CN109929875B - Construction method and application of LAG3 gene humanized animal model - Google Patents

Abstract

The invention provides a method for preparing a LAG3 humanized animal model and application thereof, the method replaces part of a mouse Lag3 gene coding extracellular region with a corresponding human LAG3 gene fragment by using a CRISPR/Cas9 gene editing technology, and simultaneously reserves an intracellular signal transduction region of the mouse Lag3 gene, so that correct conduction of cell signals is not influenced. The model is an ideal animal model for screening human LAG3 inhibitor drugs.

Description

Construction method and application of LAG3 gene humanized animal model

Technical Field

The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a construction method of a LAG3 gene modified humanized animal model.

Background

Complex biological processes often require in vivo analysis, and studies of in vivo biology in humans are severely limited by ethics and technology, and thus animal models are increasingly needed for in vivo studies of human cells, tissues and organs. Currently, scientists have developed a variety of humanized mice or human murine chimeras to overcome these limitations and have now become an important tool for in vivo studies of human cells and tissues.

In the process of clinical drug development, mice are widely used for preclinical safety and effectiveness evaluation of candidate drugs, such as in vivo effectiveness evaluation of novel antiviral drugs, immunotherapy of tumors and development of novel chemotherapeutic drugs, therapeutic effects of novel anti-autoimmune drugs, in vivo metabolism and hepatotoxicity of drugs, preliminary development and evaluation of humanized antibodies, and the like.

However, most of the candidate drugs evaluated in the preclinical stage I fail in the clinical stage I, and the difference in physiology, pathology, etc. between mice and humans is an important factor: differences in the human and mouse immune systems, germ line specificities of many pathogenic factors and drugs between human and mouse, differences in affinities of drug targets and receptors between human and mouse, differences between human and mouse derived tumors, and the like all affect the consistency of preclinical and clinical data. Therefore, the construction of a humanized mouse model with human functional genes, cells and tissues has important significance, and clinical data can be expected in a better response along with the continuous improvement of the humanized mouse.

Anti-tumor immunotherapy has raised the research enthusiasm in the last two years, and many drugs of Checkpoint have been clinically approved and have achieved favorable performance. LAG3(Lymphocyte activation protein 3), Lymphocyte activation protein 3, is expressed on activated T lymphocytes, B lymphocytes, natural killer cells, and plasmacytoid dendritic cells. The membrane protein encoded by the LAG3 gene, which comprises four immunoglobulin superfamily domains, is very similar to CD4 in structure and chromosomal localization, but binds with higher affinity to MHC II (major histocompatibility complex class II molecules) and down-regulates T cell function.

Studies have shown that knocking out the LAG3 gene or in vivo antibodies blocks LAG3 function, resulting in increased aggregation and effector function of antigen-specific CD8+ T cells in organs or tumors. Therefore, blocking LAG3 is a potential tumor treatment.

The applicant replaces part of the encoding extracellular region of the mouse Lag3 gene with a corresponding human LAG3 gene segment by using a gene editing technology, and simultaneously reserves an intracellular signal transduction region of the mouse Lag3 gene, so that the correct conduction of cell signals is not influenced. The model is an ideal animal model for screening human LAG3 inhibitor drugs.

In the process, sgRNA aiming at a mouse Lag3 gene and a vector carrying a human LAG3 gene fragment are constructed, all elements used in the CRISPR/Cas9 technology, including sgRNA and the like, are fully optimized and adjusted, and the high success rate and the high accuracy rate of preparing a humanized LAG3 gene animal model by adopting the technology are ensured.

Disclosure of Invention

The invention provides a method for preparing a humanized animal model of LAG3, which is characterized in that an extracellular region of an animal LAG3 gene is replaced by a corresponding fragment of a human LAG3 gene, and an intracellular signal transduction region of animal LAG3 is reserved in the humanized animal.

Preferably, it comprises the following steps:

(1) constructing a plasmid for expressing sgRNA aiming at the murine Lag3 gene;

(2) constructing a targeting vector of the humanized LAG3 gene;

(3) and (3) injecting sgRNA obtained by in vitro transcription of the plasmid in the step (1), the vector in the step (2) and Cas9mRNA or Cas9 protein into cytoplasm or nucleus of a mouse fertilized egg, and transplanting the sgRNA into a recipient mother mouse to produce a LAG3 gene modified humanized mouse model.

Preferably, wherein the humanized LAG3 gene comprises the chimeric sequence SEQ ID NO: 10.

preferably, the sequence of sgRNA aiming at the murine Lag3 gene is shown as SEQ ID NO. 2 and SEQ ID NO. 6.

Preferably, the method further comprises the step of identifying the genotype of the animal model using primers.

Preferably, the primers used to identify the genotype of the animal model are:

primer name	Primer sequences
		YF000087-HuLAG3-5tF1	AGTAGGGTTAAGGACTAGACAGC(SEQNo.11)
YF000087-HuLAG3-5tR1	CTTGGGTTTCTGTTTCTTCTCTG(SEQNo.12)
		YF000087-HuLAG3-3tF1	GACCCAAACGCCACAGCAATAATC(SEQNo.13)
YF000087-HuLAG3-3tR1	TGCTTTCCTTCTCTGTAGCCAG(SEQNo.14)
		YF000087-HuLAG3-wt-tF4	TCTCCATCACGTACAACCTCAAG(SEQNo.15)
YF000087-HuLAG3-wt-tR4	CATGACTGTCTGTAGGTCAAGTTC(SEQNo.16)

。

Preferably, the method further comprises sequencing verification of mouse clones positive for both-end PCR identification.

Wherein the primers used for the sequencing are as follows:

further, the invention also provides application of the method in evaluating the effectiveness of the targeted LAG3 drug, screening and developing the LAG3 targeted drug, evaluating the antitumor effect of the LAG3 targeted drug in combination with other drugs, or in toxicological studies of the LAG3 targeted drug.

The invention also provides sgRNA of the specific target mouse Lag3 gene, and the sequence of the sgRNA is shown as SEQ ID NO. 2 and SEQ ID NO. 6.

The invention also provides a chimeric LAG3 gene, the sequence of which is shown as SEQ ID NO: shown at 10.

The invention has the following positive effects:

1. the repair donor containing a mouse homologous sequence as a homologous recombination repair template is injected into a mouse fertilized egg together with the Cas9/sgRNA, so that the probability of homologous recombination repair is increased, and the probability of obtaining a positive LAG3 humanized mouse is improved; the mice prepared using this method do not contain other foreign sequences than the sequence of interest.

2. According to the LAG3 humanized mouse constructed by the method, the extracellular region of the mouse Lag3 gene is replaced by a human sequence, and the intracellular region retains the complete murine sequence. The humanized animal model with the humanized extracellular region is successfully prepared, and humanized immune checkpoint drugs (such as neutralizing antibodies) can be screened; the murine intracellular domain ensures that intracellular signal transduction is not affected, and external stimuli are faithfully transformed into intracellular behaviors (activation or inhibition).

3. The LAG3 humanized mouse can be used for evaluating the drug effect of antibody tumor and has important guiding significance for the clinical effect of drug development. The LAG3 humanized mouse can be used for single drug evaluation of an anti-human LAG3 antibody, can also be used for evaluation of the antitumor effect of the LAG3 antibody combined with other drugs (small molecules or antibody drugs), and has profound guiding significance for evaluation of the effect of a clinical prodrug.

4. The invention provides a specific operation method for preparing an optimized human LAG3 gene animal model, which optimizes the steps of sgRNA, vector preparation and the like to the maximum extent and ensures the success rate of the animal model.

Drawings

FIG. 1 is an electrophoresis chart of genotype identification at generation F0. 73/78/83/99/110/111/123 PCR was performed using the mouse tail DNA of accession number F0 as a template; b6 as negative control, using C57BL/6 mouse tail DNA as template to carry out PCR; n is blank control, no template control; p is positive plasmid control;

TRANS2K PLUS II band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp.

FIG. 2 is an electrophoresis chart of genotype identification at generation F0. 151/152/155/157/168/179/183/197/204 PCR was performed using the mouse tail DNA of accession number F0 as a template; b6 as negative control, using C57BL/6 mouse tail DNA as template to carry out PCR; n is blank control, no template control; p is positive plasmid control;

TRANS2K PLUS II band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp.

FIG. 3 is an electrophoretogram for genotyping F1. 185/186/188/203/197/199/200/191/194/195 PCR was performed using the mouse tail DNA of accession number F1 as a template; b6 as negative control, using C57BL/6 mouse tail DNA as template to carry out PCR; n is blank control, no template control; p is positive plasmid control;

TRANS2K PLUS II band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp.

Fig. 4 is a mouse LAG3 protein expression assay.

FIG. 5 shows the measurement of the T/B/NK cell ratio in mice.

Figure 6 is the antitumor efficacy reactivity results of tumor-bearing LAG3 humanized mice against human LAG3 antibody: (a) a schematic representation of tumor growth curves; (b) schematic diagram of the body weight change curve of the mouse.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

Example 1: LAG3 humanized mouse model establishment

The human LAG3 gene Exon1, Exon2, Exon3, Exon4, Exon5, Exon6 and Exon7 are replaced by mouse Lan 3 gene Exon1, Exon2, Exon3, Exon4, Exon5, Exon6 and Exon7 in a CRISPR Cas9 mode to establish a LAG3 gene humanized mouse model. The C57BL/6 mouse is a mature mouse strain in the current research, the C57BL/6 mouse is a background mouse, and a LAG3 humanized mouse model is successfully obtained.

1. Determining human source fragment replacement region and inserted human source sequence

According to the functional domain of the human LAG3 protein, selecting Exon1, Exon2, Exon3, Exon4, Exon5, Exon6 and Exon7 of a human LAG3 sequence to replace Exon1, Exon2, Exon3, Exon4, Exon5, Exon6 and Exon7 of murine LAG3, and reserving a signal peptide, a transmembrane region and an intracellular region of the mouse LAG3 sequence, wherein the selected human LAG3 gene sequence is shown as SEQ ID No. 1.

SEQ ID No.1

atgtgggaggctcagttcctgggcttgctgtttctgcagccgctttgggtggctccaggtaaaacggggatggcgggagggttgacctccagccccacaggaggggaccagcagggatctctgtggccacaaaggtcctgaggtccttagctctgtggattcttctaatcccttttttgggcagtccttccaccccgaaagcctctctgggcagagaagaaacagaaacccaagttcttcctgcaccctgtttctccctcgggaaacacccaggctccttctctacccctgcctctcggctcacgccccctccccttggcctctcttttgctcacctagtgaagcctctccagccaggggctgaggtcccggtggtgtgggcccaggagggggctcctgcccagctcccctgcagccccacaatccccctccaggatctcagccttctgcgaagagcaggggtcacttggcagcatcagccagacaggtatgcaccccaaacttgggcaacaggacctccgaatccagcactcaaccccacacccgtgccggtcctctgtcccctgccctgaggtgtcactccctctgaagccagtgacccagtctccctgccctcgcttgcaccgttcctgcccttgctctgcaatcagcgaccctcacgccagcatcccttctctccagaagtggatgcggccagtccaacagaggggtcgggcgtgaggggacggttggtggtcaagagaactcttggggcgggctttctcatcctcaacgggtggctgcctgcatcctcccgggcttcctacccctggagcttctcaactccattctctttcccgcccagtggcccgcccgctgccgcccccggccatcccctggcccccggccctcacccggcggcgccctcctcctgggggcccaggccccgccgctacacggtgctgagcgtgggtcccggaggcctgcgcagcgggaggctgcccctgcagccccgcgtccagctggatgagcgcggccggcagcgcggggacttctcgctatggctgcgcccagcccggcgcgcggacgccggcgagtaccgcgccgcggtgcacctcagggaccgcgccctctcctgccgcctccgtctgcgcctgggccaggcctcgagtatgtggggcgggacgatgggagaagggctgggaggtgggtccccatcccctgcctcccgggacgcaggaagggctggggcagaggctgcgccctaggccctgtcggagagctcccagaagagtagaggaagggggtgggcggcctgctggagtggaaggtgcccccgaagcacgtgtatggggggccctgtggagagattgtgtcacccccgagctccccttctcccacccacgcgggagtgcccagagggagggggagggggggagagcatggggctaaagtgattcatttcagatatctgtagctcagggggtgggcttcgcggggttccaggccaggaaaacggcaagggtggctgatgccaagtaaactccaggccagggacggggaaagtggtcctggggagtcttggggatccactttatgcacctccaggtgctggaagctgagatggggagagggtgatgtgggagaggagaagacaagtctaaagccaggtgcctgtttccaggagcttccggcttggcagtcctgctgtgttgggaaattgtttccagtgggctgatgaagtcttctttatccttgcacagtgactgccagccccccaggatctctcagagcctccgactgggtcattttgaactgctccttcagccgccctgaccgcccagcctctgtgcattggttccggaaccggggccagggccgagtccctgtccgggagtccccccatcaccacttagcggaaagcttcctcttcctgccccaagtcagccccatggactctgggccctggggctgcatcctcacctacagagatggcttcaacgtctccatcatgtataacctcactgttctgggtaactcccccactctgcttcacatttgaccacaactccttcctgccccccttgtcacctcccctaactatgggtccccaaaccaggttctcggcagcgagtggcctacgtcattgctgtgggtctcactgttcgacccctttatattgctggcagcctcacagctgccatcaccccttcttgcttctcccgtggccttccagcgtcattgccggccttccctctccttccggctaagcccacttgctgggtttctgagcctcctcagctcatcaccttattctgctccttagcactcttatgagccagaccatctcctgaattcttctgcctcccttccttgcagccccagcactccctccccactgcagcacccagctttaactttgggttttcttttctcttcaggtctggagcccccaactcccttgacagtgtacgctggagcaggttccagggtggggctgccctgccgcctgcctgctggtgtggggacccggtctttcctcactgccaagtggactcctcctgggggaggccctgacctcctggtgactggagacaatggcgactttacccttcgactagaggatgtgagccaggcccaggctgggacctacacctgccatatccatctgcaggaacagcagctcaatgccactgtcacattggcaatcatcacaggtcagcctcaggtgggaaaggagtagctgccctcccagggtagaaaggacagggaggaagggctggcagggcaaagactaggcaaacccaccctgtgatgccaggccactgggcacaagttccagagcctgcccatctcggcccccacttttctcacccccataataaagaaacgaaactgaaaatctcctcttgagtcacaagataaaagttccaccgttctctatgggactcccctgctctcaattggcgggagggtctgggaagttagaaggaaaggtgacaaaaattctgaatggttcgaaagaggtagaatatatttctagaatccttgtctactttgcagccagggcttgggttagagttgcaggaagtggcctggatttgggaggagtgaataaatccgtcccttggtcagcaaatatttactgagcaagggttttccaagacagtataaaacaaacacagaaaaaagaatactcagagagtatgtgttgactggttgataactatcggccatgacagattagccatgtctgcagcacgcacctgcggccactcagtagtagcaccccacggcaggtgcttaataatgtatagagattgaatgaatacgtgaacatgctaatggataatacatctcctgaaggccaatcctgagttttcacttgctttctggatacctctaactagatgttataccacctctcagccgccttaccctgaaccccagctttttctcccaccaatccccttgctgactcagcctgtcagtaatccactggtatccatccacctcgaaacctggcctcctcctctctacatcccatcagtcatcaaagtccaccagttctttctgtccaatgccctcttgaaggtcaactgttttatgtagacggctcaatgagagagatactgttaccccgaagactttctcaaaacagctgggaagtgccagactctgggtctaaactcaggtgtctgcatcaccaaatcactctccaggacactactcagatgcccttcaccctttgtctcctcccacccacccatctctcactttacaacttggagaatgctctgtctctagatgaatgggtcagttccctgttctctataccttcagaaagggggatgatgttcattcagggggcaccacaaggcaggggacactgatgtgagccgcatcatggaagttccaaaattgtgaagtagatgggggtcagctcatctgactgattttgagggttctgagcatttgagtagaaagtaatgtaaaagaatctgtaatagtatttattctgtggtctgccagggccagtttgagtgtgaaggcagtagggagcctggaaatgttttgagcaggggagggagctgactagagcattgtctgaataagataaatgtggccggcagggcatgatggctcacacctgtaatcccagcactttgaaaggctgagacgagaggagcacttgagtccaggaattccagactagcctgggcaacatggtgaaaacgcatctgtatacaatttaataaaaaaataaatagaaagaaaaagatgaacgtggccatgacccatgatgtccttccccaacctcccctggccagaacccaagtgagtgcagggtgattgagacttggggttcaacctgtgatatcacgtaaggggggcagaatcccaaaagatctcttccttctactcttttcagtgactcccaaatcctttgggtcacctggatccctggggaagctgctttgtgaggtgactccagtatctggacaagaacgctttgtgtggagctctctggacaccccatcccagaggagtttctcaggaccttggctggaggcacaggaggcccagctcctttcccagccttggcaatgccagctgtaccagggggagaggcttcttggagcagcagtgtacttcacagagctgtctagcccaggtccgaggccccagaatgccaggacccaaacgccacagcaataatctttatcctccttccagaacagcccctgccacccccatcccagcgcttttctttccagtcagggacctgatctctggcttttgcctagggttgacccgtttccgccactgatcagctgaatgaccctgggacaagtcccttaaactctctggatctcattgcatctgtaaagtctgagagaatgacaaagtgtcctttctagtcctgaccctatgcctcatcctgtactttctccataggtgcccaacgctctgggagagccccaggtgccctcccagcaggccacctc

2. sgRNA screen prepared from LAG3 humanized mice

The design of the sgRNA is not mature enough at present, is not easy to realize like the design of a primer, and needs to be screened and adjusted through a large number of experiments, so that the used sgRNA sequence is optimized to the maximum extent to ensure the accuracy and efficiency of targeting.

The approximate region of the sgRNA was determined from the replacement fragment. Several groups of sequences with the lowest off-target rate are selected for the target fragment, and the following sequences are used as sgrnas to be selected (see the following table). Designing and synthesizing and recognizing a 5 'end target site and a 3' end target site, and constructing a sgRNA expression vector. The recognition sites of the sgRNAs at two ends are respectively positioned on the first exon and the seventh exon of a mouse Lan 3 gene, and the target site sequence of each sgRNA on Lan 3 is as follows:

table 1 sgRNA information

sgRNA name	sgRNA sequence (5 '→ 3')
		YF000087-huLAG3-5S1	AGGGAGGACCTGCTCCTTGG(SEQNo.2)
YF000087-huLAG3-5S2	GTAAGGTGGAGAGTCCAGCAGG(SEQNo.3)
		YF000087-huLAG3-5S3	GAAGCTCCAGGTAAGGTGG(SEQNo.4)
YF000087-huLAG3-5S4	GACTCTCCACCTTACCTGG(SEQNo.5)
		YF000087-huLAG3-3S1	AGATTCTCCTAGCACTGTGGG(SEQNo.6)
YF000087-huLAG3-3S2	AGATTCTCCTAGCACTGTGG(SEQNo.7)
		YF000087-huLAG3-3S3	CACAGTGCTAGGAGAATCTCAGG(SEQNo.8)
YF000087-huLAG3-3S4	CACTGGGTTAGATCCGGGGTTGG(SEQNo.9)

The sgRNA transcription preparation method comprises the following steps: PCR was performed using PrimerStar Max system (Table 4), sgRNA-F, sgRNA-R as primers, and the correctly sequenced puc57-sgRNA plasmid (1: 30 dilution) as a template, and the PCR product was purified to prepare a template for sgRNA transcription. Transcription of sgRNA was performed using T7-ShortScript in vitro transcription kit (AM 1354).

sgRNA screening: pairwise pairing of the 5 'end target site and the 3' end target site sgrnas is performed to combine 10 pairs of sgrnas (5S1/3S1, 5S1/3S2, 5S1/3S3, 5S2/3S1, 5S2/3S2, 5S2/3S3, 5S3/3S1, 5S3/3S2, 5S4/3S1, 5S4/3S 2). After 10 pairs of sgrnas and Cas9 proteins are respectively incubated, the mixed solution is injected into 0.5 day fertilized eggs, and after the fertilized eggs are cultured to a blastocyst stage, the KO positive rate of mouse Lag3 gene is identified, so that sgRNA pairs with high cleavage activity are screened.

The sgRNA cleavage identification method comprises the following steps: PCR amplification is carried out on the collected blastocysts (the PCR scheme is as follows), statistics is carried out according to the PCR result (a target band can be amplified only when KO occurs), the probability of KO occurrence is counted (the identification result is shown in the following table 2), and YF000087-huLAG3-5S1+ YF000087-huLAG3-3S1 combination is finally selected according to the position of each sgRNA and various combined cutting efficiencies for subsequent experiments, and although the cutting efficiency is only 35%, the sgRNA is selected because the position of the sgRNA is more favorable for gene recombination.

Table 2 sgRNA cleavage activity

Combinations of sgRNAs	Onset of KO efficiency
		YF000087-huLAG3-5S1/3S1	7/20＝35％
YF000087-huLAG3-5S1/3S2	10/20＝50％
		YF000087-huLAG3-5S1/3S3	10/20＝50％
YF000087-huLAG3-5S2/3S1	6/20＝30％
		YF000087-huLAG3-5S2/3S2	12/20＝60％
YF000087-huLAG3-5S2/3S3	13/20＝65％
		YF000087-huLAG3-5S3/3S1	7/20＝35％
YF000087-huLAG3-5S3/3S2	13/20＝65％
		YF000087-huLAG3-5S4/3S1	10/20＝50％
YF000087-huLAG3-5S4/3S2	6/20＝30％

3. Construction of humanized targeting vector

The homologous recombination technology is used for replacing parts of mouse Exon1, Exon2, Exon3, Exon4, Exon5, Exon6 and Exon7 of human Exon1, Exon2, Exon3, Exon4, Exon5, Exon6 and Exon7 fragments, and successfully constructing targeting vector sequences as shown in SEQ No.10 (809 5796bp human sequences, 1-808bp and 5797 + 6599bp mouse sequences, 809 + 866bp human Exon1 sequence, 1148 + 1295bp human Exon2 sequence, 1653 + 1957bp human Exon3 sequence, 2551 + 2820bp human Exon4 sequence, 3229 + 4bp human Exon5 sequence, 5220 bp 545 bp human Exon 545 + 5747 sequence, 5857575747 + 5796 sequence and 5857573 sequence).

SEQ No.10

ccgtgtggctactatacttttccactgagcccttacccagatctggtaacttgtggtatggtgacacttcttgagtacctgtccaagagatgctgaaaggctaggcaggcagactgtaggacagtggcttttttttttttttttttcagtgttaatgggaaagagcaagtcaagaagaaactgtggggacagtagaggaagcttaaagatacagctgtagttctaggcagaaatgcttggcagagagagagagagagagagagagagagagagagagagagagagagagaggagacagagggggaagaggtgaagagggggcggtagggagacccgagtctgaggaagtaaacaaggggagtgccaccaccgagaggagggctcggctgctgggaatcagccccctcacactttccactgcgaagcgaaaccccgcgccttggtctgggggggcgggcagtggggaggagaagcagaaggactgggtctggaggagcagctcaagttctagctagctgcagtgggtttgcctgcactctgctctgggtcccagcccgggcctctgatcattatccatcctgctgtctccagtccccactcctggggcgtcctcttcaccctacattctttccctccgcctcacctcctccttgtagaacttctctctctctctctctctctctctctctctctctctctctctctctgtgtgtgtgtgtgtgtctgtctgtctgtctgtctctctctcctcccaggacctttttctaacctcccttggagggctggggaggcccgggccatagaggagatgtgggaggctcagttcctgggcttgctgtttctgcagccgctttgggtggctccaggtaaaacggggatggcgggagggttgacctccagccccacaggaggggaccagcagggatctctgtggccacaaaggtcctgaggtccttagctctgtggattcttctaatcccttttttgggcagtccttccaccccgaaagcctctctgggcagagaagaaacagaaacccaagttcttcctgcaccctgtttctccctcgggaaacacccaggctccttctctacccctgcctctcggctcacgccccctccccttggcctctcttttgctcacctagtgaagcctctccagccaggggctgaggtcccggtggtgtgggcccaggagggggctcctgcccagctcccctgcagccccacaatccccctccaggatctcagccttctgcgaagagcaggggtcacttggcagcatcagccagacaggtatgcaccccaaacttgggcaacaggacctccgaatccagcactcaaccccacacccgtgccggtcctctgtcccctgccctgaggtgtcactccctctgaagccagtgacccagtctccctgccctcgcttgcaccgttcctgcccttgctctgcaatcagcgaccctcacgccagcatcccttctctccagaagtggatgcggccagtccaacagaggggtcgggcgtgaggggacggttggtggtcaagagaactcttggggcgggctttctcatcctcaacgggtggctgcctgcatcctcccgggcttcctacccctggagcttctcaactccattctctttcccgcccagtggcccgcccgctgccgcccccggccatcccctggcccccggccctcacccggcggcgccctcctcctgggggcccaggccccgccgctacacggtgctgagcgtgggtcccggaggcctgcgcagcgggaggctgcccctgcagccccgcgtccagctggatgagcgcggccggcagcgcggggacttctcgctatggctgcgcccagcccggcgcgcggacgccggcgagtaccgcgccgcggtgcacctcagggaccgcgccctctcctgccgcctccgtctgcgcctgggccaggcctcgagtatgtggggcgggacgatgggagaagggctgggaggtgggtccccatcccctgcctcccgggacgcaggaagggctggggcagaggctgcgccctaggccctgtcggagagctcccagaagagtagaggaagggggtgggcggcctgctggagtggaaggtgcccccgaagcacgtgtatggggggccctgtggagagattgtgtcacccccgagctccccttctcccacccacgcgggagtgcccagagggagggggagggggggagagcatggggctaaagtgattcatttcagatatctgtagctcagggggtgggcttcgcggggttccaggccaggaaaacggcaagggtggctgatgccaagtaaactccaggccagggacggggaaagtggtcctggggagtcttggggatccactttatgcacctccaggtgctggaagctgagatggggagagggtgatgtgggagaggagaagacaagtctaaagccaggtgcctgtttccaggagcttccggcttggcagtcctgctgtgttgggaaattgtttccagtgggctgatgaagtcttctttatccttgcacagtgactgccagccccccaggatctctcagagcctccgactgggtcattttgaactgctccttcagccgccctgaccgcccagcctctgtgcattggttccggaaccggggccagggccgagtccctgtccgggagtccccccatcaccacttagcggaaagcttcctcttcctgccccaagtcagccccatggactctgggccctggggctgcatcctcacctacagagatggcttcaacgtctccatcatgtataacctcactgttctgggtaactcccccactctgcttcacatttgaccacaactccttcctgccccccttgtcacctcccctaactatgggtccccaaaccaggttctcggcagcgagtggcctacgtcattgctgtgggtctcactgttcgacccctttatattgctggcagcctcacagctgccatcaccccttcttgcttctcccgtggccttccagcgtcattgccggccttccctctccttccggctaagcccacttgctgggtttctgagcctcctcagctcatcaccttattctgctccttagcactcttatgagccagaccatctcctgaattcttctgcctcccttccttgcagccccagcactccctccccactgcagcacccagctttaactttgggttttcttttctcttcaggtctggagcccccaactcccttgacagtgtacgctggagcaggttccagggtggggctgccctgccgcctgcctgctggtgtggggacccggtctttcctcactgccaagtggactcctcctgggggaggccctgacctcctggtgactggagacaatggcgactttacccttcgactagaggatgtgagccaggcccaggctgggacctacacctgccatatccatctgcaggaacagcagctcaatgccactgtcacattggcaatcatcacaggtcagcctcaggtgggaaaggagtagctgccctcccagggtagaaaggacagggaggaagggctggcagggcaaagactaggcaaacccaccctgtgatgccaggccactgggcacaagttccagagcctgcccatctcggcccccacttttctcacccccataataaagaaacgaaactgaaaatctcctcttgagtcacaagataaaagttccaccgttctctatgggactcccctgctctcaattggcgggagggtctgggaagttagaaggaaaggtgacaaaaattctgaatggttcgaaagaggtagaatatatttctagaatccttgtctactttgcagccagggcttgggttagagttgcaggaagtggcctggatttgggaggagtgaataaatccgtcccttggtcagcaaatatttactgagcaagggttttccaagacagtataaaacaaacacagaaaaaagaatactcagagagtatgtgttgactggttgataactatcggccatgacagattagccatgtctgcagcacgcacctgcggccactcagtagtagcaccccacggcaggtgcttaataatgtatagagattgaatgaatacgtgaacatgctaatggataatacatctcctgaaggccaatcctgagttttcacttgctttctggatacctctaactagatgttataccacctctcagccgccttaccctgaaccccagctttttctcccaccaatccccttgctgactcagcctgtcagtaatccactggtatccatccacctcgaaacctggcctcctcctctctacatcccatcagtcatcaaagtccaccagttctttctgtccaatgccctcttgaaggtcaactgttttatgtagacggctcaatgagagagatactgttaccccgaagactttctcaaaacagctgggaagtgccagactctgggtctaaactcaggtgtctgcatcaccaaatcactctccaggacactactcagatgcccttcaccctttgtctcctcccacccacccatctctcactttacaacttggagaatgctctgtctctagatgaatgggtcagttccctgttctctataccttcagaaagggggatgatgttcattcagggggcaccacaaggcaggggacactgatgtgagccgcatcatggaagttccaaaattgtgaagtagatgggggtcagctcatctgactgattttgagggttctgagcatttgagtagaaagtaatgtaaaagaatctgtaatagtatttattctgtggtctgccagggccagtttgagtgtgaaggcagtagggagcctggaaatgttttgagcaggggagggagctgactagagcattgtctgaataagataaatgtggccggcagggcatgatggctcacacctgtaatcccagcactttgaaaggctgagacgagaggagcacttgagtccaggaattccagactagcctgggcaacatggtgaaaacgcatctgtatacaatttaataaaaaaataaatagaaagaaaaagatgaacgtggccatgacccatgatgtccttccccaacctcccctggccagaacccaagtgagtgcagggtgattgagacttggggttcaacctgtgatatcacgtaaggggggcagaatcccaaaagatctcttccttctactcttttcagtgactcccaaatcctttgggtcacctggatccctggggaagctgctttgtgaggtgactccagtatctggacaagaacgctttgtgtggagctctctggacaccccatcccagaggagtttctcaggaccttggctggaggcacaggaggcccagctcctttcccagccttggcaatgccagctgtaccagggggagaggcttcttggagcagcagtgtacttcacagagctgtctagcccaggtccgaggccccagaatgccaggacccaaacgccacagcaataatctttatcctccttccagaacagcccctgccacccccatcccagcgcttttctttccagtcagggacctgatctctggcttttgcctagggttgacccgtttccgccactgatcagctgaatgaccctgggacaagtcccttaaactctctggatctcattgcatctgtaaagtctgagagaatgacaaagtgtcctttctagtcctgaccctatgcctcatcctgtactttctccataggtgcccaacgctctgggagagccccaggtgccctcccagcaggccacctcgttctcgttctcatccttggtgccctctccctgttccttttggtggccggggcctttggctttcactggtggagaaaacaggtgagacagacctcagagtgctgtgtgggaccgcttccccttccgcaagaaagagaaccatactgggttcggaatttgctcccagagtccctctactgggaacaccttgtagctgggaagcttaaaggtgctgaaaggcggccctgggccaaatggagaagcctggaaagacagaagtggagctccgtgattatcttctttgttctccattgtcctttctgtctttcccccccacagttgctactgagaagattttctgccttagaacatgggattcagccatttccggctcagaggaagatagaggagctggagcgagaactggagacggagatgggacaggagccggagcccgagccggagccacagctggagccagagcccaggcagctctgacctggagccgaggcagccagcaggtctcagcagctccgcccgcccgcccgcccgcccgaataaactccctgtcagcagcatcagcaagctggagtctgtctttcttgtctgggtctggaaaggttcctgtttctcccagccactaggggctcccttctctcacttcagcctcccaacctactttctccagccaatctttctcctcattcccttttctgttgcttgcctctttgtttcttccactgttgtcccctttctgttaagtaatccgttcctctccgcctccttccccccgccccctgaaagctccaaagagccctcacttc

4. LAG3 humanized mouse model establishment

And designing and constructing repair donor carrying a human-derived sequence, namely a humanized targeting vector according to the sgRNA obtained by screening, injecting the repair donor and the Cas9/sgRNA system into a mouse fertilized egg for 0.5 day, transplanting the mouse fertilized egg into a pseudopregnant female mouse for 0.5 day, and screening a medium-target mouse by gene identification after the mouse is born (F0).

And (3) carrying out genotype identification on the humanized mouse F0 generation. Performing PCR identification on the obtained rat tail genomic DNA of the F0 mouse after target targeting by using two pairs of primers respectively, wherein the primers YF000087-HuLAG3-5tF1/YF000087-HuLAG3-5tR1 are respectively positioned outside the 5 'homology arm and in the human fragment of the repair donor, if the pair of primers is amplified to generate a PCR product, the target donor is inserted effectively in the mouse genome 5'; YF000087-HuLAG3-3tF1/YF000087-HuLAG3-3tR1 are respectively positioned in the human fragment and outside the 3 'homologous arm of the repair donor, if the pair of primers is amplified, a PCR product is generated, and the target donor is inserted effectively in the 3' of the mouse genome.

Table 3F0 identifies primers:

TABLE 4 PCR reaction System

Reagent (TakaraR045)	Volume (μ l)	Specification of
			PrimeSTAR Max Premix(2×)	12.5	\
ddH2O	9.5	\
			Primer
	1	10μM
			Primer
	1	10μM
			Template
	1

TABLE 5 PCR reaction conditions

Sequencing verification is carried out on mouse clones which are identified to be positive by PCR at two ends, and after the repair donor replaces Exon1, Exon2, Exon3, Exon4, Exon5, Exon6 and Exon7 in mouse genomes, the clones which are sequenced correctly are identified as positive F0 mice.

TABLE 6 sequencing primers

Primer name	Primer sequences
		YF000087-HuLAG3-5seqR1	CTGTCCTACAGTCTGCCTGC(SEQ No.17)
YF000087-HuLAG3-5seqF1	AGAATGAAGGTTAACACCGTG(SEQ No.18)
		YF000087-HuLAG3-5seqF2	GTTCTAGCTAGCTGCAGTGG(SEQ No.19)
YF000087-HuLAG3-5seqF3	GCCATAGAGGAGATGTGGGAG(SEQ No.20)
		YF000087-HuLAG3-5seqR2	GGACTGGAGACAGCAGGATG(SEQ No.21)
YF000087-HuLAG3-5seqR3	AGAGGCTTTCGGGGTGGAAG(SEQ No.22)
		YF000087-HuLAG3-3seqR1	CACACAGCACTCTGAGGTCTG(SEQ No.23)
YF000087-HuLAG3-3seqF1	TCTAGTCCTGACCCTATGCCTC(SEQ No.24)
		YF000087-HuLAG3-3seqF2	AGAGCCCAGGCAGCTCTGAC(SEQ No.25)

As a result: 4 positive F0 mice were obtained. As shown in fig. 1 and fig. 2, the electrophoresis images of the assay of the tail DNA of hLAG3F0 show that the 5 'and 3' of the human LAG3 gene of mice # 99, # 110, # 157 and # 183 are positive, indicating that the mice are positive mice correctly undergoing gene recombination.

F0 and a background mouse are bred to obtain F1, F1 mouse tails are subjected to gene identification, primers YF000087-HuLAG3-wt-tF4/YF000087-HuLAG3-wt-tR4 are added on the basis of F0 mouse identification and are respectively positioned in murine Exon4 and Intro4 fragments, PCR products are generated by amplification of the primers, the result of PCR experiments of F1 mice is shown in figure 3, and the results of PCR experiments of the murine LAG3 genes are positive, and meanwhile, murine detection is positive, so that the obtained mice are heterozygous positive mice which are correctly subjected to gene recombination. F1 breeding to obtain homozygote.

Example 2: expression and functional verification of LAG3 humanized mouse model

First, LAG3 expression and function verification of LAG3 humanized mouse

Protein expression detection: f1 generation heterozygote mating to obtain homozygote, selecting C57BL/6 background mouse, LAG3 humanized heterozygote and homozygous mouse spleen, grinding and digesting the tissue into single cells, using human anti-LAG 3 antibody and murine anti-LAG 3 antibody, and analyzing the expression of humanized mouse protein by flow cytometry.

The flow-type protein detection method comprises the following steps:

material taking: c57BL/6 background heterozygous and homozygous LAG3 humanized mice at 4 weeks of age are injected with 7.5ug of CD3e antibody in the abdominal cavity, and after 24 hours of stimulation, spleens are taken for flow detection, weighed and placed in a C-shaped tube.

Digestion: in the C-tube, 3ml of a precooled enzyme digest (PBS containing Ca, Mg + 2% CS +10mM HEPES +30ug DNase +1.75Mg collagenase D) was placed in a 37 ℃ water bath for digestion for 30 min. Digestion was stopped by adding 300ul of 0.1M EDTA to the spleen cells that had been digested. The undigested tissue mass was removed by filtration through a 1mL filter and EDTA neutralized by adding 2mL FACS buffer per tube. Spleens were lysed with RBCs for 5min at room temperature in the dark, washed and resuspended in FACS buffer, and aliquoted into 100uL flow tubes to prepare the incubated antibodies.

Antibody incubation: adding anti-hLAG3 and anti-mLAG3 antibodies, and incubating for 1h on ice in the dark; and (4) washing by using the FACS buffer, adding the FACS buffer, and detecting on a machine. Sytoxblue (final concentration 1:10000 dilution) was added 5min before loading to differentiate dead and live cells.

Analysis of the results shows that human LAG3 expression is not detected in C57BL/6 background mice, while only human LAG3 expression can be detected in LAG3 homozygous mice, and the expression of human LAG3 is basically consistent with that of control group mouse LAG3 (see figure 4), which indicates that the LAG3 protein can be successfully expressed in the LAG3 humanized mice prepared by the method, and the expression level is equivalent to that of wild-type mice.

Second, evaluation of immune System index of LAG3 humanized mouse

The humanized LAG3 mouse obtained by establishing the line should have a healthy immune system, and the expression of hLAG3 protein does not cause the disturbance of the immune system of the mouse (serious defect of T/NK cells and the like), and is crucial to the effectiveness of the drug evaluation of the LAG3 target. Detecting immune indexes (mainly T/B/NK cells) of the mice by flow cytometry sorting, and judging immune system indexes of the mice. A humanized mouse model that can express functional LAG3 and is immune system robust would be an important preclinical assessment tool for evaluation of antibody efficacy based on LAG3 targets.

The flow detection method comprises the following steps:

material taking: c57BL/6 background mice, LAG3 heterozygous mice and homozygous mice of 4 weeks old are injected with 7.5ug of CD3e antibody in the abdominal cavity, after 24h of stimulation, peripheral blood and spleen are taken for flow detection, weighed and placed in a C-shaped tube.

Digestion: in the C-tube, 3ml of a precooled enzyme digest (PBS containing Ca, Mg + 2% CS +10mM HEPES +30ug DNase +1.75Mg collagenase D) was placed in a 37 ℃ water bath for digestion for 30 min. Digestion was stopped by adding 300ul of 0.1M EDTA to the spleen cells that had been digested. The undigested tissue mass was removed by filtration through a 1mL filter and EDTA neutralized by adding 2mL FACS buffer per tube. Spleens were lysed with RBCs for 5min at room temperature in the dark, washed and resuspended in FACS buffer, and aliquoted into 100uL flow tubes to prepare the incubated antibodies.

Antibody incubation: adding CD3, CD4, CD8, CD19, CD335, IgM and B220 antibodies, and incubating for 1h on ice in a dark place; and (4) washing by using the FACS buffer, adding the FACS buffer, and detecting on a machine. Sytoxblue (final concentration 1:10000 dilution) was added 5min before loading to differentiate dead and live cells.

To verify whether hLAG3 is involved in the immune response of mice, spleen cells of LAG3 humanized mice were isolated in vitro and assayed for lymphocyte activation status after stimulation with Anti-hLAG3 and Anti-mLAG3 antibodies, respectively. The data for the immune cell populations in the spleen are shown in FIG. 5. LAG3 humanized mouse spleen mononuclear cells (MNC) were activated by Anti-hLAG3 and Anti-mLAG3 antibodies, the proportion of CD25+ CD69+ double positive cells was > 95%, and lymphocyte activation was significant. The number of each T, B, NK immune cell of the C57BL/6-hLAG3 homozygous mouse is basically not different from that of the C57BL/6 background mouse. The data show that hLAG3 is involved in the immune response process of mice, and the LAG3 humanized mice have normal immune system and no difference compared with common background mice, and are ideal LAG3 humanized animal models.

Thirdly, LAG3 humanized mouse is evaluated to verify the antitumor effect of anti-hLAG3

The detection method of the tumor-bearing and tumor comprises the following steps:

1) MC38 cell culture; and (3) cell recovery: taking out from liquid nitrogen tank, rapidly thawing in 37 deg.C water bath, and inoculating in 15cm dish for culture.

2) Passage; MC38 are adherent cells, usually requiring passage every 2-3 days.

3) Observing and measuring; mice were observed daily for growth and weighed 2 times per week prior to grouping. After confirming that the mice have a tumor, the tumor size is measured.

4) Grouping; 7 days after inoculating MC38 cells, the average tumor volume is 80-150mm³Mice were randomly divided into 2 groups and pharmacodynamic experiments were initiated.

5) Subcutaneous injection of MC38 cells; the cells were collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, and washed 2 times with 10ml of calcium-magnesium ion-free PBS. PBS cell resuspension, adjusting the final cell concentration to 5X 10⁶And/ml. Subcutaneous injection, 100uL of MC38 cells were injected into the right back of the mouse, above the thigh.

6) The treatment group was injected with 1mpk of anti-mPD1 drug, 10mpk of anti-hLAG3 drug and a combination of both, and the control group was injected with an equal volume of the blank solvent PBS.

7) The body weight and tumor volume of the mice were measured on the day of administration, and subsequently 2 times per week, and the body weight of the mice was measured before each administration, and the administration was performed on the day according to the body weight.

8) The tumor volume of a single mouse reaches 3000mm 24 days after the first administration or after inoculation³An euthanasia end experiment was performed.

Selecting 4-6w LAG3 humanized homozygous mouse, inoculating mouse colon cancer cell MC38 subcutaneously, and waiting for tumor average volume of 80-150mm³Then, the samples were randomly divided into four groups: the first group is injected with 1mpk of anti-mPD1 antibody intraperitoneally, the second group is injected with 10mpk of anti-hLAG3 antibody intraperitoneally, the third group is injected with 1mpk of anti-mPD1 antibody +10mpk of anti-hLAG3 antibody intraperitoneally, and the control group is injected with the same volume of blank solvent PBS; the frequency of administration was 2 times per week and 6 times per week (see table 7 for the dosing schedule); the end point of the administration is tumor regression/inhibition, or the mean tumor volume is greater than 3000mm³The mice were euthanized.

Table 7 grouping and dosing regimens

In the experimental process, all mice are in good health, the weight growth curves of the mice in the experimental group and the control group are not obviously different, the single medicine group of anti-mPD1 and the single medicine group of anti-hLAG3 have small tumor growth inhibition effect (the single medicine group of anti-mPD1 TGI is 19.73 percent, the single medicine group of anti-hLAG3 TGI is 21.13 percent), and the combined group of anti-mPD-1 and anti-hLAG3 has large tumor growth inhibition effect (the combined group of anti-mPD-1 and anti-hLAG3 TGI is 40.69 percent). Mean tumor volume ± SEM (fig. 6a), mean mouse weight ± SEM (fig. 6 b). The results prove that: LAG3 humanized mice are a powerful tool to evaluate the in vivo potency of anti-hLAG3 antibodies.

While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.

Sequence listing

<110> Jiangsu Jiejiaokang Biotech limited

<120> construction method of LAG3 gene humanized animal model

<160> 25

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4988

<212> DNA

<213> homo sapiens

<400> 1

atgtgggagg ctcagttcct gggcttgctg tttctgcagc cgctttgggt ggctccaggt 60

aaaacgggga tggcgggagg gttgacctcc agccccacag gaggggacca gcagggatct 120

ctgtggccac aaaggtcctg aggtccttag ctctgtggat tcttctaatc ccttttttgg 180

gcagtccttc caccccgaaa gcctctctgg gcagagaaga aacagaaacc caagttcttc 240

ctgcaccctg tttctccctc gggaaacacc caggctcctt ctctacccct gcctctcggc 300

tcacgccccc tccccttggc ctctcttttg ctcacctagt gaagcctctc cagccagggg 360

ctgaggtccc ggtggtgtgg gcccaggagg gggctcctgc ccagctcccc tgcagcccca 420

caatccccct ccaggatctc agccttctgc gaagagcagg ggtcacttgg cagcatcagc 480

cagacaggta tgcaccccaa acttgggcaa caggacctcc gaatccagca ctcaacccca 540

cacccgtgcc ggtcctctgt cccctgccct gaggtgtcac tccctctgaa gccagtgacc 600

cagtctccct gccctcgctt gcaccgttcc tgcccttgct ctgcaatcag cgaccctcac 660

gccagcatcc cttctctcca gaagtggatg cggccagtcc aacagagggg tcgggcgtga 720

ggggacggtt ggtggtcaag agaactcttg gggcgggctt tctcatcctc aacgggtggc 780

tgcctgcatc ctcccgggct tcctacccct ggagcttctc aactccattc tctttcccgc 840

ccagtggccc gcccgctgcc gcccccggcc atcccctggc ccccggccct cacccggcgg 900

cgccctcctc ctgggggccc aggccccgcc gctacacggt gctgagcgtg ggtcccggag 960

gcctgcgcag cgggaggctg cccctgcagc cccgcgtcca gctggatgag cgcggccggc 1020

agcgcgggga cttctcgcta tggctgcgcc cagcccggcg cgcggacgcc ggcgagtacc 1080

gcgccgcggt gcacctcagg gaccgcgccc tctcctgccg cctccgtctg cgcctgggcc 1140

aggcctcgag tatgtggggc gggacgatgg gagaagggct gggaggtggg tccccatccc 1200

ctgcctcccg ggacgcagga agggctgggg cagaggctgc gccctaggcc ctgtcggaga 1260

gctcccagaa gagtagagga agggggtggg cggcctgctg gagtggaagg tgcccccgaa 1320

gcacgtgtat ggggggccct gtggagagat tgtgtcaccc ccgagctccc cttctcccac 1380

ccacgcggga gtgcccagag ggagggggag ggggggagag catggggcta aagtgattca 1440

tttcagatat ctgtagctca gggggtgggc ttcgcggggt tccaggccag gaaaacggca 1500

agggtggctg atgccaagta aactccaggc cagggacggg gaaagtggtc ctggggagtc 1560

ttggggatcc actttatgca cctccaggtg ctggaagctg agatggggag agggtgatgt 1620

gggagaggag aagacaagtc taaagccagg tgcctgtttc caggagcttc cggcttggca 1680

gtcctgctgt gttgggaaat tgtttccagt gggctgatga agtcttcttt atccttgcac 1740

agtgactgcc agccccccag gatctctcag agcctccgac tgggtcattt tgaactgctc 1800

cttcagccgc cctgaccgcc cagcctctgt gcattggttc cggaaccggg gccagggccg 1860

agtccctgtc cgggagtccc cccatcacca cttagcggaa agcttcctct tcctgcccca 1920

agtcagcccc atggactctg ggccctgggg ctgcatcctc acctacagag atggcttcaa 1980

cgtctccatc atgtataacc tcactgttct gggtaactcc cccactctgc ttcacatttg 2040

accacaactc cttcctgccc cccttgtcac ctcccctaac tatgggtccc caaaccaggt 2100

tctcggcagc gagtggccta cgtcattgct gtgggtctca ctgttcgacc cctttatatt 2160

gctggcagcc tcacagctgc catcacccct tcttgcttct cccgtggcct tccagcgtca 2220

ttgccggcct tccctctcct tccggctaag cccacttgct gggtttctga gcctcctcag 2280

ctcatcacct tattctgctc cttagcactc ttatgagcca gaccatctcc tgaattcttc 2340

tgcctccctt ccttgcagcc ccagcactcc ctccccactg cagcacccag ctttaacttt 2400

gggttttctt ttctcttcag gtctggagcc cccaactccc ttgacagtgt acgctggagc 2460

aggttccagg gtggggctgc cctgccgcct gcctgctggt gtggggaccc ggtctttcct 2520

cactgccaag tggactcctc ctgggggagg ccctgacctc ctggtgactg gagacaatgg 2580

cgactttacc cttcgactag aggatgtgag ccaggcccag gctgggacct acacctgcca 2640

tatccatctg caggaacagc agctcaatgc cactgtcaca ttggcaatca tcacaggtca 2700

gcctcaggtg ggaaaggagt agctgccctc ccagggtaga aaggacaggg aggaagggct 2760

ggcagggcaa agactaggca aacccaccct gtgatgccag gccactgggc acaagttcca 2820

gagcctgccc atctcggccc ccacttttct cacccccata ataaagaaac gaaactgaaa 2880

atctcctctt gagtcacaag ataaaagttc caccgttctc tatgggactc ccctgctctc 2940

aattggcggg agggtctggg aagttagaag gaaaggtgac aaaaattctg aatggttcga 3000

aagaggtaga atatatttct agaatccttg tctactttgc agccagggct tgggttagag 3060

ttgcaggaag tggcctggat ttgggaggag tgaataaatc cgtcccttgg tcagcaaata 3120

tttactgagc aagggttttc caagacagta taaaacaaac acagaaaaaa gaatactcag 3180

agagtatgtg ttgactggtt gataactatc ggccatgaca gattagccat gtctgcagca 3240

cgcacctgcg gccactcagt agtagcaccc cacggcaggt gcttaataat gtatagagat 3300

tgaatgaata cgtgaacatg ctaatggata atacatctcc tgaaggccaa tcctgagttt 3360

tcacttgctt tctggatacc tctaactaga tgttatacca cctctcagcc gccttaccct 3420

gaaccccagc tttttctccc accaatcccc ttgctgactc agcctgtcag taatccactg 3480

gtatccatcc acctcgaaac ctggcctcct cctctctaca tcccatcagt catcaaagtc 3540

caccagttct ttctgtccaa tgccctcttg aaggtcaact gttttatgta gacggctcaa 3600

tgagagagat actgttaccc cgaagacttt ctcaaaacag ctgggaagtg ccagactctg 3660

ggtctaaact caggtgtctg catcaccaaa tcactctcca ggacactact cagatgccct 3720

tcaccctttg tctcctccca cccacccatc tctcacttta caacttggag aatgctctgt 3780

ctctagatga atgggtcagt tccctgttct ctataccttc agaaaggggg atgatgttca 3840

ttcagggggc accacaaggc aggggacact gatgtgagcc gcatcatgga agttccaaaa 3900

ttgtgaagta gatgggggtc agctcatctg actgattttg agggttctga gcatttgagt 3960

agaaagtaat gtaaaagaat ctgtaatagt atttattctg tggtctgcca gggccagttt 4020

gagtgtgaag gcagtaggga gcctggaaat gttttgagca ggggagggag ctgactagag 4080

cattgtctga ataagataaa tgtggccggc agggcatgat ggctcacacc tgtaatccca 4140

gcactttgaa aggctgagac gagaggagca cttgagtcca ggaattccag actagcctgg 4200

gcaacatggt gaaaacgcat ctgtatacaa tttaataaaa aaataaatag aaagaaaaag 4260

atgaacgtgg ccatgaccca tgatgtcctt ccccaacctc ccctggccag aacccaagtg 4320

agtgcagggt gattgagact tggggttcaa cctgtgatat cacgtaaggg gggcagaatc 4380

ccaaaagatc tcttccttct actcttttca gtgactccca aatcctttgg gtcacctgga 4440

tccctgggga agctgctttg tgaggtgact ccagtatctg gacaagaacg ctttgtgtgg 4500

agctctctgg acaccccatc ccagaggagt ttctcaggac cttggctgga ggcacaggag 4560

gcccagctcc tttcccagcc ttggcaatgc cagctgtacc agggggagag gcttcttgga 4620

gcagcagtgt acttcacaga gctgtctagc ccaggtccga ggccccagaa tgccaggacc 4680

caaacgccac agcaataatc tttatcctcc ttccagaaca gcccctgcca cccccatccc 4740

agcgcttttc tttccagtca gggacctgat ctctggcttt tgcctagggt tgacccgttt 4800

ccgccactga tcagctgaat gaccctggga caagtccctt aaactctctg gatctcattg 4860

catctgtaaa gtctgagaga atgacaaagt gtcctttcta gtcctgaccc tatgcctcat 4920

cctgtacttt ctccataggt gcccaacgct ctgggagagc cccaggtgcc ctcccagcag 4980

gccacctc 4988

<210> 2

<211> 20

<212> DNA

<213> homo sapiens

<400> 2

agggaggacc tgctccttgg 20

<210> 3

<211> 22

<212> DNA

<213> homo sapiens

<400> 3

gtaaggtgga gagtccagca gg 22

<210> 4

<211> 19

<212> DNA

<213> homo sapiens

<400> 4

gaagctccag gtaaggtgg 19

<210> 5

<211> 19

<212> DNA

<213> homo sapiens

<400> 5

gactctccac cttacctgg 19

<210> 6

<211> 21

<212> DNA

<213> homo sapiens

<400> 6

agattctcct agcactgtgg g 21

<210> 7

<211> 20

<212> DNA

<213> homo sapiens

<400> 7

agattctcct agcactgtgg 20

<210> 8

<211> 23

<212> DNA

<213> homo sapiens

<400> 8

cacagtgcta ggagaatctc agg 23

<210> 9

<211> 23

<212> DNA

<213> homo sapiens

<400> 9

cactgggtta gatccggggt tgg 23

<210> 10

<211> 6599

<212> DNA

<213> homo sapiens

<400> 10

ccgtgtggct actatacttt tccactgagc ccttacccag atctggtaac ttgtggtatg 60

gtgacacttc ttgagtacct gtccaagaga tgctgaaagg ctaggcaggc agactgtagg 120

acagtggctt tttttttttt ttttttcagt gttaatggga aagagcaagt caagaagaaa 180

ctgtggggac agtagaggaa gcttaaagat acagctgtag ttctaggcag aaatgcttgg 240

cagagagaga gagagagaga gagagagaga gagagagaga gagagagaga ggagacagag 300

ggggaagagg tgaagagggg gcggtaggga gacccgagtc tgaggaagta aacaagggga 360

gtgccaccac cgagaggagg gctcggctgc tgggaatcag ccccctcaca ctttccactg 420

cgaagcgaaa ccccgcgcct tggtctgggg gggcgggcag tggggaggag aagcagaagg 480

actgggtctg gaggagcagc tcaagttcta gctagctgca gtgggtttgc ctgcactctg 540

ctctgggtcc cagcccgggc ctctgatcat tatccatcct gctgtctcca gtccccactc 600

ctggggcgtc ctcttcaccc tacattcttt ccctccgcct cacctcctcc ttgtagaact 660

tctctctctc tctctctctc tctctctctc tctctctctc tctctctgtg tgtgtgtgtg 720

tgtctgtctg tctgtctgtc tctctctcct cccaggacct ttttctaacc tcccttggag 780

ggctggggag gcccgggcca tagaggagat gtgggaggct cagttcctgg gcttgctgtt 840

tctgcagccg ctttgggtgg ctccaggtaa aacggggatg gcgggagggt tgacctccag 900

ccccacagga ggggaccagc agggatctct gtggccacaa aggtcctgag gtccttagct 960

ctgtggattc ttctaatccc ttttttgggc agtccttcca ccccgaaagc ctctctgggc 1020

agagaagaaa cagaaaccca agttcttcct gcaccctgtt tctccctcgg gaaacaccca 1080

ggctccttct ctacccctgc ctctcggctc acgccccctc cccttggcct ctcttttgct 1140

cacctagtga agcctctcca gccaggggct gaggtcccgg tggtgtgggc ccaggagggg 1200

gctcctgccc agctcccctg cagccccaca atccccctcc aggatctcag ccttctgcga 1260

agagcagggg tcacttggca gcatcagcca gacaggtatg caccccaaac ttgggcaaca 1320

ggacctccga atccagcact caaccccaca cccgtgccgg tcctctgtcc cctgccctga 1380

ggtgtcactc cctctgaagc cagtgaccca gtctccctgc cctcgcttgc accgttcctg 1440

cccttgctct gcaatcagcg accctcacgc cagcatccct tctctccaga agtggatgcg 1500

gccagtccaa cagaggggtc gggcgtgagg ggacggttgg tggtcaagag aactcttggg 1560

gcgggctttc tcatcctcaa cgggtggctg cctgcatcct cccgggcttc ctacccctgg 1620

agcttctcaa ctccattctc tttcccgccc agtggcccgc ccgctgccgc ccccggccat 1680

cccctggccc ccggccctca cccggcggcg ccctcctcct gggggcccag gccccgccgc 1740

tacacggtgc tgagcgtggg tcccggaggc ctgcgcagcg ggaggctgcc cctgcagccc 1800

cgcgtccagc tggatgagcg cggccggcag cgcggggact tctcgctatg gctgcgccca 1860

gcccggcgcg cggacgccgg cgagtaccgc gccgcggtgc acctcaggga ccgcgccctc 1920

tcctgccgcc tccgtctgcg cctgggccag gcctcgagta tgtggggcgg gacgatggga 1980

gaagggctgg gaggtgggtc cccatcccct gcctcccggg acgcaggaag ggctggggca 2040

gaggctgcgc cctaggccct gtcggagagc tcccagaaga gtagaggaag ggggtgggcg 2100

gcctgctgga gtggaaggtg cccccgaagc acgtgtatgg ggggccctgt ggagagattg 2160

tgtcaccccc gagctcccct tctcccaccc acgcgggagt gcccagaggg agggggaggg 2220

ggggagagca tggggctaaa gtgattcatt tcagatatct gtagctcagg gggtgggctt 2280

cgcggggttc caggccagga aaacggcaag ggtggctgat gccaagtaaa ctccaggcca 2340

gggacgggga aagtggtcct ggggagtctt ggggatccac tttatgcacc tccaggtgct 2400

ggaagctgag atggggagag ggtgatgtgg gagaggagaa gacaagtcta aagccaggtg 2460

cctgtttcca ggagcttccg gcttggcagt cctgctgtgt tgggaaattg tttccagtgg 2520

gctgatgaag tcttctttat ccttgcacag tgactgccag ccccccagga tctctcagag 2580

cctccgactg ggtcattttg aactgctcct tcagccgccc tgaccgccca gcctctgtgc 2640

attggttccg gaaccggggc cagggccgag tccctgtccg ggagtccccc catcaccact 2700

tagcggaaag cttcctcttc ctgccccaag tcagccccat ggactctggg ccctggggct 2760

gcatcctcac ctacagagat ggcttcaacg tctccatcat gtataacctc actgttctgg 2820

gtaactcccc cactctgctt cacatttgac cacaactcct tcctgccccc cttgtcacct 2880

cccctaacta tgggtcccca aaccaggttc tcggcagcga gtggcctacg tcattgctgt 2940

gggtctcact gttcgacccc tttatattgc tggcagcctc acagctgcca tcaccccttc 3000

ttgcttctcc cgtggccttc cagcgtcatt gccggccttc cctctccttc cggctaagcc 3060

cacttgctgg gtttctgagc ctcctcagct catcacctta ttctgctcct tagcactctt 3120

atgagccaga ccatctcctg aattcttctg cctcccttcc ttgcagcccc agcactccct 3180

ccccactgca gcacccagct ttaactttgg gttttctttt ctcttcaggt ctggagcccc 3240

caactccctt gacagtgtac gctggagcag gttccagggt ggggctgccc tgccgcctgc 3300

ctgctggtgt ggggacccgg tctttcctca ctgccaagtg gactcctcct gggggaggcc 3360

ctgacctcct ggtgactgga gacaatggcg actttaccct tcgactagag gatgtgagcc 3420

aggcccaggc tgggacctac acctgccata tccatctgca ggaacagcag ctcaatgcca 3480

ctgtcacatt ggcaatcatc acaggtcagc ctcaggtggg aaaggagtag ctgccctccc 3540

agggtagaaa ggacagggag gaagggctgg cagggcaaag actaggcaaa cccaccctgt 3600

gatgccaggc cactgggcac aagttccaga gcctgcccat ctcggccccc acttttctca 3660

cccccataat aaagaaacga aactgaaaat ctcctcttga gtcacaagat aaaagttcca 3720

ccgttctcta tgggactccc ctgctctcaa ttggcgggag ggtctgggaa gttagaagga 3780

aaggtgacaa aaattctgaa tggttcgaaa gaggtagaat atatttctag aatccttgtc 3840

tactttgcag ccagggcttg ggttagagtt gcaggaagtg gcctggattt gggaggagtg 3900

aataaatccg tcccttggtc agcaaatatt tactgagcaa gggttttcca agacagtata 3960

aaacaaacac agaaaaaaga atactcagag agtatgtgtt gactggttga taactatcgg 4020

ccatgacaga ttagccatgt ctgcagcacg cacctgcggc cactcagtag tagcacccca 4080

cggcaggtgc ttaataatgt atagagattg aatgaatacg tgaacatgct aatggataat 4140

acatctcctg aaggccaatc ctgagttttc acttgctttc tggatacctc taactagatg 4200

ttataccacc tctcagccgc cttaccctga accccagctt tttctcccac caatcccctt 4260

gctgactcag cctgtcagta atccactggt atccatccac ctcgaaacct ggcctcctcc 4320

tctctacatc ccatcagtca tcaaagtcca ccagttcttt ctgtccaatg ccctcttgaa 4380

ggtcaactgt tttatgtaga cggctcaatg agagagatac tgttaccccg aagactttct 4440

caaaacagct gggaagtgcc agactctggg tctaaactca ggtgtctgca tcaccaaatc 4500

actctccagg acactactca gatgcccttc accctttgtc tcctcccacc cacccatctc 4560

tcactttaca acttggagaa tgctctgtct ctagatgaat gggtcagttc cctgttctct 4620

ataccttcag aaagggggat gatgttcatt cagggggcac cacaaggcag gggacactga 4680

tgtgagccgc atcatggaag ttccaaaatt gtgaagtaga tgggggtcag ctcatctgac 4740

tgattttgag ggttctgagc atttgagtag aaagtaatgt aaaagaatct gtaatagtat 4800

ttattctgtg gtctgccagg gccagtttga gtgtgaaggc agtagggagc ctggaaatgt 4860

tttgagcagg ggagggagct gactagagca ttgtctgaat aagataaatg tggccggcag 4920

ggcatgatgg ctcacacctg taatcccagc actttgaaag gctgagacga gaggagcact 4980

tgagtccagg aattccagac tagcctgggc aacatggtga aaacgcatct gtatacaatt 5040

taataaaaaa ataaatagaa agaaaaagat gaacgtggcc atgacccatg atgtccttcc 5100

ccaacctccc ctggccagaa cccaagtgag tgcagggtga ttgagacttg gggttcaacc 5160

tgtgatatca cgtaaggggg gcagaatccc aaaagatctc ttccttctac tcttttcagt 5220

gactcccaaa tcctttgggt cacctggatc cctggggaag ctgctttgtg aggtgactcc 5280

agtatctgga caagaacgct ttgtgtggag ctctctggac accccatccc agaggagttt 5340

ctcaggacct tggctggagg cacaggaggc ccagctcctt tcccagcctt ggcaatgcca 5400

gctgtaccag ggggagaggc ttcttggagc agcagtgtac ttcacagagc tgtctagccc 5460

aggtccgagg ccccagaatg ccaggaccca aacgccacag caataatctt tatcctcctt 5520

ccagaacagc ccctgccacc cccatcccag cgcttttctt tccagtcagg gacctgatct 5580

ctggcttttg cctagggttg acccgtttcc gccactgatc agctgaatga ccctgggaca 5640

agtcccttaa actctctgga tctcattgca tctgtaaagt ctgagagaat gacaaagtgt 5700

cctttctagt cctgacccta tgcctcatcc tgtactttct ccataggtgc ccaacgctct 5760

gggagagccc caggtgccct cccagcaggc cacctcgttc tcgttctcat ccttggtgcc 5820

ctctccctgt tccttttggt ggccggggcc tttggctttc actggtggag aaaacaggtg 5880

agacagacct cagagtgctg tgtgggaccg cttccccttc cgcaagaaag agaaccatac 5940

tgggttcgga atttgctccc agagtccctc tactgggaac accttgtagc tgggaagctt 6000

aaaggtgctg aaaggcggcc ctgggccaaa tggagaagcc tggaaagaca gaagtggagc 6060

tccgtgatta tcttctttgt tctccattgt cctttctgtc tttccccccc acagttgcta 6120

ctgagaagat tttctgcctt agaacatggg attcagccat ttccggctca gaggaagata 6180

gaggagctgg agcgagaact ggagacggag atgggacagg agccggagcc cgagccggag 6240

ccacagctgg agccagagcc caggcagctc tgacctggag ccgaggcagc cagcaggtct 6300

cagcagctcc gcccgcccgc ccgcccgccc gaataaactc cctgtcagca gcatcagcaa 6360

gctggagtct gtctttcttg tctgggtctg gaaaggttcc tgtttctccc agccactagg 6420

ggctcccttc tctcacttca gcctcccaac ctactttctc cagccaatct ttctcctcat 6480

tcccttttct gttgcttgcc tctttgtttc ttccactgtt gtcccctttc tgttaagtaa 6540

tccgttcctc tccgcctcct tccccccgcc ccctgaaagc tccaaagagc cctcacttc 6599

<210> 11

<211> 23

<212> DNA

<213> homo sapiens

<400> 11

agtagggtta aggactagac agc 23

<210> 12

<211> 23

<212> DNA

<213> homo sapiens

<400> 12

cttgggtttc tgtttcttct ctg 23

<210> 13

<211> 24

<212> DNA

<213> homo sapiens

<400> 13

gacccaaacg ccacagcaat aatc 24

<210> 14

<211> 22

<212> DNA

<213> homo sapiens

<400> 14

tgctttcctt ctctgtagcc ag 22

<210> 15

<211> 23

<212> DNA

<213> homo sapiens

<400> 15

tctccatcac gtacaacctc aag 23

<210> 16

<211> 24

<212> DNA

<213> homo sapiens

<400> 16

catgactgtc tgtaggtcaa gttc 24

<210> 17

<211> 20

<212> DNA

<213> homo sapiens

<400> 17

ctgtcctaca gtctgcctgc 20

<210> 18

<211> 21

<212> DNA

<213> homo sapiens

<400> 18

agaatgaagg ttaacaccgt g 21

<210> 19

<211> 20

<212> DNA

<213> homo sapiens

<400> 19

gttctagcta gctgcagtgg 20

<210> 20

<211> 21

<212> DNA

<213> homo sapiens

<400> 20

gccatagagg agatgtggga g 21

<210> 21

<211> 20

<212> DNA

<213> homo sapiens

<400> 21

ggactggaga cagcaggatg 20

<210> 22

<211> 20

<212> DNA

<213> homo sapiens

<400> 22

agaggctttc ggggtggaag 20

<210> 23

<211> 21

<212> DNA

<213> homo sapiens

<400> 23

cacacagcac tctgaggtct g 21

<210> 24

<211> 22

<212> DNA

<213> homo sapiens

<400> 24

tctagtcctg accctatgcc tc 22

<210> 25

<211> 20

<212> DNA

<213> homo sapiens

<400> 25

agagcccagg cagctctgac 20

Claims (6)

1. A method for preparing a humanized animal model of LAG3, wherein in the humanized animal, an extracellular region of animal Lag3 gene is replaced by a corresponding fragment of human LAG3 gene, and an intracellular signal transduction region of animal LAG3 is reserved, comprising the following steps:

(1) constructing a plasmid for expressing sgRNA aiming at the murine Lag3 gene; the selected sgRNA is located at a position which is more favorable for gene recombination;

(2) constructing a targeting vector of the humanized LAG3 gene;

(3) injecting sgRNA obtained by in vitro transcription of the plasmid in the step (1), the vector in the step (2) and Cas9mRNA or Cas9 protein into cytoplasm or nucleus of a mouse fertilized egg, and transplanting the sgRNA into a recipient mother mouse to produce a LAG3 gene modified humanized mouse model, wherein the targeting vector of the humanized LAG3 gene comprises a chimeric sequence SEQ ID NO: 10, wherein 1-808bp and 5797-5799 bp are murine sequences, and 809-5796bp are human sequences; wherein the sequence of sgRNA aiming at the murine Lag3 gene is shown as SEQ ID NO:2 and SEQ ID NO: and 6.

2. The method of claim 1, further comprising the step of identifying a genotype of the animal model using primers.

3. The method of claim 2, wherein the primers used to identify the genotype of the animal model are:

4. the method of claim 3, further comprising sequencing verification of mouse clones positive for double-ended PCR identification.

5. The method of claim 4, wherein the sequencing uses the following primers:

primer name Primer sequences YF000087-HuLAG3-5seqR1 CTGTCCTACAGTCTGCCTGC(SEQ No.17) YF000087-HuLAG3-5seqF1 AGAATGAAGGTTAACACCGTG(SEQ No.18) YF000087-HuLAG3-5seqF2 GTTCTAGCTAGCTGCAGTGG(SEQ No.19) YF000087-HuLAG3-5seqF3 GCCATAGAGGAGATGTGGGAG(SEQ No.20) YF000087-HuLAG3-5seqR2 GGACTGGAGACAGCAGGATG(SEQ No.21) YF000087-HuLAG3-5seqR3 AGAGGCTTTCGGGGTGGAAG(SEQ No.22) YF000087-HuLAG3-3seqR1 CACACAGCACTCTGAGGTCTG(SEQ No.23) YF000087-HuLAG3-3seqF1 TCTAGTCCTGACCCTATGCCTC(SEQ No.24) YF000087-HuLAG3-3seqF2 AGAGCCCAGGCAGCTCTGAC(SEQ No.25)

。

6. Use of the humanized animal model of LAG3 gene made by the method of any one of claims 1-5 in assessing LAG 3-targeted drug efficacy, LAG 3-targeted drug screening development, evaluation of anti-tumor efficacy of LAG 3-targeted drugs in combination with other drugs, or toxicology studies of LAG 3-targeted drugs for non-disease diagnostic and non-disease treatment purposes.

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