Construction and application of novel humanized site systemic lupus erythematosus mouse model

Abstract

The invention discloses construction and application of a novel humanized locus systemic lupus erythematosus mouse model, and provides a brand-new humanized systemic lupus erythematosus mouse model for the first time, and a spontaneous lupus mouse Fas / lpr is hybridized with an hCD38 humanized locus mouse, so that the mouse model is obtained. According to the present invention, the stable mouse model carrying the humanized site (hCD38) and the lupus mutant gene (lpr) is obtained, the mouse model can well simulate the typical autoimmune response of the SLE, and the invention provides the SLE treatment related target drug research model and the disease mechanism research model for the field, and the method can be directly used for verifying the effectiveness of the medicine designed aiming at the hCD38 target spot.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular, relates to the construction and application of a novel humanized site-specific systemic lupus erythematosus mouse model. Background Art

[0002] Systemic lupus erythematosus (SLE) is a typical autoimmune disease that causes organ damage due to immune disorders. The prevalence in my country is 30-70 / 100,000. SLE affects a wide range of systems, and the disease progresses rapidly during the attack period, seriously affecting the life and health of patients. Current treatments for SLE include hormones, immunosuppressants, and biological agents, but these drugs cannot cure patients, and the treatment effect on some severe patients is also poor. Therefore, continuously improving the precise diagnosis and treatment of SLE has important theoretical and practical significance, and is also a problem that needs to be urgently solved in clinical practice.

[0003] CD38 has been found to play a key role in the study of SLE targets. CD38 is a cell surface protein expressed on plasma cells, early B cells, and subsets of T cells and NK cells. Compared with existing CD19-targeted therapies, targeting CD38 provides a different target cell spectrum and combines the advantages of targeting antibody-producing cells (plasmablasts and plasma cells) and eliminating B cells in the early stages of differentiation without affecting mature B cells (including memory B cells). Related studies have also observed that CD38 expression is widely dysregulated in SLE and can be found in a variety of cell subsets in peripheral blood. A type of CD38-highly expressed CD8+T cell was found in the peripheral blood of SLE patients, whose cytotoxic ability was reduced and associated with an increased tendency to infection. In the serum of SLE patients with inactive disease, increased levels of anti-CD38 autoantibodies were observed, and SLE patients who produced anti-CD38 antibodies had relatively good disease control. In some cases of refractory lupus, the use of anti-CD38 antibodies (Daratumumab) has also achieved good therapeutic effects. Therefore, CD38 is a promising and advantageous target for the treatment of SLE. Further experimental verification is still needed. It is very important to select an appropriate lupus model to directly verify the effectiveness of drugs designed for the human CD38 target. However, there is currently no verifiable lupus mouse model.

[0004] At present, there are two main types of lupus mouse models used to study the pathogenesis of SLE and develop new treatments. These mouse models mainly show lupus-like nephritis and the production of autoantibodies, including spontaneous models and induced models. Spontaneous models include New Zealand black × white F1 mice (NZB / W), Murphy Roths Large / Lpr mice (MRL / Lpr) and BXSB / Yaa mice, and induced models are mainly pristane-induced mouse models. However, there are differences in the immune systems and related targets between humans and mice, and neither of the above two model mice can be used as disease models that can be directly verified for drug treatment research applied to humans. At present, the construction of SLE humanized mouse model mainly involves transplanting human peripheral blood mononuclear cells (PBMCs) or peripheral blood lymphocytes (PBLs) of SLE patients into immunodeficient mice. The PBLs / PBMCs humanized mouse model is the most widely used, but the individual differences of SLE patients are large, which often leads to inconsistent model parameters and poor consistency. Although this model can be used to study human T cells well, the reconstruction effect of human B cells is poor, the level of human NK cells is low, and there is a lack of differentiation of human myeloid cells. The model induction results are unstable, the onset time is inconsistent, and the disease conditions vary greatly, which cannot meet the needs of scientific research. Therefore, in order to better verify the development and application of targeted drugs and explore the pathogenesis of SLE disease progression, it is urgent to develop a fast and stable systemic lupus erythematosus mouse model. Summary of the invention

[0005] In order to overcome the technical problems existing in the prior art, the purpose of the present invention is to provide the construction and application of a novel humanized site-specific systemic lupus erythematosus mouse model.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a method for constructing a CD38 humanized systemic lupus erythematosus mouse model.

[0008] Further, the method comprises the following steps:

[0009] (1) MRL-lpr systemic lupus erythematosus mice were crossed with hCD38 homozygous mice to generate F1 mice;

[0010] (2) pairing the F1 generation mice with males and females to produce F2 generation mice;

[0011] (3) Identify the genotype of F2 mice and select mice that are homozygous for humanized CD38 and homozygous for Fas mutation, which is the CD38 humanized systemic lupus erythematosus mouse model.

[0012] Furthermore, the method further comprises the following steps:

[0013] Pairing the mice homozygous for humanized CD38 and homozygous for Fas mutation described in step (3) to produce F3 mice;

[0014] The genotype of F3 mice was identified, and the F3 mice were paired with males and females to obtain a CD38 humanized systemic lupus erythematosus mouse model with a stable genotype.

[0015] Furthermore, the hCD38 homozygous mice in step (1) are hCD38-C57BL / 6J mice.

[0016] Furthermore, the age of the male and female pairs is 6-8 weeks.

[0017] In some embodiments, after the F3 generation mice are paired with males and females, the 4-6 generation mice are preferably identified to ensure the stable genotype of the prepared CD38 humanized systemic lupus erythematosus mouse model.

[0018] Furthermore, in the step (3) of identifying the mouse genotype, the primer sequence for wild-type Fas is shown in SEQ ID NO: 1-2;

[0019] The primer sequences for mutant Fas are shown in SEQ ID NO: 3-4.

[0020] Furthermore, the MRL-lpr systemic lupus erythematosus mouse described in step (1) is a commercial spontaneous lupus mouse model purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.

[0021] Furthermore, the hCD38 homozygous mice described in step (2) are commercial hCD38-C57BL / 6J mice carrying hCD38, purchased from Jicui Pharmaceutical Co., Ltd.

[0022] In some embodiments, the CD38 humanized systemic lupus erythematosus mouse model can also be constructed by the following method: humanizing the MRL-lpr systemic lupus erythematosus mouse, knocking out the mCD38 gene, and knocking in hCD38.

[0023] In some embodiments, PCR can be used to detect whether the constructed mouse is a homozygous Fas mutation mouse, that is, to identify the genotype of the mouse. The basic principle of PCR detection of gene mutation is to use a pair of primers to bind to the sequences at both ends of the DNA fragment to be amplified, and then under the action of DNA polymerase, use the primers as the starting point to synthesize a new complementary DNA chain. Since each newly synthesized chain can be used as a template for the next round of replication, the number of target DNA fragments will be rapidly amplified, thereby facilitating subsequent detection and analysis.

[0024] In some embodiments, flow cytometry can be used to detect whether the obtained mouse is a hCD38 homozygous mouse. In a specific embodiment of the present invention, the present invention uses the following method to detect whether the obtained mouse is a hCD38 homozygous mouse: take tail blood from the mouse to be tested, lyse the red blood cells and then wash, stain with antibodies FITC-Anti-mouse-CD38, APC-anti-human-CD38, PE-mCD19, and detect. After circling the lymphocytes, analyze the expression of mCD38 and hCD38 in CD19 positive cells.

[0025] In some embodiments, gene knockout refers to the use of specific technical means to make a gene in a cell or organism lose its function. Common methods such as CRISPR / Cas9 technology, by designing a guide RNA (sgRNA) for the target gene, guide the nuclease to cut the gene, and then the cell repairs the DNA damage through non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanism. NHEJ repair usually introduces insertion or deletion mutations, resulting in changes in the gene's reading frame, so that the gene cannot express protein normally, achieving gene knockout.

[0026] In some embodiments, gene knock-in is the integration of exogenous genes or gene fragments into the genome of cells or organisms. CRISPR / Cas9 technology can also be used to cause double-stranded DNA breaks at the target location, and then provide exogenous gene fragments containing homologous arms, and the cell accurately integrates the exogenous gene into the genome through the HDR mechanism.

[0027] In the present invention, the humanized mouse refers to a mouse carrying functional human genes, cells, tissues or organs, immune system or microorganisms, which is used for biomedical research and development of clinical treatment programs. Including but not limited to: expressing a human molecule in the mouse (such as transgenic mice), transplanting human cells, growing human tissues (such as human liver), etc. In a specific embodiment of the present invention, the humanized mouse is a systemic lupus erythematosus mouse model carrying a functional human gene (hCD38).

[0028] In a specific embodiment of the present invention, the CD38 humanized systemic lupus erythematosus mouse model is a mouse model that stably carries the humanized site (hCD38) and the lupus mutant gene (lpr).

[0029] In the present invention, the MRL / lpr mouse is an experimental mouse model commonly used to study autoimmune diseases, especially in systemic lupus erythematosus (SLE) and other autoimmune diseases. These mice carry a mutant gene called Fas (or lpr, lymphoproliferation), which leads to the loss of Fas receptor function. Fas receptor plays a key role in regulating cell apoptosis, and its loss of function can lead to abnormal immune cell proliferation and autoimmune reactions.

[0030] MRL-lpr mice are produced by complex mating of LG / J, AKR / J, C3H / Di and C57BL / 6 mice. Due to the apoptosis-related mutation in the Fas gene, i.e., lpr (lymphoproliferation), T cells, B cells and macrophages show apoptosis defects, leading to T cell hyperplasia, systemic lymphadenopathy, erosive arthritis, anti-DNA, anti-Sm, anti-Su, anti-nucleoside P antibodies, high titer ANA, hypergammaglobulinemia and rheumatoid factor.

[0031] Fas (also known as CD95 or APO-1) is a cell surface receptor that belongs to the tumor necrosis factor (TNF) receptor family. The Fas receptor induces apoptosis by binding to its ligand FasL (fas Ligand), which is crucial in regulating the immune system and clearing damaged or harmful cells. MRL / lpr mice are the main animal model for studying SLE, which exhibits many symptoms similar to humans, such as autoantibody production, immune complex deposition, nephritis, etc.

[0032] In a specific embodiment of the present invention, the present invention proves through comparative experiments that the hCD38-lpr systemic lupus erythematosus mouse constructed by the method described in the first aspect of the present invention can develop a stable lupus phenotype earlier than the hCD38 systemic lupus erythematosus mouse model induced by pristane. This technical effect is a technical effect that is unexpected by a person skilled in the art based on the contents disclosed in the prior art.

[0033] The second aspect of the present invention provides a CD38 humanized systemic lupus erythematosus mouse model.

[0034] Furthermore, the mouse model is a CD38 humanized systemic lupus erythematosus mouse model prepared according to the method of the first aspect of the present invention.

[0035] The third aspect of the present invention provides any of the following applications:

[0036] (1) Use of a CD38 humanized systemic lupus erythematosus mouse model prepared according to the method of the first aspect of the present invention in screening drugs for treating systemic lupus erythematosus;

[0037] (2) The fourth aspect of the present invention provides the use of the CD38 humanized systemic lupus erythematosus mouse model prepared by the method according to the first aspect of the present invention in screening drugs for treating diseases related to the CD38 gene;

[0038] (3) The fifth aspect of the present invention provides the use of the CD38 humanized systemic lupus erythematosus mouse model prepared by the method according to the first aspect of the present invention in studying the functions and mechanisms of action of the CD38 gene;

[0039] (4) The sixth aspect of the present invention provides the use of a CD38 humanized systemic lupus erythematosus mouse model prepared according to the method of the first aspect of the present invention in screening drugs targeting CD38 or verifying the effectiveness of drugs designed for the CD38 target.

[0040] The fourth aspect of the present invention provides a system for constructing a CD38 humanized systemic lupus erythematosus mouse model, the system comprising the following units:

[0041] In the first treatment unit, MRL-lpr systemic lupus erythematosus mice were crossed with hCD38 homozygous mice to generate F1 mice;

[0042] The second processing unit is to pair the F1 generation mice with males and females to produce F2 generation mice;

[0043] The third processing unit is to identify the genotype of F2 mice and select mice that are homozygous for humanized CD38 and homozygous for Fas mutation, which is the CD38 humanized systemic lupus erythematosus mouse model.

[0044] Furthermore, the system further comprises the following units:

[0045] The fourth processing unit is to pair the humanized CD38 homozygous and Fas mutation homozygous mice to produce F3 generation mice;

[0046] The fifth processing unit is to identify the genotype of F3 mice, pair the F3 mice with males and females, and obtain a CD38 humanized systemic lupus erythematosus mouse model with a stable genotype;

[0047] Optionally, the hCD38 homozygous mouse is a hCD38-C57BL / 6J mouse;

[0048] Optionally, the male-female pairing is 6-8 weeks old;

[0049] Optionally, in identifying the mouse genotype, the primer sequences for wild-type Fas are shown as SEQ ID NOs: 1-2, and the primer sequences for mutant Fas are shown as SEQ ID NOs: 3-4.

[0050] The fifth aspect of the present invention provides a device for constructing a CD38 humanized systemic lupus erythematosus mouse model, the device comprising:

[0051] A single or multiple processors, and a memory, the memory being used to store a single or multiple computer programs, which when executed by the single or multiple processors implement:

[0052] Operation 1, MRL-lpr systemic lupus erythematosus mice were crossed with hCD38 homozygous mice to generate F1 mice;

[0053] Operation 2, pairing the F1 generation mice with males and females to produce F2 generation mice;

[0054] Operation 3, identify the genotype of F2 mice, select mice that are homozygous for humanized CD38 and homozygous for Fas mutation, which is the CD38 humanized systemic lupus erythematosus mouse model;

[0055] Optionally, the device further comprises:

[0056] Operation 4, pairing the humanized CD38 homozygous and Fas mutation homozygous mice to produce F3 generation mice;

[0057] Operation 5, identifying the genotype of F3 mice, pairing the F3 mice by sex, and obtaining a CD38 humanized systemic lupus erythematosus mouse model with a stable genotype.

[0058] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0059] (1) The present invention provides a new humanized systemic lupus erythematosus mouse model for the first time. By hybridizing spontaneous lupus mice Fas / lpr with hCD38 humanized site mice, a stable mouse model carrying the humanized site (hCD38) and lupus mutant gene (lpr) is obtained. The mouse model can better simulate the typical autoimmune response of SLE. The present invention provides a drug research model and disease mechanism research model for SLE treatment-related targets in the field, which can be directly used to verify the effectiveness of drugs designed for the hCD38 target.

[0060] (2) Compared with the currently widely used non-humanized systemic lupus erythematosus mouse model, the humanized systemic lupus erythematosus mouse model provided by the present invention has a humanized site CD38, and the hCD38 target plays an important role in the pathogenesis and treatment research of systemic lupus erythematosus. It can be directly used to verify whether therapeutic drugs targeting the human CD38 target are effective, and the research results are more real and reliable.

[0061] (3) Compared with the current method of constructing humanized mice by transplanting PBMCs from SLE patients, the construction method provided by the present invention has the advantages of low model construction cost, simple and easy operation, good model stability, and short cycle. Compared with other feasible methods for constructing disease humanized models (for example, chemical drug-induced humanized mice), the construction method provided by the present invention has the advantages of short model cycle, easy promotion, and more obvious and intuitive disease phenotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the result diagram corresponding to the mouse genotype identification;

[0063] Figure 2 This is the result diagram of flow cytometry detection of whether hCD38 is homozygous;

[0064] Figure 3 This is a result diagram corresponding to the lupus phenotype of the mouse model constructed by the method provided in Example 2 of the present invention;

[0065] Figure 4 This is the result of antinuclear antibody (ANA) test;

[0066] Figure 5 This is a graph showing the results of anti-ds-DNA antibody testing;

[0067] Figure 6 A schematic diagram of a system for constructing a CD38 humanized systemic lupus erythematosus mouse model provided in an embodiment of the present invention;

[0068] Figure 7 A schematic diagram of a device for constructing a CD38 humanized systemic lupus erythematosus mouse model provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0069] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0070] The reagents and raw materials used in the present invention are easily obtained by ordinary technicians in the field and can be obtained from commercial channels unless otherwise specified. The experimental methods in the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way.

[0071] Example 1 Construction of a humanized systemic lupus erythematosus mouse model

[0072] 1. Experimental Materials

[0073] MRL / MpJ-Faslpr / J (MRL-lpr spontaneous lupus mouse model) was purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.;

[0074] hCD38-C57BL / 6J mice (humanized hCD38 locus mice) were purchased from Jicui Pharmaceutical Co., Ltd.;

[0075] Rat tail genotype rapid identification kit: Bimake: B40013;

[0076] Flow cytometry antibody detection: FITC-Anti-mouse-CD38 (Biolegend: 165608), APC-anti-human-CD38 (Biolegend: 303510), PE-anti-mouse-CD19 (Biolegend: 152408).

[0077] 2. Experimental methods

[0078] (1) Crossbreeding to obtain stable mice

[0079] MRL-lpr lupus mice were crossed with hCD38 homozygous mice to generate the first generation of two-locus mice (F1);

[0080] Pair the male and female first generation mice to produce the second generation mice (F2);

[0081] The genotype of the second generation was identified, and mice homozygous for the hCD38 and Fas loci were selected for male-female pairing to produce the third generation of mice (F3);

[0082] After the genotype of the third generation of mice is identified, the males and females are paired again (preferably 4-6 generations) to obtain hCD38-lpr mice with stable genotypes. The age of the paired mice is 6-8 weeks.

[0083] (2) Mouse genotype identification

[0084] 1) Take 2-3 mm of the tail of a 4-week-old offspring mouse, digest the tail tissue using an identification kit and further inactivate the protease, then centrifuge and take the supernatant as a PCR template.

[0085] 2) PCR primers

[0086] The primer sequences for wild-type Fas are as follows:

[0087] Forward primer (F1): 5'-GTAAATAATTGTGCTTCGTCAG-3' (SEQ ID NO: 1);

[0088] Reverse primer (R1): 5'-CAAATCTAGGCATTAACAGTG-3' (SEQ ID NO: 2).

[0089] The primer sequences for mutant Fas are as follows:

[0090] Forward primer (F1): 5'-GTAAATAATTGTGCTTCGTCAG-3' (SEQ ID NO: 3);

[0091] Reverse primer (R1): 5'-TAGAAAGGTGCACGGGTGTG-3' (SEQ ID NO: 4).

[0092] 3) PCR amplification

[0093] 4) DNA gel running

[0094] (3) Flow cytometry to detect whether hCD38 is homozygous

[0095] The tail blood was collected, the red blood cells were lysed and washed, and then stained with antibodies FITC-Anti-mouse-CD38, APC-anti-human-CD38, and PE-mCD19 for detection. After the lymphocytes were circled, the expression of mCD38 and hCD38 in CD19 positive cells was analyzed.

[0096] 3. Experimental results

[0097] The corresponding results of mouse genotype identification are as follows Figure 1 As shown in the figure, the gene detection result of the hybrid F4 generation of hCD38-lpr mice shows that the obtained hCD38-lpr mice are homozygous mice for the Fas mutation. The flow cytometry test results of whether hCD38 is homozygous are shown in Figure 2 As shown, the results show that compared with wild-type mice, the homozygous mice constructed in this example express the humanized target hCD38. The above results show that this example successfully constructed a homozygous humanized lupus mouse model (hCD38-lpr mouse) expressing hCD38 and carrying Fas mutation. Example 2 Comparison of lupus phenotypes of humanized lupus mouse models constructed by different methods

[0098] 1. Experimental Materials

[0099] Blood and urine of humanized lupus mouse models constructed in different ways;

[0100] ANA detection kit: Shanghai ELISA, Cat#ml002245;

[0101] dsDNA detection kit: Alpha Diagnostic, Cat#5120;

[0102] Urine protein detection kit: Jiangcai Biotechnology, Cat# JL-T0030.

[0103] 2. Experimental methods

[0104] (1) Construction of humanized lupus mouse models by different methods: Pristane was injected intraperitoneally into hCD38-C57BL / 6J mice at 6-8 weeks to induce autoimmunity, and urine was collected 3, 4, 5, and 6 months after induction. Blood was collected from the extraorbital venous plexus after anesthesia, and plasma was collected after centrifugation to detect autoantibodies.

[0105] (2) Detection methods for autoantibodies and urine protein: Elisa method for detecting autoantibodies and CBB method for detecting urine protein.

[0106] 1) Elisa: ANA test / dsDNA test

[0107] ① The kit should be taken out of the refrigerated environment and equilibrated at room temperature for 15-30 minutes, and the standard wells and sample wells should be designed.

[0108] ② Before use, mix all reagents thoroughly. Do not allow the liquid to produce a large amount of foam, so as to avoid adding a large number of bubbles when adding samples, resulting in errors in adding samples.

[0109] ③ Add samples to standard, sample and blank wells. Add 50μL of standard of different concentrations to each standard well; blank well (blank control well does not add sample and enzyme labeling reagent, the rest of the steps are the same); test sample well: dilute the sample with specimen diluent 1:5 and add 50μL to the reaction well. Add the sample to the bottom of the well of the ELISA plate, try not to touch the well wall, and gently shake to mix.

[0110] ④ Add 50 μL of biotin-labeled antibody, cover the plate, shake gently to mix, and incubate at 37°C for 1 hour.

[0111] ⑤ Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, shake for 30 seconds and then spin dry, repeat 3-5 times and pat dry.

[0112] ⑥ Add 50 μL of substrate A and B to each well, gently shake to mix, and incubate at 37°C in the dark for 15 minutes.

[0113] ⑦Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow). The results should be measured immediately after adding the stop solution.

[0114] ⑧ Determination: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0115] 2) Urine protein test

[0116] ① Preparation of CBB application solution: Prepare CBB reagent at a ratio of 1:4, i.e. 5-fold dilution.

[0117] ② Add 3 mL of CBB application solution to the sample group, standard group, and blank group, and add 500 μL of sample, standard, and distilled water, respectively.

[0118] ③ Mix thoroughly, let stand for 5 minutes, wavelength 595nm, light path 1cm, double water zero adjustment, and measure the absorbance value of each tube.

[0119] 3. Experimental results

[0120] The results are as follows Figure 3 As shown, the results show that the humanized lupus mouse model constructed by the method provided in this example has manifestations such as skin, lymphocyte proliferation, and blood system involvement.

[0121] Antinuclear antibody (ANA) test results Figure 4 As shown, hCD38 refers to a humanized mouse model constructed by inducing humanized CD38 mice with pristane; lpr mice refer to a humanized mouse model constructed by the method described in Example 1. The results show that the model constructed by the hybridization method described in Example 1 can produce autoantibodies earlier (3 months old) and stably compared with the induction method. The difference between the hybridization method and the WT group is statistically significant, while the induction method can only show an increasing trend of autoantibodies after 5 months of age, with no statistical difference.

[0122] Anti-ds-DNA antibody test results Figure 5 As shown, hCD38 refers to a humanized mouse model constructed by inducing humanized CD38 mice with pristane; lpr mice refer to a humanized mouse model constructed by the method described in Example 1. The results show that the model constructed by the hybridization method described in Example 1 can produce lupus-specific antibodies earlier and more stably than the induction method, and the difference is statistically significant, proving the successful construction of the lupus phenotype. However, the induction method did not produce specific antibodies at 5 months of age.

[0123] In addition, the present invention also demonstrates through experiments that the humanized lupus mouse model (hCD38-lpr mouse) constructed by the method described in Example 1 of the present invention can develop a stable lupus phenotype earlier than the pristane-induced hCD38 lupus mouse model constructed by the method described in Example 2 of the present invention, and has a better effect.

[0124] The results of the above comparative experiments prove that the hybridization method described in Example 1 of the present invention can successfully construct a humanized lupus mouse model, and its effect is significantly better than the conventional induction method reported in the prior art.

[0125] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiment of the present invention. In some of the processes described in the specification and claims of the present invention and the above-mentioned drawings, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this document or executed in parallel. The serial numbers of the operations, such as 101, 102, 103, etc., are only used to distinguish between different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel.

[0126] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0127] Figure 6 Schematic diagram of a system for constructing a CD38 humanized systemic lupus erythematosus mouse model provided in an embodiment of the present invention. Specifically, the system includes the following units:

[0128] In the first treatment unit, MRL-lpr systemic lupus erythematosus mice were crossed with hCD38 homozygous mice to generate F1 mice;

[0129] The second processing unit is to pair the F1 generation mice with males and females to produce F2 generation mice;

[0130] The third processing unit is to identify the genotype of F2 mice and select mice that are homozygous for humanized CD38 and homozygous for Fas mutation, which is the CD38 humanized systemic lupus erythematosus mouse model.

[0131] In one embodiment, the system further comprises the following units:

[0132] The fourth processing unit is to pair the humanized CD38 homozygous and Fas mutation homozygous mice to produce F3 generation mice;

[0133] The fifth processing unit is to identify the genotype of F3 mice, pair the F3 mice with males and females, and obtain a CD38 humanized systemic lupus erythematosus mouse model with a stable genotype;

[0134] In one embodiment, the hCD38 homozygous mouse is a hCD38-C57BL / 6J mouse;

[0135] In one embodiment, the male-female pair is 6-8 weeks old;

[0136] In one embodiment, in identifying the mouse genotype, the primer sequences for wild-type Fas are shown as SEQ ID NOs: 1-2, and the primer sequences for mutant Fas are shown as SEQ ID NOs: 3-4.

[0137] Figure 7 Schematic diagram of a device for constructing a CD38 humanized systemic lupus erythematosus mouse model provided in an embodiment of the present invention. Specifically, the device comprises:

[0138] A single or multiple processors, and a memory, the memory being used to store a single or multiple computer programs, which when executed by the single or multiple processors implement:

[0139] Operation 1, MRL-lpr systemic lupus erythematosus mice were crossed with hCD38 homozygous mice to generate F1 mice;

[0140] Operation 2, pairing the F1 generation mice with males and females to produce F2 generation mice;

[0141] Operation 3, identify the genotype of F2 mice, select mice that are homozygous for humanized CD38 and homozygous for Fas mutation, which is the CD38 humanized systemic lupus erythematosus mouse model;

[0142] In some embodiments, the device further comprises:

[0143] Operation 4, pairing the humanized CD38 homozygous and Fas mutation homozygous mice to produce F3 generation mice;

[0144] Operation 5, identifying the genotype of F3 mice, pairing the F3 mice by sex, and obtaining a CD38 humanized systemic lupus erythematosus mouse model with a stable genotype.

[0145] The validation results of this validation example show that assigning intrinsic weights to indications can moderately improve the performance of the method relative to the default setting.

[0146] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0147] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0148] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0149] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0150] A person of ordinary skill in the art can understand that all or part of the steps in the various methods of the above embodiments can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium, and the storage medium may include: a read-only memory (ROM), a random access memory (RAM), a disk or an optical disk, etc.

[0151] A person skilled in the art can understand that all or part of the steps in the above-mentioned embodiment method can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned storage medium can be a read-only memory, a disk or an optical disk, etc.

[0152] The above is a detailed introduction to a computer device provided by the present invention. For a person skilled in the art, according to the concept of the embodiments of the present invention, there may be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A method for constructing a CD38 humanized systemic lupus erythematosus mouse model, characterized in that: The method comprises the following steps: (1) MRL-lpr systemic lupus erythematosus mice were crossed with hCD38 homozygous mice to generate F1 mice; (2) pairing the F1 generation mice with males and females to produce F2 generation mice; (3) Identify the genotype of F2 mice and select mice that are homozygous for humanized CD38 and homozygous for Fas mutation, which is the CD38 humanized systemic lupus erythematosus mouse model.

2. The method according to claim 1, characterized in that The method further comprises the steps of: Pairing the mice homozygous for humanized CD38 and homozygous for Fas mutation described in step (3) to produce F3 mice; The genotype of F3 mice was identified, and the F3 mice were paired with males and females to obtain a CD38 humanized systemic lupus erythematosus mouse model with a stable genotype.

3. The method according to claim 1, characterized in that The hCD38 homozygous mice in step (1) are hCD38-C57BL / 6J mice.

4. The method according to claim 1, characterized in that: The male-female pairs were 6-8 weeks old.

5. The method according to claim 1, characterized in that: In the step (3) of identifying the mouse genotype, the primer sequence for wild-type Fas is shown in SEQ ID NO: 1-2; The primer sequences for mutant Fas are shown in SEQ ID NO: 3-4.

6. A CD38 humanized systemic lupus erythematosus mouse model, characterized in that: The mouse model is a CD38 humanized systemic lupus erythematosus mouse model prepared according to the method of any one of claims 1-5.

7. Any of the following applications: (1) Use of the CD38 humanized systemic lupus erythematosus mouse model prepared according to any one of claims 1 to 5 in screening drugs for treating systemic lupus erythematosus; (2) Use of the CD38 humanized systemic lupus erythematosus mouse model prepared by the method according to any one of claims 1 to 5 in screening drugs for treating diseases related to the CD38 gene; (3) Use of the CD38 humanized systemic lupus erythematosus mouse model prepared by the method according to any one of claims 1 to 5 in studying the functions and mechanisms of action of the CD38 gene; (4) Use of the CD38 humanized systemic lupus erythematosus mouse model prepared by the method according to any one of claims 1 to 5 in screening drugs targeting CD38 or verifying the effectiveness of drugs designed for the CD38 target.

8. A system for constructing a CD38 humanized systemic lupus erythematosus mouse model, characterized in that: The system comprises the following units: In the first treatment unit, MRL-lpr systemic lupus erythematosus mice were crossed with hCD38 homozygous mice to generate F1 mice; The second processing unit is to pair the F1 generation mice with males and females to produce F2 generation mice; The third processing unit is to identify the genotype of F2 mice and select mice that are homozygous for humanized CD38 and homozygous for Fas mutation, which is the CD38 humanized systemic lupus erythematosus mouse model.

9. The system for constructing a CD38 humanized systemic lupus erythematosus mouse model according to claim 8, characterized in that: The system also includes the following units: The fourth processing unit is to pair the humanized CD38 homozygous and Fas mutation homozygous mice to produce F3 generation mice; The fifth processing unit is to identify the genotype of F3 mice, pair the F3 mice with males and females, and obtain a CD38 humanized systemic lupus erythematosus mouse model with a stable genotype; Optionally, the hCD38 homozygous mouse is a hCD38-C57BL / 6J mouse; Optionally, the male-female pairing is 6-8 weeks old; Optionally, in identifying the mouse genotype, the primer sequences for wild-type Fas are shown as SEQ ID NOs: 1-2, and the primer sequences for mutant Fas are shown as SEQ ID NOs: 3-4.

10. A device for constructing a CD38 humanized systemic lupus erythematosus mouse model, characterized in that: The device comprises: A single or multiple processors, and a memory, the memory being used to store a single or multiple computer programs, which when executed by the single or multiple processors implement: Operation 1, MRL-lpr systemic lupus erythematosus mice were crossed with hCD38 homozygous mice to generate F1 mice; Operation 2, pairing the F1 generation mice with males and females to produce F2 generation mice; Operation 3, identify the genotype of F2 mice, select mice that are homozygous for humanized CD38 and homozygous for Fas mutation, which is the CD38 humanized systemic lupus erythematosus mouse model; Optionally, the device further comprises: Operation 4, pairing the humanized CD38 homozygous and Fas mutation homozygous mice to produce F3 generation mice; Operation 5, identifying the genotype of F3 mice, pairing the F3 mice by sex, and obtaining a CD38 humanized systemic lupus erythematosus mouse model with a stable genotype.

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