CN116376976A - Construction method and application of humanized IL32 gamma conditional knock-in mouse model - Google Patents

Abstract

The invention discloses a construction method and application of a human IL32 gamma conditional knock-in mouse model. The method comprises the steps of specifically introducing IL32 gamma protein gene of human interleukin 32 spliceosome into a receptor mouse to obtain a transgenic mouse, hybridizing the transgenic mouse with a Cre enzyme transgenic mouse to obtain a mouse model, wherein the mouse model is in CD4 ⁺ IL32 gamma protein is specifically expressed in T cells. The amino acid sequence of the IL32 gamma protein is a sequence 1 in a sequence table. Experiments prove that the inventionMouse model constructed in the Ming method in CD4 ⁺ The successfully knocked-in human IL32 gamma gene in the T cells expresses IL32 gamma protein, so that the Th1 cells are expressed in a large quantity, and the ratio of the Th1 cells in the thoracic cavity flushing fluid of the mouse model is obviously higher than that of a control mouse. The construction method and the mouse model constructed by the method can be applied to clinical study of pathogenesis of pleurisy and clinical guidance medication.

Description

Construction method and application of human IL32γ conditional knock-in mouse model

technical field

The invention relates to the field of medical biotechnology, in particular to a method for constructing a human IL32γ conditional knock-in mouse model and its application.

Background technique

Interleukin-32 (interleukin-32, IL-32) is a pro-inflammatory cytokine with multiple subtypes, which is not homologous to the known IL family. IL-32 is widely expressed in various cells, especially T cells and NK cells, and no clear IL-32 receptor has been found. A total of 9 secreted IL-32 types were found, among which IL-32α, IL-32β, IL-32γ and IL-32δ were the most abundant. Compared with other subtypes, IL-32γ has the strongest activity, however, the function of IL-32γ is not fully understood. The expression level of IL-32 is different in the same kind of cells in different microenvironments, and the expression level of IL-32 is also different in different cells in the same kind of microenvironment. Therefore, it is important to study the function of specific cells expressing IL-32 in diseases. It is of great significance to understand the role of IL-32 in the occurrence and development of diseases.

IL-32 is not expressed in all animals. It is known that IL-32 is expressed in humans, but there is no expression of IL-32 in model animals such as mice and rats, which brings certain difficulties to disease research.

Tuberculosis (TB) is a global public health problem that seriously endangers human health. According to the "Global Tuberculosis Report 2021" released by the World Health Organization, there will be nearly 2 billion Mycobacterium tuberculosis latent infections and 9.87 million new cases of TB in the world in 2020. TB prevention and control will still face severe challenges for a long time in the future. Previous studies have found that the levels of IL-32 expressed by T cells, B cells, and macrophages in tuberculous pleurisy are very different, and the expression level is the highest in CD4 ⁺ T cells. It is of great significance to elucidate the mechanism of action of IL-32 ⁺ CD4 ⁺ T cells on TB immune regulation.

Based on the above analysis, it is necessary to construct a tool mouse that expresses IL-32 in CD4 ⁺ T cells, which is of great significance for elucidating the mechanism of IL-32 regulating the immune microenvironment of TB and developing new diagnostic and intervention methods.

Human interleukin-32 (interleukin-32, IL-32) splice body IL32γ (NCBI reference sequence: NM_001308078.4) is located on human chromosome 16.

Contents of the invention

The technical problem to be solved by the present invention is how to construct a mouse model specifically expressing human interleukin-32 splice body IL32γ in CD4 ⁺ T cells.

In order to solve the above-mentioned technical problems, the present invention first constructs a mouse model that specifically expresses the splice body IL32γ protein of human interleukin-32 in immune cells.

The method includes specifically introducing the splice body IL32γ protein gene of human interleukin 32 into immune cells of recipient mice to obtain transgenic mice, and crossing the transgenic mice with Cre enzyme transgenic mice to obtain the small mouse model. The Cre enzyme transgenic mouse can specifically express Cre enzyme in immune cells. The mouse model can specifically express the IL32γ protein in the immune cells.

In the above method, the IL32γ protein can be selected from any of the following:

A1) The amino acid sequence is sequence 1 in the sequence listing;

A2) A protein having more than 80% identity with the protein shown in A1) obtained by substituting and/or deleting and/or adding amino acid residues to the protein shown in A1) and having glutamic acid decarboxylase activity;

A3) A fusion protein obtained by linking protein tags at the N-terminus or/and C-terminus of A1) or A2).

In the above method, the introduction may be specific knock-in.

The coding sequence of the IL32γ protein can be the 3632-4333 position of the sequence 2 in the sequence listing.

In the above method, the specific knock-in may be knocking the interleukin-32 splice body IL32γ protein conditional overexpression cassette into the intron of the ROSA26 gene of the recipient mouse.

The ROSA26 gene is the complementary sequence of the 113044389-113054205th nucleotide of the mouse genome GenBank Accession No.NC_000072.7 (UpdateDate 18-Oct-2022), and the 113054205th nucleotide of the above-mentioned genome is recorded as the ROSA26 gene The first nucleotide, wherein, the 1210th-1211th nucleotide is the insertion site of the interleukin 32 splice body IL32γ expression frame, and the site is located on the intron of the ROSA26 gene.

In the above method, the specific knock-in may include the step of using the CRISPR/Cas9 system to knock the gene into the intron of the ROSA26 gene of the recipient mouse.

In the above method, the knock-in may include introducing a nucleic acid molecule expressing a gRNA targeting the intron of the ROSA26 gene, a Cas9 protein, and a nucleic acid molecule encoding a gene of the human interleukin 32 splice body IL32γ protein The steps of obtaining transgenic mice from the recipient mice.

The nucleic acid molecule may be a conditional overexpression cassette comprising human Il32γ.

In the above method, the target sequence of the gRNA may correspond to the complementary sequence of nucleotides 113,052,985-113,053,007 of GenBank Accession No.NC_000072.7 (Update Date 18-Oct-2022) of the mouse genome.

The immune cells mentioned above may be CD4 ⁺ T lymphocytes.

In the above method, the method may include the following steps:

Introduce the nucleic acid molecule of the gRNA targeting the intron of the ROSA26 gene of the recipient mouse, the Cas9 protein and the nucleic acid molecule of the mutant coding gene of the human interleukin 32 splice body IL32γ protein into the recipient mouse The F ₀ generation transgenic mice were obtained.

F ₀ generation transgenic mice were crossed with wild-type recipient mice to obtain F ₁ generation heterozygous mice, compared with the recipient mice, the F ₁ generation heterozygous knock-in ROSA26 gene on one chromosome of the mice The complementary sequence between nucleotides 113,052,996-113,052,997 of GenBank Accession No.NC_000072.7 was inserted into the human IL32γ conditional expression cassette shown in sequence 2 in the sequence listing;

The F ₁ generation heterozygous knock-in mice were crossed with CD4-Cre mice to obtain double-gene heterozygous mice targeted to knock-in human interleukin 32 gene and Cre recombinase to obtain the mouse model .

The Cre transgenic mouse may contain a Cre recombinase expression cassette, and may specifically express the Cre recombinase in CD4 ⁺ T lymphocytes.

The Cre enzyme transgenic mice can be CD4-Cre mice donated by Saiye (Suzhou) Biotechnology Co., Ltd. The T lymphocytes can be CD4 ⁺ T cells.

Any of the following applications also belongs to the protection scope of the present invention:

B1) Application of the method described above and/or the mouse model described above in the preparation, development or research of human interleukin-32 to regulate TB immune microenvironment-related drugs;

B2) Application of the method described above and/or the mouse model described above in the preparation of drugs for clinical experiments related to human interleukin-32-related diseases.

Description of drawings

Figure 1 is a schematic illustration of the construction strategy of the human interleukin 32 splice body IL32γ conditional knock-in mouse model. Wildtype allele represents the wild type ROSA26 site, Targeting vector represents the targeting vector containing the IL32γ conditional expression cassette, Mutant allele 1 (Targeted allele) represents the ROSA26 site successfully inserted into the IL32γ conditional expression cassette, Mutant allele 2 (After Cre recombination) The ROSA26 site of overexpressed IL32γ after CD4-Cre recombination. Exon represents the exon of the ROSA26 gene, Homology arm represents the homology arm for site-directed integration, CAG promoter represents the strong promoter for widespread expression of CAG throughout the body, loxP site represents the Cre recombinase recognition site loxP, 6*SV40 pA represents the six Strong termination element composed of SV40 late polyadenylation signal, Kozak is an element that enhances human IL32γ protein expression, human IL32γ CDS represents the CDS region of human interleukin-32 spliced IL32γ, rBG pA represents rabbit β-globin polyadenylation signal .

Fig. 2 is a schematic diagram of the primer scheme for PCR identification of genotypes of F ₁ generation mice in the embodiment of the present invention. Mutant allele 1 (Targeted allele) represents the ROSA26 site where the IL32γ conditional expression cassette was successfully inserted.

Fig. 3 is the genotype identification result of the F1 _generation mouse in the embodiment of the present invention. A is the agarose gel electrophoresis picture of F1/R1 primers, B is the agarose gel electrophoresis picture of F2/R2 primers. Lanes 17, 24, and 27 represent the detection results of the positive mice numbered 17, 24, and 27 in F1 _generation mice, M represents Marker, Water represents the water control, and WT represents wild-type mice.

Fig. 4 is a schematic diagram of enzyme digestion, 5'probe and 3'probe design for Southern blot identification of F1 _generation mice in the embodiment of the present invention.

Fig. 5 is the Southern hybridization detection result of the genotype of the F1 _generation mouse in the embodiment of the present invention. A is the Southern hybridization detection result of sample No. 17 of F ₁ generation using restriction endonuclease Bsu36I and 5'arm probe. WT represents the lane of wild-type mice, and a ~4.48kb band was detected, and 17 represents F ₁ The lanes of mouse No. 17 in generation 1, the bands of ~4.48kb and 7.81kb were detected; B is the sample of No. 17 in generation _F1 , the detection result of Southern hybridization using restriction endonuclease BstEII and 3'arm probe, WT represents the lane of wild-type mouse, with ~4.77kb bands detected; 17 represents the lane of _F1 generation mouse No. 17, with bands of ~4.77kb and 9.66kb detected; C is _F1 generation No. 24 and No. 27 Samples, using restriction endonuclease Bsu36I digestion and Southern hybridization detection results of 5'arm probes, WT represents the lane of wild-type mice, a ~4.48kb band was detected, 24 and 27 represent the F ₁ generation24, 27 The lanes of mouse No. 4.48kb and 7.81kb were detected; D is the results of No. 24 and No. 27 samples of _F1 generation, using restriction endonuclease BstEII digestion and 3'arm probe Southern hybridization detection results, WT represents the lanes of wild-type mice, and a ~4.77kb band was detected; 24 and 27 represent the lanes of _F1 generation 24 and 27 mice, and the bands of ~4.77kb and 9.66kb were detected; the above results indicate that human The source IL32γ was successfully inserted into the ROSA26 safety site.

Fig. 6 is the genotype identification scheme of F2 _generation mice in the embodiment of the present invention. Wildtype allele represents wild-type mice, and Mutant allele 2 (After Cre recombination) represents the finally obtained mutant human IL32γCD4+ T cell-specific knock-in mice. Exon stands for ROSA26 gene exon, rBG pA stands for rabbit β-globin polyadenylation signal, loxP site stands for loxP site sequence, CAG promoter stands for CAG promoter, Kozak-Human IL32 CDS stands for alternative splicing of human interleukin-32 The CDS region of the body IL32γ protein, Homology arm represents the homology arm. F3/R3 was used to detect the WT site, and F5/R6 was used to detect the overexpressed IL32γ site after Cre recombinase action.

Fig. 7 is the PCR identification scheme of the F2 _generation mouse in the embodiment of the present invention. A is the identification result (345bp) of PCR amplification of F3 ^/ R3 using primers for IL32γ flox/- CD4 ^Cre mice; B is PCR for IL32γ ^flox/- CD4 ^Cre mice using primers for Cre-F/Cre-R The identification result of amplification (336bp); C is the identification result (323bp) of PCR amplification of IL32γ ^flox/- CD4 ^Cre mouse using primer pair F5/R6.

Figure 8 shows the detection results of IL32γ protein expression in CD4 ⁺ T or CD4 ⁻ T cells of KI (IL32γ ^flox/- CD4 ^Cre ) mice and IL32γ ^flox/- mice. The ordinate is the concentration of IL32γ protein. A represents the detection results of CD4 ⁺ T cells in KI mice, B represents the detection results of CD4 ^- T cells in KI mice, and C represents the detection results of CD4 ⁺ T cells in IL32γ ^flox/- mice.

Figure 9 is the detection of the proportion of Th1 cells in CD4 ⁺ T cells in pleurisy mice constructed by using KI (IL32γ ^flox/- CD4 ^Cre ) mice and IL32γ ^flox/- mice respectively. The vertical axis is the percentage of Th1 cells in CD4 ⁺ T cells. *** represents P<0.001.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1. Construction of human interleukin 32 splice body IL32γ protein conditional knock-in mouse model

Mouse ROSA26 gene (Gene ID: 14910) is located on mouse chromosome 6, and its genome sequence is the complementary sequence of nucleotides 113044389-113054205 of GenBank Accession No.NC_000072.7 (Update Date 18-Oct-2022) , a total of 9817 nucleotides. Nucleotides at position 1033-1034 of intron 1 of the ROSA26 gene (complementary sequence between nucleotide positions 113,052,996-113,052,997 of mouse genome GenBank Accession No.NC_000072.7 (Update Date18-Oct-2022)) The conditional expression cassette of human IL32γ was knocked in.

1. Construction of RNP injection complex targeting mouse ROSA26 gene

1.1 gRNA target sequence design

The gRNA target sequence designed according to the intron sequence of the mouse ROSA26 gene is as follows:

5'-CTCCAGTCTTTCTAGAAGAT-3'

The target sequence of the gRNA may correspond to the complementary sequence of nucleotides 113,052,985-113,053,007 of the mouse genome GenBank Accession No.NC_000072.7 (UpdateDate 18-Oct-2022).

1.2 Synthesis of CrRNA and tracrRNA

Artificially synthesized CrRNA (CRISPR RNA) sequence (IDT Company) and tracrRNA (trans-activatingcrRNA) sequence (Gentry Biotechnology). The combination of CrRNA and tracrRNA will form a gRNA sequence.

CrRNA:

5'-CUCCAGUCUUUCUAGAAGAUGUUUUAGAGCUAUGCUGUUUUG-3';

tracrRNA:

5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3'.

1.3 Preparation of exogenous donor vector

Artificially synthesized exogenous donor vector Donor plasmid, Donor plasmid is a human IL32 gamma conditional expression cassette (named CAG Promoter-loxP-6xSV40 pA-loxP-Kozak-Human IL32 CDS-rBG pA) (sequence 2 in the sequence listing). Including the CAG promoter sequence (the 1st-1726th nucleotide of sequence 2 in the sequence listing), the loxP site sequence (the 1728th-1761st nucleotide and the 3565-3598th nucleotide of the sequence 2 in the sequence listing acid), six SV40 virus late polyA sequences (the 1762-3564th nucleotide of sequence 2 in the sequence listing) and the CDS nucleotide sequence of human IL32γ (the 3632-4336th nucleotide of the sequence 2 in the sequence listing ). The amino acid sequence of human IL32γ is shown in sequence 1 in the sequence listing.

Sequence 1 (5'-3'):

MCFPKVLSDDMKKLKARMIMLLPTSAQGLGAWVSACDTEDTVGHLGPWRDKDPALWCQLCLSSQHQAIERFYDKMQNAESGRGQVMSSLAELEDDFKEGYLETVAAYYEEQHPELTPLLEKERDGLRCRGNRSPVPDVEDPATEEPGESFCDKVMRWFQAMLQRLQTWWHGVLAWV KEKVVALVHAVQALWKQFQSFCCSLSELFMSSFQSYGAPRGDKEELTPQKCSEPQSSK.

Sequence 2 (5'-3'):

attgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccatgacgtcaatgggtggagtatttacggta aactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagccccacgttctgcttcactctccccatctccccccctcc ccacccccaattttgtattttttttttaattattttgtgcagcgatgggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcgg cggcggccctataaaaagcgaagcgcgcggcgggcgggagtcgctgcgcgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttactcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagcgcttggttaatg acggcttgtttcttttctgtggctgcgtgaaagccttgaggggctccgggagggccctttgtgcggggggagcggctcggggggtgcgtgcgtgtgtgtgtgcgtggggagcgccgcgtgcggctccgcgctgcccggcggctgtgagcgctgcgggcgcggcgcggggct ttgtgcgctccgcagtgtgcgcgaggggagcgcggccggggggcggtgccccgcggtgcggggggggggctgcgggggaacaaaggctgcgtgcggggtgtgtgcgtgggggggtgagcaggggggtgtgggcgtcggtcggggctgcaaccccccctgcaccccccctccccgagt tgctgagcacggcccggcttcgggtgcggggctccgtacggggcgtggcgcggggctcgccgtgccgggcggggggtggcggcaggtgggggtgccgggcggggcggggccgcctcgggccggggagggctcgggggaggggcgcggcggcccccggagcgccggcggctgtcgaggcgcggcgagccg cagccattgccttttatggtaatcgtgcgagagggcgcagggacttcctttgtcccaaatctgtgcggagccgaaatctgggaggcgccgccgcaccccctctagcgggcgcggggcgaagcggtgcggcgccggcaggaaggaaatgggcggggagggccttcgtgcgtcgccgcgccgccgt ccccttctccctctccagcctcggggctgtccgcggggggacggctgccttcggggggacggggcagggcggggttcggcttctggcgtgaccggcggctctagagcctctgctaaccatgttcatgccttcttctttttcctacagctcctgggcaacgtgctggttattgtgctgtctcatcattttggca aagaattcataacttcgtatagcatacattatacgaagttattcttctgatattgacttgcggcgcctccgcgatcgcgatcagcttgatggggatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaatgctttattgtgaaatttgtgatgctattgctttattt gtaaccattataagctgcaataaacaagttaacaacaacaattgcattcatttttgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatcctctagagtcgcagatccagacatgataagatcattgatgagtttggacaaaccacaactagaatg cagtgaaaaaaatgctttattgtgaaatttgtgatgctattgctttattgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatccctagag tcgcagatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttattgtgaaatttgtgatgctattgctttattgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttgtttcaggttcagggggaggtgtgggaggtt ttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatcctctagagtcgcagatcctctagagtcgcagatctgcaagctaattcctgcaggtcgaggggatatcgccacctgagttactgggagatgcgctgatgttgaggccccttgggttctcgagctggatatgctctagaaaggcaggcc ctgagcagcttagcaaatgatgcatgatcagcttgatggggatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttattgtgaaatttgtgatgctattgctttattgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttg tttcaggttcagggggaggtgtgggaggtttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatcctctagagtcgcagatccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaatgctttattgtgaaatttgtgatgctattgcttt atttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcatttttgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatcctctagagtcgcagatccagacatgataagatacattgatgagtttggacaaaccacaactaga atgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtatggctgattatgatccctct agagtcgcagatcctctagagtcgcagatctgcaagctaattcctgcaggtcgagggacctaataacttcgtatagcatacattatacgaagttatattaagggttccggatccactacacgtgccaccatgtgcttcccgaaggtcctctctgatgacatgaagaagctgaaggcccgaatgataatgctcctccctacttctgctcagggg ttgggggcctgggtctcagcgtgtgacactgaggacactgtgggacacctgggaccctggagggacaaggatccggccctttggtgccaactctgcctctcttcacagcaccaggccatagaaagattttatgataaaatgcaaaatgcagaatcaggacgtggacaggtgatgtcgagcctggcagagctggaggacgact tcaaagaggggctacctggagacagtggcggcttattatgaggagcagcacccagagctcactcctctacttgaaaaagaaagagatggattacggtgccgaggcaacagatcccctgtcccggatgttgaggatcccgcaaccgaggagcctggggagagcttttgtgacaaggtcatgagatggttccaggccatgctgcagcggct gcagacctggtggcacggggttctggcctgggtgaaggagaaggtggtggccctggtccatgcagtgcaggccctctggaaacagttccagagtttctgctgctctctgtcagagctcttcatgtcctctttccagtcctacggagccccacggggggacaaggaggagctgacaccccagaagtgctctgaaccccaatcctca aaatgacttaagtcctcaggtgcaggctgcctatcagaaggtggtggctggtgtggccaatgccctggctcacaaataccactgagatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaattttttcattgcaatagtgtgttggaattttttgtgtgt ctctcactcggaaggacatatgggagggcaaatcatttaaaacatcagaatgagtatttggtttagagtttggcaacatatgcccatatgctggctgccatgaacaaaggttggctataaagaggtcatcagtatatgaaacagccccctgctgtccattccttatccatagaaaagccttgacttgaggttagatttttttatatt ttgttttgtgttatttttttctttaacatccctaaaattttccttacatgttttactagccagatttttcctcctctcctgactactcccagtcatagctgtccctcttctcttatggagatc.

1.4 Preparation of RNP injection complexes

Tube 1 solution: add 0.8uL 100pmol/uL CrRNA to 5.2uL RNase-free water, then add 0.6uL 100pmol/uL TracRNA, mix and incubate for 5min, then add 0.2uL Cas9 protein (NEB, catalog number: M0646M) and mix and incubate 10min to obtain tube 1 solution;

Tube 2 solution: Donor plasmid with a final concentration of 15ng/uL;

Mix tube 1 solution and tube 2 solution to get RNP injection complex.

2. Preparation of Human Interleukin-32 Alternative Splicing Body IL32γ Protein Conditional Knock-in Mice

2.1 Preparation of mouse fertilized oocytes

2.1.1 Superovulation

One 3-4 week old male C57BL/6J mouse and 3 female C57BL/6 mice were screened. Female mice were injected intraperitoneally with pregnant horse serum gonadotropin PMSG (5 U/rat) at 14:00 on the first day, and human chorionic gonadotropin hCG (5 U/rat) at 14:00 on the third day, and the time interval between the two injections was 46-48h; then immediately put 3 female mice and 1 male mouse in the same cage, check the vaginal plugs of the female mice at 8:00-9:00 on the fourth day, and select the female mice whose vaginal plugs were detected.

2.1.2 Collection and culture of fertilized eggs

Euthanize the female mouse whose vaginal thrombus was detected in the above step 2.1.1, collect the oviduct aseptically, put it into a 35mm Petri dish (drop culture medium made with M2 culture medium and hyaluronidase solution, pre-prepared at 37°C temperature), tear the ampulla of the fallopian tube with tweezers under a stereomicroscope, release the zygote mass surrounded by cumulus cells, move it into the hyaluronidase droplet and place it for a few minutes, collect the fertilized eggs, and transfer them to the M2 droplet In the culture medium, fertilized eggs with normal morphology were selected and cultured at 37° C. and 5% CO ₂ for later use.

2.1.3 Pronuclear microinjection and intra-fallopian embryo transfer

Prepare microinjection injection needles and fixation needles (brand Sutter; model number BF100-78-15) and fixation needles (brand Sutter; model number: B100-75-15);

Add the RNP injection complex prepared in step 1 (3-5uL) into the microinjection needle;

The fertilized eggs with normal morphology collected in the screening step 2.1.2 are placed in an injection dish, and the RNP injection complex is injected into the nucleus of the fertilized egg by microinjection under a 200-400× magnification inverted microscope;

The injected fertilized eggs were transferred to M16 medium, and placed in a 37°C constant temperature 5% CO ₂ incubator for overnight culture, and the embryos developed to the 2-cell stage were selected and implanted into surrogate mice.

The specific steps of embryo transfer are as follows: Prepare pseudopregnant female mice: select fertile CD-1 female mice (Weitonglihua) of the appropriate age (6-8 weeks old, 25-33g body weight) and sterilized CD-1 mice after vasectomy. 1 Male mice (Victoria) were mated, and a series of pregnancy changes were stimulated in the female mice to obtain pseudopregnant female mice, which were used as surrogate mice after fertilized eggs were transgenic;

For CD-1 female mice with pseudopregnancy 0.5 days (the vaginal plug was detected in the morning), an incision of about 1 cm was made on the back after anesthesia to expose the ovaries and fallopian tubes, and cultured to the 2-cell stage after microinjection under a stereo microscope. Embryos were implanted in the ampulla of fallopian tubes, the ovaries and fallopian tubes were put back into the abdominal cavity and then sutured, and both left and right fallopian tubes were transplanted, and 20 2-cell stage embryos were implanted into each mouse to obtain CD-1 surrogate mice.

2.1.4 Identification of positive F ₀ generation mice

1) About 20 days after transplantation, the CD-1 surrogate mice gave birth to obtain F ₀ generation mice. After the F ₀ generation mice were born, their tails were cut at 1-2 weeks of age, and the obtained tissues were used for the extraction of genomic DNA, and wild-type C57BL/6J mice were used as controls.

2) Using the extracted genomic DNA as a template, using primer pairs F1/R1 and F2/R2 to carry out PCR amplification to obtain the PCR product 1 (2.8kb) of the F1/R1 primer pair and the PCR product 2 (2.8kb) of the F2/R2 primer pair 2.6 kb), PCR product 1 was sequenced using F3 primers, and PCR product 2 was sequenced using R3 primers. The primer sequences are as follows:

F1: 5'-AAAGATCGCTCTCCACGCCCTAG-3';

R1: 5'-GATGGGGAGAGTGAAGCAGAACG-3';

F2: 5'-GCATCTGACTTCTGGCTAATAAAG-3';

R2: 5'-ATGGGAAGTTAGTAGCAAACAAGAG-3';

F3: 5'-CACTTGCTCTCCCAAAGTCGCTC-3';

R3: 5'-ATACTCCGAGGCGGATCACAA-3'.

The results of PCR identification showed that compared with wild-type C57BL/6J mice, positive F ₀ generation heterozygous mice (containing 2.8kb PCR product 1 and 2.6kb PCR product 2) in the two homologous chromosomes of chromosome 6 The DNA molecule shown in Sequence 2 in the sequence listing (i.e. the expression cassette of the IL32γ mutant) was inserted between the 1033-1034 nucleotides of intron 1 of the ROSA26 gene on one homologous chromosome, and the other chromosome No mutation occurred.

2.1. The acquisition and identification of 5F _1st generation mice

2.1.5.1 F ₁ generation mouse PCR detection and sequencing analysis

The positive F ₀ generation mice were crossed with wild-type C57BL/6J mice to obtain F ₁ generation heterozygous mice.

Use the same detection method as the F ₀ generation mice for PCR detection and sequencing analysis of the F ₁ generation heterozygous mice, including the use of primer pairs F1/R1 and F2/R2 for PCR amplification detection (Figure 2), and the use of F3 Sequencing analysis with R3 primer: When the F ₁ generation heterozygous mice were 1-2 weeks old, tail-cutting genotype identification was performed to screen for mice with the same genotype as the positive F ₀ generation heterozygous mice, that is, the F ₁ generation positive heterozygotes Between nucleotides 1033-1034 of intron 1 of the ROSA26 gene on one of the two homologous chromosomes of the mouse (containing 2.8kb PCR product 1 and 2.6kb PCR product 2) The DNA molecule shown in Sequence 2 in the sequence listing (i.e. the expression cassette of IL32γ) was inserted, and the other chromosome was not mutated, and a total of 3 F ₁ generation positive heterozygous targeted knock-in mice (IL32 ^flox/- , code number 17, 24 and 27, respectively) (Fig. 3).

2.1.5.2 F ₁ generation mouse Southern hybridization detection:

Southern blot analysis was performed on the tail DNA samples (tail cut at 3-4 weeks of age and genomic DNA extracted from the obtained tissue) of 3 _F1 generation positive targeted knock-in mice (17, 24 and 27), Correct gene knock-in targeting was confirmed. The strategy of Southern blot analysis is shown in Figure 4, using restriction enzymes Bsu36I and BstEII to digest and use the genomic DNA of wild-type mice (WT) and _F1 generation positive targeted knock-in mice (MT) respectively. 5'arm probe and 3'arm probe were identified.

The results of restriction endonuclease Bsu36I digestion and Southern hybridization of 5'arm probes showed that the wild-type mice (WT) could detect a 4.48kb band, and the _F1 generation positive targeted knock-in mice (MT) ( 17, 24 and 27) can detect bands of 4.48kb and 7.81kb; Southern hybridization detection results using restriction endonuclease BstEII and 3'arm probes show that wild-type mice (WT) can detect A 4.77kb band was detected, and the 4.77kb and 9.66kb bands could be detected in the _F1 generation of positive targeted knock-in mice (MT) (17, 24 and 27) (Fig. 5). The test results showed that human IL32γ was successfully inserted into the ROSA26 safety site in the _F1 generation positive targeted knock-in mice.

5' forward primer: 5'-AAACGTGGAGTAGGCAATACCCAGG-3'

5' reverse primer: 5'-AAAGAAGGGTCACCTCAGTCTCCCCT-3'

3' forward primer: 5'-TTCTGGGCAGGCTTAAAGGCTAAC-3'

3' reverse primer: 5'-AGGAGCGGGAGAAATGGATATGAAG-3'.

2.1.6 The mouse model of CD4 ⁺ T cell-specific overexpression of IL32γ was obtained

2.1.6.1 Breeding of IL32 ^flox/- CD4 ^Cre mice

F ₁ generation heterozygous mice (IL32 ^flox/- ) and CD4-Cre mice (Cd4-Cre containing the expression cassette of Cre recombinase gene, specifically express Cre recombinase in T lymphocytes expressing CD4, Saiye (Suzhou) Presented by Biotechnology Co., Ltd., JaxStrain#:022071, RRID:IMSR_JAX:022071) After reaching sexual maturity, they were mated to obtain F ₂ generation mice. After 5-7 days, follow the same method as step 2.1.5 for identification and screening of tail-truncated genotypes The F ₂ generation heterozygous mice were IL32 ^flox/- CD4 ^Cre , and the F ₂ generation IL32 ^flox/- CD4 ^Cre heterozygous mice carried IL32γ conditional expression cassette and Cd4-Cre at the same time. Cd4-Cre contains the CD4 enhancer, promoter and silencer sequences that drive the expression of the Cre recombinase gene. These mice can be used to generate conditional mutations in CD4 expressing tissue.

F ₂ generation IL32 ^flox/- CD4 ^Cre heterozygous mice were identified using three pairs of primers:

F3:5'-GTGTTGCAATACCTTTTCTGGGAGTT-3';

R3:5'-ATACTCCGAGGCGGATCACAA-3';

Use the primer pair F3/R3 to perform PCR to detect the wild-type band at the ROSA26 site, and the size is 353bp.

F5:5'-GGCAACGTGCTGGTTATTGTG-3';

R6: 5'-TTTCTATGGCCTGGTGCTGTGAAG-3';

The primer pair F5/R6 was used to perform PCR to detect the overexpressed band of IL32γ after Cre action, and the size was 323bp.

Cre recombinase PCR identification:

Cre-F: 5'-GTTCTTTGTATATATTGAATGTTAGCC-3'

Cre-R: 5'-CTTTGCAGAGGGCTAACAGC-3'

The size of the amplification product is: 336bp

Using the genomic DNA of the mouse as a template, carry out PCR, and select the mouse that contains the three bands of 353bp, 323bp and 336bp in the amplified product as the target mouse, that is, the targeted knock-in IL32γ conditional overexpression cassette heterozygote and Mice heterozygous for the Cre transgene (cKI mice).

Example 2, Verification of human IL32γ conditional knock-in mouse model

Human IL32γ is a secreted protein, so the present invention detects the expression of IL32γ in the culture supernatant of human IL32γ conditional knock-in mice (cKI mice) CD4 cells constructed in Example 1 to confirm the conditional knock-in of human IL32γ Successful establishment of the mouse model.

Three IL32γ ^flox/- CD4 ^Cre mice (cKI mice) and three IL32γ ^flox/- mice were selected, and each mouse was tested separately, killed by neck dislocation, soaked and disinfected in alcohol for 5 minutes, the spleen was taken and grinded, and the lymphocyte separation medium ( MP Biomedicals, Cat. No.: 50494X) to extract mononuclear cells, lyse red blood cells (TONBO, Cat. No.: TNB-4300-L100) for 6 min and wash with PBS. CD4 ⁺ T cells were obtained by mouse magnetic bead positive separation kit (Miltenyi Biotec, catalog number: 130-117-043), and CD4 ⁻ cells of cKI mice were collected at the same time. After counting the cells, take 1× ¹⁰⁶ cells/well and inoculate them in a 48-well plate, add 250uL RPMI1640 medium to each well, and add 0.5uL/well of cell stimulation mixture (Thermo Fisher, catalog number: 00-4970-03) at the same time , after culturing in a 37°C incubator for 4 hours, the cell culture supernatant was collected. Use IL32 ELISA kit (CUSABIO, product number: CSB-E12074h) to detect the concentration of IL32γ in the medium supernatant according to the instructions. The detection results are shown in Figure 8. IL32γ was detected in CD4 ⁺ T cells in cKI mice (represented by IL32 ^flox/- CD4 ^Cre CD4 ⁺ T cells in Figure 8), while IL32γ was detected in CD4 + T cells in cKI mice. IL32γ protein was not detected in ^- cells (represented by IL32 ^flox/- CD4 ^Cre CD4 ^- cells in Figure 8) and CD4 ⁺ T cells of IL32γ ^flox/- mice (represented by IL32 ^flox/- CD4 ⁺ T cells in Figure 8) expression. Therefore, the human IL32γ conditional knock-in mouse model was successfully constructed.

Example 3: Construction of a pleurisy model using human IL32γ conditional knock-in CD4 ⁺ T cell mice

It is well known that the proportion of Th1 cells increases in pleurisy and plays an important role in immune regulation. In this example, the cKI mice constructed in Example 1 were injected with live BCG bacteria to construct a mouse model of pleurisy, and the ratio of Th1 cells in the mouse model of pleurisy was observed.

Select 6-8 weeks old experimental group IL-32γ ^flox/ -CD4 ^Cre mice (cKI mice) constructed in Example 1 and 9 littermate control group IL-32 ^flox/- mice each, The mice in each group were randomly divided into 3 groups, 3 in each group, anesthetized with 0.5% pentobarbital sodium intraperitoneally, and each mouse was injected with 5×10 ⁶ BCG live bacteria (100 μL) intrapleurally to establish a pleurisy model .

On the 7th day after modeling, the cells in the pleural washing fluid of the experimental group and the control group were collected, and the cells in the pleural washing fluid of each group were mixed to obtain the total pleural washing fluid and mononuclear nuclei were extracted, and detected by flow cytometry Ratio of Th1 to CD4 ⁺ T cells in the pleural washings of mice in the experimental group and the control group.

The experimental results found that the proportion of Th1 cells in the pleural washings of cKI mice (represented by IL32 ^flox/- CD4 ^Cre mouse in Figure 9) was significantly higher than that in the pleural washings of IL-32γ ^flox/- mice (IL32 ^flox/- in Figure 9 mouse, p<0.001). Therefore, the cKI mice constructed in Example 1 successfully knocked in the human IL32γ gene to express IL32γ protein in CD4 ⁺ T cells, which affected the expression of Th1 cells, so that the cKI mice expressed a large number of Th1 cells and successfully obtained pleurisy mouse model. The human IL32γ conditional knock-in CD4 ⁺ T cell mouse model and the pleurisy mouse model constructed in the present invention can be applied to clinical research on the pathogenesis of pleurisy and clinical guidance of medication.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application.

Claims (10)

1. The method for constructing a tool mouse model that specifically expresses human interleukin 32 splice body IL32γ protein in immune cells, including specifically introducing the gene of human source interleukin 32 splice body IL32γ protein into recipient mice to obtain transgenic small mouse, the mouse model is obtained by crossing the transgenic mouse with the Cre enzyme transgenic mouse; the Cre enzyme transgenic mouse specifically expresses Cre enzyme in the immune cells; the mouse model specifically expresses the Cre enzyme in the immune cells Express the IL32γ protein. 2. The method according to claim 1, characterized in that: the IL32γ protein is selected from any of the following proteins: A1) The amino acid sequence is sequence 1 in the sequence listing; A2) A protein having more than 80% identity with the protein shown in A1) obtained by substituting and/or deleting and/or adding amino acid residues to the protein shown in A1) and having glutamic acid decarboxylase activity; A3) A fusion protein obtained by linking protein tags at the N-terminus or/and C-terminus of A1) or A2). 3. The method according to claim 1 or 2, characterized in that: the introduction is a specific knock-in, and the coding nucleotide sequence of the gene is the 3632-4333 position of sequence 2 in the sequence listing. 4. The method according to any one of claims 1-3, wherein the specific knock-in is knocking the gene into an intron of the ROSA26 gene of the recipient mouse. 5. The method according to any one of claims 1-4, characterized in that: the specific knock-in comprises using the CRISPR/Cas9 system to knock the gene into the inclusion of the ROSA26 gene of the recipient mouse son. 6. according to the method described in any one of claim 1-5, it is characterized in that: described knock-in comprises the nucleic acid molecule that will express the gRNA targeting the intron of described ROSA26 gene, Cas9 protein and described The step of introducing the nucleic acid molecule of the coding gene of human interleukin 32 splice body IL32γ protein into the recipient mice to obtain transgenic mice. 7. The method according to claim 6, wherein the target sequence of the gRNA corresponds to the complementary sequence of nucleotides 113,052,985-113,053,007 of the mouse genome GenBank Accession No.NC_000072.7. 8. The method according to any one of claims 6 or 7, wherein said method comprises the steps of: Introduce the nucleic acid molecule of the gRNA targeting the intron of the ROSA26 gene of the recipient mouse, the Cas9 protein and the nucleic acid molecule of the mutant coding gene of the human interleukin 32 splice body IL32γ protein into the recipient mouse Obtain F ₀ generation transgenic mice; F ₀ generation transgenic mice were crossed with wild-type recipient mice to obtain F ₁ generation heterozygous mice, compared with the recipient mice, the F ₁ generation heterozygous knock-in ROSA26 gene on one chromosome of the mice The complementary sequence between nucleotides 113,052,996-113,052,997 of GenBank Accession No.NC_000072.7 was inserted into the human IL32γ conditional expression cassette shown in sequence 2 in the sequence listing; The F ₁ generation heterozygous knock-in mice were crossed with CD4-Cre mice to obtain double-gene heterozygous mice targeted to knock-in human interleukin 32 gene and Cre recombinase to obtain the mouse model ; The Cre transgenic mouse contains an expression cassette of Cre recombinase, and specifically expresses Cre recombinase in CD4+T lymphocytes. 9. The method of any one of claims 1-8 and/or the mouse model described in any one of claims 1-8 is used in the preparation, development or research of human interleukin 32 to regulate TB Application in drugs related to immune microenvironment. 10. The method described in any one of claims 1-8 and/or the mouse model described in any one of claims 1-8 is used for clinical trials of diseases related to human interleukin 32 in the application.

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