Construction method and application of mouse model with B cell-specific knockout of Ewsr1 gene

Abstract

Antigen immunization of mice with B cell specific knockout of Ewsr1 gene can significantly enhance their humoral immune response, increase the proportion of B cells in the germinal center and the antibody level in the serum. This patent relates to the method and application of constructing an animal model with B cell specific knockout of Ewsr1 gene, specifically including: using C57BL / 6J mice with B cell specific knockout of Ewsr1 gene as experimental animals, immunizing with various antigens (including viruses, pathogens, tumor antigens and biomarkers, etc.) and related adjuvants, separating the antibodies or B cells obtained after immunization for subsequent applications, especially separating B cells for subsequent screening of monoclonal antibodies and CAR scFv sequences.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and relates to a method for constructing a mouse model for specifically knocking out the Ewsr1 gene in B cells and an application thereof, in particular to an application in the screening of therapeutic antibodies and CAR-T scFv sequences. Background Art

[0002] Traditional treatment methods, especially in the field of cancer treatment, are limited by their specificity and efficacy. The development of gene editing technology has made significant progress in cell therapy. CAR is the core component of CAR-T, and how to obtain CAR that specifically recognizes tumor antigens is the key to development. In addition, antibody drugs are also an important part of the biopharmaceutical field. Currently, there are more than 100 antibody drugs approved by the US FDA for the treatment of a variety of human diseases. Since 2014, the US FDA or the European EMA has approved 6-13 antibody drugs each year. Similar to CAR, the screening of antibody production sequences is also the key to antibody drug research and development.

[0003] At present, the commonly used screening technologies for scFv and antibody sequences in the market mainly include hybridoma, phage antibody library, and flow cytometry single B cell. The traditional hybridoma platform requires the fusion of B cells and tumor cells for immortalization before further screening, subcloning, etc. First of all, a large amount of B cell diversity will be lost in the fusion step-even if calculated at the high fusion efficiency of 1 / 1000 believed by the industry. Obviously, such a large-scale loss will directly affect the diversity of candidate antibody molecules and hinder the discovery of new antibody molecules. Secondly, due to the need for multiple complex and time-consuming steps such as fusion, screening, subcloning, and rescreening, the hybridoma screening cycle often takes 2-3 months (excluding animal immunization time). Third, this fusion requirement limits some species (such as chickens, camels, and sharks).

[0004] The whole human library combined with the phage display platform does not require animal immunization and can quickly screen antibody molecules within 1-2 months, which is also the main reason why this technology attracts R&D personnel. However, the whole human library platform also has obvious limitations: first, whether the target antibody can be screened depends largely on the quality and size of the library itself, and the screening strategy and even the operation method will affect the results. Secondly, since the antibody has not undergone the in vivo antibody maturation process, the screened antibody sequences often have limited affinity, and a single point mutation library needs to be established to complete affinity maturation-in a sense, it means a secondary large-scale screening, which undoubtedly increases the workload; third, due to the random combination of antibody heavy chains and light chains in the library platform, the natural pairing of antibody heavy chains and light chains cannot be maintained.

[0005] It can be seen that compared with the hybridoma platform, the single B cell sorting platform frees researchers from the limitations and negative effects of fusion. At the same time, most platforms integrate high-throughput modes for screening, which can screen tens of thousands of B cells within one day, greatly improving efficiency. In addition, the use of a full human antibody library for antibody screening has a higher random success rate. The single B cell platform mostly uses samples after antigen immunization. The B cells have undergone in vivo maturation, the secreted antibodies have high affinity, and the heavy and light chains are naturally paired. In addition, the platform can also use transgenic animals and combine rapid immunization technology to screen out full human antibody molecules within 1 month at the fastest. In short, the single B cell screening platform can better retain B cell diversity, while being unique in screening efficiency.

[0006] B lymphocytes play a vital role in the immune response against pathogens and tumors by producing protective antibodies. B lymphocytes are continuously produced in the bone marrow through an orderly lymphopoiesis process. Newly mature B cells enter the peripheral circulation, recognize antigens, and play an immune protective role. After receiving antigen immunization, CD4 T lymphocytes that recognize the same antigen interact with antigen-activated B cells in an antigen-specific manner, provide cytokines and other factors to promote clonal B cell expansion, and form germinal centers (GC). Germinal centers GC are the places where B cells undergo positive selection and somatic hypermutation (SHM), inducing affinity maturation to produce high-affinity memory B cells and long-lived plasma cells.

[0007] The main processes of the single B cell screening platform include: immunization of experimental animals, flow cytometry sorting of antigen-specific B cells, culture and ELISA identification of single B cells, amplification of single B cell variable regions, rapid expression of monoclonal antibodies and ELISA identification, and monoclonal antibody expression and functional identification. Immunization of experimental animals is the first step in the process and is also a key step in determining the quality of subsequent B cells. In order to enhance the immune effect of mice, the methods used by researchers mainly include: 1. Combining antigens with macromolecules with strong immunogenicity; 2. Selecting strong immune adjuvants; 3. Improving the immune site and immune method (such as "primer-boost"). These methods can enhance the immune effect of mice to a certain extent, but the effects of these methods have certain limitations, and the influence of the nature of the antigen must be considered during the application process.

[0008] The Ewsr1 gene, full name: Ewing sarcoma breakpoint region 1 gene, is a member of the TET family, located between base pairs 29663998 and 29696515 on chromosome 22q12, and was first studied in Ewing sarcoma. The Ewsr1 gene encodes a multifunctional protein involved in a variety of cellular mechanisms, including gene expression, meiosis and mitosis, cell signaling, DNA repair, and cell aging. Ewsr1 has 17 exon coding sequences, encoding a 656 amino acid nuclear protein, including a carboxyl-terminal 87 amino acid RNA binding domain (exons 11-13), which is involved in protein RNA binding, transcription, and RNA metabolism.

[0009] Ewsr1 Flox mice are mice with the Loxp sequence located on one side of the Ewsr1 exon. These Ewsr1 Flox mice are obtained by designing and constructing targeting vectors, embryonic stem cell recombination, blastocyst microinjection, and chimeric mouse passage. However, systemic Ewsr1 gene knockout in mice can easily cause embryonic lethality and the effects on other immune cells or tissues, resulting in a low acquisition rate of model mice. Summary of the invention

[0010] The purpose of the present invention is to provide a method for constructing an animal model for knocking out the Ewsr1 gene in B cells specifically and its application, in particular, a gene-edited experimental animal that can significantly improve the efficiency of monoclonal antibody and CAR scFv sequence screening in single B cell screening technology. Immunization of this type of mouse can significantly increase the proportion of antigen-specific B cells in the B cell screening process, save research and development costs, and have high drug research and development application value.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] The first object of the present invention is to provide a method for constructing a model for specifically knocking out the Ewsr1 gene in animal B cells, the method comprising the following steps:

[0013] (1) Ewsr1 flox / + After crossing the animal with the Mb1-Cre animal, the genotype obtained was Ewsr1 flox / + Mb1-Cre+ and Ewsr1 flox / + Mb1-Cre-F1 animals;

[0014] (2) mating the two animals obtained in step (1) to obtain F2 generation animals;

[0015] (3) Select the genotype Ewsr1 from the F2 generation flox / flox Mb1-Cre+ animals with the genotype Ewsr1 flox / floxMb1-Cre+ animals are animals in which the Ewsr1 gene is specifically knocked out in B cells.

[0016] Furthermore, step (3) also includes the genotype Ewsr1 flox / flox Mb1-Cre- animals, the Ewsr1 flox / flox Mb1-Cre- animals can be used as control animals.

[0017] Furthermore, in step (1), Ewsr1 flox / + The animal was constructed by inserting Loxp sites at both ends of the fourth exon of the EWSR1 gene.

[0018] Preferably, the EWSR1 gene is site-specifically modified by homologous recombination using CRISPR / Cas9 technology to insert Loxp sites at both ends of the fourth exon of the EWSR1 gene;

[0019] Further preferably, the specific process of the CRISPR / Cas9 technology includes: designing and transcribing gRNA in vitro, constructing homologous recombination Donor at the same time, injecting Cas9 mRNA, gRNA, and Donor into animal fertilized eggs at the same time, Cas9 mRNA translates Cas9 protein in vivo, Cas9 protein binds to the target site under the guidance of gRNA to cause DNA double-strand breaks, and the broken double-strands are repaired in the repair process using the donor Donor as a template, thereby achieving modification of the target gene. The gRNA sequence is as follows:

[0020] gRNA-1: 5'-TCGTGGAAACATCCTAGGACAGG-3' (SEQ ID NO. 1);

[0021] gRNA-2: 5'-CCCTACTTACAAGATTCACAGAA-3' (SEQ ID NO. 2).

[0022] Furthermore, the Mb1-Cre animal in step (2) is constructed by inserting Cre recombinase into the Mb1 locus. The construction method of the Mb1-Cre animal is referenced in: PNAS September 12, 2006 vol. 103 no. 37 13789-13794.

[0023] Furthermore, in step (4), the genotype selected from the F2 generation is Ewsr1 flox / floxMb1-Cre+ specifically adopts the PCR method and uses the F2 generation animal mouse tail genomic DNA as a template for PCR amplification. Animals that meet the following requirements are animals with specific knockout of the Ewsr1 gene in B cells: homozygous Ewsr1 gene amplification product containing loxp sites, Cre recombinase transgenic amplification product, and internal reference amplification product.

[0024] The Ewsr1 gene primer is Ewsr1 flox / flox The upstream primer and the downstream primer, the Mb1-Cre recombinase gene primers are Mb1-Cre primers 1, 2, 3, 4; the animal that meets the following requirements is an animal in which the Ewsr1 gene is specifically knocked out in B cells: using Ewsr1 flox / flox The upstream primer and the downstream primer amplified a band of 301 bp, and the Mb1-Cre primers 1, 2, 3, and 4 amplified two bands of 230 bp and 197 bp.

[0025] Furthermore, the Ewsr1 flox / flox The upstream and downstream primers are:

[0026] Ewsr1 flox / flox Upstream primer: 5'-AAAGCCAAGGATGACAGACTCTA-3' (SEQ ID NO. 3);

[0027] Ewsr1 flox / flox Downstream primer: 5'-TAAAGCCTTTGAGAAGCACGACT-3' (SEQ ID NO. 4).

[0028] Furthermore, the Mb1-Cre primers 1, 2, 3, and 4 are:

[0029] Mb1-Cre primer 1: 5′-CTAGGCCACAGAATTGAAAGATCT-3′ (SEQ ID NO. 5);

[0030] Mb1-Cre primer 2: 5'-GTAGGTGGAAATTCTAGCATCC-3' (SEQ ID NO. 6);

[0031] Mb1-Cre primer 3: 5′-AGATGCCAGGACATCAGGAACCTG-3′ (SEQ ID NO. 7);

[0032] Mb1-Cre primer 4: 5′-ATCAGCCACACCAGACACAGAGATC-3′ (SEQ ID NO. 8).

[0033] The second object of the present invention is to provide an animal model of Ewsr1 gene specifically knocked out in B cells constructed by the aforementioned method.

[0034] The Ewsr1 gene is highly conservative, and the method of the present invention can be used to achieve specific knockout of the Ewsr1 gene in B cells in animals. In a specific embodiment, the animal is a mouse.

[0035] The third object of the present invention is to provide the use of the aforementioned method or an animal with Ewsr1 gene specifically knocked out in B cells constructed by the aforementioned method in screening or preparing drugs for diseases regulated by B cell immunity.

[0036] Furthermore, the drug is a therapeutic antibody and / or CAR-T.

[0037] Advantages and beneficial effects of the present invention:

[0038] Mb1-cre specifically expresses Cre recombinase in B cells. In the present invention, Mb1-cre is bred with Ewsr1 Flox mice to obtain Ewsr1 flox / flox Mb1-Cre mice are mice with B cell-specific knockout of Ewsr1.

[0039] By constructing Ewsr1 gene B cell knockout mice and immunizing them with model antigen NP, it was found that compared with normal wild-type mice, Ewsr1 flox / flox The proportion of GCB cells and the level of antigen-specific antibodies in the serum of Mb1-Cre+ mice increased, that is, Ewsr1 flox / flox Mb1-Cre+ mice have a strong response ability to antigens. Selecting experimental animals with strong response ability as immunized animals for the B cell screening platform can significantly promote the production of B cells, significantly reduce the number of immunizations, and shorten the time used for immunizing animals.

[0040] The present invention discloses for the first time a method for constructing an animal model by gene knockout, successfully conditionally knocks out the Ewsr1 gene using the Cre-loxp system, and successfully establishes an animal model of specifically knocking out the Ewsr1 gene in mouse B cells, so as to avoid embryonic lethality caused by systemic gene knockout in mice and the effects produced in other immune cells or tissues, and lays a foundation for in-depth research on the role of the Ewsr1 gene in B cells. It is suitable for medicine, life sciences, and experimental scientific research, and provides a good research model for exploring the function and mechanism of action of Ewsr1 in B cells, especially for research related to the Ewsr1 gene and B cells in the fields of immunology and oncology. The method of the present invention is scientific, reliable, feasible, easy to operate, and worthy of promotion.

[0041] The technology covered by this patent can be applied to cancer treatment, immunotherapy and other fields to produce efficient and specific cells and antibodies for treatment and scientific research. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a schematic diagram of Ewsr1 gene targeting.

[0043] Figure 2 This is a diagram of the breeding strategy for mice with conditional knockout of the Ewsr1 gene in B cells.

[0044] Figure 3 This is the agarose gel electrophoresis diagram of PCR identification of Ewsr1 and Mb1-Cre during the breeding process.

[0045] Figure 4 WB analysis Ewsr1 flox / flox Mb1-Cre+(BKO) and Ewsr1 flox / flox EWSR1 protein expression in B cells of Mb1-Cre-(WT) mice.

[0046] Figure 5 Flow cytometry was used to detect NP-OVA immune Ewsr1 flox / flox Mb1-Cre+(BKO) and Ewsr1 flox / flox Flow cytometry and statistical plots of GCB cells in the spleen of Mb1-Cre-(WT) mice.

[0047] Figure 6 Immunohistochemical detection of NP-OVA-immunized Ewsr1 flox / flox Mb1-Cre+(BKO) and Ewsr1 flox / flox Histochemistry and statistical graph of GCB cells in the spleen of Mb1-Cre-(WT) mice.

[0048] Figure 7 ELISA to detect NP-OVA immune Ewsr1 flox / flox Mb1-Cre+(BKO) and Ewsr1 flox / flox NP-specific IgM / IgG / IgG1 antibody levels in the serum of Mb1-Cre-(WT) mice. Figure 7 A, B, and C are IgM, IgG, and IgG1 antibody levels, respectively. DETAILED DESCRIPTION

[0049] The present invention is further explained below with reference to the examples, but the examples do not limit the present invention in any form.

[0050] Example 1

[0051] Experimental animals

[0052] Ewsr1 flox / + Mice were obtained from Saiye Biotechnology Co., Ltd., and Mb1-Cre mice were obtained from Jackson Laboratory in the U.S. After introduction, both strains of mice were raised in a specific pathogen-free environment (SPF grade) at the Experimental Animal Center of Nanjing Medical University.

[0053] As an alternative, Ewsr1 flox / + The mouse was constructed by using CRISPR / Cas9 technology to modify the target gene at a specific site through the principle of homologous recombination ( Figure 1 ). The specific process includes: designing and transcribing gRNA in vitro, and constructing homologous recombination Donor at the same time. Cas9 mRNA, gRNA, and Donor are injected into the fertilized egg at the same time. Cas9 mRNA translates Cas9 protein in vivo, and Cas9 protein binds to the target site under the guidance of gRNA to cause DNA double-strand breaks. The broken double-strands are repaired during the repair process using the donor Donor as a template, thereby modifying the target gene. We used the following gRNA sequences:

[0054] gRNA-1: 5'-TCGTGGAAACATCCTAGGACAGG-3' (SEQ ID NO. 1);

[0055] gRNA-2: 5'-CCCTACTTACAAGATTCACAGAA-3' (SEQ ID NO. 2).

[0056] Primers and main reagents

[0057] Ewsr1 flox / flox Upstream primer: 5'-AAAGCCAAGGATGACAGACTCTA-3' (SEQ ID NO. 3);

[0058] Ewsr1 flox / flox Downstream primer: 5'-TAAAGCCTTTGAGAAGCACGACT-3' (SEQ ID NO. 4);

[0059] Mb1-Cre primer 1: 5′-CTAGGCCACAGAATTGAAAGATCT-3′ (SEQ ID NO. 5);

[0060] Mb1-Cre primer 2: 5'-GTAGGTGGAAATTCTAGCATCC-3' (SEQ ID NO. 6);

[0061] Mb1-Cre primer 3: 5′-AGATGCCAGGACATCAGGAACCTG-3′ (SEQ ID NO. 7);

[0062] Mb1-Cre primer 4: 5′-ATCAGCCACACCAGACACAGAGATC-3′ (SEQ ID NO. 8).

[0063] TaKaRa MiniBEST Universal Genomic DNAExtraction kit(Takara,9765)

[0064] Premix Taq Polymerase(Vazyme,P222)

[0065] method

[0066] 1. Breeding of conditional B cell Ewsr1 knockout mice ( Figure 2 )

[0067] The mice were first mated at 8 weeks old, with a 2:1 ratio of male to female and long-term cohabitation. The offspring were weaned at 3 weeks old. Figure 1 shown.

[0068] Ewsr1 flox / + Crossing with Mb1-Cre mice resulted in the acquisition of the Ewsr1 genotype. flox / + Mb1-Cre+ and Ewsr1 flox / + The F1 generation mice were then hybridized to obtain the F2 generation mice.

[0069] Ewsr1 was identified by PCR flox / flox The F2 generation mice have the Mb1-Cre+ genotype, which is the mouse with conditional knockout of the Ewsr1 gene in B cells.

[0070] 2. Identification of Ewsr1 gene and Mb1-Cre recombinase gene

[0071] The PCR identification described in "1" is as follows: when the mouse grows to 2 weeks old, 4mm of the mouse tail is cut, the mouse tail genomic DNA is extracted, and the genotype is identified by the PCR method. At the same time, the mouse tail genomic DNA is taken and amplified using Ewsr1 gene primers and Mb1-Cre recombinase gene primers, respectively, to perform relevant gene identification. The length of the homozygous Ewsr1 gene amplification product containing loxp sites is 301bp, the length of the Ewsr1 gene (wild type) amplification product without loxp sites is 221bp, and the heterozygous products are 301bp and 221bp. The length of the Cre recombinase transgenic product is 230bp, and the length of the internal reference is 197bp.

[0072] result

[0073] 1. Mouse genotype identification

[0074] According to Mendel's law of gene segregation and independent combination, the F1 generation of mice Ewsr1 flox / + Mb1-Cre+ and Ewsr1 flox / + The genotype of the F2 mice obtained by hybridization with Mb1-Cre- may be: Ewsr1 flox / flox Mb1-Cre+, Ewsr1 flox / flox Mb1-Cre-, Ewsr1 flox / + Mb1-Cre+, Ewsr1 flox / + Mb1-Cre-, Ewsr1 + / + Mb1-Cre+, Ewsr1 + / + Mb1-Cre-. The genotype is Ewsr1 flox / flox Mb1-Cre+ mice are conditional Ewsr1 gene knockout mice in B cells.

[0075] The PCR identification results of various genotypes of F2 mice are shown in Figure 3 .

[0076] 2. Identification of the Mb1-Cre gene

[0077] When the Loxp site binds to the Cre recombinase, gene deletion will occur. The genomic product obtained by PCR amplification with Ewsr1 primers has only one band at 301bp for flox homozygotes, only one band at 221bp for wild type, and one band at 301bp and 221bp for heterozygotes. Then, the genome of the candidate mouse is amplified by PCR with Cre primers, and the product is 230bp. It can be considered that the genome of the mouse carries the Mb1-Cre gene. If there is no 230bp in the amplified product, the mouse does not carry the Cre gene.

[0078] Example 2 Detection of cell-specific EWSR1 protein expression levels in B cell-specific Ewsr1 gene knockout mice

[0079] Take Ewsr1 prepared in Example 1 flox / flox Mb1-Cre+(Ewsr1-BKO)Ewsr1 flox / flox Spleen tissue of Mb1-Cre-(WT) mice is enriched for B cells.

[0080] Extraction of B cell protein: Add 200 μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors to the centrifuge tube containing B cells, lyse on ice for 30 minutes, vortex every 5 minutes; centrifuge at 4°C, 12,000 rpm for 15 minutes, collect the supernatant, take an appropriate amount of supernatant and add a certain volume of 5× protein loading buffer, boil the protein for 5 minutes to denature the protein. Add protein samples to the wells of SDS-PAGE gel, and the electrophoresis program is: 115V, 30min; 175V, 1h.

[0081] Add transfer buffer to the electrophoresis tank, and transfer the protein to the PVDF membrane using the wet transfer method. The electrophoresis program is: 100v, 1.5h. Use 5% skim milk powder as the blocking solution and incubate at room temperature for 1.5h at 160rpm. Wash away the milk with PBST, and incubate the diluted rabbit EWSR1 primary antibody and mouse α-Actin primary antibody on a shaker at 4℃ overnight. Wash the membrane 3 times with PBST, 10min each time, and incubate the diluted goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies at room temperature for 1h. After washing the membrane 3 times with PBST, develop and take pictures.

[0082] The results are as follows Figure 4 As shown, the expression level of EWSR1 protein in B cells of Ewsr1-BKO mice was significantly decreased compared with that of control WT mice.

[0083] Example 3 Phenotypic analysis of B cell-specific Ewsr1 gene knockout mice

[0084] Take Ewsr1 prepared in Example 1 flox / flox Mb1-Cre+(Ewsr1-BKO)Ewsr1 flox / flox Ten Mb1-Cre-(WT) mice aged 8 weeks were immunized by intraperitoneal injection of 0.2 mL of NP-OVA + aluminum adjuvant mixed by shaking. The mice were treated 4 and 14 days after immunization, and four pairs of non-immunized mice were taken as controls. The treatment process is as follows:

[0085] (I) Flow staining

[0086] Take part of the spleen tissue of the mouse and place it in a six-well plate containing 1640 culture medium (R2) containing 2% newborn calf serum. Take a 0.22μm mesh and place it on the spleen tissue. Then use a 1mL syringe piston head to gently grind the tissue until there is no content. Gently blow the cell suspension evenly, and then transfer the cell suspension to a 4mL centrifuge tube. Count the cells and take 5 million total cells for each sample for flow staining. Place the cells in a round-bottom 96-well plate, centrifuge at 1000g for 1min, discard the supernatant, add the prepared flow antibody mixture (take (number of samples + 1) x 50μL of FACS flow staining buffer, add B220-APC, Fas-FITC, GL7-PB antibodies at 1:200 respectively), resuspend the cells in 50μL per well, avoid bubbles, and place on ice away from light for 25min for flow staining.

[0087] After staining, resuspend and wash the cells twice with FACS flow staining buffer before flow cytometry detection. During the whole process, the cells should be kept away from light and on ice.

[0088] The results are as follows Figure 5 As shown, the proportion of GCB in the spleen of non-immunized mice was extremely low and similar in WT and Ewsr1 BKO mice. Four days after NP-KLH immunization, the proportion of GCB in the spleen of WT mice was 1.02% while that in the spleen of Ewsr1 BKO mice was 2.57%. Fourteen days after NP-KLH immunization, the proportion of GCB in the spleen of WT mice was 1.58% while that in the spleen of Ewsr1BKO mice was 3.21%. The results show that under the condition of NP-KLH immunization, the proportion of GCB in the spleen of Ewsr1 BKO mice is more than twice that of WT mice.

[0089] (II) Immunohistochemistry

[0090] Take 1 / 4 spleen tissue of mice and fix it in 4% PFA overnight, then put it into embedding machine for series of tissue dehydration. The procedure is as follows: 70% ethanol, 30min; 80% ethanol, 30min; 90% ethanol, 30min; 95% ethanol, 30min; anhydrous ethanol, 30min; anhydrous ethanol, 30min; xylene, 15min; xylene, 15min. Put the dehydrated tissue into paraffin wax for 2h, change the wax twice, and put it in a 60℃ constant temperature incubator. Put the tissue into the preheated embedding frame, position it with tweezers, and solidify the hot wax. Fix the paraffin-embedded tissue on the paraffin slicer and start slicing. The slice thickness is 5μm. Use a brush to unfold the slice on the 42℃ water surface and stick it on the slide. Incubate at 37℃ overnight for subsequent staining.

[0091] The prepared paraffin tissue sections were placed in a 60°C constant temperature incubator and baked for 2 h.

[0092] The paraffin tissue sections were dewaxed and rehydrated with a series of ethanol: xylene, 10 min; xylene, 20 min; xylene, 20 min; anhydrous ethanol, 15 min; anhydrous ethanol, 15 min; 95% ethanol, 5 min; 90% ethanol, 5 min; 80% ethanol, 5 min; 70% ethanol, 5 min.

[0093] Wash the tissue sections with PBS for 5 minutes. Place the tissue sections in a pressure cooker filled with citrate buffer for antigen repair. Wash the sections with distilled water and incubate with 3% hydrogen peroxide at room temperature for 15 minutes to eliminate the activity of endogenous peroxidase. Wash with PBS for 2 minutes × 3 times and soak in PBS for 5 minutes. Depending on the species of the primary antibody, use blocking solutions from different sources. The present invention uses goat serum (#KIT-9710; MXB) and blocks in a 37°C incubator for 40 minutes. Pour off the serum, add a rabbit-derived PNA primary antibody (1:250, Vector Laboratories) at a certain dilution ratio, and incubate at 4°C overnight. Wash away the primary antibody with PBS for 5 minutes × 3 times. Add HRP-labeled goat anti-rabbit IgG secondary antibody to the tissue sections and incubate in a 37°C constant temperature incubator for 1 hour. Wash away the secondary antibody with PBS for 5 minutes × 3 times, and place the tissue sections in PBS. Use TSA Plus Fluorescein working solution to develop color under a microscope, and the brown area is the GC area.

[0094] The results are as follows Figure 6 As shown, 14 days after NP-KLH immunization, the number of GCs in the spleen of Ewsr1 BKO mice was significantly increased compared with that of WT mice.

[0095] 3. ELISA

[0096] Blood was collected from the ocular vein of unimmunized mice and mice on days 7 and 14 after immunization. The blood was placed in an untreated 1.5 mL EP tube. After being placed at room temperature for 1 hour, the blood was centrifuged at 12000 rpm for 5 minutes and the supernatant was placed in a new EP tube. Centrifugation was performed again and the supernatant was placed in a new EP tube. The light yellow supernatant was serum. The serum was diluted 1:100 with antibody diluent (PBS + 1% BSA) for ELISA detection, and the remaining serum was stored in a -80°C refrigerator.

[0097] Take out the ELISA plate (Corning) coated with 2.5μg / mL NP30-BSA (Biosearch) antigen 12 hours in advance from the 4℃ refrigerator. Discard the supernatant, wash the plate 3 times with PBST, add 100μL blocking solution (PBS+2% BSA) to each well, and block at room temperature for 2 hours. Discard the supernatant, add the diluted serum accordingly, and leave at room temperature for 2 hours. Discard the supernatant, wash the plate 10 times with PBST, add the primary antibody (goat anti-mouse IgM / G / G1-HRP) (Southern Biotech) diluted with antibody diluent, and incubate at room temperature for 1 hour. Discard the supernatant, wash the plate 10 times with PBST, add TMB colorimetric solution (Vector Laboratories), and after appropriate color development, add an equal volume of 2M sulfuric acid to terminate the reaction. Use an enzyme reader to read the absorbance (wavelength is OD450, and OD570 is selected as the reference wavelength).

[0098] The results are as follows Figure 7 As shown, at 7 and 14 days after NP-KLH immunization, the concentrations of NP-specific IgM, IgG, and IgG1 in the serum of Ewsr1 BKO mice were increased compared with those of WT mice, especially IgG and IgG1. flox / flox Mb1-Cre+ mice have a stronger ability to respond to antigens.

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a model for specifically knocking out the Ewsr1 gene in animal B cells, characterized in that: The method comprises the following steps: (1) Ewsr1 flox / + After crossing the animal with the Mb1-Cre animal, the genotype obtained was Ewsr1 flox / + Mb1-Cre+ and Ewsr1 flox / + F1 animals of Mb1-Cre-;Ewsr1 flox / + The animals were constructed by inserting Loxp sites at both ends of the fourth exon of the EWSR1 gene; (2) mating the two animals obtained in step (1) to obtain F2 generation animals; (3) Select the Ewsr1 genotype from the F2 generation flox / flox Mb1-Cre+ animals, the genotype is Ewsr1 flox / flox Mb1-Cre+ animals are a model for specifically knocking out the Ewsr1 gene in B cells.

2. The method according to claim 1, characterized in that In step (1), the insertion of Loxp sites at both ends of the fourth exon of the EWSR1 gene is performed by using CRISPR / Cas9 technology to perform site-specific modification on the EWSR1 gene through homologous recombination, thereby inserting Loxp sites at both ends of the fourth exon of the EWSR1 gene.

3. The method according to claim 2, characterized in that The specific process of the CRISPR / Cas9 technology includes: designing and transcribing gRNA in vitro, constructing homologous recombination Donor at the same time, injecting Cas9 mRNA, gRNA, and Donor into animal fertilized eggs at the same time, Cas9 mRNA translates Cas9 protein in vivo, Cas9 protein binds to the target site under the guidance of gRNA to cause DNA double-strand breaks, and the broken double-strands are repaired in the repair process using the donor Donor as a template, thereby modifying the target gene. The gRNA sequence is as follows: gRNA-1: 5'-TCGTGGAAACATCCTAGGACAGG-3'; gRNA-2: 5'-CCCTACTTACAAGATTCACAGAA-3'.

4. The method according to claim 1, characterized in that: The genotype selected from the F2 generation in step (3) is Ewsr1 flox / flox Mb1-Cre + specifically adopts the PCR method, uses the genomic DNA of F2 animal tissue as a template, and uses Ewsr1 gene primers and Mb1-Cre recombinase gene primers for PCR amplification. Animals that meet the following requirements are animals with specific knockout of the Ewsr1 gene in B cells: homozygous Ewsr1 gene amplification product containing loxp sites, Cre recombinase transgenic amplification product, and internal reference amplification product.

5. The method according to claim 4, characterized in that The Ewsr1 gene primer is Ewsr1 flox / flox The upstream primer and the downstream primer, the Mb1-Cre recombinase gene primers are Mb1-Cre primers 1, 2, 3, 4; the animal that meets the following requirements is an animal in which the Ewsr1 gene is specifically knocked out in B cells: using Ewsr1 flox / flox The upstream primer and the downstream primer amplified only one band of 301 bp, and the Mb1-Cre primers 1, 2, 3, and 4 amplified two bands of 230 bp and 197 bp.

6. The method according to claim 5, characterized in that The Ewsr1 flox / flox The upstream and downstream primers are: Ewsr1 flox / flox Upstream primer: 5′-AAAGCCAAGGATGACAGACTCTA-3′; Ewsr1 flox / flox Downstream primer: 5′-TAAAGCCTTTGAGAAGCACGACT-3′.

7. The method according to claim 5, characterized in that The Mb1-Cre primers 1, 2, 3, and 4 are: Mb1-Cre primer 1: 5′-CTAGGCCACAGAATTGAAAGATCT-3′; Mb1-Cre primer 2: 5′-GTAGGTGGAAATTCTAGCATCATCC-3′; Mb1-Cre primer 3: 5′-AGATGCCAGGACATCAGGAACCTG-3′; Mb1-Cre Primer 4: 5′-ATCAGCCACACCAGACACAGAGATC-3′.

8. Use of the animal model constructed by the method according to any one of claims 1 to 7 in screening or preparing drugs for diseases regulated by B cell immunity.

9. The use according to claim 8, characterized in that: The drug is a therapeutic antibody and / or CAR-T.

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