Construction method and application of mouse model with pathological IgA deposition in glomerular mesangial region

Abstract

The invention discloses a construction method and application of a glomerular mesangial region pathological IgA deposition mouse model, and belongs to the technical field of biologication.The construction method comprises the following steps that 1, gRNA is designed; step 2, constructing a Donor vector, recovering gel, connecting and converting, screening bacterial colonies, extracting positive clone plasmids, performing enzyme digestion identification and sequencing, and preparing plasmids for injection; step 3, synthesizing gRNA (guide Ribonucleic Acid); step 4, preparing an RNP compound; step 5, preparing fertilized eggs; step 6, carrying out pronucleus microinjection; 7, preparing a pregnant rat and transplanting an embryo; step 8, identifying F0 generation mice; and 9, breeding the transgenic mouse and establishing a line. According to the method disclosed by the invention, starting from simulating the pathogenesis of IgAN, a simple and feasible animal model is provided for screening drugs capable of decomposing immune complex deposition in a mesangial region, exploring an early IgA removal mechanism and removing abnormally deposited IgA by adopting a biological method so as to achieve the research of treating IgA nephropathy.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region and application thereof. Background Art

[0002] IgAN animal models are mainly divided into immune-induced, secondary lesion and spontaneous lesion types. Immune-induced animal models can be mainly divided into three categories: models caused by immune complexes, models induced by pathogenic microorganisms, and models stimulated by heterologous proteins. IgAN mice induced by CCL4 mixtures are heterologous protein-induced. The spontaneous lesion type is mainly the Japanese ddY mouse strain, which is similar to human IgAN in terms of the occurrence and development of IgAN. In particular, the subsequently developed Higa mice and GddY mice are closer to human IgAN in terms of phenotype and pathology. Higa mice show stable expression of high levels of IgA. However, there is no obvious correlation between the level of IgA in serum and the degree of glomerular damage and the incidence of nephropathy. In contrast, GddY mice can detect statistically significant proteinuria at about 8 weeks of birth, and at 24 weeks, there will be a significant increase in blood creatinine levels, accompanied by severe glomerular damage, mesangial cell proliferation, mesangial matrix proliferation and tubulointerstitial damage. Obvious co-deposition of IgA, IgG and C3 occurs in the mesangial area of ​​the glomerulus. Currently, this mouse is patented and there is no ddY mouse strain in China. The secondary lesion type is rarely used due to the high mortality rate of model animals.

[0003] At present, the animal model used by domestic scholars to study IgAN is the traditional CCl4 mouse model. The main principle of establishing this animal model is based on the following understanding: oral administration of BSA (bovine serum albumin) can increase the production of IgA in mice; LPS (lipopolysaccharide) as an immune adjuvant can destroy the endothelial reticular system of the mouse liver, thereby reducing the body's clearance of IgA; and the CCL4 (carbon tetrachloride) mixture is mainly used to induce liver cirrhosis, thereby further reducing the ability to clear IgA. However, there is still some controversy about the length of time required to build this animal model and the specific amount of BSA, etc., resulting in inconsistent severity of the constructed models.

[0004] TANK-binding kinase 1 (TBK1) is generally regarded as an activator of the innate immune response, acting as a key node protein in the innate immune signaling pathway and is essential for maintaining the body's immune balance. NF-κB is a family of proteins that regulate DNA transcription, cytokine production, and cell survival. It consists of five members: RelA (p65), RelB, c-Rel, p50 / p105 (NF-κB1), and p52 / p100 (NF-κB2). These proteins can form homodimers or heterodimers and bind to specific DNA sequences (called κB sites) to initiate or regulate the transcription of target genes. NF-κB is involved in a variety of cellular responses, including but not limited to inflammation, immune response, cell growth, apoptosis, and stress response. NF-κB is usually kept in an inactive state in the cytoplasm by binding to an inhibitory protein called IκB (κB inhibitor). The activation of NF-κB is a complex process that can be initiated by a variety of stimuli such as cytokines, free radicals, ultraviolet radiation, bacteria, and viral infections. There are two main activation pathways for NF-κB: the classical pathway and the atypical pathway. The atypical pathway is also called the non-classical pathway, which is mainly activated by certain cytokines (such as CD40L or BAFF). This pathway depends on the activation of NF-κB-inducing kinase (NIK) and IKKα, which leads to partial degradation of p100 to p52. This pathway is slower and more persistent than the classical pathway, and plays a vital role in processes such as B cell maturation and lymphoid organ formation. The upstream kinase in the non-classical pathway is NF-κB-inducing kinase (NIK). TBK1 can promote the phosphorylation of NIK at the Ser862 site, which happens to be located in the degradation area of ​​NIK. The phosphorylation event enhances the degradation of NIK, thereby preventing the activation of the non-classical NF-κB signaling pathway. Previous studies have shown that the classical NF-kB pathway regulates IgA class switching and plays an important role in regulating IgA formation. Jin and other immunology teams identified TBK1 as a key negative regulator of the classical NF-kB pathway. TBK1 gene deletion leads to abnormally high serum IgA levels. Summary of the invention

[0005] In order to overcome the defects of the prior art, the present invention provides a method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region and its application. The TBK1-BKO mice in the present invention have no difference in growth, development and survival compared with wild-type mice. The TBK1-BKO mouse model has high stability and can obtain an animal model with completely consistent genotypes. At the same time, its phenotype is closer to human IgA nephropathy.

[0006] The technical solution of the present invention is as follows:

[0007] A method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region comprises the following steps:

[0008] Step 1. gRNA design

[0009] Step 2: Donor vector construction, gel recovery, ligation and transformation, colony screening, positive clone plasmid extraction, enzyme digestion identification and sequencing, and injection plasmid preparation;

[0010] Step 3: gRNA synthesis

[0011] Step 4, RNP complex preparation:

[0012] Step 5: Fertilized egg preparation:

[0013] Step 6: Pronuclear microinjection:

[0014] Step 7: Preparation of surrogate mice and embryo transfer:

[0015] Step 8, identification of F0 generation mice:

[0016] Step 9: Breeding and establishing transgenic mice.

[0017] In the above technical scheme, in step 1, by visiting the website http: / / crispor.tefor.net / , gRNA is designed and a sequence with a higher score is selected.

[0018] In the above technical scheme, in step 2, Donor vector construction: fragment amplification (5'arm, cKO, 3'arm): using P515 high-fidelity enzyme of Novagen, PCR amplification was performed in a volume of 50μl, and the number of cycles was set to 30 times; gel recovery: the PCR product was subjected to electrophoresis analysis, and the target fragment was recovered using Qiagen gel recovery kit (No.: 28706); ligation and transformation: using C115 ligase of Novagen to ligate the recovered fragment, and using Takara Stellar competent cells to introduce the ligation product into Escherichia coli, and cultured overnight at 37°C; colony screening: 16 well-grown colonies were selected, and P222 Taq enzyme of Novagen was used for PCR amplification in a volume of 25μl, and the colonies with the correct inserted fragments were confirmed and inoculated into 4mL Small-scale fermentation was performed in LB liquid medium; plasmid extraction of positive clones: plasmid DNA was extracted by alkaline lysis method; restriction enzyme identification and sequencing: appropriate NEB restriction endonucleases were selected, and restriction enzyme identification was performed in a 20 μl system, and the correct plasmid was confirmed to be sent for sequencing; preparation of plasmid for injection: large-scale fermentation (22.5 mL LB medium) was performed on the clones with correct sequencing, and the plasmid was extracted using Qiagen plasmid extraction kit (No.: 27106) for injection.

[0019] In the above technical scheme, in step 3, crRNA (CRISPR RNA) is synthesized by IDT; tracrRNA (trans-activating crRNA) is provided by GenScript Biotech Co., Ltd.; crRNA and tracrRNA are combined to form gRNA.

[0020] In the above technical scheme, in step 4, solution in tube 1: 0.8 μL 100 pmol / μL CrRNA and 0.6 μL 100 pmol / μL tracrRNA were dissolved in 5.2 μL RNase-free water, mixed and incubated at room temperature for 5 minutes, and then 0.2 μL NEB's Cas9 protein (No.: M0646M) was added, and incubated at room temperature for 10 minutes to obtain solution in tube 1; solution in tube 2: Donor plasmid was diluted to 15 ng / μL; solution in tube 1 was mixed with solution in tube 2 to prepare an RNP injection complex.

[0021] In the above technical scheme, in step 5, 3-4 week old C57BL / 6 female mice are selected, and pregnant mare serum (PMSG) and human chorionic gonadotropin (HCG) are injected respectively, with an interval of 46-48 hours between the two injections; after the HCG injection, the female mice are mated with adult male mice; the next day, the female mice are euthanized, and fertilized eggs are collected from the oviducts and placed in an incubator at 37°C and 5% CO2 for later use.

[0022] In the above technical scheme, in step 6, a microinjection needle and a fixing needle are prepared; the RNP complex is mixed with plasmid DNA, diluted and loaded into the microinjection needle; under an inverted microscope (magnification 200-400 times), the exogenous gene is injected into the nucleus of the fertilized egg; the injected fertilized egg is transferred to M16 culture medium, and continues to be cultured in an incubator at 37°C and 5% CO2 for 0.5-1 hour, and then transplanted or cultured to the 2-cell stage and then transplanted.

[0023] In the above technical scheme, in step 7, female mice of appropriate age are used to mate with ligated male mice to create a pseudo-pregnancy state; the fertilized eggs carrying exogenous genes are transplanted into the fallopian tubes of the pseudo-pregnant female mice; after the transplantation, the female mice are placed in a warm environment until they recover; 19-20 days after the operation, the female mice give birth to pups; and the mice are marked and preliminarily identified one week after birth.

[0024] In the above technical scheme, in step 8, tissue samples of 1-2 week old mice are collected; genomic DNA is extracted and PCR amplification and electrophoresis are performed; offspring with integrated exogenous genes are screened; integrated individuals (i.e., founder mice) are confirmed, and subsequent breeding and phenotypic analysis are performed.

[0025] In the above technical scheme, in step 9, mice carrying exogenous genes are mated with wild-type mice; the genotype of F1 generation mice is identified; F1 generation positive mice can be used for experiments and further breeding; to obtain homozygotes, F1 generation positive individuals can be hybridized to produce F2 generation, 25% of which may be homozygous; mice expressing the target gene are confirmed and strains are established, and the breeding conditions and pedigree information are recorded.

[0026] Beneficial Effects

[0027] IgA nephropathy is the most common cause of idiopathic glomerulonephritis in developed countries. In the early days, it was believed that the course of IgAN was benign, but with clinical observation and research, it was found that at least 20% of patients have the risk of progressing to chronic kidney disease stage 5. The occurrence of IgAN is the result of the combined action of multiple factors. Its progression and prognosis are related to the patient's blood pressure, age, urine protein level, severity of hematuria, race, renal pathological changes, eGFR at the time of consultation, and whether normal treatment is received. The establishment of an animal model is of great significance in exploring its pathogenesis. In this study, slight IgA deposition can be observed in the mesangial area of ​​some 2-month-old and 3-month-old TBK1-BKO mice, and clumps of IgA and IgG deposition can be seen in the mesangial area of ​​the 5-month-old, 8-month-old, and 12-month-old mice. Compared with WT mice and traditional CCl4 mice, some glomeruli of TBK1-BKO mice began to show mesangial cell proliferation and increased mesangial matrix at 2 months of age, and the changes in the mesangial area of ​​the glomeruli gradually worsened with age. In this study, renal function damage in mice occurred later than renal pathological changes. The differences in blood creatinine and urine albumin-creatinine ratio from 8 months of age were statistically significant compared with WT mice. TBK1-BKO mice showed no difference in growth and survival compared with wild-type mice. The TBK1-BKO mouse model has high stability and can obtain an animal model with completely consistent genotypes. At the same time, its phenotype is closer to human IgA nephropathy. The method of the present invention starts from simulating the pathogenesis of IgAN, and provides a simple and feasible animal model for screening drugs that can decompose immune complex deposits in the mesangial region, exploring the early IgA clearance mechanism, and using biological methods to remove abnormally deposited IgA to achieve the treatment of IgA nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a statistical chart of clinical examination of 2-month-old TBK1-BKO mice.

[0029] Figure 2 This is a statistical chart of clinical examination of 3-month-old TBK1-BKO mice.

[0030] Figure 3 This is a statistical chart of clinical examination of 5-month-old TBK1-BKO mice.

[0031] Figure 4 This is a statistical chart of clinical examinations of 8-month-old TBK1-BKO mice.

[0032] Figure 5 This is a statistical chart of clinical examination of 12-month-old TBK1-BKO mice.

[0033] Figure 6 Results of IgA and IgG immunofluorescence staining of kidneys of 2-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400).

[0034] Figure 7 Results of immunofluorescence staining of IgA and IgG in the kidneys of 3-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400).

[0035] Figure 8 Results of immunofluorescence staining of IgA and IgG in the kidneys of 5-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400).

[0036] Fig. 9 Results of IgA and IgG immunofluorescence staining of kidneys of 8-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400).

[0037] Fig.10 Results of immunofluorescence staining of IgA and IgG in the kidneys of 12-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400).

[0038] Fig.11 HE and PAS staining results of kidney tissues of 2-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400).

[0039] Fig.12 HE and PAS staining results of kidney tissues of 3-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400).

[0040] Fig.13 HE and PAS staining results of kidney tissues of 5-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400).

[0041] Fig.14 HE and PAS staining results of kidney tissues of 8-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400).

[0042] Fig.15 HE and PAS staining results of kidney tissues of 12-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400). DETAILED DESCRIPTION

[0043] In order to explain the technical content, structural features, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0044] Example 1 - Preparation of TBK1-BKO Animal Model

[0045] 1. In this example, a TBK1-BKO mouse model was established by cooperating with Saiye (Suzhou) Biotechnology Co., Ltd.

[0046] (1) gRNA design: Design gRNA by visiting http: / / crispor.tefor.net / and select sequences with higher scores.

[0047] (2) Donor vector construction: Fragment amplification (5'arm, cKO, 3'arm): Use P515 high-fidelity enzyme from Novagen, perform PCR amplification in a volume of 50 μl, and set the number of cycles to 30 times;

[0048] Gel recovery: The PCR products were analyzed by electrophoresis, and the target fragments were recovered using the Qiagen gel recovery kit (No. 28706);

[0049] Ligation and transformation: Use C115 ligase from Novagen to ligate the recovered fragments, and use Takara Stellar competent cells to introduce the ligation products into E. coli and culture them overnight at 37°C.

[0050] Colony screening: 16 colonies with good growth were selected and PCR amplified in a volume of 25 μl using P222 Taq enzyme from Novagen. The colonies with the correct inserted fragments were confirmed and inoculated into 4 mL LB liquid medium for small-scale fermentation.

[0051] Plasmid extraction of positive clones: plasmid DNA was extracted using alkaline lysis method;

[0052] Enzyme digestion identification and sequencing: Select appropriate NEB restriction endonucleases and perform enzyme digestion identification in a 20μl system to confirm the correct plasmid for sequencing;

[0053] Preparation of plasmid for injection: The clones with correct sequencing were fermented on a large scale (22.5 mL LB medium), and the plasmid was extracted using Qiagen plasmid extraction kit (No.: 27106) for injection.

[0054] (3) gRNA synthesis: crRNA (CRISPR RNA) was synthesized by IDT; tracrRNA (trans-activating crRNA) was provided by GenScript Biotech Co., Ltd.; crRNA and tracrRNA combined to form gRNA.

[0055] (4) Preparation of RNP complex:

[0056] Tube 1 solution: Dissolve 0.8 μL 100 pmol / μL crRNA and 0.6 μL 100 pmol / μL tracrRNA in 5.2 μL RNase-free water, mix well and incubate at room temperature for 5 minutes, then add 0.2 μL NEB Cas9 protein (No.: M0646M), incubate at room temperature for 10 minutes to obtain Tube 1 solution;

[0057] Tube 2 solution: dilute the Donor plasmid to 15 ng / μL;

[0058] Mix the solution in tube 1 with the solution in tube 2 to prepare the RNP injection complex.

[0059] (5) Preparation of fertilized eggs:

[0060] 3-4 week old C57BL / 6 female mice were selected and injected with pregnant mare serum (PMSG) and human chorionic gonadotropin (HCG), respectively, with an interval of 46-48 hours between the two injections;

[0061] After HCG injection, the female mice were mated with adult male mice;

[0062] The next day, the female mice were euthanized, and the fertilized eggs were collected from the oviduct and placed in an incubator at 37°C and 5% CO2 for later use.

[0063] (6) Pronuclear microinjection:

[0064] Prepare microinjection needles and fixation needles;

[0065] The RNP complex is mixed with plasmid DNA, diluted and loaded into a microinjection needle;

[0066] Under an inverted microscope (magnification 200-400 times), the foreign gene was injected into the nucleus of the fertilized egg;

[0067] After injection, the fertilized eggs are transferred to M16 medium and cultured in an incubator at 37°C and 5% CO2 for 0.5-1 hour before transplantation or culture to the 2-cell stage and then transplantation.

[0068] (7) Preparation of surrogate mice and embryo transplantation:

[0069] Use female mice of appropriate age to mate with neutered male mice to create a pseudopregnancy state;

[0070] The fertilized eggs carrying the foreign gene were transplanted into the oviducts of pseudo-pregnant female mice;

[0071] After transplantation, the mother mice were placed in a warm environment until they recovered;

[0072] 19-20 days after surgery, the mother mice gave birth;

[0073] The mice were marked and preliminarily identified one week after birth.

[0074] (8) Identification of F0 generation mice:

[0075] Collect tissue samples from 1-2 week old mice;

[0076] Extract genomic DNA and perform PCR amplification and electrophoresis detection;

[0077] Screening offspring that have integrated foreign genes;

[0078] Confirm the integrated individuals (i.e., founder mice) and perform subsequent breeding and phenotypic analysis.

[0079] (9) Transgenic mouse breeding and strain establishment:

[0080] Mice carrying the foreign gene were mated with wild-type mice;

[0081] The genotype of F1 mice was identified;

[0082] F1 generation positive mice can be used for experiments and further breeding;

[0083] To obtain homozygotes, positive individuals of the F1 generation can be crossed to produce the F2 generation, of which 25% may be homozygous;

[0084] Identify mice expressing the gene of interest and establish strains, and record breeding status and pedigree information.

[0085] The blood urea nitrogen, blood creatinine, urine microalbumin-to-creatinine ratio, and kidney and spleen tissues of TBK1-BKO mice at 2, 3, 5, 8, and 12 months of age were collected and analyzed.

[0086] Example 2 Preparation of traditional CCl4 mouse model

[0087] 1. Twelve 8-week-old C57BL / 6 mice weighing 20-25g were selected. BSA+LPS+CCL4 was used to prepare the IgAN animal model. The specific method is as follows: from the beginning of the experiment, from the 1st to the 9th week, BSA was gavaged 0.8mg / g, qod; from the 1st to the 9th week, 100uL of CCl4 castor oil solution (CCL4: castor oil was diluted at 1:5) was injected subcutaneously every week, and 80μL of CCl4 castor oil solution was injected intraperitoneally every two weeks; 0.05mg LPS was injected once through the tail vein on the 6th and 8th week; 2 mice were randomly selected in the 9th week to verify the renal pathology. If the modeling was successful, the remaining mice were killed and the specimens were kept.

[0088] 2. Prepare C57BL / 6 mice and collect blood, urine, kidney tissue, and spleen tissue specimens.

[0089] Example 3 Postoperative sampling

[0090] Before the mice were killed, 0.3% sodium pentobarbital solution was used for intraperitoneal anesthesia, and the administration volume was 0.1 ml / 10 g. It can be observed that the chest of the mouse was up and down, and the heartbeat and breathing were not suppressed. Blood was collected by removing the eyeball. After the blood was collected, the heart was immediately perfused by fine needle puncture with room temperature PBS solution until clear perfusion fluid flowed out of the eyeball. At this time, the organ perfusion was completed, and the kidney was removed. After removing the capsule, the surface moisture was dried with absorbent paper. Next, one-third of the kidney tissue was taken and immersed in 4% paraformaldehyde solution for 24 hours, and then made into paraffin sections for HE (hematoxylin-eosin staining) and PAS (Periodic Acid-Schiff staining). Another one-third of the kidney tissue was embedded in OCT medium and frozen by dry ice, and the remaining one-third of the kidney tissue was stored in a -80 ° C refrigerator for transcriptome sequencing. For spleen processing, first gently lift the peritoneum under the spleen, cut it open and fold it upward to expose the spleen. Use forceps to clamp the spleen, cut the connective tissue below with scissors, and carefully remove the spleen. Place the removed spleen in a culture dish containing 5 ml of complete culture medium to wash and peel off the surrounding connective tissue. Finally, store the processed spleen tissue in a -80℃ refrigerator for subsequent transcriptome sequencing analysis.

[0091] Example 4 Collection and treatment of mouse blood and urine

[0092] After the mice are anesthetized, 0.8 ml ± of blood is collected from their eyeballs. After blood collection, the blood needs to be left at room temperature to allow the blood to coagulate naturally. This process takes about 15-30 minutes, and the purpose is to separate the red blood cells and plasma in the blood. The coagulated blood is then centrifuged in a centrifuge. During centrifugation, the blood should be placed in a centrifuge tube and centrifuged at an appropriate speed and time to separate the serum and clots. Usually the centrifugal speed is between 3000-5000g, and the centrifugation time is about 10-15 minutes. The subsequent serum is used for renal function testing, etc. The mice were fasted but not watered the day before being killed. Metabolic cages were used to retain urine for 24 hours. The collected urine was centrifuged for 15 minutes, the supernatant was taken, and frozen in a -80°C refrigerator for subsequent urine protein and urine creatinine testing to evaluate the urine protein / creatinine ratio.

[0093] Example 5 Observation of Mouse Kidney Pathology

[0094] Pathological changes in mouse kidneys: Hematoxylin-eosin staining (HE staining) and periodic acid-Schiff reaction (PAS staining) were used to observe the cell structure, general tissue morphology and pathological changes in the kidneys of each group of mice. Immunofluorescence was used to observe the deposition of immune complexes. In IgAN, mesangial cell proliferation and matrix hyperplasia are the main pathological changes, and the mass-like deposition of IgA in the mesangial region is a characteristic manifestation.

[0095] Example 6 ELISA to measure urine protein level

[0096] (1) Reagent preparation: Return all reagents to room temperature and dilute and prepare according to the instructions.

[0097] (2) Urine sample processing: Collect urine samples from mice, remove the precipitate by centrifugation, and take the supernatant for dilution (the specific dilution multiple is determined according to the instructions of the kit).

[0098] (3) Sample addition: Add the diluted urine sample and standard into the corresponding wells of the ELISA plate, 100 μl per well, and shake gently.

[0099] (4) Incubation: Place the ELISA plate in a 37°C constant temperature water bath and incubate for 30 minutes to allow the antigen and antibody to fully bind.

[0100] (5) Washing: Discard the liquid in the wells and wash five times with washing solution. Add 200 μl of washing solution to each well each time, let it stand for 30 seconds, then discard and pat dry.

[0101] (6) Add enzyme: Add 100 μl of enzyme-labeled secondary antibody to each well, shake gently, and incubate in a 37°C constant temperature water bath for 30 minutes.

[0102] (7) Wash again: Repeat the washing operation in step 5.

[0103] (8) Color development: Add 50 μl of substrate solution A or B to each well, shake gently, and incubate at 37°C in the dark for 15 minutes for color development.

[0104] (9) Termination: Add 50 μl of stop solution to each well to terminate the color development reaction.

[0105] (10) Measurement: The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader.

[0106] (11) Calculation of results: Draw a standard curve with the concentration of the standard substance as the horizontal axis and the OD value as the vertical axis. According to the OD value of the urine sample, find the corresponding protein content on the standard curve.

[0107] During the experiment, care should be taken to avoid cross contamination and ensure that each step is performed accurately. Avoid violent shaking of the ELISA plate during incubation and color development to avoid affecting the experimental results. The washing process should be thorough to ensure that unbound antibodies and impurities are removed. Reagents should be stored under specified conditions to avoid expiration.

[0108] Example 7 Kidney Specimen Pathology

[0109] 1. Paraffin sections of kidney specimens

[0110] (1) Sampling: Fresh tissue material should be collected as much as possible after surgery. If the time is too long, tissue autolysis may occur.

[0111] (2) Fixation: The tissue block was immediately immersed in 4% paraformaldehyde fixative to maintain a thickness of <2 mm for 6 hours at 4°C. The volume ratio of fixative to tissue block was 20:1.

[0112] (3) Dehydration: The fixed material is placed in alcohols of varying concentrations for dehydration, including 75% ethanol overnight, followed by 85%, 95%, and 100% ethanol, with each concentration treated for 30 minutes to gradually remove moisture from the tissue block.

[0113] (4) Transparency: Place the dehydrated tissue block in xylene for transparency treatment to remove residual water.

[0114] (5) Wax dipping and embedding: Place the transparent tissue block in the melted paraffin wax for wax dipping. Then use heated elbow forceps to gently pick up the tissue block and place it flatly in the center of the embedding base mold. Add melted paraffin wax and quickly place it on a cold table. After the melted wax in the embedding base mold solidifies, take out the integrated wax block and embedding box from the embedding mold and set aside.

[0115] (6) Sectioning: Use a rotary slicer to slice the embedded wax block into thin slices with a thickness of 4-7 μm. Cut out wax strips one by one, and then gently place them on paper with a brush. Cut the wax slices that can be used, place them in warm water for spreading, and place the spread wax slices on a glass slide using the water-scooping method.

[0116] (7) Staining: Staining includes HE staining and PAS staining

[0117] 2. HE staining steps

[0118] (1) HE staining: The sections were dewaxed with xylene, then hydrated with different concentrations of alcohol, and then stained in HE staining solution for 10 minutes, and then the residual stain was rinsed off with water.

[0119] (2) Sealing and observation: The stained sections were sealed with neutral gum, and then observed and recorded using an optical microscope.

[0120] 3. PAS staining steps

[0121] (1) PAS staining: The sections were dewaxed with xylene, then hydrated with different concentrations of alcohol, and then stained using a PAS staining kit. Periodic acid was added to the tissue, left to stand at room temperature for 8 minutes, and then rinsed with tap water.

[0122] (2) Sealing: The stained sections were sealed with neutral resin, and then observed and recorded using an optical microscope.

[0123] 4. Preparation of frozen sections

[0124] The tissue was embedded in OCT, quickly frozen in liquid nitrogen, and stored in a -20°C refrigerator. The slices were sliced ​​with a freezing microtome to a thickness of 8 μm and stored in a -20°C refrigerator for later use.

[0125] 5. Immunofluorescence staining

[0126] (1) Thawing and fixation: The frozen sections were placed at room temperature for 1 hour to thaw. They were then fixed with 4% paraformaldehyde at room temperature for 20 minutes.

[0127] (2) Washing: Discard excess paraformaldehyde and wash three times with PBS, 10 minutes each time.

[0128] (3) Blocking: Use 5% BSA to block the membrane for 1 hour at room temperature.

[0129] (4) Adding primary antibody: discard the excess BSA and dilute the IgA and IgG antibodies with 1% BSA, add the antibodies to the tissue, store in dark, and incubate at 4°C overnight.

[0130] (5) Sealing: Finally, use anti-fluorescence quenching sealing agent to seal the slices to protect the fluorescent signal from damage.

[0131] (6) Filming: Images were acquired using a Leica laser confocal microscope.

[0132] result

[0133] 1. Blood and urine test results of 2-month-old TBK1-BKO mice

[0134] TBK1-BKO mice were obtained using CRISPR / Cas9 gene editing technology. After the model was successfully constructed, blood, urine, kidney tissue, and spleen tissue were collected at 2, 3, 5, 8, and 12 months of age, and serum was extracted to detect blood urea nitrogen (BUN) and blood creatinine (SCr); urine was used to detect the mouse urine albumin creatinine ratio (UACR), and some kidney tissues were taken for light microscopy HE and PAS staining, and immunofluorescence IgA and IgG staining. The differences in blood and urine tests between TBK1-BKO mice at 2, 3, 5, 8, and 12 months of age were compared with traditional CCl4 mice and WT mice, and statistical analysis was performed.

[0135] The results of blood and urine tests showed that the blood urea nitrogen (10.60±1.17mmol / L), blood creatinine (25.75±3.59umol / L), and urine albumin-creatinine ratio (13.38±2.57mg / gCr) of 2-month-old TBK1-BKO mice were significantly higher than those of WT mice (11.83±1.55mmol / L), blood creatinine (23.00±4.74umol / L), and urine albumin-creatinine ratio (22.89±7.43mg / gCr). / gCr) had no significant difference (P>0.05), but had significant differences in blood urea nitrogen (15.67±5.28mmol / L), blood creatinine (36.2±19.75umol / L), and urine albumin-creatinine ratio (65.36±56.33mg / gCr) compared with traditional CCl4 mice (P<0.05); the levels of BUN, SCr, and UACR in WT mice were significantly different from those in traditional CCl4 mice (P<0.05) ( Figure 1 ).

[0136] Figure 1 :Statistical chart of clinical examination of 2-month-old TBK1-BKO mice. The levels of BUN, SCr, and UACR in TBK1-BKO mice were not significantly different from those in WT mice (P>0.05), but were significantly different from those in traditional CCl4 mice (P<0.05).

[0137] 2. Blood and urine test results of 3-month-old TBK1-BKO mice

[0138] The results of blood and urine tests showed that the blood urea nitrogen (10.32±0.96mmol / L), blood creatinine (24.50±3.44umol / L), and urine albumin-creatinine ratio (22.89±7.43mg / gCr) of 3-month-old TBK1-BKO mice were not significantly different from those of WT mice (P>0.05), but were significantly different from those of traditional CCl4 mice (P<0.05). The levels of BUN, SCr, and UACR in WT mice were significantly different from those in traditional CCl4 mice (P<0.05). Figure 2 ).

[0139] Figure 2 :Statistical chart of clinical examination of 3-month-old TBK1-BKO mice. The levels of BUN, SCr, and UACR in TBK1-BKO mice were not significantly different from those in WT mice (P>0.05), but were significantly different from those in traditional CCl4 mice (P<0.05).

[0140] 3. Blood and urine test results of 5-month-old TBK1-BKO mice

[0141] The results of blood and urine tests showed that the blood urea nitrogen (10.79±1.10mmol / L), blood creatinine (26.88±1.75umol / L), and urine albumin-creatinine ratio (22.38±8.65mg / gCr) of 5-month-old TBK1-BKO mice were not significantly different from those of WT mice (P>0.05), but were significantly different from those of traditional CCl4 mice (P<0.05). The levels of BUN, SCr, and UACR in WT mice were significantly different from those in traditional CCl4 mice (P<0.05). Figure 3 ).

[0142] Figure 3 :Statistical chart of clinical examination of 5-month-old TBK1-BKO mice. The levels of BUN, SCr, and UACR in TBK1-BKO mice were not significantly different from those in WT mice (P>0.05), but were significantly different from those in traditional CCl4 mice (P<0.05).

[0143] IV. Blood and urine test results of 8-month-old TBK1-BKO mice

[0144] The results of blood and urine tests showed that the blood urea nitrogen (11.52±1.86mmol / L) of 8-month-old TBK1-BKO mice was not significantly different from that of WT mice (P>0.05), nor was it significantly different from that of traditional CCl4 mice (P>0.05).

[0145] The serum creatinine of TBK1-BKO mice (32.75±4.02umol / L) was significantly different from that of WT mice (P<0.01), but not significantly different from that of traditional CCl4 mice (P>0.05).

[0146] The urine albumin-creatinine ratio of TBK1-BKO mice (27.47±10.39mg / gCr) was significantly different from that of WT mice (P<0.05) and was significantly different from that of traditional CCl4 mice (P<0.01). Figure 4 ).

[0147] Figure 4 :Statistical chart of clinical examination of 8-month-old TBK1-BKO mice. There was no significant difference in BUN between TBK1-BKO mice and WT mice and traditional CCl4 mice (P>0.05); there was no significant difference in SCr between TBK1-BKO mice and traditional CCl4 mice (P>0.05), but there was a significant difference between TBK1-BKO mice and WT mice (P<0.01); there was a significant difference in UACR between TBK1-BKO mice and WT mice (P<0.05), and there was a significant difference between TBK1-BKO mice and traditional CCl4 mice (P<0.01).

[0148] 5. Blood and urine test results of 12-month-old TBK1-BKO mice

[0149] The results of blood and urine tests showed that the blood urea nitrogen (14.34±1.57mmol / L) of 12-month-old TBK1-BKO mice was significantly different from that of WT mice (P<0.05), but not significantly different from that of traditional CCl4 mice (P>0.05).

[0150] The serum creatinine of TBK1-BKO mice (34.13±7.49umol / L) was significantly different from that of WT mice (P<0.05), but not significantly different from that of conventional CCl4 mice (P>0.05);

[0151] The urine albumin-creatinine ratio of TBK1-BKO mice (31.84±10.95 mg / gCr) was significantly different from that of WT mice (P<0.05) and that of traditional CCl4 mice (P<0.05). Figure 5 ).

[0152] Figure 5:Statistical chart of clinical examination of 12-month-old TBK1-BKO mice. The levels of BUN, SCr, and UACR in TBK1-BKO mice were significantly different from those in WT mice (P<0.05); there were no significant differences in BUN and SCr compared with traditional CCl4 mice (P>0.05), but there was a significant difference in UACR compared with traditional CCl4 mice (P<0.05).

[0153] VI. Immunofluorescence staining of kidney tissue of 2-month-old TBK1-BKO mice

[0154] Immunofluorescence staining of frozen kidney sections showed weakly positive IgA deposition and negative IgG deposition in the glomerular mesangium of 2-month-old TBK1-BKO mice, no IgA and IgG deposition in the glomerular mesangium of WT mice, and obvious IgA and IgG clumps in the glomerular mesangium of traditional CCl4 mice ( Figure 6 ).

[0155] Figure 6 :IgA and IgG immunofluorescence staining results of kidneys of 2-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400)

[0156] VII. Immunofluorescence staining of kidney tissue of 3-month-old TBK1-BKO mice

[0157] Immunofluorescence staining of frozen kidney sections showed weakly positive IgA deposition and negative IgG deposition in the glomerular mesangial region of 3-month-old TBK1-BKO mice, and more significant IgA deposition than that of 2-month-old mice. No IgA and IgG deposition was observed in the glomerular mesangial region of WT mice, and obvious IgA and IgG clumps were deposited in the glomerular mesangial region of traditional CCl4 mice ( Figure 7 ).

[0158] Figure 7 :IgA and IgG immunofluorescence staining results of kidneys of 3-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400)

[0159] 8. Immunofluorescence staining of kidney tissue of 5-month-old TBK1-BKO mice

[0160] Immunofluorescence staining of frozen kidney sections showed that IgA and IgG were deposited in the glomerular mesangium of 5-month-old TBK1-BKO mice, but no IgA and IgG were deposited in the glomerular mesangium of WT mice. However, IgA and IgG were deposited in the glomerular mesangium of traditional CCl4 mice. Figure 8 ).

[0161] Figure 8 :IgA and IgG immunofluorescence staining results of kidneys of 5-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400)

[0162] IX. Immunofluorescence staining of kidney tissues of 8-month-old TBK1-BKO mice

[0163] Immunofluorescence staining of frozen kidney sections showed that IgA and IgG were deposited in the glomerular mesangium of 8-month-old TBK1-BKO mice, but no IgA and IgG were deposited in the glomerular mesangium of WT mice. However, IgA and IgG were deposited in the glomerular mesangium of traditional CCl4 mice. Fig. 9 ).

[0164] Fig. 9 :IgA and IgG immunofluorescence staining results of kidneys of 8-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400)

[0165] 10. Immunofluorescence staining of kidney tissue of 12-month-old TBK1-BKO mice

[0166] Immunofluorescence staining of frozen kidney sections showed that IgA and IgG were deposited in the glomerular mesangium of 12-month-old TBK1-BKO mice, but no IgA and IgG were deposited in the glomerular mesangium of WT mice. However, IgA and IgG were deposited in the glomerular mesangium of traditional CCl4 mice. Fig.10 ).

[0167] Fig.10 :IgA and IgG immunofluorescence staining results of kidneys of 12-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400)

[0168] XI. HE and PAS staining of kidney tissue of 2-month-old TBK1-BKO mice

[0169] HE and PAS staining of kidney tissue showed mild proliferation of mesangial cells and matrix in the mesangial region of 2-month-old TBK1-BKO mice, no obvious proliferation of mesangial cells and matrix in the mesangial region of WT mice, and proliferation of mesangial cells and matrix in the mesangial region of traditional CCl4 mice ( Fig.11 ).

[0170] Fig.11 :HE and PAS staining results of kidney tissues of 2-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400)

[0171] 12. HE and PAS staining of kidney tissue of 3-month-old TBK1-BKO mice

[0172] HE and PAS staining of kidney tissue showed mild proliferation of mesangial cells and matrix in the mesangial region of 3-month-old TBK1-BKO mice, no obvious proliferation of mesangial cells and matrix in the mesangial region of WT mice, and proliferation of mesangial cells and matrix in the mesangial region of traditional CCl4 mice ( Fig.12).

[0173] Fig.12 :HE and PAS staining results of kidney tissues of 3-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400)

[0174] HE and PAS staining of kidney tissue of TBK1-BKO mice at 13-15 months old

[0175] HE and PAS staining of kidney tissue showed mild proliferation of mesangial cells and matrix in the mesangial region of 5-month-old TBK1-BKO mice, no obvious proliferation of mesangial cells and matrix in the mesangial region of WT mice, and proliferation of mesangial cells and matrix in the mesangial region of traditional CCl4 mice ( Fig.13 ).

[0176] Fig.13 :HE and PAS staining results of kidney tissues of 5-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400)

[0177] 14. HE and PAS staining of kidney tissue of 8-month-old TBK1-BKO mice

[0178] HE and PAS staining of kidney tissue showed proliferation of mesangial cells and matrix in the glomerular mesangial region of 8-month-old TBK1-BKO mice and traditional CCl4 mice, but no obvious proliferation of mesangial cells and matrix in the glomerular mesangial region of WT mice ( Fig.14 ).

[0179] Fig.14 :HE and PAS staining results of kidney tissues of 8-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400)

[0180] 15. HE and PAS staining of kidney tissue of 12-month-old TBK1-BKO mice

[0181] HE and PAS staining of kidney tissue showed proliferation of mesangial cells and matrix in the glomerular mesangial region of 12-month-old TBK1-BKO mice and traditional CCl4 mice, but no obvious proliferation of mesangial cells and matrix in the glomerular mesangial region of WT mice ( Fig.14 ).

[0182] Fig.15 :HE and PAS staining results of kidney tissues of 12-month-old TBK1-BKO mice, WT mice and traditional CCl4 mice (×400)

[0183] discuss

[0184] This study chose to observe and verify TBK1-BKO mice (TBK1 conditional defective mice in B cells). This model mouse can be born according to the expected Mendelian ratio and has no difference in growth, development and survival compared with wild-type mice. Previous studies have determined that TBKI gene knockout in mice can lead to increased serum IgA levels, and it is speculated that the TBK1 gene is an important inhibitor of IgA production in mice. In this study, slight IgA deposition can be observed in the mesangial area of ​​some 2-month-old and 3-month-old TBK1-BKO mice, and clumps of IgA and IgG deposition can be seen in the mesangial area of ​​5-month-old, 8-month-old, and 12-month-old mice. Compared with WT mice and traditional CCl4 mice, some glomeruli of TBK1-BKO mice began to show mesangial cell proliferation and increased mesangial matrix at 2 months of age, and the changes in the mesangial area of ​​the glomeruli gradually worsened with age. In this study, renal function damage in mice was later than renal pathological changes. The serum creatinine and urine albumin-creatinine ratio were statistically different from those of WT mice at 8 months of age. In summary, TBK1-BKO mice have no difference in growth, development and survival compared with wild-type mice. The TBK1-BKO mouse model is highly stable and can obtain an animal model with completely consistent genotypes. At the same time, its phenotype is closer to human IgA nephropathy.

[0185] It should be noted that, although the above embodiments have been described in this article, the patent protection scope of the present invention is not limited thereby. Therefore, based on the innovative concept of the present invention, changes and modifications made to the embodiments described herein, or equivalent structures or equivalent process changes made using the contents of the present invention specification and drawings, directly or indirectly applying the above technical solutions to other related technical fields, are all included in the patent protection scope of the present invention.

Claims

1. A method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region, characterized in that: The steps include: Step 1. gRNA design Step 2: Donor vector construction, gel recovery, ligation and transformation, colony screening, positive clone plasmid extraction, enzyme digestion identification and sequencing, and injection plasmid preparation; Step 3: gRNA synthesis Step 4, RNP complex preparation: Step 5: Fertilized egg preparation: Step 6: Pronuclear microinjection: Step 7: Preparation of surrogate mice and embryo transfer: Step 8, identification of F0 generation mice: Step 9: Breeding and establishing transgenic mice.

2. The method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region according to claim 1, characterized in that: In step 2, Donor vector construction: fragment amplification uses P515 high-fidelity enzyme from Novagen, PCR amplification is performed in a volume of 50 μl, and the number of cycles is set to 30 times; gel recovery: the PCR product is analyzed by electrophoresis, and the target fragment is recovered using Qiagen gel recovery kit; ligation and transformation: the recovered fragment is ligated using C115 ligase from Novagen, and the ligation product is introduced into Escherichia coli with the help of Takara Stellar competent cells, and cultured overnight at 37°C; colony screening: 16 well-growing colonies are selected, and PCR amplification is performed in a volume of 25 μl using P222 Taq enzyme from Novagen, and the colonies with the correct inserted fragment are confirmed and inoculated into 4 mL Small-scale fermentation was performed in LB liquid medium; plasmid extraction of positive clones: plasmid DNA was extracted by alkaline lysis method; restriction enzyme identification and sequencing: appropriate NEB restriction endonucleases were selected, and restriction enzyme identification was performed in a 20 μl system to confirm the correct plasmid for sequencing; preparation of plasmid for injection: large-scale fermentation was performed on the clones with correct sequencing, and the plasmid was extracted using Qiagen plasmid extraction kit for injection.

3. The method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region according to claim 1, characterized in that: In step 3, crRNA is synthesized by IDT; tracrRNA is provided by GenScript Biotech Co., Ltd.; crRNA and tracrRNA are combined to form gRNA.

4. The method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region according to claim 1, characterized in that: In step 4, solution in tube 1: dissolve 0.8 μL 100 pmol / μL CrRNA and 0.6 μL 100 pmol / μL tracrRNA in 5.2 μL RNase-free water, mix well and incubate at room temperature for 5 minutes, then add 0.2 μL NEB's Cas9 protein and incubate at room temperature for 10 minutes to obtain solution in tube 1; solution in tube 2: dilute the Donor plasmid to 15 ng / μL; mix the solution in tube 1 with the solution in tube 2 to prepare an RNP injection complex.

5. The method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region according to claim 1, characterized in that: In step 5, 3-4 week old C57BL / 6 female mice were selected and injected with pregnant mare serum and human chorionic gonadotropin, respectively, with an interval of 46-48 hours between the two injections; after HCG injection, the female mice were mated with adult male mice; the next day, the female mice were euthanized, and the fertilized eggs were collected from the oviducts and placed in an incubator at 37°C and 5% CO2 for later use.

6. The method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region according to claim 1, characterized in that: In step 6, prepare a microinjection needle and a fixing needle; mix the RNP complex with the plasmid DNA, dilute and load into the microinjection needle; inject the exogenous gene into the nucleus of the fertilized egg under an inverted microscope (magnification 200-400 times); the injected fertilized egg is transferred to M16 culture medium, and continues to be cultured in an incubator at 37°C and 5% CO2 for 0.5-1 hour, and then transplanted or cultured to the 2-cell stage and then transplanted.

7. The method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region according to claim 1, characterized in that: In step 7, female mice of appropriate age are used to mate with sterilized male mice to create a pseudo-pregnancy state; the fertilized eggs carrying the exogenous gene are transplanted into the fallopian tubes of the pseudo-pregnant female mice; after the transplantation, the female mice are placed in a warm environment until they recover; 19-20 days after the operation, the female mice give birth to pups; the mice are marked and preliminarily identified one week after birth.

8. The method for constructing a mouse model of pathological IgA deposition in the glomerular mesangial region according to claim 1, characterized in that: In step 8, tissue samples from 1-2 week old mice are collected; genomic DNA is extracted and PCR amplified and electrophoresed; offspring with integrated exogenous genes are screened; integrated individuals are confirmed, and subsequent breeding and phenotypic analysis are performed.

9. An animal model prepared by the method according to claims 1-8.

10. Use of the animal model according to claim 9 in screening drugs for IgA nephropathy.

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