CN112646783A - Expression gene, method for constructing genetically modified fertilized egg, and method for constructing mouse model - Google Patents

Abstract

The invention relates to a construction method of an expression gene and a genetically modified fertilized egg and a construction method of a mouse model, wherein the construction method of the genetically modified fertilized egg comprises the following steps: the expressed gene of the human interferon receptor is knocked in and expressed on the interferon receptor gene of a mouse by utilizing a CRISPR/Cas9 mediated homologous recombination technology, and the fertilized egg modified by the gene is prepared. The mouse model prepared from the fertilized eggs prepared by the construction method can identify human interferon and normally respond to the stimulation of the human interferon.

Description

Expression gene, method for constructing genetically modified fertilized egg, and method for constructing mouse model

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a construction method of an expression gene, a genetically modified fertilized egg and a construction method of a mouse model.

Background

According to data published by the World Health Organization (WHO), 20 million people worldwide are infected with Hepatitis B Virus (HBV), of which 2.4 million people are chronic HBV infected, and HBV infection can cause various acute and chronic liver diseases, and about 60 million people worldwide die each year from HBV-associated liver failure, cirrhosis and hepatocellular carcinoma.

For antiviral treatment of chronic hepatitis b, two major classes of drugs are currently approved for clinical use: small molecular nucleoside (acid) analogues (NA) targeting HBV (hepatitis B virus) reverse transcription from pre-genomic RNA (pgRNA) to DNA link of progeny virus can completely inhibit the generation of new virus in a short time, but cannot clear cccDNA (deoxyribonucleic acid), and has limited effect of reducing HBsAg (hepatitis B Virus); the other class of drugs is interferon-alpha (IFN-alpha) with direct antiviral and immunoregulation and a pegylation form thereof ((PegIFN-alpha). Studies show that interferon can increase antiviral immune response, inhibit viruses from entering liver cells, induce partial degradation of cccDNA of HBV, and inhibit cccDNA transcription and inhibit secretion of viruses after transcription through epigenetics, so that the interferon becomes one of potential drugs capable of realizing an ideal end point of hepatitis B curing due to important regulation and control on multiple links of HBV infected liver cells, but the action mechanism of interferon in vivo is not clear, and further research on the action mechanism of the interferon on an animal model is needed.

The currently available animal models for curing HBV by interferon can be divided into the following categories: the first is the animal model for HBV infection replication naturally existing in nature, such as gorilla, Mauritius monkey, tree shrew and woodchuck, but these HBV infection animal models have high experimental cost, and their interferon receptors are far from the human interferon receptor from the evolution point of view, so the action effect of interferon in these animal models has a certain difference from the action effect of interferon in human body; the second is a liver humanized mouse model that can support high-level HBV replication and can test drugs that act directly on HBV (like DAA class drugs of hepatitis c), but there are fundamental drawbacks for assessing the effect of interferon on HBV: 1) the cost for constructing the double humanized mouse with the liver and the immune system is high; 2) except the liver and immune system, other organs only express the receptor of the murine interferon and cannot normally respond to the stimulation of the human interferon.

Disclosure of Invention

Therefore, there is a need for a method for constructing a genetically modified fertilized egg, and a mouse model prepared from the fertilized egg prepared by the construction method can correctly recognize human interferon and normally respond to the stimulation of human interferon.

In addition, it is necessary to provide a construction method of an expression gene and a mouse model and a kit for constructing the mouse model, the construction cost of the construction method of the mouse model is low, and the mouse model constructed according to the construction method can correctly recognize human interferon and normally respond to the stimulation of the human interferon.

A method for constructing a genetically modified fertilized egg, comprising the steps of:

knocking in an expression gene on a type I interferon receptor gene of a wild mouse by using a CRISPR/Cas9 mediated homologous recombination technology to prepare a genetically modified fertilized egg, wherein the expression gene comprises:

a first functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor 1, and a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor 1;

a second functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor2, and a transcription terminator; and

a linking region for linking said first functional region to said second functional region, said linking region comprising a nucleic acid fragment encoding a protein having self-cleavage function.

The construction method of the gene modification has lower cost, and the obtained fertilized eggs can identify human interferon when used for preparing a mouse model and normally respond to the stimulation of the human interferon.

A method for constructing a genetically modified fertilized egg, comprising the steps of:

in one embodiment, the step of preparing a genetically modified fertilized egg comprises:

constructing a homologous recombination vector containing an expressed gene according to a target site, wherein the target site is positioned on the type I interferon receptor gene of the mouse;

designing a gRNA according to the target site; and

introducing the homologous recombination vector, the gRNA and the Cas9 protein into a fertilized egg of the wild-type mouse, and preparing a genetically modified fertilized egg.

In one embodiment, the target site is located in the region following the start codon of the second exon on the IFNAR2 gene of the mouse.

In one embodiment, the nucleotide sequence of the target site is shown as SEQ ID No. 1;

and/or the nucleotide sequence of the gRNA is shown as SEQ ID NO. 2;

and/or the nucleotide sequence of the homologous arm of the homologous recombinant vector is shown as SEQ ID NO. 3-SEQ ID NO. 4.

In one embodiment, the wild-type mouse is a C57BL/6J mouse.

An expressed gene comprising:

a first functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor 1, and a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor 1;

a second functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor2, and a transcription terminator; and

a linking region for linking said first functional region to said second functional region, said linking region comprising a nucleic acid fragment encoding a protein having self-cleavage function.

A method for constructing a mouse model comprises the following steps:

constructing a genetically modified fertilized egg by adopting the construction method of the genetically modified fertilized egg;

transplanting the genetically modified fertilized egg into a surrogate mouse to prepare a mouse model.

In one embodiment, after the step of transplanting the genetically modified fertilized egg into a surrogate mouse, the method further comprises:

breeding the mouse for surrogate pregnancy to obtain an F0 mouse; and

and (4) screening homozygote of the F0 mouse to obtain a mouse model.

A kit for constructing a mouse model, comprising:

a homologous recombination vector comprising two homology arms capable of undergoing homologous recombination with a target site and an expressed gene located between the two homology arms, the expressed gene comprising a first functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor 1 and a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor 1, a second functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor2 and a transcription terminator, and a joining region for joining the first functional region and the second functional region, the joining region comprises a nucleic acid fragment encoding a protein having a self-cleaving function; and

a gRNA designed according to the target site.

In one embodiment, a Cas9 protein is also included.

In one embodiment, the nucleotide sequence of the target site is shown as SEQ ID No. 1;

and/or the nucleotide sequence of the gRNA is shown as SEQ ID NO. 2;

and/or the nucleotide sequence of the homologous arm of the homologous recombinant vector is shown as SEQ ID NO. 3-SEQ ID NO. 4.

Drawings

FIG. 1 is a schematic diagram of a transgenic strategy for a mouse model according to one embodiment;

FIG. 2 is a schematic diagram showing the structure of a human murine type I interferon chimeric receptor formed after expression of an expressed gene in the embodiment shown in FIG. 1;

FIG. 3 is a schematic diagram of the structure of a wild-type mouse type I interferon receptor;

FIG. 4 is a schematic diagram of primer design in step 2 of test validation;

FIG. 5 is a gel electrophoresis of the PCR product of step 2 of the assay validation;

FIG. 6 is the results of sequencing validation of the Knockin at the 5' end of the homozygote mouse at step 3;

FIG. 7 is the results of sequencing validation of the Knockin at the 3' end of the homozygote mouse at step 3;

FIG. 8 shows the expression of the extracellular domain of human IFNAR2 in IFNAR2 mouse;

FIG. 9 shows the expression of MX1 gene in liver, blood and spleen of IFNAR2 mouse after stimulation by human interferon;

FIG. 10 shows the expression of ISG15 gene in liver, blood and spleen of IFNAR2 mouse after stimulation with human interferon.

Detailed Description

The present invention will now be described more fully hereinafter for the purpose of facilitating an understanding thereof, and may be embodied in many different forms and not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. The terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Interferons are active proteins with broad-spectrum antiviral functions on the same cells, and the activity of the active proteins is regulated and controlled by cell genes. Interferons include type I interferons and type II interferons. The type I interferons include IFN-alpha and IFN-beta, wherein the IFN-alpha has more than twenty subtypes and the IFN-beta has only one subtype. In vivo, the receptor that binds IFN-a and IFN- β is the interferon type I receptor (IFNAR). IFNAR contains a and beta two subunits, called interferon type I receptor 1(interferon alpha receptor 1, IFNAR1) and interferon type I receptor 2(interferon alpha/beta receptor2, IFNAR2), and the structure of IFNAR1 and IFNAR2 includes domains for extracellular, transmembrane and intracellular domains.

Referring to fig. 1, an embodiment of the present invention provides a method for constructing a mouse model, which includes steps a to b:

step a: a CRISPR/Cas9 mediated homologous recombination technology is utilized to knock in an expression gene on a type I interferon receptor gene of a wild mouse to prepare a fertilized egg modified by the gene.

Specifically, the expressed gene includes a first functional region, a second functional region and a joining region.

The first functional region comprises a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1, and a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor 1 and a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor 1, and the first functional region is for expressing the extracellular domain of human type I interferon receptor 1, the transmembrane domain of mouse type I interferon receptor 1 and the intracellular domain of mouse type I interferon receptor 1.

Alternatively, in the first functional region, a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor 1, and a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor 1 are sequentially linked.

In this embodiment, the nucleotide sequence of the nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1 is shown in SEQ ID NO. 5; the nucleotide sequence of the nucleic acid segment of the transmembrane domain of the coding mouse type I interferon receptor 1 is shown as SEQ ID NO. 6; the nucleotide sequence of the nucleic acid segment of the intracellular domain of the mouse type I interferon receptor 1 is shown as SEQ ID NO. 7.

The second functional region comprises a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor2, and a transcription terminator, and is used for expressing the extracellular domain of human type I interferon receptor2, the transmembrane domain of mouse type I interferon receptor2, and the intracellular domain of mouse type I interferon receptor 2.

Alternatively, in the second functional region, a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor2, and a transcription terminator are sequentially linked. The terminator is used to stop transcription. Optionally, the terminator is polyA.

In this embodiment, the nucleotide sequence of the nucleic acid fragment encoding the extracellular domain of human type I interferon receptor2 is shown in SEQ ID NO. 8; the nucleotide sequence of the nucleic acid segment of the transmembrane structural domain of the coding mouse type I interferon receptor2 is shown as SEQ ID NO. 9; the nucleotide sequence of the nucleic acid segment of the intracellular structural domain of the mouse type I interferon receptor2 is shown as SEQ ID NO. 10; the nucleotide sequence of the terminator is shown as SEQ ID NO. 11.

The joining region includes a nucleic acid fragment encoding a protein having a self-cleavage function for joining the first functional region and the second functional region. Optionally, the linking region comprises a nucleic acid fragment encoding a2A peptide. In one embodiment, the nucleotide sequence of the nucleic acid fragment encoding the 2A peptide is set forth in SEQ ID NO.12

More specifically, steps a1 to a3 of preparing a genetically modified fertilized egg:

step a 1: constructing a homologous recombination vector containing the expressed gene according to the target site.

Specifically, the target site is located on the mouse type I interferon receptor gene. Alternatively, the target site is located on the type I interferon 2 gene.

In this embodiment, the target site is located in the region following the start codon of the second exon on the type I interferon receptor 2(IFNAR2) gene. In a specific example, the nucleotide sequence of the target site is shown in SEQ ID No. 1. Of course, in other embodiments, the target site is not limited to the above, but may be located at other positions of the mouse type I interferon receptor gene. So long as it is capable of expressing the human mouse type I interferon chimeric receptor (human type I interferon receptor2 ectodomain-mouse type I interferon receptor2 transmembrane domain-mouse type I interferon receptor2 endodomain and human mouse type I interferon receptor 1 ectodomain-mouse type I interferon receptor 1 transmembrane domain-mouse type I interferon receptor 1 endodomain, resulting in a human mouse type I interferon chimeric receptor) after the expressed gene is knocked in, and does not express the mouse type I interferon receptor.

Specifically, the homologous recombination vector containing the expressed gene includes two homology arms and the above-described expressed gene located between the homology arms. The homology arms are used for homologous recombination with the target site. The homology arms are designed according to target sites. In this embodiment, the nucleotide sequences of the homology arms are shown in SEQ ID NO.3 to SEQ ID NO. 4.

The step of constructing a homologous recombination vector containing an expressed gene includes: the homology arms and the expression genes are inserted into the empty space, and a homologous recombination vector containing the expression genes is prepared.

Step a 2: grnas were designed according to the target site.

In this embodiment, the target site is located close to the second exon of IFNAR2 gene, and the nucleotide sequence of the target site is shown in SEQ ID NO. 1. Therefore, the gRNA in this embodiment is designed based on the target site having the sequence shown in SEQ ID NO.1, wherein the PAM sequence is 5 '-CGG-3'. Alternatively, the nucleotide sequence of the gRNA is shown in SEQ ID No. 2.

Of course, in other embodiments, the nucleotide sequence of the gRNA is not limited to the above, and may be other.

Step a 3: the homologous recombination vector, the gRNA, and the Cas9 protein were introduced into a fertilized egg of a type I interferon receptor-deficient mouse, and a fertilized egg containing the expression gene was prepared.

Specifically, under the guidance of the gRNA, Cas9 protein reaches the target site and breaks the double-stranded DNA of the target site, and simultaneously the repair mechanism of the cell causes homologous recombination between the homologous arm on the homologous recombination vector and the two fragments formed after the target site is broken, so that the expressed gene is connected to the junction of the target site, and the expressed gene knocks into the target site.

In this embodiment, C57BL/6 mice were used as wild-type mice. The C57BL/6 mouse strain is stable and easy to reproduce, is the first mouse strain for completing genome sequencing, is pure in heredity, and can ensure high stability on the genetic background when being applied to gene knockout.

Referring to FIGS. 2 and 3, the extracellular domain of the chimeric human murine type I interferon receptor formed by gene expression (see FIG. 2) is that of the human interferon receptor, compared to the structure of the wild-type mouse type I interferon receptor (see FIG. 3).

Step b: and breeding fertilized eggs containing the expression genes to prepare a mouse model.

Specifically, the step of breeding a genetically modified fertilized egg to prepare a mouse model includes steps b1 to b 2: step b 1: fertilized eggs containing the expressed gene were cultured to prepare F0-generation mice.

Transferring the fertilized eggs into a surrogate mouse body and culturing to obtain an F0 surrogate mouse.

Step b 2: homozygote screening was performed on mice of the F0 generation to prepare a mouse model.

Specifically, homozygous mice capable of stably inheriting the shape were obtained by screening homozygous mice from the F0 generation.

Alternatively, screening for homozygotes is performed by a combination of classical breeding and PCR identification. Specifically, F0-generation mice were bred with wild-type mice to generate F1-generation mice, and positive mice among the F1-generation mice were identified by PCR. F2 generation mice were then generated by sibling mating of positive mice in the F1 generation. Pairs of positive mice were selected from F2 generation mice and mated siblings to yield F3 generation mice. According to the integration rate of each nest mouse and the PCR sequencing result, homozygote mice are selected.

The construction method of the mouse model at least has the following advantages:

through a CRISPR/Cas9 mediated homologous recombination technology, the extracellular domain of the human type I interferon receptor is expressed in a mouse, and the transmembrane domain and the intracellular domain of the mouse type I interferon receptor are reserved, so that the constructed mouse can correctly recognize the human interferon and induce the response of the downstream gene of the mouse. Compared with the traditional liver humanized mouse model, the method for constructing the mouse model has low cost, and the constructed mouse model can respond to the stimulation of human interferon; compared with the traditional transgenic mouse model, the I-type interferon receptor of the mouse model constructed by the mouse model is inactivated, does not interfere the human I-type interferon receptor to recognize human interferon, and can reflect the natural expression mode of the human I-type interferon receptor. Compared with other traditional animal models with farther affinities, the mouse model has closer affinities with human beings and can better reflect the effect of human interferon in vivo.

One embodiment of the present invention provides an expression gene for expressing and forming a chimeric receptor of human murine type I interferon. That is, the expression genes are used to express the extracellular domain of human type I interferon receptor 1, the transmembrane domain of mouse type I interferon receptor 1 and the intracellular domain of mouse type I interferon receptor 1, and to express the extracellular domain of human type I interferon receptor2, the transmembrane domain of mouse type I interferon receptor2 and the intracellular domain of mouse type I interferon receptor2, thereby forming a chimeric human-mouse type I interferon receptor. The specific composition of the expressed gene is as described above.

In addition, an embodiment of the present invention further provides a kit for constructing a mouse model, which corresponds to the method for constructing the mouse model, that is, the kit can be used for constructing the mouse model. Specifically, the kit comprises: homologous recombination vectors and grnas.

Specifically, the homologous recombination vector includes two homology arms and an expressed gene located between the two homology arms. The design and expression of the homology arms are as described above and will not be described in detail here.

In some embodiments, the above kits further comprise a Cas9 protein.

The kit for constructing the mouse model is simple and convenient, and the mouse model prepared by using the kit can truly reflect the natural expression mode of the human interferon receptor and normally respond to the stimulation of the human interferon.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.

Example 1

A schematic diagram of the transgene strategy of Hu-IFNAR2 mice is shown in FIG. 1, and specifically comprises the following steps.

(1) Constructing a homologous recombinant vector: a homologous recombination vector is prepared by inserting an expression gene comprising two homology arms and located between the two homology arms into an empty vector. Wherein: the empty vector is a conventional T vector (pUC-57), the nucleotide sequence of a homologous arm is shown as SEQ ID No. 3-SEQ ID No.4, the expression gene comprises a nucleic acid segment (the nucleotide sequence is shown as SEQ ID No. 5) for coding the extracellular domain of the human type I interferon receptor 1, a nucleic acid segment (the nucleotide sequence is shown as SEQ ID No. 6) for coding the transmembrane domain of the mouse type I interferon receptor 1, a nucleic acid segment (the nucleotide sequence is shown as SEQ ID No. 7) for coding the intracellular domain of the mouse type I interferon receptor 1, a nucleic acid segment (the nucleotide sequence is shown as SEQ ID No. 12) for coding the 2A peptide, a nucleic acid segment (the nucleotide sequence is shown as SEQ ID No. 8) for coding the extracellular domain of the human type I interferon receptor2, a nucleic acid (the nucleotide sequence is shown as SEQ ID No. 9) for coding the transmembrane domain of the mouse type I interferon receptor2, which are connected in sequence, A nucleic acid segment (the nucleotide sequence is shown as SEQ ID NO. 10) for coding the intracellular structural domain of the mouse type I interferon receptor2 and a transcription terminator (the nucleotide sequence is shown as SEQ ID NO. 11).

(2) In vitro, a homologous recombination vector, a gRNA (nucleotide sequence is shown as SEQ ID NO. 2) and a Cas9 protein are introduced into a fertilized egg of mouse C57BL/6J to prepare a fertilized egg containing an expression gene.

(3) The fertilized eggs are transferred into the body and then cultured to obtain F0 generation mice.

(4) Mice from the F0 generation were bred with wild-type mice to generate mice from the F1 generation, and positive mice among the F1 generation of mice were identified by PCR. F2 generation mice were then generated by sibling mating of positive mice in the F1 generation. Pairs of positive mice were selected from F2 generation mice and mated siblings to yield F3 generation mice. And selecting homozygous mice according to the integration rate of each nest mouse and the PCR sequencing result.

Test verification

1. DNA was extracted from a homozygote mouse (hereinafter referred to as IFNAR2 mouse), a heterozygote mouse, and a wild type mouse (WT mouse), and the knock-in position of the homozygote mouse was confirmed:

step 1: extracting DNA of homozygote mice by using a tissue genome DNA extraction kit (Tiangen Biochemical technology), wherein the operation of three types of mice comprises the following steps: 1) taking 20mg of mouse muscle tissue, processing the muscle tissue into cell suspension by using a grinder, centrifuging the cell suspension for 1min at 10000rpm (-11200 Xg), pouring out the supernatant, and adding 200 mu L of buffer solution GA; oscillating until completely suspending; 2) adding 20uL of protease K, mixing uniformly, and standing at 56 ℃ until the tissue is dissolved; 3) adding 200 μ L buffer solution GB, fully reversing and mixing, standing at 70 deg.C for 10min, and cleaning the solution; 4) adding 200 μ L of anhydrous ethanol, shaking thoroughly, mixing for 15s to obtain flocculent precipitate, and centrifuging briefly to remove water drop on the inner wall of the tube cover; 5) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column, centrifuging at 12000rpm (-13400 Xg) for 30s, pouring the waste liquid, and putting the adsorption column back into the collecting pipe; 6) adding 500 mu L of buffer GD into an adsorption column, centrifuging at 12000rpm for 30s, pouring waste liquid, and putting the adsorption column into a collection tube; 7) adding 600 μ L of rinsing solution PW (anhydrous ethanol is added before use) into adsorption column, centrifuging at 12000rpm for 30s, pouring off waste liquid, and placing adsorption column into collection tube; 8) repeating the operation step 7); 9) the adsorption column was returned to the collection tube, centrifuged at 12000rpm for 2min and the waste liquid was decanted. Placing the adsorption column at room temperature for several minutes to thoroughly dry the residual rinsing liquid in the adsorption material; 10) transferring the adsorption column into a clean centrifuge tube, suspending and dropwise adding 50 μ L of RNase-free water to the middle part of the adsorption membrane, standing at room temperature for 3min, centrifuging at 12000rpm for 2min, and collecting the solution into the centrifuge tube.

Step 2: PCR amplification of the DNA fragment to be sequenced:

first, different inserts (5 'end, 3' end and full sequence, the specific schematic diagram is shown in fig. 4, for example, "Knock in fragment" in 4 refers to an expressed gene) are amplified from the extracted DNA by using the PCR technique, and the PCR primer information is shown in table 1 below.

TABLE 1

Then, utilize

Taq 2 Xmix kit (NEB, M0287S) amplifies the corresponding DNA fragment. Wherein, the amplification system is shown in table 2, and the PCR amplification program is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 seconds; annealing at 65 ℃ for 30 seconds; extension is carried out for 72 ℃ (the extension time of the reaction system with primer numbers (i) and (ii) is 3 minutes, and the extension time of the reaction system with primer number (iii) is 5 minutes); the number of cycles was 30.

TABLE 2

Finally, the PCR product was subjected to agarose gel electrophoresis, and the results are shown in FIG. 5. Wild type (Wildtype, WT), homozygote (Homo), and heterozygote (Heterozygous, Heter) have been indicated in the figure).

As can be seen from FIG. 5, the homozygote mouse was successfully constructed and it was confirmed that the genetically modified homozygote mouse can be normally bred.

And step 3: agarose gel DNA recovery: the PCR product is recovered by using a common agarose gel DNA recovery kit (Tiangen Biotechnology), and the specific operations comprise: 1) column balancing: adding 500 μ L of balance liquid BL into the adsorption column, centrifuging at 12000rpm for 1min, pouring off waste liquid in the collection tube, and replacing the adsorption column into the collection tube; 2) cutting a single target DNA band from the agarose gel (cutting off redundant parts as much as possible), putting the single target DNA band into a clean centrifugal tube, and weighing; 3) adding equal volume of PN sol solution (if the weight of the gel is 0.1g, and the volume can be regarded as 100 muL, then 100 muL of PN sol solution) into the gel block, placing in a water bath at 50 ℃, and continuously and gently turning the centrifugal tube up and down during the placement to ensure that the gel block is fully dissolved; 4) adding the solution obtained in the previous step into an adsorption column (the adsorption column is placed into a collecting pipe), standing at room temperature for 2min, centrifuging at 12000rpm for 60s, pouring off waste liquid in the collecting pipe, and placing the adsorption column into the collecting pipe; 5) adding 600 mu L of rinsing liquid PW into the adsorption column, centrifuging at 12000rpm for 60s, pouring off waste liquid in the collecting pipe, and putting the adsorption column into the collecting pipe; 6) repeating the operation step 5); 7) the adsorption column was returned to the collection tube and centrifuged at 12000rpm for 2min to remove the rinse as much as possible. Placing the adsorption column at room temperature for several minutes, and completely drying; 8) placing the adsorption column in a clean centrifuge tube, suspending and dropwise adding 50 μ L of RNase-free water to the middle position of the adsorption membrane, and standing at room temperature for 2 min. The DNA solution was collected by centrifugation at 12000rpm for 2 min.

And 4, step 4: the recovered product was sent to the test, and the results are shown in FIGS. 6 to 7. FIG. 6 is the results of Knock in at the 5 'end of homozygote mice, and FIG. 7 is the results of Knock in at the 3' end of homozygote mice.

As can be seen from FIGS. 6 and 7, the homozygote Knock in is correct.

2. For the expression of the extracellular domain of human IFNAR2 in homozygous mice (IFNAR2 mice):

extracting liver tissue RNA of a homozygote mouse and a WT mouse by using a tissue total RNA extraction kit (Tiangen biochemical technology), and specifically comprising the following operations:

1) approximately 20g of each of the liver tissues from IFNAR2 and WT mice were removed, the tissues were thoroughly ground with a pestle, 300. mu.L of lysis solution RL (1% β -mercaptoethanol) was added, and 590. mu.L of RNase-Free ddH was added to the homogenate₂Mixing O and 10 μ L protease K, and treating at 56 deg.C for 20 min; 2) centrifuging at 12000rpm for 5min, collecting supernatant, slowly adding 0.5 times of anhydrous ethanol, mixing, transferring the obtained solution and precipitate into adsorption column, centrifuging at 12000rpm for 60s, removing waste liquid in the collection tube, and placing the adsorption column back into the collection tube; 3) adding 350 μ L deproteinized solution RW1 into adsorption column, centrifuging at 12000rpm for 360s, discarding waste liquid, and placing adsorption column back into collection tube; 4) add 80. mu.L of DNase I working solution to the center of the adsorption column (preparation of DNase I working solution: putting 10 μ L DNase I stock solution into a new RNase-Free centrifuge tube, adding 70 μ L RDD buffer solution, gently mixing, and standing at room temperature for 15 min; 5) adding 350 μ L deproteinized solution RW1 into adsorption column, centrifuging at 12000rpm for 60s, discarding waste liquid, and placing adsorption column back into collection tube; 6) adding 500 μ L of rinsing solution RW into adsorption column, standing at room temperature for 2min, centrifuging at 12000rpm for 60s, discarding the waste liquid, and placing the adsorption column back into the collection tube; 7) repeating step 6); 8) centrifuging at 12000rpm for 2min, and discarding waste liquid. Placing the adsorption column at room temperature for several minutes to thoroughly air-dry the residual rinsing liquid in the adsorption material; 9) transferring the adsorption column into a new RNase-Free centrifuge tube, suspending and dropwise adding 50 μ L of RNase-Free ddH2O into the middle part of the adsorption membrane, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to obtain RNA solution.

2) The level of human IFNAR2 RNA in homozygous mice (IFNAR2 mice) was determined by the steps of:

a. extracting IFNAR2 and WT mouse liver RNA by using an RNA extraction kit;

b. reverse transcribing each RNA to cDNA, respectively;

c. detection was performed using Bio-rad SYBR detection reagent. Wherein the primer sequence of human IFNAR2 is F: CACAAGCCTGAGATCAAG (SEQ ID NO.19), R: TAGACAGAGACACAGTAGTT (SEQ ID NO. 20). The results are shown in FIG. 8, the ordinate represents the human IFNAR2 gene expression fold.

As can be seen from FIG. 8, human IFNAR2 was expressed in homozygous mice.

3. Functional identification of the human IFNAR2 receptor in homozygous mice (IFNAR2 mice):

(1) homozygote mice were injected subcutaneously with interferon PEG-IFNa2a (trade name: Peruoxin, Roche) at a dose of 10ug/kg, and 16 hours after injection, the mice were euthanized and blood and organs were collected.

(2) The RNA extraction kit is used for extracting the RNA of blood, liver and spleen of IFNAR2 mice.

(3) Each RNA was reverse transcribed to cDNA, respectively.

(4) Detection was performed using Bio-rad SYBR detection reagent. Wherein: MX1 gene and ISG15 gene are target genes, and the upstream primer of MX1 gene is: AAGATGGTCCAAACTGCCTTCG (SEQ ID NO. 21); downstream primer of MX1 gene: GCCTTGGTCTTCTCTTTCTCAGC (SEQ ID NO. 22); upstream primer of ISG15 gene: AACTGCAGCGAGCCTCTGA (SEQ ID NO. 23); downstream primer of ISG15 gene: CACCTTCTTCTTAAGCGTGTCTACAG (SEQ ID NO.24), and GAPDH (upstream primer: AGGTCGGTGTGAACGGATTTG (SEQ ID NO. 25); downstream primer: TGTAGACCATGTAGTTGAGGTCA (SEQ ID NO.26)) as an internal reference gene. The reaction liquid for RT-qPCR was as follows:

the RT-qPCR reaction system is as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 s; annealing at 60 ℃; 30 s; extension at 65 ℃ for 30 s; 35 cycles.

As a result, as shown in FIGS. 9 and 10, FIG. 9 shows the expression of MX1 gene in liver (liver), blood (blood) and spleen (spleens) in IFNAR2 mice after human interferon stimulation, and FIG. 10 shows the expression of ISG15 gene in liver (liver), blood (blood) and spleen (spleens) in IFNAR2 mice after human interferon stimulation. In FIG. 9, the ordinate represents the expression fold of MX1 gene, and in FIG. 10, the ordinate represents the expression fold of ISG15 gene.

As can be seen from FIGS. 9 and 10, IFNAR2 mouse constructed in example 1 can activate the response of mouse downstream gene under the stimulation of human interferon, i.e. can normally respond to the stimulation of human interferon.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> eighth national hospital in Guangzhou City

GUANGZHOU FURONG BIOTECHNOLOGY Co.,Ltd.

<120> expression gene, method for constructing genetically modified fertilized egg, and method for constructing mouse model

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agctgagcag gatgcgttca 20

<210> 2

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agctgagcag gatgcgttca cgg 23

<210> 3

<211> 304

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

caaacttgct atgtagccga ggatgacctt gaatgcctga cctttccccc acctctctgc 60

ttagaggaca gatgtgacac gcacagtaaa ctcatgcaag tttaacccta atcctaacca 120

atccagggct accacggggc cgcatctgca gctaaatctg gctcgttctt actcgtctct 180

cgttagcgtg tatgtgtcta tcatgtaaat tacaatataa ttgggtgctt ctgagttttg 240

accaactcaa tattgatctc tttcaggtgt gagagcagaa aaacggactt aagagctgag 300

cagg 304

<210> 4

<211> 420

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttgtgggtaa gggctacttc tcagcacagc ccttagagga gaaagcctct gtttctgtca 60

tcacagagag ccctggtgtg gagcagcaca ctgatgtcca tatctggaga acccagatca 120

gcacggccag catcaggcac cccacggggg tcttcccctt cattttagct aagccagaat 180

aatataggct acagccatat tcaggaaacg gccttgttta taattcaaag ggttgcggct 240

ctgcacaccc tgaatctcac gcccggtggc gtttagaagg tggccatccc tttatctctt 300

cccatataaa ctaacttgaa aaatccatcc ctacacattg atttatactc ttcctttctt 360

tttagccctt atcttttcta tatctgtatt tcttcacgtc ctttctgttc cttcctctga 420

<210> 5

<211> 1296

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgatggtgg tgctgctggg cgccaccacc ctggtgctgg tggctgtggc cccatgggtg 60

ctgtctgctg ctgctggcgg caagaacctg aagtcccccc agaaggtgga ggtggacatc 120

attgatgaca acttcatcct gaggtggaac aggtctgatg agtctgtggg caatgtgacc 180

ttctcctttg actaccagaa gacaggcatg gacaactgga tcaagctgtc tggctgccag 240

aacatcacct ccaccaagtg caacttctcc tccctgaaac tgaatgtcta tgaggagatc 300

aagctgagga tcagggctga gaaggagaac acctcctcct ggtatgaggt ggactccttc 360

accccattcc gcaaggccca gattggcccc cctgaagtgc atctggaggc tgaggacaag 420

gccattgtga tccacatctc ccctggcacc aaggactctg tgatgtgggc tctggatggc 480

ctgtccttca cctactccct ggtgatctgg aagaactcct ctggcgtgga ggagaggatt 540

gagaacatct actccaggca caagatctac aagctgtccc ctgagaccac ctactgcctg 600

aaggtgaagg ctgccctgct gacctcctgg aagattggcg tctactcccc tgtgcactgc 660

atcaagacca cagtggagaa tgagctgccc ccccctgaga acattgaggt ctctgtgcag 720

aaccagaact atgtgctgaa gtgggactac acctatgcca acatgacctt ccaagtgcag 780

tggctgcatg ccttcctgaa gaggaaccct ggcaaccatc tgtacaagtg gaagcagatc 840

cctgactgtg agaatgtgaa gaccacccag tgtgtcttcc cccaaaatgt cttccagaag 900

ggcatctacc tgctgagggt gcaggcctct gatggcaaca acacctcctt ctggtctgag 960

gagatcaagt ttgacacaga gatccaggcc ttcctgctgc cccctgtctt caacatccgc 1020

tccctgtctg actccttcca catctacatt ggcgccccca agcagtctgg caacacccct 1080

gtgatccagg actaccccct gatctatgag atcatcttct gggagaacac ctccaatgct 1140

gagaggaaga tcattgagaa gaagacagat gtgacagtgc ccaacctgaa gcccctgaca 1200

gtctactgtg tgaaggccag ggctcacacc atggatgaga agctgaacaa gtcctctgtc 1260

ttctctgatg ctgtctgtga gaagaccaag cctggc 1296

<210> 6

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tccttctcca ccatctggat catcaccggc ctgggcgtgg tcttc 45

<210> 7

<211> 450

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttctctgtga tggtgctgta tgccctgagg tctgtctgga agtacctgtg ccatgtctgc 60

ttcccccccc tgaaaccccc ccgctccatt gatgagttct tctctgagcc cccatccaag 120

aacctggtgc tgctgacagc tgaggagcac acagagcgct gcttcatcat tgagaacaca 180

gacacagtgg ctgtggaggt gaagcatgcc cctgaggagg acctgaggaa gtactcctcc 240

cagacctccc aagactctgg caactactcc aatgaggagg aggagtctgt gggcacagag 300

tctggccagg ctgtgctgtc caaggcccca tgtggcggcc catgctctgt gccatccccc 360

cctggcaccc tggaggatgg cacctgcttc ctgggcaatg agaagtacct gcagtcccct 420

gccctgagga cagagcctgc cctgctgtgc 450

<210> 8

<211> 693

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgctgctgt cccagaatgc cttcatcttc cgctccctga acctggtgct gatggtctac 60

atctccctgg tctttggcat ctcctatgac tcccctgact acacagatga gtcctgcacc 120

ttcaagatct ccctgaggaa cttcaggtcc atcctgtcct gggagctgaa gaaccactcc 180

attgtgccca cccactacac cctgctgtac accatcatgt ccaagcctga ggacctgaag 240

gtggtgaaga actgtgccaa caccaccagg tccttctgtg acctgaccga tgagtggagg 300

tccacccatg aggcctatgt gacagtgctg gagggcttct ctggcaacac caccctgttc 360

tcctgctccc acaacttctg gctggccatt gacatgtcct ttgagccccc tgagtttgag 420

attgtgggct tcaccaacca catcaatgtg atggtgaagt tcccatccat tgtggaggag 480

gagctgcagt ttgacctgtc cctggtgatt gaggagcagt ctgagggcat tgtgaagaag 540

cacaagcctg agatcaaggg caacatgtct ggcaacttca cctacatcat tgacaagctg 600

atccccaaca ccaactactg tgtctctgtc tacctggagc actctgatga gcaggctgtg 660

atcaagtccc ccctgaagtg caccctgctg ccc 693

<210> 9

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cctggccagg agtctgagtc tgctgagtct gccattgtgg gcatc 45

<210> 10

<211> 801

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

accacctcct gcctggtggt gatggtcttt gtctccacca ttgtgatgct gaagaggatt 60

ggctacatct gcctgaagga caacctgccc aatgtgctga acttcaggca cttcctgacc 120

tggatcatcc ctgagaggtc cccatctgag gccattgaca ggctggagat catccccacc 180

aacaagaaga agaggctgtg gaactatgac tatgaggatg gctctgactc tgatgaggag 240

gtgcccacag cctctgtgac aggctacacc atgcatggcc tgacaggcaa gcccctgcag 300

cagacctctg acacctctgc ctcccctgag gaccccctgc atgaggagga ctctggcgct 360

gaggagtctg atgaggctgg cgctggcgct ggcgctgagc ctgagctgcc cacagaggct 420

ggcgctggcc catctgagga ccccacaggc ccatatgaga ggaggaagtc tgtgctggag 480

gactccttcc ccagggagga caactcctcc atggatgagc ctggcgacaa catcatcttc 540

aatgtgaacc tgaactctgt cttcctgagg gtgctgcatg atgaggatgc ctctgagacc 600

ctgtccctgg aggaggacac catcctgctg gatgagggtc cccagaggac agagtctgac 660

ctgaggattg ctggcggcga caggacccag ccccccctgc catccctgcc atcccaagac 720

ctgtggacag aggatggctc ctctgagaag tctgacacct ctgactctga tgctgatgtg 780

ggcgatggct acatcatgag g 801

<210> 11

<211> 537

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa tgccctggct 60

cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat catgaagccc 120

cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg 180

aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag 240

aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa 300

ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc 360

catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt 420

tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc 480

tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga cctgcag 537

<210> 12

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggctctggcg ccaccaactt ctccctgctg aagcaggctg gcgatgtgga ggagaaccct 60

gggccc 66

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ggtggctacc gtaatgtcgg ta 22

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

agcctccaga tgcacttcag gg 22

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tggacaactg gatcaagctg tc 22

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggcccagact gtcaacatta ctct 24

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

cttagaggac agatgtgaca cgc 23

<210> 18

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gacatcagtg tgctgctcca cac 23

<210> 19

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cacaagcctg agatcaag 18

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tagacagaga cacagtagtt 20

<210> 21

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

aagatggtcc aaactgcctt cg 22

<210> 22

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gccttggtct tctctttctc agc 23

<210> 23

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

aactgcagcg agcctctga 19

<210> 24

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

caccttcttc ttaagcgtgt ctacag 26

<210> 25

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

aggtcggtgt gaacggattt g 21

<210> 26

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

tgtagaccat gtagttgagg tca 23

Claims (10)

1. A method for constructing a genetically modified fertilized egg, comprising the steps of:

knocking in an expression gene on a type I interferon receptor gene of a wild mouse by using a CRISPR/Cas9 mediated homologous recombination technology to prepare a genetically modified fertilized egg, wherein the expression gene comprises:

a first functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor 1, and a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor 1;

a second functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor2, and a transcription terminator; and

a linking region for linking said first functional region to said second functional region, said linking region comprising a nucleic acid fragment encoding a protein having self-cleavage function.

2. The method of constructing according to claim 1, wherein the step of preparing the genetically modified fertilized egg comprises:

constructing a homologous recombination vector containing an expressed gene according to a target site located on the type I interferon receptor gene of the wild type mouse;

designing a gRNA according to the target site; and

introducing the homologous recombination vector, the gRNA and the Cas9 protein into a fertilized egg of the wild-type mouse, and preparing a genetically modified fertilized egg.

3. The method of claim 2, wherein the target site is located in the region of the mouse after the start codon of the second exon in IFNAR2 gene.

4. The construction method according to claim 3, wherein the nucleotide sequence of the target site is shown in SEQ ID No. 1;

and/or the nucleotide sequence of the gRNA is shown as SEQ ID NO. 2;

and/or the nucleotide sequence of the homologous arm of the homologous recombinant vector is shown as SEQ ID NO. 3-SEQ ID NO. 4.

5. The method of constructing a recombinant human factor according to any one of claims 1 to 4, wherein the wild-type mouse is a C57BL/6J mouse.

6. An expressed gene, comprising:

a first functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor 1, and a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor 1;

a second functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor2, and a transcription terminator; and

a linking region for linking said first functional region to said second functional region, said linking region comprising a nucleic acid fragment encoding a protein having self-cleavage function.

7. A method for constructing a mouse model is characterized by comprising the following steps:

constructing a genetically modified fertilized egg by the method for constructing a genetically modified fertilized egg according to any one of claims 1 to 5;

transplanting the genetically modified fertilized egg into a surrogate mouse to prepare a mouse model.

8. The method of constructing according to claim 7, further comprising, after the step of transplanting the genetically modified fertilized egg into a surrogate mouse:

breeding the mouse for surrogate pregnancy to obtain an F0 mouse; and

and (4) screening homozygote of the F0 mouse to obtain a mouse model.

9. A kit for constructing a mouse model, comprising:

a homologous recombination vector comprising two homology arms capable of undergoing homologous recombination with a target site and an expressed gene located between the two homology arms, the expressed gene comprising a first functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor 1, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor 1 and a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor 1, a second functional region comprising a nucleic acid fragment encoding the extracellular domain of human type I interferon receptor2, a nucleic acid fragment encoding the transmembrane domain of mouse type I interferon receptor2, a nucleic acid fragment encoding the intracellular domain of mouse type I interferon receptor2 and a transcription terminator, and a joining region for joining the first functional region and the second functional region, the joining region comprises a nucleic acid fragment encoding a protein having a self-cleaving function; and

a gRNA designed according to the target site.

10. The kit according to claim 9, wherein the nucleotide sequence of the target site is shown in SEQ ID No. 1;

and/or the nucleotide sequence of the gRNA is shown as SEQ ID NO. 2;

and/or the nucleotide sequence of the homologous arm of the homologous recombinant vector is shown as SEQ ID NO. 3-SEQ ID NO. 4.

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