Construction method of mouse model for expressing human III-type interferon receptor gene

Abstract

The invention relates to a construction method of a mouse model for expressing a human III-type interferon receptor gene, and belongs to the technical field of gene engineering. According to the invention, a humanized mouse model is constructed by introducing a human type III interferon receptor gene into a wild mouse. The humanized mouse has good response to the human type III interferon, can better reflect the function of the type III interferon receptor in the human body, provides a more suitable experimental model for researching the signal path of the human type III interferon, and in addition, the humanized mouse can be used for preparing the human type III interferon. The humanized mouse can also be used for evaluating the curative effect and antiviral mechanism of the human type III interferon in treating hepatitis caused by HCV, and can provide a platform for research and development of antiviral and vaccines related to the human type III interferon.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a method for constructing a mouse model expressing the human type III interferon receptor gene. Background Art

[0002] Type III interferon (IFN-λ) is a new type of interferon discovered in 2003, mainly including IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B) and IFN-λ4. They bind to specific cell surface receptors (IFNLR1 and IL-10RB), activate intracellular signaling pathways, and play various biological functions such as antiviral, immunomodulatory and anti-tumor effects. In the early stage of IFN-λ discovery, it was always thought that IFN-λ had redundant effects with type I interferon. However, in recent years, more and more studies have found that IFN-λ plays an important role in antiviral immune regulation. For example, in the treatment of HCV infection, IFN-λ shows significant antiviral potential, which improves the virus clearance rate and does not cause strong type I interferon-related inflammatory reactions (Tsui et al., 2008); in the early stage of influenza infection, IFN-λ is often induced earlier to participate in the initial antiviral response, and then induces the expression of type I interferon and inflammatory factors to further prevent the spread of the virus (Galani et al., 2017); in SARS-CoV-2 infection, IFN-λ stimulates the expression of downstream interferon-stimulated genes (ISGs) to play an antiviral role. In addition, in the early stage of SARS-CoV-2 infection, IFN-λ has antiviral activity and regulates the immune response, inhibits the inflammatory response, and early intervention and reasonable application of IFN-λ may help control the severity of COVID-19 (Schuhenn et al., 2021). In addition, IFN-λ is also involved in immunomodulatory effects: 1. IFN-λ can promote the proliferation and antibody secretion of B cells, and at the same time can regulate the differentiation of B cells, such as inducing the differentiation of plasma cells and enhancing the humoral immune response of the body (Krammer et al., 2021); 2. IFN-λ can promote the activation and proliferation of T cells and regulate the differentiation of T cells (Krammer et al., 2021); 3. Promote the maturation and antigen presentation ability of dendritic cells, thereby enhancing the adaptive immune response of the body (Borodovsky et al., 2020); 4. Enhance the cytotoxicity of NK cells and improve their killing ability against pathogens and tumor cells, thereby enhancing the innate immune function of the body (Bachiller et al., 2020).

[0003] Interferons are species-specific. In mice, only IFN-λ2 and IFN-λ3 exist, and the sequence similarity between murine IFN-λ receptor mIFNLR1 and human IFN-λ receptor huIFNLR1 is approximately 50%. This difference limits the direct use of human IFN-λ for research in mice. Summary of the Invention

[0004] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for constructing a mouse model expressing the human type III interferon receptor gene.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In the first aspect, the present invention provides a gene targeting vector, which is used to insert the coding sequence of the extracellular domain of the human type III interferon receptor protein in front of the coding sequences of the transmembrane domain and the intracellular structural domain of the corresponding murine type III interferon receptor protein, while retaining the promoter and regulatory sequences encoding the murine type III interferon receptor.

[0007] As a preferred embodiment of the present invention, the gene targeting vector includes a 5' homologous arm and a 3' homologous arm, and a target gene located between the 5' homologous arm and the 3' homologous arm.

[0008] As a preferred embodiment of the present invention, the target gene includes the following nucleotide sequences connected in sequence:

[0009] The nucleotide sequence encoding the extracellular domain of human type III interferon receptor 1, the nucleotide sequence encoding the transmembrane domain and the intracellular domain of murine type III interferon receptor 1, the nucleotide sequence encoding the P2A peptide segment, the nucleotide sequence encoding the extracellular domain of human type III interferon receptor 2, and the nucleotide sequence encoding the transmembrane domain and the intracellular domain of murine type III interferon receptor 2.

[0010] As a preferred embodiment of the present invention, the nucleotide sequence encoding the extracellular domain of human type III interferon receptor 1 is as shown in SEQ ID NO: 4; and / or, the nucleotide sequence encoding the transmembrane domain and the intracellular domain of murine type III interferon receptor 1 is as shown in SEQ ID NO: 5; and / or, the nucleotide sequence encoding the P2A peptide segment is as shown in SEQ ID NO: 6; and / or, the nucleotide sequence encoding the extracellular domain of human type III interferon receptor 2 is as shown in SEQ ID NO: 7; and / or, the nucleotide sequence encoding the transmembrane domain and the intracellular domain of murine type III interferon receptor 2 is as shown in SEQ ID NO: 8.

[0011] As a preferred embodiment of the present invention, the nucleotide sequence of the 5' homologous arm is as shown in SEQ ID NO: 3; and / or, the nucleotide sequence of the 3' homologous arm is as shown in SEQ ID NO: 9.

[0012] As a preferred embodiment of the present invention, it further includes gRNA and Cas9RNA.

[0013] In a second aspect, the present invention provides a kit prepared from the gene targeting vector described in the first aspect.

[0014] In a third aspect, the present invention provides a method for constructing a gene-modified fertilized egg, comprising the following steps:

[0015] Using the CRISPR / Cas9-mediated homologous recombination technology, an expression gene is knocked into the type III interferon receptor gene of a wild-type mouse to prepare a gene-modified fertilized egg;

[0016] Among them, the step of preparing the gene-modified fertilized egg includes: according to the target site, constructing the gene targeting vector described in any one of claims 1-5, and the target site is located in the region after the start codon of the second exon of the Ifnlr1 gene of the wild-type mouse; designing gRNA according to the target site; and introducing the gene targeting vector, the gRNA and Cas9 protein into the fertilized egg of the wild-type mouse to prepare a gene-modified fertilized egg.

[0017] In a fourth aspect, the present invention provides a method for constructing a mouse model with partial humanization of the type III interferon receptor, comprising the steps:

[0018] S1 Transplant the fertilized egg described in claim 7 into a pseudopregnant mouse, and wait for it to become pregnant and give birth to obtain the F0 generation;

[0019] S2 Mate the positive F0 generation mice with wild mice to obtain F1 generation heterozygous mice;

[0020] S3 Self-cross the F1 generation heterozygous mice to obtain F2 generation homozygous mice, namely the mouse model with partial humanization of the type III interferon receptor.

[0021] As a preferred embodiment of the present invention, it further includes the step of performing PCR identification on the DNA extracted from the mouse model with partial humanization of the type III interferon receptor, wherein the primer sequences for PCR identification are as shown in SEQ ID NO: 11-16.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] By introducing the human type III interferon receptor gene into mice, the present invention can construct a "humanized" mouse model. Such humanized mice can better reflect the function of the human type III interferon receptor in the human body, provide a more suitable experimental model for studying the human type III interferon signaling pathway. In addition, such humanized mice can also be used to evaluate the efficacy and antiviral mechanism of human type III interferon in the treatment of hepatitis caused by HCV, and can provide a platform for the research and development of antiviral drugs and vaccines related to human type III interferon. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the transgenic strategy for the humanized mouse model of type III interferon receptor;

[0025] Figure 2 Schematic diagram of the structure of the human-mouse chimeric receptor of type III interferon (the green part is the extracellular domain of the human type III interferon receptor huIFNLR1 that replaces the mouse type III interferon receptor mIFNLR1, the purple part is the extracellular domain of the human type III interferon receptor huIL10RB that replaces the mouse type III interferon receptor mIFNLR1, the orange part is the mouse type III interferon receptor mIFNLR1, and the blue part is the transmembrane region and intracellular domain of mIL10RB);

[0026] Figure 3 Schematic diagram of the structure of the type III interferon receptor of normal mice;

[0027] Figure 4 Schematic diagram of the design of primers for genotype identification;

[0028] Figure 5 Gel electrophoresis diagram of PCR products for genotype identification (where the numbers are the mouse tail numbers, WT is C57BL / 6JGpt, N is the blank control, and M is the DNA Marker; among them, the mouse tail numbers 29, 31, 40, 41, 44, 45, and 48 are positive mice identified by PCR);

[0029] Figure 6 Schematic diagram of the sequencing results of the 5'arm knock-in part of the homozygous type III interferon receptor humanized mouse;

[0030] Figure 7 Schematic diagram of the sequencing results of the 3'arm knock-in part of the homozygous type III interferon receptor humanized mouse;

[0031] Figure 8 Schematic diagram of the expression results of the human hIFNLR1 and hIL10RB receptor genes in the type III interferon receptor humanized mouse;

[0032] Figure 9Schematic diagram of the expression results of Isg15 and Ifit1 genes in nasal turbinates, trachea and lung tissues of humanized mice with type III interferon receptor after stimulation with human interferon (hIFNλ1). Detailed implementation manners

[0033] To better illustrate the objectives, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0034] Example 1 Transgenic construction method of humanized mouse model with type III interferon receptor (IFNLR1 and IL10RB)

[0035] In this example, a transgenic targeting method is used to construct a partially humanized mouse model with type III interferon receptor. The transgenic strategy in this example is as follows: the extracellular domain sequence of human type III interferon receptor 1 (huIFNLR1) is connected to the transmembrane domain and intracellular domain sequence of mouse type III interferon receptor 1 (mIFNLR1), and the extracellular domain sequence of human type III interferon receptor 2 (huIL10RB) and the transmembrane domain and intracellular domain sequence of mouse type III interferon receptor 2 (mIL10RB) are connected with a P2A sequence as the target gene to be expressed; the target gene to be expressed is transferred into exon 2 of the Ifnlr1 gene of C57BL / 6JGpt background mice by the CRISPR / Cas9 method to obtain a partially humanized mouse model with type III interferon receptor. The transgenic strategy of the humanized mouse model with type III interferon receptor is as Figure 1 shown, and specifically includes the following steps:

[0036] 1. Construct a homologous recombination vector:

[0037] Connect the target gene containing two homologous arms and located between the two homologous arms to a conventional T vector (pUC-57) to prepare a homologous recombination vector.

[0038] The homologous arm sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 9 respectively.

[0039] The order of the target gene is as follows:

[0040] The nucleotide sequence encoding the extracellular domain of human type III interferon receptor 1 (huIFNLR1) (shown in SEQ ID NO: 4),

[0041] The nucleotide sequence encoding the transmembrane domain and intracellular domain of mouse type III interferon receptor 1 (mIFNLR1) (shown in SEQ ID NO: 5),

[0042] The nucleotide sequence encoding the P2A peptide segment (shown in SEQ ID NO: 6),

[0043] The nucleotide sequence of the extracellular domain of human type III interferon receptor 2 (huIL10RB) encoding a human (as shown in SEQ ID NO.7).

[0044] The nucleotide sequence of the transmembrane and intracellular domains of mouse type III interferon receptor 2 (mIL10RB) encoding a mouse (as shown in SEQ ID NO:8).

[0045] 2. Based on the CRISPR / Cas9 technology, design the gRNA sequence using the website http: / / crispr.mit.edu / , clone it into the pU57-T7-GDNA vector, and identify it by PCR sequencing to obtain the gRNA expression plasmid. Use T7 RNA polymerase for in vitro transcription to obtain gRNA and Cas9RNA.

[0046] 3. By means of gene targeting, introduce the gRNA, Cas9RNA and the above homologous recombination vector into the fertilized eggs of C57BL / 6JGpt mice by microinjection. Then, take the surviving fertilized eggs after injection and transplant them into the body of pseudopregnant female mice. Wait for them to become pregnant and give birth to obtain the F0 generation. Tail and toe clippings of the F0 generation pups are numbered at 5 - 7 days, and genomic DNA is extracted for PCR and sequencing identification to confirm the genotype.

[0047] 4. Mate the positive F0 generation mice with WT mice to obtain F1 generation heterozygous mice, and self-cross the F1 generation mice to obtain F2 generation homozygous IFN-λ receptor humanized mice.

[0048] Table 1 Information table of target genes, gRNA and target sequences

[0049]

[0050]

[0051]

[0052]

[0053] Example 2 Identification of humanized mice with type III interferon receptor

[0054] Extract the genomic DNA of humanized mice with type III interferon receptor for genotype identification and sequencing identification.

[0055] Step 1: Extract the DNA of homozygous mice using a tissue genomic DNA extraction kit (Tiangen Biochemical Technology). The specific operations are as follows: 1) Take 20 mg of mouse tissue, use a grinder to process it into a cell suspension, then centrifuge at 10000 rpm (~11200×g) for 1 min, pour out the supernatant, and add 200 μL of buffer GA; shake until completely suspended; 2) Add 20 μL of Proteinase K, mix well, and place at 56 °C until the tissue dissolves; 3) Add 200 μL of buffer GB, invert thoroughly to mix, place at 70 °C for 10 min, and the solution becomes clear; 4) Add 200 μL of absolute ethanol, shake well for 15 s, and visible flocculent precipitates appear in the tube. Briefly centrifuge to remove the water droplets on the inner wall of the tube cap; 5) Add the solution and flocculent precipitates obtained in the previous step into the adsorption column, centrifuge at 12000 rpm (~13400×g) for 30 s, pour out the waste liquid, and put the adsorption column back into the collection tube; 6) Add 500 μL of buffer GD to the adsorption column, centrifuge at 12000 rpm for 30 s, pour out the waste liquid, and place the adsorption column into the collection tube; 7) Add 600 μL of washing solution PW (add absolute ethanol before use) to the adsorption column, centrifuge at 12000 rpm for 30 s, pour out the waste liquid, and place the adsorption column into the collection tube; 8) Repeat operation step 7); 9) Put the adsorption column back into the collection tube, centrifuge at 12000 rpm for 2 min, pour out the waste liquid. Place the adsorption column at room temperature for several minutes to completely dry the residual washing solution in the adsorption material; 10) Transfer the adsorption column into a clean centrifuge tube, suspend and drop 50 μL of RNase-free water in the middle of the adsorption membrane, place at room temperature for 3 min, centrifuge at 12000 rpm for 2 min, and collect the solution into the centrifuge tube.

[0056] Step 2: PCR amplify the DNA fragment to be sequenced. The PCR primer information is shown in the following table:

[0057] Table 2 Primer sequences for genotyping PCR

[0058]

[0059] The primer design ideas for genotyping in the above table are as Figure 4 shown. Among them, primers F1 (huIFNLR-KI-F1) and R1 (huIFNLR-KI-R1) are primer pair ①, which is used to identify the knock-in situation of huIFNLR1; primers F2 (huIFNLR-KI-F2) and R2 (huIFNLR-KI-R2) are primer pair ②, which is used to identify the knock-in situation of huIL10RB; primers F3 (huIFNLR-WT-F3) and R3 (huIFNLR-WT-R3) are primer pair ③, which is used to identify WT.

[0060] The identification results of the wild type (WT) can show that no Target band was obtained in the PCR reaction with primer pairs ① and ②, while a single WT band was obtained in the PCR reaction with primer pair ③.

[0061] The identification results of the heterozygote can show that Target bands were obtained in the PCR reaction with primer pairs ① and ②, and a single WT band was obtained in the PCR reaction with primer pair ③.

[0062] The identification results of the homozygote can show that Target bands were obtained in the PCR reaction with primer pairs ① and ②, and no single WT band was obtained in the PCR reaction with primer pair ③.

[0063] The PCR system is shown in the following table:

[0064] Table 3 PCR System

[0065]

[0066]

[0067] The PCR program is shown in the following table:

[0068] Table 4 PCR Program

[0069]

[0070] Prepare a 1% agarose gel. After loading the samples, electrophorese at 120V for 20 - 25 minutes, and then take ultraviolet photos to confirm the results.

[0071] Step 3: Agarose Gel DNA Recovery: Use a common agarose gel DNA recovery kit (Tiangen Biochemical Technology) to recover the PCR products. The specific operations include: 1) Column equilibration: Add 500 μL of equilibration buffer BL to the adsorption column, centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube; 2) Cut the single target DNA band from the agarose gel (try to remove the excess as much as possible) and put it into a clean centrifuge tube, weigh it; 3) Add an equal volume of solution PN solubilization solution to the gel block (if the gel weighs 0.1 g, its volume can be regarded as 100 μL, then add 100 μL of PN solubilization solution), place it in a 50 °C water bath, and gently invert the centrifuge tube up and down continuously during this period to ensure that the gel block is fully dissolved; 4) Add the solution obtained in the previous step to an adsorption column (the adsorption column is placed in the collection tube), let it stand at room temperature for 2 min, centrifuge at 12,000 rpm for 60 s, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube; 5) Add 600 μL of wash buffer PW to the adsorption column, centrifuge at 12,000 rpm for 60 s, discard the waste liquid in the collection tube, and place the adsorption column in the collection tube; 6) Repeat operation step 5); 7) Place the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 2 min to remove as much of the wash buffer as possible. Place the adsorption column at room temperature for several minutes to dry it thoroughly; 8) Place the adsorption column into a clean centrifuge tube, suspend and add 50 μL of RNase-free water to the middle position of the adsorption membrane, let it stand at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min to collect the DNA solution. Then send the recovered product to a sequencing company for sequencing.

[0072] The results are as Figure 5 shown. The mice with tail numbers 29, 31, 40, 41, 44, 45, and 48 were positive mice identified by PCR, successfully expressing the humanized type III interferon receptor, indicating the successful construction of the humanized type III interferon receptor mice.

[0073] The results are as Figure 6 shown. The sequencing results of the 5’ arm Knock in part of the homozygous humanized type III interferon receptor mice indicate that the human IFNLR1 gene was successfully inserted into the genome of the homozygous humanized type III interferon receptor mice, further confirming that this mouse is the target humanized type III interferon receptor mouse.

[0074] The results are as Figure 7 shown. The sequencing results of the 3’ arm Knock in part of the homozygous humanized type III interferon receptor mice indicate that the human IL10RB gene was successfully inserted into the genome of the homozygous humanized type III interferon receptor mice, further confirming that this mouse is the target humanized type III interferon receptor mouse.

[0075] Example 3 Identification of the expression of the humanized mouse receptor of type III interferon receptor and functional verification of pathway response - I. Receptor expression identification

[0076] 1. Select 6 - week - old WT mice and humanized mice of type III interferon receptor. After anesthesia with isoflurane, decapitate and sacrifice them;

[0077] 2. Fix the mice on a rack and spray - disinfect with 75% alcohol; then use surgical scissors to cut open the peritoneum of the mice and open the thoracic cavity;

[0078] 3. Use a 10 - ml syringe to aspirate 10 ml of sterile PBS, and adopt the method of cardiac perfusion to inject PBS into the mouse heart from the apex of the heart to remove the blood in the tissue;

[0079] 4. Then collect the lung tissue of the mice into 1.5 - ml EP tubes, add 1 ml of trizol, and grind twice at 65 HZ for 1 min to fully lyse the tissue samples;

[0080] 5. Take 500 μl of tissue lysate, add 200 μl of ddH2O in a biosafety cabinet, let it stand at 4°C for 10 min, and then centrifuge at 12000 rpm at 4°C for 15 min.

[0081] 6. Transfer the supernatant to a new 1.5 - ml EP tube, add an equal volume of pre - cooled isopropanol, mix well by inverting up and down, let it stand at - 20°C for at least 20 min, and centrifuge at 12000 rpm at 4°C for 15 min;

[0082] 7. Remove the upper liquid, and then add pre - cooled 75% ethanol to wash twice;

[0083] 8. Finally, remove the upper liquid, air - dry, and then add an appropriate amount of ddH2O to dissolve the RNA;

[0084] 9. Denature the RNA at 65°C for 10 min;

[0085] 10. Then measure the concentration and reverse - transcribe it into cDNA according to the RNA reverse - transcription kit of Toyobo;

[0086] 11. Then perform qPCR to detect the mRNA expression levels of hIFNLR1 and hIL10RB receptors in the tissue.

[0087] The results are as Figure 8 shown. The quantitative results of the expression of hIFNLR1 and hIL10RB genes in the mouse lung tissue show that huIFNLR mice highly express the human hIFNLR1 and hIL10RB genes, while WT mice do not express the human hIFNLR1 and hIL10RB genes.

[0088] II. Functional verification

[0089] 1. Select 6-week-old interferon receptor III humanized mice, randomly divide them into groups and administer 2ug hIFNλ1 or PBS by nasal drops for 24h;

[0090] 2. The mice were then anesthetized with isoflurane, killed by cervical dislocation, fixed on the operating table, and disinfected with 75% alcohol;

[0091] 3. Use surgical scissors to open the abdomen of the mouse, gently use forceps to push aside the intestinal tissue to expose the dorsal collateral aorta, then cut it off with surgical scissors, and then absorb the blood with sterile cotton balls;

[0092] 4. Open the chest cavity, remove the blood in the tissue by cardiac perfusion, then collect the mouse's nasal concha, trachea and lung tissues, lyse them with trizol, extract RNA, and perform RT-qPCR to detect the expression of ISGs (interferon-stimulated genes) downstream of III interferon. If III interferon can be successfully expressed, its downstream products sg15 and Ifit1 genes can also be successfully expressed. Primer information is shown in the table below:

[0093] Table 5 qPCR primer information

[0094]

[0095]

[0096] qPCR system, see the table below:

[0097] Table 6 qPCR system preparation table

[0098] Reaction components Volume (ul) Template cDNA (2 ng) 4 SYRB green mix 5 Primer-F (2.5 umol) 0.5 Primer-R (2.5 umol) 0.5

[0099] qPCR program, see the table below:

[0100] Table 7 qPCR program table

[0101]

[0102] The results are as follows Figure 9 As shown, the expression of Isg15 ( Figure 9 A) and Ifit1( Figure 9 B) Gene expression results, in which Isg15 and Ifit1 gene expressions were upregulated in the samples stimulated by hIFNλ1, and Isg15 and Ifit1 gene expressions were low in the PBS group. Black represents the PBS group, and blue represents the hIFNλ1 group. It can be seen that the IFN-λ receptor partially humanized mice constructed by the present invention can successfully respond to stimulation from human hIFNλ1.

[0103] Comparative Example 1

[0104] In this comparative example, other sequence fragments of huIFNLR1 were used to replace the sequence shown in SEQ ID NO: 4 in Example 1, and the others were the same as in Example 1. The sequence in Comparative Example 1 is as follows:

[0105] MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGN

[0106] PQDVTYFVAYQSSPTRRRWREVEECAGTKELLCSMMCLKKQDLYNKFKGRV

[0107] RTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANATYQLPPCMPPL

[0108] DLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVP

[0109] KYSKFSKPTCFLLEVPEANWAFLVLPSLLILLLVIAAGGVIWKTLMGNPWFQR

[0110] AKMPRALELTRGVRPTPRVRAPATQQTRWKKDLAEDEEEEDEEDTEDGVSFQ

[0111] PYIEPPSFLGQEHQAPGHSEAGGVDSGRPRAPLVPSEGSSAWDSSDRSWASTV

[0112] DSSWDRAGSSGYLAEKGPGQGPGGDGHQESLPPPEFSKDSGFLEELPEDNLSS

[0113] WATWGTLPPEPNLVPGGPPVSLQTLTFCWESSPEEEEEARESEIEDSDAGSWG

[0114] AESTQRTEDRGRTLGHYMAR(SEQ ID NO: 27).

[0115] Comparative Example 2

[0116] In this comparative example, other sequence fragments of huIFNLR1 were used to replace the sequence shown in SEQ ID NO: 4 in Example 1, and the others were the same as in Example 1. The sequence in Comparative Example 2 is as follows:

[0117] MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGN

[0118] PQDVTYFVAYQSSPTRRRWREVEECAGTKELLCSMMCLKKQDLYNKFKGRV

[0119] RTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANATYQLPPCMPPL

[0120] DLKYEVAFWKEGAGNKVGSSFPAPRLGPLLHPFLLRFFSPSQPAPAPLLQEVFP

[0121] VHS (SEQ ID NO: 28).

[0122] Comparative Example 3

[0123] In this comparative example, other sequence fragments of huIFNLR1 were used to replace the sequence shown in SEQ ID NO: 4 in Example 1, and the others were the same as in Example 1. The sequence in Comparative Example 3 is as follows:

[0124] MAGPERWGPLLLCLLQAAPGRPRLAPPQNVTLLSQNFSVYLTWLPGLGN

[0125] PQDVTYFVAYQSSPTRRRWREVEECAGTKELLCSMMCLKKQDLYNKFKGRV

[0126] RTVSPSSKSPWVESEYLDYLFEVEPAPPVLVLTQTEEILSANATYQLPPCMPPL

[0127] DLKYEVAFWKEGAGNKTLFPVTPHGQPVQITLQPAASEHHCLSARTIYTFSVP

[0128] KYSKFSKPTCFLLEVPGLFWTHTPCGNLSAQQTRVRE (SEQ ID NO: 29).

[0129] Comparative Example 4

[0130] In this comparative example, other sequence fragments of huIL10RB were used to replace the sequence shown in SEQ ID NO: 7 in Example 1, and the others were the same as in Example 1. The sequence in Comparative Example 4 is as follows:

[0131] MAWSLGSWLGGCLLVSALGMVPPPENVRMNSVNFKNILQWESPAFAKG

[0132] NLTFTAQYLSYRIFQDKCMNTTLTECDFSSLSKYGDHTLRVRAEFADEHSDW

[0133] VNITFCPVDDTIIGPPGMQVEVLADSLHMRFLAPKIENEYETWTMKNVYNSW

[0134] TYNVQYWKNGTDEKFQITPQYDFEVLRNLEPWTTYCVQVRGFLPDRNKAGE

[0135] WSEPVCEQTTHDETVPSWMVAVILMASVFMVCLALLGCFALLWCVYKKTKY

[0136] AFSPRNSLPQHLKEE (SEQ ID NO: 30).

[0137] Comparative Example 5

[0138] In this comparative example, other sequence fragments of huIL10RB were used to replace the sequence shown in SEQ ID NO: 7 in Example 1, and the others were the same as in Example 1. The sequence in Comparative Example 5 is as follows:

[0139] MAWSLGSWLGGCLLVSALGMVPPPENVRMNSVNFKNILQWESPAFAKG

[0140] NLTFTAQYLSYRIFQDKCMNTTLTECDFSSLSKYGDHTLRVRAEFADEHSDW

[0141] VNITFCPVDDTIIGPPGMQVEVLADSLHMRFLAPKIENEYETWTMKNVYNSW

[0142] TYNVQYWKNGTDEKFQITPQYDFEVLRNLEPWTTYCVQVRGFLPDRNKAGE

[0143] WSEPVCEQTTHDETVPSWMVAVILMASVFMVCLALLGCFALLWCVYKKTKY

[0144] AFSPRNSLPQHLKELSGINFSTSQKHFGPQIPKGTLFFILENTMPTYQNKGQIG

[0145] AFASK (SEQ ID NO: 31).

[0146] Comparative Example 6

[0147] In this comparative example, other sequence fragments of huIL10RB were used to replace the sequence shown in SEQ ID NO: 7 in Example 1. Other conditions were the same as in Example 1. The sequence in Comparative Example 6 was as follows:

[0148] MAWSLGSWLGGCLLVSALGMVPPPENVRMNSVNFKNILQWESPAFAKG

[0149] NLTFTAQYLSYRIFQDKCMNTTLTECDFSSLSKYGDHTLRVRAEFADEHSDW

[0150] VNITFCPVDDTIIGPPGMQVEVLADSLHMRFLAPKIENEYETWTMKNVYNSW

[0151] TYNVQYWKNGTDEKFQITPQYDFEVLRNLEPWTTYCVQVRGFLPDRNKAGE

[0152] WSEPVCEQTTHDVFGPSSS (SEQ ID NO: 32).

[0153] The constructed mice in Comparative Examples 1 - 6 were administered 2 μg of hIFNλ1 by nasal drip. The results showed that the Isg15 and Ifit1 genes in the nasal turbinates, tracheas, and lung tissues of the mice were lowly expressed, indicating that the sequences in Comparative Examples 1 - 6 could not successfully respond to human hIFNλ1.

[0154] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A gene targeting vector, characterized in that, The gene targeting vector is used to insert the coding sequence of the extracellular domain of the human type III interferon receptor protein in front of the coding sequences of the transmembrane domain and the intracellular structural domain of the corresponding murine type III interferon receptor protein, while retaining the promoter and regulatory sequences encoding the murine type III interferon receptor.

2. The gene targeting vector according to claim 1, characterized in that, The gene targeting vector includes a 5' homologous arm and a 3' homologous arm, and a target gene located between the 5' homologous arm and the 3' homologous arm.

3. The gene targeting vector according to claim 2, wherein The target gene includes the following nucleotide sequences connected in sequence: The nucleotide sequence encoding the extracellular domain of human type III interferon receptor 1, the nucleotide sequence encoding the transmembrane domain and the intracellular domain of murine interferon receptor 1, the nucleotide sequence encoding the P2A peptide segment, the nucleotide sequence encoding the extracellular domain of human type III interferon receptor 2, and the nucleotide sequence encoding the transmembrane domain and the intracellular domain of murine type III interferon receptor 2.

4. The gene targeting vector according to claim 3, wherein The nucleotide sequence encoding the extracellular domain of human type III interferon receptor 1 is as shown in SEQ ID NO: 4; and / or, The nucleotide sequence encoding the transmembrane domain and the intracellular domain of murine interferon receptor 1 is as shown in SEQ ID NO: 5; and / or, The nucleotide sequence encoding the P2A peptide segment is as shown in SEQ ID NO: 6; and / or, The nucleotide sequence encoding the extracellular domain of human type III interferon receptor 2 is as shown in SEQ ID NO: 7; and / or, The nucleotide sequence encoding the transmembrane domain and the intracellular domain of murine type III interferon receptor 2 is as shown in SEQ ID NO:

8.

5. The gene targeting vector according to claim 2, wherein The nucleotide sequence of the 5' homologous arm is as shown in SEQ ID NO: 3; and / or, the nucleotide sequence of the 3' homologous arm is as shown in SEQ ID NO:

9.

6. The gene targeting vector according to claim 1, wherein It also includes gRNA and Cas9RNA.

7. A kit prepared from the gene targeting vector according to any one of claims 1-6.

8. A method for constructing a genetically modified fertilized egg, characterized in that, It includes the following steps: Using the CRISPR / Cas9-mediated homologous recombination technology, an expression gene is knocked into the type III interferon receptor gene of wild-type mice to prepare genetically modified fertilized eggs. Among them, the step of preparing the genetically modified fertilized eggs includes: constructing the gene targeting vector according to any one of claims 1-5 according to the target site, where the target site is located in the region after the start codon of the second exon of the Ifnlr1 gene of the wild-type mice; designing gRNA according to the target site; and introducing the gene targeting vector, the gRNA and Cas9 protein into the fertilized eggs of the wild-type mice to prepare genetically modified fertilized eggs.

9. A method for constructing a partially humanized mouse model of type III interferon receptor, characterized in that, It includes the steps: S1 Transplant the fertilized eggs as described in claim 7 into pseudopregnant mice, wait for them to become pregnant and give birth to obtain the F0 generation. S2 Mate the positive F0 generation mice with wild mice to obtain F1 generation heterozygous mice. S3 Self-cross the F1 generation heterozygous mice to obtain F2 generation homozygous mice, namely the murine model with partial humanization of the type III interferon receptor.

10. The construction method according to claim 8, wherein It also includes the step of performing PCR identification on the DNA of a mouse model with a partially humanized type III interferon receptor, wherein the primer sequences for PCR identification are as shown in SEQ ID NO: 11-16.

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