3-H) Clones of 10F11 (FL 1-H, SSC-H subset 62.3%) were clones with relatively weak signal, poor specificity or less antibody secretion;

FIG. 10: FACS detection results of mAbs produced by hybridomas derived from mouse No. 38, in which NC (FL 1-H, SSC-H subset 1.69%) was a negative control, PC (FL 1-H, SSC-H subset 88.6%) was a positive control, numbered 1A2 (FL 1-H, SSC-H subset 85.2%), 2G4 (FL 1-H, SSC-H subset 86.6%), 2H7 (FL 1-H, SSC-H subset 85.9%), 3C8 (FL 1-set H, SSC-H subset 86.2%), 4H3 (FL 1-H, SSC-H subset 85.4%), 8A1 (FL 1-H, SSC-H subset 85.3%), 7 positive clones numbered 2B 3-H3%, and 7 positive clones (FL 1-H, SSC-H subset 99.3%), and numbered 7 positive clones were selected, Clones of 7D10 (FL 1-H, SSC-H subset 60.8%) were clones with relatively weak signal, poor specificity or less antibody secretion;

FIG. 11: FACS detection results of mAbs generated by hybridomas derived from mouse No. 40, in which NC (FL 1-H, SSC-H subset 2.22%) was a negative control, PC (FL 1-H, SSC-H subset 75.1%) was a positive control, clones numbered 3C8 (FL 1-H, SSC-H subset 78.3%), 6C8 (FL 1-H, SSC-H subset 69.9%), 6C9 (FL 1-H, SSC-H subset 69%) were 3 positive clones screened, and clones numbered 1E2 (FL 1-H, SSC-H subset 47.1%) were clones with relatively poor signal, poor specificity or less antibody secretion;

FIG. 12: FACS detection results of mAbs generated from hybridomas derived from mouse No. 41, in which NC (FL 1-H, SSC-H subset 0.894%) was a negative control, PC (FL 1-H, SSC-H subset 63.1%) was a positive control, and clones numbered 10D12 (FL 1-H, SSC-H subset 62.8%) were screened positive clones;

FIG. 13: the results of monoclonal antibody cross-reaction generated from hybridomas derived from mouse No. 7, in which NC (FL 1-H, SSC-H subset 1.73%) was a negative control, PC (FL 1-H, SSC-H subset 7.50%) was a positive control, and all clone detection results were negative, indicating that these antibodies 4C3 (FL 1-H, SSC-H subset 2.88%), 5B10 (FL 1-H, SSC-H subset 2.48%), 5D9 (FL 1-H, SSC-H subset 1.43%), 5G11 (FL 1-H, SSC-H subset 1.05%), 9B12 (FL 1-H, SSC-H subset 1.77%), 9E6 (FL 1-H, SSC-H subset 2.51%), 7 (FL 1-SSC, SSC subset 2.10%), all were not bound to human Cln1.18.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

In each of the following examples, the equipment and materials were obtained from several companies as indicated below:

BALB/c mice were purchased from Experimental animals technology, Inc. of Wei Tongli, Beijing;

ambion in vitro transcription kit purchased from Ambion, cat # AM 1354;

escherichia coli TOP10 competent cells were purchased from Tiangen, Inc. under the accession number CB 104-02;

EcoRI, BamHI, BbsI enzymes were purchased from NEB, and the respective cargo numbers are; R3101M, R3136M, R0539L;

kanamycin was purchased from Amresco under cat number 0408;

cas9mRNA source SIGMA, cat # CAS9MRNA-1 EA;

the AIO kit is from Beijing Baiosaixi map gene biotechnology limited company with the cargo number BCG-DX-004;

the UCA kit is sourced from Beijing Baiosai chart gene biotechnology limited company with the cargo number of BCG-DX-001;

flow cytometer manufacturer BD, model Calibur;

fluorescein (FITC) -Affinipure Goat Anti-Mouse IgG source Jackson, cat # 115-.

Example 1: cldn18 gene sgRNA

The target sequence determines the targeting specificity of the sgRNA and the efficiency of inducing Cas9 to cleave the gene of interest. Therefore, efficient and specific target sequence selection and determination are a prerequisite for constructing sgRNA expression vectors.

Multiple sgRNAs were obtained for the mouse Cldn18 gene (GeneBANK: 56492), in which each sgRNA targets the following target site sequence:

sgRNA-1 target site sequence (SEQ ID NO: 1): 5'-agttgaatacagcggtcaccggg-3'

sgRNA-2 target site sequence (SEQ ID NO: 2): 5'-gtgtcactgatcgggtttgcggg-3'

sgRNA-3 target site sequence (SEQ ID NO: 3): 5'-tagttgaatacagcggtcaccgg-3'

sgRNA-4 target site sequence (SEQ ID NO: 4): 5'-atgtcggtgaccgcctgccaggg-3'

sgRNA-5 target site sequence (SEQ ID NO: 5): 5'-ccgggttgttgtataaatcctgg-3'

sgRNA-6 target site sequence (SEQ ID NO: 6): 5'-ccgctgtattcaactaccaaggg-3'

sgRNA-7 target site sequence (SEQ ID NO: 7): 5'-gctccactggtccatacaagtgg-3'

sgRNA-8 target site sequence (SEQ ID NO: 8): 5'-cagccacttgtatggaccagtgg-3'

sgRNA-9 target site sequence (SEQ ID NO: 9): 5'-catcattgcagccacttgtatgg-3'

The target sequences are located in exon 1 of the Cldn18 gene.

Example 2: screening of Cldn18 Gene sgRNA

The activity of multiple sgrnas is detected by using a UCA kit, and the results show that the sgrnas have different activities, wherein although the activities of the sgrnas 4 and 9 are relatively low, which may be caused by the specificity of a target site sequence, according to our experiments, the values of the sgrnas 4 and 9 are still significantly higher than those of a control group, and the sgrnas 4 and 9 can still be judged to be active, so that the activity meets the requirements of gene targeting experiments, except for SEQ ID NO: results for sgrnas other than 1-9 that did not have activity were not shown. sgRNA7 was randomly selected from the sgrnas for subsequent experiments, and specific detection results are shown in fig. 1. According to the activity detection result, sgRNA-7 is randomly selected from the sequence, TAGG is added to the 5 'end of the upstream sequence of the sgRNA-7 to obtain a forward oligonucleotide, AAAC is added to the 5' end of a complementary strand (downstream sequence) of the sgRNA-7 to obtain a reverse oligonucleotide, and subsequent experiments are carried out after the forward oligonucleotide and the reverse oligonucleotide are synthesized.

Upstream of the sgRNA-7 sequence: 5'-CTCCACTGGTCCATACAAG-3' (SEQ ID NO: 18)

Forward oligonucleotide: 5'-TAGGCTCCACTGGTCCATACAAG-3' (SEQ ID NO: 10)

Downstream of the sgRNA-7 sequence: 5'-CTTGTATGGACCAGTGGAG-3' (SEQ ID NO: 19)

Reverse oligonucleotide: 5'-AAACCTTGTATGGACCAGTGGAG-3' (SEQ ID NO: 11)

Example 3: construction of pT7-sgRNA G2 plasmid

A fragment DNA containing a T7 promoter and sgRNA scaffold is synthesized by a plasmid synthesis company, is sequentially connected to a skeleton vector pHSG299 through enzyme digestion (EcoRI and BamHI), and is verified by sequencing of a professional sequencing company, and the result shows that a target plasmid is obtained: the plasmid map of pT7-sgRNAG2, pT7-sgRNAG2 is shown in FIG. 2.

Fragment DNA containing the T7 promoter and sgRNA scaffold (SEQ ID NO: 12):

5’-gaattctaatacgactcactatagggggtcttcgagaagacctgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttaaaggatcc-3’

example 4: construction of pT7-Cldn18-7 plasmid

The forward and reverse oligonucleotides obtained in example 2 were annealed and then ligated to pT7-sgRNA G2 plasmid (the plasmid was first linearized with BbsI), respectively.

The ligation reaction system is shown in Table 1:

table 1: ligation reaction System

Reaction conditions are as follows:

ligation was performed at room temperature for 10-30min, transformed into 30. mu.L of TOP10 competent cells, and 200. mu.L of the cells were plated on Kan-resistant plates, cultured at 37 ℃ for at least 12 hours, and 2 clones were selected and inoculated into LB medium (5 mL) containing Kan resistance, and cultured at 37 ℃ with shaking at 250rpm for at least 12 hours.

Randomly picked clones were sent to a sequencing company for sequencing verification, and correctly ligated expression vectors pT7-Cldn18-7 were selected for subsequent experiments.

Example 5: microinjection, embryo transfer and breeding

Taking BALB/c mouse prokaryotic fertilized eggs, pre-mixing in vitro transcription products of pT7-Cldn18-7 plasmid (using Ambion in vitro transcription kit, and performing transcription according to the method of the instruction) and Cas9mRNA by using a microinjection instrument, and injecting the mixture into mouse fertilized egg cytoplasm or cell nucleus. Microinjection of fertilized eggs is carried out according to the method in the experimental manual for mouse embryo manipulation (third edition), antrraga, shinagaji, chemical industry publishers, 2006), the fertilized eggs after injection are transferred to a culture solution for short-term culture, then the fertilized eggs are transplanted to the oviduct of a recipient mother mouse for development, the obtained Cldn18 gene mutation mouse is hybridized and selfed, the population number is enlarged, and a stable Cldn18 gene mutation mouse strain is established.

Example 6: genotyping of mice with Cldn18 gene mutation

1. Genotype identification of F0 generation

To verify whether F0 generation Cldn18 gene mutation heterozygous mice were obtained, the F0 generation mice obtained in example 5 were tailed to extract genomic DNA, and then fragments of about 600bp near the target site were amplified using the extracted DNA as a template and primers MSD-F (SEQ ID NO: 13) and MSD-R (SEQ ID NO: 14), with MSD-F located at the left side of the target site and MSD-R located at the right side of the target site, and the specific primer sequences were as follows:

MSD-F（SEQ ID NO：13）：5’-gcatcaagcttggtaccgatggcggaaaacaaatggctcttggta-3’

MSD-R（SEQ ID NO：14）：5’-acttaatcgtggaggatgatatcattgtggcagtgactgttgctc-3’

the reaction system and amplification conditions are shown in tables 2 and 3. The PCR product was sent to the sequencing company for sequencing.

Table 2: PCR reaction System (20. mu.L)

Table 3: PCR amplification reaction conditions

Sequencing results of F0 mouse generation (Table 4) show that a plurality of Cldn18 gene mutant mice are obtained by the method and have a plurality of mutation types. Exemplary sequencing results are shown in FIGS. 3-4, where FIG. 3 is the sequencing result for numbers F0-7 and FIG. 4 is the sequencing result for numbers F0-25.

Table 4: f0 generation gene detection result

Description of the drawings: Δ is a knockout, such as 17bp for Δ 17 and 3bp for Δ 3; in is knock-in, in1 is knock-in of 1bp

2. Genotype identification of F1 generation

The F0 generation mice identified as positive were mated with wild type mice to give F1 generation mice.

1) Mating F0-22 with wild type Balb/c mice to obtain 17 mice, wherein the numbers of the mice are F1-1 to F1-17;

2) f0-25 was mated with wild type Balb/c mice to give 12 mice numbered F1-18 through F1-29.

Sequencing verification is carried out on the mice, the sequencing method is the same as the F0 generation genotype identification method, and the sequencing result shows that 17F 1 generation mice obtained by mating F0-22 and wild type mice are all positive heterozygote mice; in contrast, 11 positive heterozygous mice out of 12 mice obtained by crossing F0-25 with wild type mice were sequenced as shown in Table 5.

Table 5: f1 generation gene detection result

3. Genotype identification of F2 generation

The heterozygote mice of the same genotype in the F1 generation are mutually mated, the later generation (F2 generation) is screened, and the gene mutation homozygote can be obtained with a certain probability according to the Mendelian inheritance rule.

1) Any one of F1-2, F1-3, F1-6 and F1-7 is mated with any one of F1-8, F1-9 and F1-14 to theoretically obtain a homozygote with 17bp knockout;

2) a homozygote of about 2bp knock-in could be theoretically obtained by mating any one of F1-18, F1-19, and F1-21 with any one of F1-22, F1-24, or F1-25.

Sequencing and screening homozygotes for the F2 generation obtained in the 1) and the 2), wherein the sequencing method is the same as the genotype identification method for the F0 generation, the detection result of the F2 generation homozygote is shown in figures 5 and 6, wherein the figure 5 is the sequencing result of the descendant of F1-6 XF 1-14, the figure 6 is the sequencing result of the descendant of F1-19 XF 1-25, the specific DNA sequence and the comparison of the specific DNA sequence and the target site sequence are shown in Table 6, and the obtained mutant individuals are shown to be mutated at the target sites.

Table 6: f2 generation gene detection result and sequence comparison

Description of the drawings: wherein in { } is a knock-in sequence, and in < > is a knock-out sequence

The experiments show that the Cldn18 gene mutant mouse capable of being stably passaged can be constructed by using the method.

Example 7: application of Cldn18 gene mutant mouse

5F 2 generation homozygote mice prepared by the method are selected, the serial numbers of the mice are 7, 36, 38, 40 and 41 respectively, wherein the mouse with the serial number of 7 is the genotype with 2bp inserted, and the rest mice are knocked out by 17 bp. Mice were immunized with a plasmid containing the DNA sequence of human Cldn18 (NCBI Gene ID: 51208) according to the protocol: on days 1, 12 and 30, hind limb muscle injections (shock pulses) were performed with 60. mu.g of plasmid DNA containing a DNA sequence encoding normal human Cldn18.2 protein (SEQ ID NO: 20). 1 week after the second immunization, mouse sera were evaluated for cldn18.2 protein binding by FACS, and the results of the test (fig. 7) showed that antibodies targeting cldn18.2 protein were produced in mice after both immunizations. 2 weeks after the third immunization (i.e., 4 days before removal of the spleen), all mice were boosted, CHO cells transfected with nucleic acids of the aforementioned plasmids were injected via tail vein, and then Mouse spleen cells were isolated for hybridoma fusion, hybridoma supernatants were added to the transfected CHO cells, after incubation on ice for 15min, antibody fluorescin (fitc) -affinipur coat Anti-Mouse IgG (Anti-Mouse IgG FcFITC) was added for labeling, incubation on ice for 15min was continued, and then flow analysis (fluorescing Activated Cell Sorting, FACS) was performed to screen hybridomas producing antibodies specific to cldn18.2, and finally 22 positive hybridoma clones were co-screened using 5 mice. FIG. 8 shows the results of FACS detection of monoclonal antibodies produced by hybridomas derived from mouse No. 7, in which clones numbered 4C3, 5B10, 5D9, 5G11, and 9E6 were 5 positive clones screened; FIG. 9 shows the results of FACS detection of monoclonal antibodies produced by hybridomas derived from mouse # 36, wherein clones numbered 1B6, 1E1, 1E4, 3E7, 7H7, and 8D11 are 6 positive clones screened, and FIG. 10 shows the results of FACS detection of monoclonal antibodies produced by hybridomas derived from mouse # 38, wherein clones numbered 1A2, 2G4, 2H7, 3C8, 4H3, 8A1, and 9D11 are 7 positive clones screened; FIG. 11 shows FACS detection results of monoclonal antibodies generated from hybridomas of mouse 40, in which clones numbered 3C8, 6C8 and 6C9 were 3 positive clones screened; FIG. 12 shows FACS detection of monoclonal antibodies produced by hybridomas derived from mouse # 41, in which clone No. 10D12 was the positive clone screened. Furthermore, we further examined the binding of the cldn18.2 monoclonal antibody produced by mouse # 7 to human cldn18.1 protein by transfection of CHO cells expressing human cldn18.1 protein, and found that none of these antibodies bound to cldn18.1 protein (see fig. 13 for results). The fact that an antibody which is prepared by using a Cldn18 gene knockout mouse and targets the human Cldn18.2 protein has high target recognition specificity is shown.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Sequence listing

<110> Beijing Baiosai map Gene Biotechnology Co., Ltd

<120> method for constructing animal model and application

<130> 1

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agttgaatac agcggtcacc ggg 23

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gtgtcactga tcgggtttgc ggg 23

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tagttgaata cagcggtcac cgg 23

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atgtcggtga ccgcctgcca ggg 23

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ccgggttgtt gtataaatcc tgg 23

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ccgctgtatt caactaccaa ggg 23

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gctccactgg tccatacaag tgg 23

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cagccacttg tatggaccag tgg 23

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catcattgca gccacttgta tgg 23

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taggctccac tggtccatac aag 23

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aaaccttgta tggaccagtg gag 23

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gaattctaat acgactcact atagggggtc ttcgagaaga cctgttttag agctagaaat 60

agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct 120

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ccacttgtat ggaccagtgg agc 23

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ccacttatgt atggaccagt ggagc 25

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ctccactggt ccatacaag 19

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acagctgttt tcaactacca ggggctgtgg cgctcctgtg tccgagagag ctctggcttc 180

accgagtgcc ggggctactt caccctgctg gggctgccag ccatgctgca ggcagtgcga 240

gccctgatga tcgtaggcat cgtcctgggt gccattggcc tcctggtatc catctttgcc 300

ctgaaatgca tccgcattgg cagcatggag gactctgcca aagccaacat gacactgacc 360

tccgggatca tgttcattgt ctcaggtctt tgtgcaattg ctggagtgtc tgtgtttgcc 420

aacatgctgg tgactaactt ctggatgtcc acagctaaca tgtacaccgg catgggtggg 480

atggtgcaga ctgttcagac caggtacaca tttggtgcgg ctctgttcgt gggctgggtc 540

gctggaggcc tcacactaat tgggggtgtg atgatgtgca tcgcctgccg gggcctggca 600

ccagaagaaa ccaactacaa agccgtttct tatcatgcct caggccacag tgttgcctac 660

aagcctggag gcttcaaggc cagcactggc tttgggtcca acaccaaaaa caagaagata 720

tacgatggag gtgcccgcac agaggacgag gtacaatctt atccttccaa gcacgactat 780

gtgtaa 786

Claims (7)

1. A method of constructing an animal model comprising introducing a loss-of-function mutation in an animal model Cldn18 gene using a sgRNA directed against the Cldn18 gene such that the animal model does not express Cldn18.2 protein, wherein the sgRNA targets a target site as set forth in SEQ ID NO: 1-9, wherein the loss-of-function mutation is introduced in exon 1 of the Cldn18 gene, and the animal model is a mouse.

2. A method as claimed in claim 1, comprising the steps of:

the first step is as follows: preparing a polypeptide comprising SEQ ID NO: 1-9 of any one of sgRNA vectors;

the second step is that: mixing an in-vitro transcription product of the sgRNA vector and Cas9mRNA to obtain a mixed solution, injecting the mixed solution into cytoplasm or nucleus of mouse fertilized eggs, transferring the injected fertilized eggs into a culture solution for culture, and then transplanting the fertilized eggs into an oviduct of a receptor mother mouse for development to obtain an F0 generation mouse.

3. A Cldn18 gene mutant cell, comprising introducing a loss-of-function mutation in the Cldn18 gene of the cell using a sgRNA directed to the Cldn18 gene such that the cell does not express Cldn18.2 protein, wherein the sgRNA targets a target site as set forth in SEQ ID NO: 1-9, wherein said loss-of-function mutation introduces exon 1 of the Cldn18 gene, and wherein said cell is derived from a mouse.

4. A sgRNA targeting the Cldn18 gene, wherein the sgRNA targets a target site as set forth in SEQ ID NO: 1-9.

5. A sgRNA vector comprising the sgRNA of claim 4.

6. Use of an animal model obtained by the method of any one of claims 1-2 or the cell of claim 3 for the preparation of an antibody for the treatment of a tumor or an immune-related disease.

7. A method of producing an antibody, comprising constructing an animal model using the method of any one of claims 1-2.

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