Construction method and application of pulmonary fibrosis animal model

Abstract

The invention relates to a construction method and application of a pulmonary fibrosis animal model. The construction method of the pulmonary fibrosis animal model comprises the following steps: (1) constructing a gene editing system for targeted knockout of a mouse Nlrp3 gene; (2) performing gene editing on fertilized eggs of the mice by using the gene editing system in the step (1), and then performing hybridization to construct Flox mice; and (3) carrying out mating propagation on the Flox mouse and a Cdh16-Cre tool mouse, screening an Nlrp3 [flox / flox, Cdh16-Cre] mouse to obtain a mouse of which the kidney cell Nlrp3 gene is specifically knocked out, and culturing to obtain the pulmonary fibrosis animal model. According to the invention, the Nlrp3 gene of mouse kidney cells is specifically knocked out to obtain a pulmonary fibrosis mouse, which has important application value in the aspects of mechanism exploration of pulmonary fibrosis diseases and drug preparation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing an animal model of pulmonary fibrosis and application thereof. Background Art

[0002] Pulmonary fibrosis includes a series of chronic lung diseases that affect the lung interstitium and lead to pulmonary gas exchange disorders, dyspnea and even respiratory failure. The disease is characterized by abnormal scar repair of lung epithelial tissue caused by persistent damage to the alveolar structure. Pulmonary fibrosis is a terminal manifestation of interstitial lung disease, which is characterized by fibroblast proliferation and a large amount of extracellular matrix deposition. The lung interstitial tissue is composed of collagen, elastin and proteoglycans. When fibroblasts are chemically or physically damaged, they will secrete collagen to repair the lung interstitial tissue, resulting in the destruction of lung tissue structure.

[0003] There are many factors that cause pulmonary fibrosis, such as occupational or home environment factors, drugs, radiotherapy, high-concentration oxygen therapy, smoking, disease factors, and genetic factors. The main clinical symptoms of patients with pulmonary fibrosis include dry cough and progressive dyspnea, which are generally obvious after activities. Some patients may show clubbing of fingers / toes. Clinical symptoms generally have an insidious onset and slow progression. Possible complications include pulmonary hypertension, emphysema, lung cancer, and gastroesophageal reflux. Treatments for pulmonary fibrosis include oxygen therapy, the use of glucocorticoids, immunosuppressants and anti-fibrotic drugs, and the prevention of deep vein thrombosis.

[0004] At present, a variety of methods for inducing animal pulmonary fibrosis models have been established, including gene-related models and non-gene-related models. Gene-related models refer to animals that are genetically engineered to become pulmonary fibrosis models, while non-gene-related models refer to pulmonary fibrosis models induced by environmental factors, drug poisons or other factors under the same or similar genetic background. For example, CN105148254A discloses a method for making a rat unilateral pulmonary fibrosis animal model, including the steps of: anesthetizing animals, endotracheal intubation, unilateral pulmonary bleomycin perfusion, postoperative care, and unilateral pulmonary bleomycin perfusion concentrations of 1.5, 3.0, and 5.0 mg / kg, respectively.

[0005] Conditional gene knockout animal models are mainly achieved through the specific recombinase system Cre-LoxP. By crossing transgenic mice with specific flox sites with transgenic mice that specifically express Cre enzymes, mice with gene knockout in specific tissue cells can be obtained. This conditional gene knockout method requires the construction of two transgenic mice (Flox mice and Cre mice) at the same time, and replacing the gene promoter of the Cre enzyme can achieve gene knockout under a variety of conditions (such as drug induction, specific time, specific tissue, etc.).

[0006] In summary, how to establish an animal pulmonary fibrosis model through genetic engineering methods has become one of the urgent problems to be solved in this field. Summary of the invention

[0007] To solve the above technical problems, the present invention provides a method for constructing an animal model of pulmonary fibrosis and its application. By specifically knocking out the Nlrp3 gene in mouse kidney cells, a pulmonary fibrosis mouse is obtained, which has important application value in the exploration of the mechanism of pulmonary fibrosis disease and the preparation of drugs.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for constructing an animal model of pulmonary fibrosis, the method for constructing an animal model of pulmonary fibrosis comprising the following steps:

[0010] (1) Construction of a gene editing system for targeted knockout of the mouse Nlrp3 gene;

[0011] (2) using the gene editing system described in step (1) to perform gene editing on mouse fertilized eggs, and then performing hybridization to construct Flox mice;

[0012] (3) Flox mice were bred with Cdh16-Cre mice, and Nlrp3[flox / flox, Cdh16-Cre] mice were selected to obtain mice with specific knockout of the Nlrp3 gene in kidney cells, and the animal model of pulmonary fibrosis was obtained after culture.

[0013] The target gene of conditional gene knockout must contain a loxP sequence that can be recognized by the Cre recombinase. This gene is called a floxed gene, and mice with a floxed target gene are called flox mice.

[0014] The Cdh16 gene encodes a calcium-dependent membrane-associated glycoprotein that promotes the formation of renal tubules and is specifically and highly expressed in renal tubular epithelial cells in the kidney and developing urogenital tract. Experiments have shown that carrying the Cdh16 gene promoter can reproduce the complete expression pattern of the gene in the developing kidney and urogenital tract, simulating the expression of endogenous genes. The present invention inserts the Cre recombinase gene expression element into the endogenous Cdh16 gene of mice to obtain Cdh16-Cre tool mice, thereby achieving specific knockout of the Nlrp3 gene in mouse kidney cells.

[0015] The Nlrp3 gene is located on chromosome 11 of mice, and its gene ID in CNBI is 216799. The present invention utilizes CRISPR / Cas9 technology, based on the principle of homologous recombination, to perform site-directed modification of the target gene by high-throughput electroporation of fertilized eggs, and obtains Nlrp3 gene conditional knockout mice.

[0016] The specific process includes: selection and design of sgRNA targeting the mouse Nlrp3 gene, construction of sgRNA vector, transcription and purification of sgRNA, construction of donor vector containing loxP site, simultaneous electroporation of Cas9 mRNA, sgRNA and donor vector containing loxP site into mouse fertilized eggs, translation of Cas9 protein by Cas9 mRNA in vivo, binding of Cas9 protein to target site under the guidance of sgRNA to cause DNA double-strand break, repair of broken double-strand using donor vector containing loxP site as template during repair process, thus achieving modification of target gene. After obtaining positive F0 mice, subsequent homozygous breeding was continued, and breeding with Cdh16-Cre mice was finally constructed to obtain Nlrp3[flox / flox, Cdh16-Cre] mice.

[0017] In the Nlrp3[flox / flox, Cdh16-Cre] mouse obtained by the present invention, both Nlrp3 alleles carry loxP sequences that can be recognized by Cre recombinase, and the genotype is flox / flox. At the same time, a Cre recombinase coding sequence is also inserted into the Cdh16 gene of the mouse.

[0018] The method for constructing an animal model of pulmonary fibrosis provided by the present invention solves the problems of high gene off-target rate and low animal survival rate in traditional gene knockout technology, and avoids the problem of gene embryonic lethality. The obtained gene-edited mouse model has important clinical transformation value for the exploration of the mechanism of pulmonary fibrosis disease and the preparation of drugs.

[0019] Preferably, the gene editing system comprises sgRNA targeting the mouse Nlrp3 gene.

[0020] Preferably, the nucleic acid sequence of the sgRNA includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.2.

[0021] SEQ ID NO. 1: TAGGTGGTATGACCGGACAGAGG.

[0022] SEQ ID NO. 2: CGTCTCAGAACGACGGAATCAGG.

[0023] The sgRNA action sites targeting the mouse Nlrp3 gene provided by the present invention are respectively located upstream and downstream of the fourth exon of the Nlrp3 gene, and the sgRNA editing efficiency is high and the off-target rate is low.

[0024] Preferably, the gene editing system further comprises Cas9 mRNA and a donor vector containing a loxP site.

[0025] Preferably, the nucleic acid sequence of the donor vector containing the loxP site includes the sequence shown in SEQ ID NO.3.

[0026] SEQ ID NO. 3: ATAACTTCGTATAGCATACATTATACGAAGTTAT.

[0027] The donor vector containing loxP sites provided by the present invention comprises left and right homology arms with a length of about 1.5 kbp, two loxP elements in the same direction and a protective base with a length of about 30 bp outside the loxP element.

[0028] Preferably, the culturing method in step (3) comprises: feeding Nlrp3[flox / flox, Cdh16-Cre] mice with a mouse feed containing 0.2% to 0.3% (for example, 0.2%, 0.22%, 0.24%, 0.25%, 0.26%, 0.28% or 0.3%) adenine by mass.

[0029] Preferably, the culturing method in step (3) specifically comprises: taking 18 to 22 weeks old (for example, it can be 18, 19, 20, 21 or 22, etc.) Nlrp3[flox / flox, Cdh16-Cre] mice, feeding them with a mouse feed containing adenine by mass fraction of 0.2% to 0.3% (for example, it can be 0.2%, 0.22%, 0.24%, 0.25%, 0.26%, 0.28% or 0.3%, etc.), and feeding them to 25 to 29 weeks of age (for example, it can be 25, 26, 27, 28 or 29, etc.), to obtain the pulmonary fibrosis animal model.

[0030] Preferably, the method for constructing the animal model of pulmonary fibrosis comprises the following steps:

[0031] (a) sgRNA, Cas9 mRNA and donor vector containing loxP sites targeting the mouse Nlrp3 gene were prepared respectively;

[0032] (b) sgRNA, Cas9 mRNA and a donor vector containing loxP sites were simultaneously electroporated into mouse fertilized eggs, and gene integration was performed to obtain gene-recombined fertilized eggs with a loxP element fragment inserted into the Nlrp3 gene. The fertilized eggs that survived the electroporation were transplanted into the uterus of pseudo-pregnant female mice to obtain F0 generation mice;

[0033] (c) breeding the positive mice in the F0 generation with wild-type mice to obtain F1 generation mice, and hybridizing the heterozygous F1 generation mice to obtain homozygous Flox mice and heterozygous Flox mice of the F2 generation;

[0034] (d) F2 generation homozygous Flox mice or F2 generation heterozygous Flox mice were bred with Cdh16-Cre mice to obtain F3 generation mice;

[0035] (e) F3 mice were backcrossed with F2 homozygous Flox mice, and Nlrp3[flox / flox, Cdh16-Cre] mice were selected to obtain mice with specific knockout of the Nlrp3 gene in kidney cells;

[0036] (f) 18-22 week old Nlrp3[flox / flox, Cdh16-Cre] mice were fed with a mouse feed containing 0.2% to 0.3% adenine by mass until they were 25 to 29 weeks old, thereby obtaining the pulmonary fibrosis animal model.

[0037] In a second aspect, the present invention provides a kit for constructing an animal model of pulmonary fibrosis, the kit comprising sgRNA targeting the mouse Nlrp3 gene, Cas9 mRNA, and a donor vector containing a loxP site.

[0038] Preferably, the nucleic acid sequence of the sgRNA includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.2.

[0039] Preferably, the kit further comprises a mouse feed containing 0.2% to 0.3% (eg, 0.2%, 0.22%, 0.24%, 0.25%, 0.26%, 0.28% or 0.3%) adenine by mass.

[0040] Preferably, the kit also includes primers for PCR identification of the genotype of offspring mice.

[0041] Preferably, the nucleic acid sequence of the primer includes the sequence shown in SEQ ID NO.4 to SEQ ID NO.7.

[0042] SEQ ID NO. 4: TTGTGGAGGATGGGAAGTCTAAAG.

[0043] SEQ ID NO. 5: CTCAGATAGACACCATCGTCTCAG.

[0044] SEQ ID NO. 6: GCAGATCTGGCTCTCCAAAG.

[0045] SEQ ID NO. 7: AGGCAAATTTTGGTGTACGG.

[0046] In a third aspect, the present invention provides a screening device for therapeutic and / or preventive drugs for pulmonary fibrosis diseases, wherein the screening device uses a pulmonary fibrosis animal model for drug screening, and the pulmonary fibrosis animal model is constructed by the method for constructing a pulmonary fibrosis animal model described in the first aspect and / or the kit for constructing a pulmonary fibrosis animal model described in the second aspect.

[0047] In a fourth aspect, the present invention provides the use of the method for constructing an animal model of pulmonary fibrosis as described in the first aspect, the kit for constructing an animal model of pulmonary fibrosis as described in the second aspect, or the screening device for treating and / or preventing drugs for pulmonary fibrosis as described in the third aspect in the preparation of drugs for treating pulmonary fibrosis.

[0048] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The present invention provides a method for constructing an animal model of pulmonary fibrosis. The Nlrp3 gene of mouse kidney cells is specifically knocked out, and the mice are further fed with a feed containing 0.2% to 0.3% adenine by mass fraction until the mice are 25 to 29 weeks old, so as to obtain a mouse with pulmonary fibrosis. The successful establishment of the model is confirmed by Masson's pathological staining, and the method has important application value in the exploration of the mechanism of pulmonary fibrosis disease and the preparation of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the result of PCR identification of loxP sites.

[0052] Figure 2 This is the result of PCR identification of the gene encoding Cre enzyme.

[0053] Figure 3 Figure 2 shows the results of immunohistochemical identification of lung tissue in the model group and the normal control group.

[0054] Figure 4 The results of Masson's staining of lung tissue in the model group and the normal control group. DETAILED DESCRIPTION

[0055] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0056] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0057] The present invention can use the method provided in the examples to prepare mice with specific knockout of the Nlrp3 gene in kidney cells, or commercial mice can be used, such as Cyagen's Nlrp3[flox / flox, Cdh16-Cre] mice, the strain name of which is C57BL / 6NCya-Nlrp3 em1flox / Cya, the strain number is CKOCMP-216799-Nlrp3-B6N-VA, the strain background is C57BL / 6Ncya, and the modification method is conditional gene knockout.

[0058] Example 1

[0059] This embodiment provides a mouse with a specific knockout of the Nlrp3 gene in kidney cells. The mouse with a specific knockout of the Nlrp3 gene in kidney cells is purchased from Cyagen. The preparation process includes the following steps:

[0060] (1) sgRNA, Cas9 mRNA and a donor vector containing a loxP site targeting the mouse Nlrp3 gene were prepared respectively, the nucleic acid sequence of the sgRNA was shown in SEQ ID NO.1 to SEQ ID NO.2, and the nucleic acid sequence of the loxP site was shown in SEQ ID NO.3.

[0061] SEQ ID NO. 1: TAGGTGGTATGACCGGACAGAGG.

[0062] SEQ ID NO. 2: CGTCTCAGAACGACGGAATCAGG.

[0063] SEQ ID NO. 3: ATAACTTCGTATAGCATACATTATACGAAGTTAT.

[0064] (2) The sgRNA, Cas9 mRNA and a donor vector containing loxP sites were simultaneously electroporated into mouse fertilized eggs. After gene integration, genetically recombinant fertilized eggs with a loxP element fragment inserted into the Nlrp3 gene were obtained. The fertilized eggs that survived the electroporation were transplanted into the uterus of pseudopregnant female mice to obtain F0 generation mice.

[0065] (3) The positive mice in the F0 generation are bred with wild-type mice to obtain F1 generation mice, and the heterozygous F1 generation mice are hybridized to obtain F2 generation homozygous Flox mice and F2 generation heterozygous Flox mice.

[0066] (4) F2 homozygous Flox mice or F2 heterozygous Flox mice were bred with Cdh16-Cre mice to obtain F3 mice.

[0067] (5) F3 mice were backcrossed with F2 homozygous Flox mice, and Nlrp3[flox / flox, Cdh16-Cre] mice were selected to obtain mice with specific knockout of the Nlrp3 gene in kidney cells.

[0068] Example 2

[0069] This embodiment provides a method for constructing an animal model of pulmonary fibrosis, comprising the following steps:

[0070] Mice with specific knockout of the Nlrp3 gene in kidney cells were cultured to 18-22 weeks of age, and were allowed to eat and drink freely in an SPF environment. The room temperature was controlled at (21±2)°C, and the relative humidity was controlled at (50±15)%. They were fed with mouse feed containing 0.25% adenine by mass for 7 weeks until the mice were 25-29 weeks of age, thereby obtaining the pulmonary fibrosis animal model.

[0071] Example 3

[0072] This embodiment provides a kit for constructing an animal model of pulmonary fibrosis, the kit comprising sgRNA targeting the mouse Nlrp3 gene, Cas9 mRNA and a donor vector containing a loxP site; the nucleic acid sequence of the sgRNA comprises the sequences shown in SEQ ID NO.1 to SEQ ID NO.2; the kit also comprises a mouse feed containing a mass fraction of 0.2% to 0.3% adenine and primers for PCR identification of the genotype of offspring mice.

[0073] Test Example 1

[0074] In this test example, PCR identification and screening were performed on F4 mice of the F3 generation mice and the F2 generation homozygous Flox mice in Example 1. The PCR primers were shown in SEQ ID NO.4 to SEQ ID NO.5. The length of the amplified fragment with loxP site inserted was 251 bp, and the length of the amplified fragment without loxP site inserted was 191 bp. Figure 1 As shown, homozygous Flox mice have only one 251bp band, heterozygous Flox mice have two bands of 251bp and 191bp, and mice that have not undergone gene editing have only one 191bp band. Then, PCR identification of the Cre enzyme encoding gene was performed, and the PCR primers are shown in SEQ ID NO.6 to SEQ ID NO.7. Figure 2 As shown, the size of Cre amplicon is about 420 bp. Homozygous Flox and Cre enzyme encoding gene were selected, namely Nlrp3[flox / flox, Cdh16-Cre] mice provided by the present invention.

[0075] SEQ ID NO. 4: TTGTGGAGGATGGGAAGTCTAAAG.

[0076] SEQ ID NO. 5: CTCAGATAGACACCATCGTCTCAG.

[0077] SEQ ID NO. 6: GCAGATCTGGCTCTCCAAAG.

[0078] SEQ ID NO. 7: AGGCAAATTTTGGTGTACGG.

[0079] Test Example 2

[0080] This test example uses the qRT-PCR method to identify the expression of the Nlrp3 gene in various tissues of the pulmonary fibrosis model mice prepared in Example 2, and extracts the total RNA of the heart, liver, spleen, lung, and kidney of the pulmonary fibrosis mice and ordinary C57BL / 6 mice, respectively, and identifies the expression of the Nlrp3 gene in different tissues by qRT-PCR. The expression of the Nlrp3 gene in the kidney of the model group was significantly lower than that of the normal control group, while in other tissues, there was no significant difference in the expression of the Nlrp3 gene between the model group and the normal control group, indicating that the present invention has obtained mice with specific knockout of the Nlrp3 gene in kidney cells.

[0081] Test Example 3

[0082] This test example uses immunohistochemistry (IHC) to identify the expression of α-SMA gene in the lungs of the pulmonary fibrosis model mice prepared in Example 2. In the lesions of pulmonary fibrosis, myofibroblasts are the main component, of which α-SMA is a marker protein in the formation process of myofibroblasts. Myofibroblasts have the ability to actively secrete collagen, so detecting the expression of α-SMA protein can reveal whether the mouse has pulmonary fibrosis.

[0083] Total RNA was extracted from the model group mice and normal C57BL / 6 mice, and the expression of α-SMA protein was identified by IHC. The results are shown in Figure 3 As shown, the expression of α-SMA protein in the model group was significantly upregulated, the proliferation of myofibroblasts in the model group was activated, a large amount of collagen was accumulated, and pulmonary fibrosis occurred.

[0084] Test Example 4

[0085] In this test example, the pulmonary fibrosis model mice prepared in Example 2 were taken, lung tissues were embedded, sliced ​​and stained with Masson's pathology, and ordinary C57BL / 6 mice were used as the normal control group. The staining steps were as follows:

[0086] 1. Dewax the paraffin sections to water: Dewax the paraffin sections in xylene, treat them with different concentrations of alcohol gradient (100%, 95%, 70%), and finally rinse them with distilled water for hydration.

[0087] 2. Nuclear staining: Use hematoxylin to stain the cell nucleus for 5-10 minutes, and then treat with tap water or bluing solution (to make the staining stable and color developing).

[0088] 3. Collagen and cytoplasm staining: Use acid fuchsin to stain collagen fibers and cytoplasm for 5-10 minutes. Treat the sample with phosphotungstic acid / phosphomolybdic acid for 5-10 minutes to help differentiation and increase the contrast of collagen fiber staining. Wash with distilled water.

[0089] 4. Staining and differentiation: Use aniline blue or brilliant green to stain collagen fibers or muscle fibers for 5-10 minutes. Wash with distilled water and pay attention to the differentiation effect.

[0090] 5. Dehydration, transparency and sealing: Dehydrate with different concentrations of alcohol (70%, 95%, 100%). Transparent with xylene several times. Finally, seal with neutral gum or sealing agent and observe under a microscope.

[0091] The staining results are as follows Figure 4 As shown, muscle fibers are red and collagen fibers are blue. Masson staining of normal C57BL / 6 mouse lung tissue showed no obvious fibrosis changes, while the lung tissue of Nlrp3 conditional knockout model mice was darkly stained with obvious fibrosis changes, showing pulmonary fibrosis.

[0092] In summary, the present invention provides a method for constructing an animal model of pulmonary fibrosis, by specifically knocking out the Nlrp3 gene of mouse kidney cells, and further feeding the mice with a feed containing 0.2% to 0.3% adenine by mass fraction until the mice are 25 to 29 weeks of age, a pulmonary fibrosis mouse is obtained, and the successful modeling is confirmed by Masson pathological staining, which has important application value in the exploration of the mechanism of pulmonary fibrosis disease and the preparation of drugs.

[0093] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for constructing an animal model of pulmonary fibrosis, characterized in that: The method for constructing the pulmonary fibrosis animal model comprises the following steps: (1) Construction of a gene editing system for targeted knockout of the mouse Nlrp3 gene; (2) using the gene editing system described in step (1) to perform gene editing on mouse fertilized eggs, and then performing hybridization to construct Flox mice; (3) Flox mice were bred with Cdh16-Cre mice, and Nlrp3[flox / flox, Cdh16-Cre] mice were selected to obtain mice with specific knockout of the Nlrp3 gene in kidney cells, and the animal model of pulmonary fibrosis was obtained after culture.

2. The method for constructing an animal model of pulmonary fibrosis according to claim 1, characterized in that: The gene editing system includes an sgRNA targeting the mouse Nlrp3 gene; Preferably, the nucleic acid sequence of the sgRNA includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.

2.

3. The method for constructing an animal model of pulmonary fibrosis according to claim 1 or 2, characterized in that: The gene editing system also includes Cas9 mRNA and a donor vector containing loxP sites; Preferably, the nucleic acid sequence of the donor vector containing the loxP site includes the sequence shown in SEQ ID NO.

3.

4. The method for constructing an animal model of pulmonary fibrosis according to any one of claims 1 to 3, characterized in that: The culturing method in step (3) comprises: feeding Nlrp3[flox / flox, Cdh16-Cre] mice with mouse feed containing 0.2% to 0.3% adenine by mass.

5. The method for constructing an animal model of pulmonary fibrosis according to any one of claims 1 to 4, characterized in that: The culture method of step (3) specifically includes: taking 18-22 week old Nlrp3[flox / flox, Cdh16-Cre] mice, feeding them with mouse feed containing 0.2% to 0.3% adenine by mass fraction, and feeding them until they are 25 to 29 weeks old to obtain the pulmonary fibrosis animal model.

6. The method for constructing an animal model of pulmonary fibrosis according to any one of claims 1 to 5, characterized in that: The method for constructing the pulmonary fibrosis animal model comprises the following steps: (a) sgRNA, Cas9 mRNA and donor vector containing loxP sites targeting the mouse Nlrp3 gene were prepared respectively; (b) sgRNA, Cas9 mRNA and a donor vector containing loxP sites were simultaneously electroporated into mouse fertilized eggs, and gene integration was performed to obtain gene-recombined fertilized eggs with a loxP element fragment inserted into the Nlrp3 gene. The fertilized eggs that survived the electroporation were transplanted into the uterus of pseudo-pregnant female mice to obtain F0 generation mice; (c) breeding the positive mice in the F0 generation with wild-type mice to obtain F1 generation mice, and hybridizing the heterozygous F1 generation mice to obtain homozygous Flox mice and heterozygous Flox mice of the F2 generation; (d) F2 generation homozygous Flox mice or F2 generation heterozygous Flox mice were bred with Cdh16-Cre mice to obtain F3 generation mice; (e) F3 mice were backcrossed with F2 homozygous Flox mice, and Nlrp3[flox / flox, Cdh16-Cre] mice were selected to obtain mice with specific knockout of the Nlrp3 gene in kidney cells; (f) 18-22 week old Nlrp3[flox / flox, Cdh16-Cre] mice were fed with a mouse feed containing 0.2% to 0.3% adenine by mass until they were 25 to 29 weeks old, thereby obtaining the pulmonary fibrosis animal model.

7. A kit for constructing an animal model of pulmonary fibrosis, characterized in that: The kit includes sgRNA targeting the mouse Nlrp3 gene, Cas9 mRNA and a donor vector containing a loxP site.

8. The kit according to claim 7, characterized in that The nucleic acid sequence of the sgRNA includes the sequences shown in SEQ ID NO.1 to SEQ ID NO.2; Preferably, the kit further comprises a mouse feed containing 0.2% to 0.3% adenine by mass; Preferably, the kit further comprises primers for PCR identification of the genotype of offspring mice; Preferably, the nucleic acid sequence of the primer includes the sequence shown in SEQ ID NO.4 to SEQ ID NO.

7.

9. A screening device for drugs for treating and / or preventing pulmonary fibrosis, characterized in that: The screening device uses a pulmonary fibrosis animal model for drug screening, and the pulmonary fibrosis animal model is constructed by the method for constructing a pulmonary fibrosis animal model according to any one of claims 1 to 6 and / or the kit for constructing a pulmonary fibrosis animal model according to claim 7 or 8.

10. Use of the method for constructing an animal model of pulmonary fibrosis according to any one of claims 1 to 6, the kit for constructing an animal model of pulmonary fibrosis according to claim 7 or 8, or the device for screening drugs for treating and / or preventing pulmonary fibrosis according to claim 9 in the preparation of drugs for treating pulmonary fibrosis.

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