CN109486860A - A kind of construction method of TIGIT humanized mouse model and its application - Google Patents

Abstract

The present invention provides a kind of methods for preparing TIGIT humanized mouse model, this method utilizes CRISPR/Cas9 technology, the introne of mouse TIGIT extracellular region and Extracellular domain sequence is part-humanised, all sequences in mouse transmembrane region and intracellular region including introne and UTR are still source of mouse, which remains the inside regulating and controlling sequence of gene, and complete intracellular signal transduction ability, the humanized model constructed using this method, realize the original endogenous expression characterization of TIGIT, meet source of people region (antibody binding domain) maximization, avoid missing effective antibody, it can be used for screening and evaluating the drug for being directed to mankind TIGIT gene, it is ideal Preclinical Drug test model.

Description

A kind of construction method of TIGIT humanized mouse model and its application

Technical field

The invention belongs to animal genetic engineerings and gene genetic to modify field, specifically, being related to based on gene editing skill The construction method of TIGIT gene modification humanized animal's model of art.

Background technique

Immunotherapy of tumors was fast-developing in recent years, already became the forth generation conventional treatments of oncotherapy.Tumour The thinking of immunization therapy mainly carries out in terms of target tumor cell and activating immune cell two.The treatment master of activating immune cell There are 4 classes: tumor vaccine, non-specific cell factor in treatment, cell therapy and immunologic test point inhibitor.Wherein, it is immunized Checkpoint inhibitor is the main force of oncotherapy new drug development, including PD1, PDL1 and CTLA4 etc. including immunologic test point Inhibitor all shows significant antitumor curative effect.Although these immunologic test points obtained in immunotherapy of tumors it is not small at Function, but only fraction patient shows lasting response to single immunologic test point, or even have some patients at present The checkpoint inhibitor of listing is simultaneously not responding to.Therefore, the more different immunologic test point inhibitor of screening, or utilize different pharmaceutical Synergistic function improve response rate have become industrial trend and hot spot.

TIGIT (T-cell immunoreceptor with Ig and ITIM domains) be 2009 by Research Team's first discovery of Genentech company, at the same they find one can be with the albumen PVR in conjunction with TIGIT (CD155), TIGIT is expressed on CD4+, CD8+, NK cell, and obvious expression is not detected in B cell；Functional experiment is shown TIGIT is active (inhibiting effect) by the interaction regulatory T-cell of the PVR with surface of dendritic cells, by PVR to dendron The control of the cytokine secretion of shape cell is realized.They were by further investigation discovery TIGIT in tumor-infiltrated T later Height expression in the surface cell blocks TIGIT and PD1 to be capable of the antitumous effect of specificity enhancing CD8+T cell simultaneously with antibody.18 Year, " Nature Immunology " above chapter article report TIGIT was related to the exhaustion of NK cell at tumor tissues position, blocked The function of TIGIT can prevent the exhaustion of NK cell and enhance the antitumor work of PDL1 antibody in such a way that NK cell relies on Property.These research achievements prove feasibility of the TIGIT as treatment of cancer novel targets, and it is adjustable a variety of to target a kind of target spot Tumour immunity relevant cell is expected to realize effect enhancing.

The monoclonal antibody project of the existing 6 targeting TIGIT into the clinical verification stage at present.Including having entered clinic Second phase is applied to the BMS-986207 (BMS) and the MTIG-7192A applied to non-small cell lung cancer of solid tumor indication (Genentech), and still in MK-7684 (MSD), the AB-154 (Arcus Bioscience), OMP- of a clinical phase 313M32(OncoMed),ASP8374(Potenza Therapeutics).Above into the medicine of the targeting TIGIT of clinical test The main pesticide application strategy of object is used in combination with the antibody drug of targeting PD1/PDL1, its treatment effect in entity tumor is tested Fruit.

The study of incident mechanism of human diseases screens effective therapeutic agent, is required to carry out a large amount of preclinical tests.By It is limited in terms of directly carrying out preclinical correlative study by ethics using people's cell and tissue, animal model just becomes the mankind The alternative of biological study.Mouse easily maintains and operates, the breeding cycle is short, with people in genome and physiology because small in size Etc. characteristic aspects are similar, and have had the gene modification technology of corresponding maturation, become widely applied mammal Model biological system.But since there are more differences with people for mouse physiological characteristic, have using the experimental result that animal model obtains Shi Buneng is adapted on human body.However the method for utilizing gene modification or cell and tissue transplantation, by human gene or groups of cells " placement " humanized mouse model prepared on mouse model is knitted, human gene or cell tissue are largely simulated Correlated activation substantially increases this kind of mouse model as the validity for simulating certain human diseases.Here it is our usual institutes The humanized mouse model said has the mouse model of mankind's functioning gene, human cell or tissue.

TIGIT humanization mouse refers to the method using gene modification, on the mouse that immune system perfects, by source of mouse TIGIT gene replacement is the gene of source of people, and building can be with anti-human source TIGIT monoclonal antibody interaction mouse model.It is most of immune Checkpoint is transmembrane protein, in conjunction with the extracellular portion of its ligand, submission downstream signal.The extracellular bond area collection of immunologic test point In albumen IgV domain functional domain, therefore antibody screening is concentrated mainly on the screening of this part.But 17 years Nature Mono- article discovery of Communications is located at the N-terminal loop region (N- outside the extracellular IgV domain functional domain of PD-1 molecule Loop itself and PD1 human antibody) have been mediated(nivolumab) main interaction of hydrogen bond, and by surface etc. from Sub-resonance technology (SPR) analyze nivolumab and PD-1 affinity, discovery N-loop missing PD-1 completely lost with The ability that nivolumab is combined, therefore, N-loop have played vital work in PD-1 and nivolumab interaction With.According to different antibodies combination feature, how to select suitable animal model with regard to most important in preclinical pharmacodynamic test.

The key of suitable humanized animal's model is the strategy of modifying gene, now prepared TIGIT people on the market Source mouse model, which has plenty of, only carries out humanization for the IgV domain of extracellular regions；Have plenty of source of people TIGIT extracellular region Coded sequence and mouse cross-film and coded sequence intracellular are inserted in behind mouse initiation codon ATG together with polyA, and which is same Source of mouse extracellular region is replaced, but rejects the sequence of all controlling genes expression, is existed unavoidably and the deviation of the original expression-form of gene. , according to the protein structure feature (Fig. 1) of TIGIT, the strategy of selection is then that mouse TIGIT whole extracellular regions are (including interior for we Containing son) it replaces with source of people TIGIT (including introne), this model has as follows compared with other above-mentioned models and common mouse Advantage: 1. will do humanization with the bond area of source of people TIGIT antibody, so that antibody is in conjunction with TIGIT in analog human body Binding pattern, better than antibody in conjunction with wild mouse TIGIT.2. extracellular whole region is done humanization, it is (anti-to realize source of people region Body binding domain) it maximizes, the complete three-dimensional conformation for retaining source of people protein extracellular domain avoids missing effective antibody.3. retaining All inherence regulating and controlling sequences (introne and 3 ' UTR), avoid damage to the intrinsic expression characteristic of gene.4. retain source of mouse trans-membrane region with Intracellular region, it is ensured that TIGIT is as interference-free as possible in signal transduction intracellular, it is ensured that antibody Composition analyzed middle and lower reaches signal Conduction and the effect expression of drug effect are interference-free.Compared with common mouse, crucial target molecule realizes humanization and changes the model It makes, and remains complete immune system, can be used for screening and evaluating the drug for being directed to human gene, be ideal face Bed prodrug test model.Simultaneously we the humanization mouse mate with the PD1 humanization mouse of independent research obtain PD1 and The equal humanized mouse model of TIGIT can be used in evaluating the cancer resistant effect that PD1 and TIGIT inhibitor are used in combination, better mould The quasi- existing pesticide application strategy of TIGIT drug.

For this purpose, we construct the sgRNA for mouse TIGIT gene and carry human TIGIT genetic fragment Carrier, using CRISPR/Cas9 technology and blastaea injection technique, using Exon1, intron1 of source of people TIGIT sequence, The portion Exon1, intron1, Exon2, intron2 and Exon3 of Exon2, intron2 and Exon3 partial replacement source of mouse TIGIT Point, retain mouse TIGIT transmembrane region, intracellular region full sequence and UTR sequence.Meanwhile we are for CRISPR/Cas9 technology In each original part for using include that gRNA etc. has carried out sufficient optimization and adjustment, ensure that and humanization is prepared using the technology The high success rate and high-accuracy of TIGIT genetic animal model.Finally, we are also by TIGIT humanization mouse and PD1 humanization Mouse mating, obtains the mouse model of two common humanizations of target spot.

Summary of the invention

The present invention provides a kind of methods for preparing TIGIT humanized mouse model using CRISPR/Cas9 technology, special Sign is, in the humanization mouse, whole extracellular regions of mouse TIGIT gene are replaced by the gene of source of people TIGIT Homologous segment retains mouse TIGIT transmembrane region and intracellular region.

Specifically, itself the following steps are included:

(1) plasmid of the building expression for the sgRNA of mouse TIGIT gene；

(2) expression vector of humanization TIGIT gene is constructed；

(3) step (1) plasmid is transcribed in vitro the sgRNA and step (2) that obtain carrier and Cas9mRNA or Cas9 protein injection is implanted into receptor female rat production TIGIT gene modification humanization into mouse fertilized egg cell matter or nucleus Mouse model；

The extracellular region of source of mouse TIGIT gene can be replaced with the extracellular region of source of people TIGIT gene by the carrier, be remained The transmembrane region and intracellular region of mouse TIGIT gene.

Preferably, humanization TIGIT gene includes sequence SEQ ID NO:1.

Preferably, the sequence such as SEQ ID NO:2 of the chimeric TIGIT albumen of humanization TIGIT gene coding.

Preferably, wherein the sequence such as SEQ ID NO:3 and SEQ ID NO:4 institute of the sgRNA for source of mouse TIGIT gene Show.

Further, wherein the process of carrier construction is as follows in step (2): being expanded respectively using mouse C57BL/6 genome Increase the homology arm segment at 1 end of mouse TIGIT gene extron and the homology arm segment at exon 3 end, with Human TIGIT's BAC is template, and above-mentioned segment composition is then attached on PMD18T carrier by amplification source of people replacement segment, constructs humanization The carrier of TIGIT gene.

Preferably, the primer that the homology arm at amplification 1 end of mouse TIGIT gene extron uses is SEQ ID NO:5 and SEQ ID NO:6, the primer that the homology arm at amplification 3 end of mouse TIGIT gene extron uses is SEQ ID NO:7 and SEQ ID NO: 8, the primer that amplification source of people replacement segment uses is SEQ ID NO:9 and SEQ ID NO:10 primer pair and SEQ ID NO:11 With SEQ ID NO:12 primer pair.

It preferably, further comprise the step of identifying the genotype of the animal model using primer.

The present invention also provides a kind of methods for preparing the bis- humanization mouse models of TIGIT/PD1, include the following steps:

(a) TIGIT gene humanization mouse is obtained using any one of claim 1-7 the method；

(b) the humanization mouse that step (a) obtains is mated and is screened with PD1 humanization mouse, obtain double source Change mouse model.

Further, drug effectiveness, the TIGIT for providing method described in any of the above embodiments in assessment targeting TIGIT are targeted The toxicity of drug screening exploitation, the antitumor evaluating drug effect of TIGIT targeted drug joint other drugs or TIGIT targeted drug is ground Application in studying carefully.

In addition, the present invention also provides the sgRNA of selectively targeted mouse TIGIT gene, sequence such as SEQ ID NO:3 and Shown in SEQID NO:4.

In addition, nucleic acid sequence includes SEQ ID NO:1, humanization the present invention also provides humanization TIGIT gene The chimeric protein sequence SEQ ID NO:2 of TIGIT gene coding.

The present invention has the positive effect that:

1, all extracellular regions of mouse TIGIT are carried out humanization by us, and intracellular signal transduction part still retains mouse sequence. Introne and 5 ' UTR and 3 ' UTR sequences between exon used in retaining simultaneously, protect regulating and controlling sequence controlling gene Intrinsic characteristic when expression makes to express on its space-time as close possible to original expression characterization.Furthermore the people of the construction of strategy is applied Source model realizes source of people region (antibody binding domain) maximization, avoids missing effective antibody.For more different demands Antibody screening, cover all TIGIT inhibitor manufacturers for antibody drug effect primary dcreening operation demand.

2, we mate TIGIT humanization mouse with PD1 humanization mouse, obtain the small of two common humanizations of target spot Mouse model is not only available for the evaluation of TIGIT antibody list medicine, while can be used for the antibody combined antitumor drug effect of PD1 antibody of TIGIT and commenting Valence has far-reaching directive significance to preclinical evaluating drug effect.

It is small in the target spot that the B6-hTIGIT and B6-hPD1/hTIGIT humanized mouse model that we establish has filled up market The blank of mouse model, while providing TIGIT inhibitor evaluation platform.Significant effect is economically brought with social perspective Benefit.

3, the present invention provides the concrete operation method of the preparation source of people TIGIT genetic animal model of optimization, in this method The maximum preparation for optimizing sgRNA, carrier and etc., it is ensured that the success rate of animal model.

Detailed description of the invention

Fig. 1 is source of people TIGIT protein structure domain.

Fig. 2 is humanization TIGIT construction strategy figure.

Fig. 3 is F0 for 5 end mouse TIGIT-KI-target and 3 ends identification electrophoretogram.

Fig. 4 is F0 for mouse TIGIT-KI- wild type identification electrophoretogram.

Fig. 5 is that electrophoretogram is identified at 5 end F1 generation TIGIT-KI-target and 3 ends.

Fig. 6 is F1 generation TIGIT-KI- wild type identification electrophoretogram.

Fig. 7 is TIGIT detection of expression result in spleen.

Fig. 8 is spleen medium size lymphocyte testing result.

Fig. 9 is that electrophoretogram is identified at 5 end TIGIT-KI-target and 3 ends in B6-hPD1/hTIGIT mouse preparation process.

Figure 10 is TIGIT-KI- wild type identification electrophoretogram in B6-hPD1/hTIGIT mouse preparation process.

Figure 11 is that electrophoretogram is identified at 5 end PDCD1-KI-target and 3 ends in B6-hPD1/hTIGIT mouse preparation process.

Figure 12 is PDCD1-KI- wild type identification electrophoretogram in B6-hPD1/hTIGIT mouse preparation process.

Figure 13 is PD1 and TIGIT detection of expression result in spleen.

Figure 14 is the fluidic cell result of spleen medium size lymphocyte.

Figure 15 is each group mouse weight variation diagram.

Figure 16 is each group mice tumors grew curve.

Specific embodiment

Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art referring to specification text Word can be implemented accordingly.

Embodiment 1:C57BL/6-TIGIT humanized mouse model is established

1, the source of people sequence of source of people segment replacement region and insertion is determined

According to source of people TIGIT and PVR protein binding functional domain, we choose source of people TIGIT gene order (Gene ID: 201633) Exon1, intron1, Exon2, intron2 and Exon3 (transcript NM_173799.3) replaces source of mouse TIGIT Exon1, intron1, Exon2, intron2 and Exon3 (transcript NM_001146325.1), retain mouse TIGIT cross-film Area and intracellular region, replacement policy figure are as shown in Figure 2.Replaced albumen chimeric sequences are as follows, and underscore part is source of people replacement Area:

MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAIC NADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLGGTM AAVLGLICLMVTGVTVLARKKSIRMHSIESGLGRTEAEPQEWNLRSLSSPGSPVQTQTAPAGPCGEQAEDDYADPQE YFNVLSYRSLESFIAVSKTG

Gene order after humanization includes sequence shown below, and leukorrhagia dashed part is people's source sequence, from mouse Exon1 Sequence starts, and 1-97 are sequence of mouse source, and 98-5664 are people's source sequence, and 5665-5739 are source of mouse Exon3 sequence Column.

cttttcaccagcttggtttcaccttacagctgcagtgagccagtttcagttggaggagaggccacatc cactttgctgtaggcctctggttagaagcatgcgctggtgtctcctcctgatctgggcccaggggctgaggcaggc tcccctcgcctcaggtaaggcctgaaacccagcagagcagcagggaggaaaaacaaggctaagcttggttggagac ttgggtgcttgtgtagggaagactccaagagcatgccacagctgcttgagagaaagaaattgcaacttttagagtc ctgtgtactcttatttaagaaaggacaatctctgagaatgaggtggggttctgtttttggtggcagatgaaggact ttgcagacttccggggttggaggagcctgactggccagcacaagaaggagcaggagaagaagaagggctgggcagg aggcattgccagtttgcttttagaagctcattcctgctccagcttcttttctgttgcctttcaggaactttccagg aaaacattataagttctctgtgaattgaaaatgtctttttcacagattccagaggttctaccactagagactgatt ttttgttaaatttatttcttctatattaaaaatcagagtcaaatatttctgagggaggggaacacaaatatttaaa aactgtggtataaattagtttttatgttgccttctgaactatctgaggatggagatgtgcccagggttgctgggga gaggttaagtgctgagagggtggttatgcctgggagaagcagggtttcctgctaagcttcctttgctattggattt cttggctttcctgaaggttttttcaggcactgcaaatgacaatcaaccaaacagcaaggttttatttcaaaggata atgcatccagcatagaactaggccgcaggaagaaacataaaaacaaaacaaaataaaaacagaagaggtggtatca gctgacaagggcttatattctaagtgggtagacaaaataggcctacgaaaataacaagctggccatgaagctacat agcagaatatgcgagggggcaacgtgctaagttgtgtagtcataagccaggcatttagtaaggtgctaattataca atacaggcagtgaatgtgaaaggaaaagggaggttgagatgggctgaggtcttctaggaggcggggagggagtgca gccttgacaagcctccctgtgggcgaggtgtaaagaggggcagaagtcagctctggaagcatagggcagtgggtgg ggaggagatgggctgggctgggctggagtagagatgtgtggagaagggtgagaagactggaaagacaacctgaatg ggggactgggagccttgaataacaggcatggagaggagcgtctcttgaaatggaagaaacaggaaataattacagc ctctatggaggagcaacaggatggactggagaaactatcattccaaaatccagttggggcctcaaaggcccttaga atttttctaggaaggttgaaggccagctgctgacccaggactcacatgtgcttcgtcctcttccctaggaatgatg acaggcacaatagaaacaacggggaacatttctgcagagaaaggtggctctatcatcttacaatgtcacctctcct ccaccacggcacaagtgacccaggtcaactgggagcagcaggaccagcttctggccatttgtaatgctgacttggg gtggcacatctccccatccttcaaggatcgagtggccccaggtcccggcctgggcctcaccctccagtcgctgacc gtgaacgatacaggggagtacttctgcatctatcacacctaccctgatgggacgtacactgggagaatcttcctgg aggtcctagaaagctcaggtattcctgctggagcaagttggtggataaacctctccctctagcatagaaaatgcaa tcctgaaacactgcacagcagggcttctcaattcgggatcacatttgaatcacctgaggagattttaaatcatact gatgccgaggcctcacccagaccaattcaatcagaatccctaatagcagagctaaacaagggtaaggtctaaaagc atttccaggtgattctaatgggcagccaatactgagaaccactgttcttatgtaagaagcacatcttacctatatt tcctaggaagaccagttgatgaggtcatatgcaaaagttcccatttattggtttagtataattgtgcaaattagaa ttaacccctaagtgtataaagagtagagctggttaaaaacatagcctgtgctaagtttaattgtacagtaatttac atttgtgtggtacttccaagtttcccaaatgccctcatatttgttatccacctgtacatttaaaccaattcccaga ggttagaaaaggattcttatcttatctgagagacagtatagcaccatggttaaaagctcagactctgacaccagac agtctgggtgcaaatcctggctctgctgttgccctcagacaggatttcttagcttctctgtgtatcagttttctca tctgtgaaaggaggatgaatataagaccttatgcgatgctaatgtaggattaaattccctactacaagaaaatggt cttagaacaatgcctggaacatggtaagtgctgtataagtgttaactattagtatgattcccatttgtttgatgca ggcagaagctggagcttggagttgctcagagtggtagagctgattttaacacagatgtttttgttctactaaactg catcctcagaactcctatttactgctgtcagttatgttgaagattaagttatatggttgcaaaatatgtgtaaact gagaagataaaaggttaccttaaagtttcctttcaaatgttacttctttttttttttttttttttttgagacggag tttcgctcttgttgcccaggctggagtgcaatggcgcaatctcagctcactgcaacttccgcctcctgagttcaag cgattctctcctgcctcagcctcctgagtatctgggattacaggcacccaccaccatgcccggctaattttgtatt tttagtagagacagggtttcttcacgttggtcaggctggtctcaaactcccgacctccagtgatctgcccgcctca gcctcccaaagtgttacttcttaactgatttcacataggccaaacaaagactggttaagagttgaggctctggaat tgcactgtctggggttgactattgcagttgaccccacgaagtctgggtaaattatgtactttttttctcagttttc ttgtcgataaaatgtggcaaataatttcatgagctgatacaagtaaggtcttacaacagtgctagataggtaataa gcttaacttcataagggctagtttttatgctatttattggttcatggttagaggaggcctgggacaataagcaggg caggggacctagattctggcaaagtaaccaagtgatggccttgaccattctgggctctttaccccagtgtcatatg ccagaggacccatcaccagacggcaggaccgggaacagggagggagttcatcaggcagaaggcccctgtggagtgg tagtaccagcaactgaagaccaaatgggagctatgtctggttgctaatctccaggccaggccattcatggagatcg gtggtcctgacatgcacactcctccccaagaccacagtggcacggctatttcccagcctgaacctggggaaatgaa ggtgcaaatttcttctctgtggctcatggacccctggggagagagatcagaagatgttctattggacccctgtgca gcctccccacaaagcctgtatgtttccacccttttgccattgcagactccagggaacaccattggtcactcggctc ttccctcttgtttcccaagtggtcagttgcttatgtggaatgcaaactctccttaattacttgcattagcacaaac cctggcttctttttgaacctaatctcaaaccttagtccaagtccctatcctaatactatctgtaatatttaagcta ttcctaaccttaatgctaaaattgtacttaaccccaaagtcaacctggttaataggcagaagttttaagtaaccct tatacagagtctgccccttccccaccctgcatgtagtgatgtcatgcttactcagggctgcttctgtttcaaagac cttgatatctttgttctatgaccataactgtgaatggagacattggtgcttttatttcaaaccaaaaattccaaat gaaattgatacatctaatatcccaagttccactctggtcccaattgtatctcgaacttatccttaaacctaaccaa actcattatcataatcttccctaaacctcaacatgcttttcactccatctatcttcatctgtccccaaggtgtaag agaacccaggcctaaccctgcctccaatctggctctgtccccaaactgatcatatatcctttcctggttttatgga tagccctctgcagcccctcttacctgctgcagtgtgttctctatgacccctgtgctttccatctgtcctctttact ccagagctaactgtatggctatggctaaccagaagccgtcctggccaactgtgactgagtccctcaaggcaggagg aacatgttctggggaatttgaattgtgcaggcaccacccttgtcttatggattggattcctaagccaccttttagc aacccagacccttaggtagagggatgagatgtgaggcttctgctgcatcctttgaagactcccctggatacctgag ccctacctgagctgctggggcagactggaaacctacctacagcgcagcatgcatgggggtgatggtgttctttttc tcacattcttcttgggtcagatcttactcatctcttcaaaactcccctcctgcatcttcttgcttccacttacatg ttatctcttaatgaaatgcactagttcatgatttcacttatgccacaaatatttatatttactgagcattcgctat gtgtcagatgctgagaaaacagcaaaggataagagtccagagcctcatgccctccagggtttcacagcctagctgg aaataagtgttataggcttaagggaaaaggttacatactttttttcatttttgtaatttaatgttaactttatttt aattttgaattaaatatttttattgatatgatttcatatttgaagttcaataaaagaaaatattcactttctgagt ttttttctggaactactatttgtttcatttcatgattaccaaaaatgattttgtcaaatagaagaaacgaatgtta aaatgacctgctctggtatcaaatatacaaggtacaccactgctgaaaatatcagtcattgatttcatgatgggcc agatggatactctgctgttcaaagtgttgggcttaaaggctccagtcccatggttacacaaagggcttggccaatc ctcccctctctgccgtgaagctcaggctgccctggggaaatcagcttcacttctggcttctgccactcatctctgt tttgtcctccctctagtggctgagcacggtgccaggttccagattccacttggaggaaccatggctgctgtgctgg gactcatttgcttaatggtcacaggagtgactgtactggctagaaag

2, the sgRNA sequence for model preparation is determined

The design of sgRNA is not mature enough at present, is easily achieved unlike design of primers, is needed by largely testing Screening and adjustment, for this purpose, we utmostly optimize used sgRNA sequence, to guarantee the accuracy and efficiency practiced shooting.

SgRNA general area is determined according to replacement segment.Several groups of minimum sequences of off-target rate are chosen for target fragment, such as Lower sequence (see the table below) as sgRNA to be selected.It designs and synthesizes identification 5 ' and holds target site and 3 ' end target sites, and construct sgRNA Expression vector.Both ends sgRNA recognition site is located on first of mouse TIGIT gene and third exon, respectively Target site sequence of the sgRNA on TIGIT are as follows:

SgRNA title	Sequence
		TIGIT-5S1	CAGCCTGTATCAGCCCC
TIGIT-5S2	GTTAGAAGCATGCATGGC
		TIGIT-3S1	GAACTGAGCCACTACA
TIGIT-3S2	AGTTCCAGACTGCCCCGCT

SgRNA transcribes preparation method: with PrimerStar or PrimerStar Max system, sgRNA-F, sgRNA-R are Primer, it is that template carries out PCR that correct puc57-sgRNA plasmid (1:30 dilution), which is sequenced, and PCR product is purified, and is prepared SgRNA transcription preparation template.The transcription of sgRNA is carried out using T7-ShortScript in-vitro transcription kit (AM1354).

SgRNA screening: 5 ' end target sites and 3 ' end target site sgRNA are matched two-by-two, are combined into 4 couples of sgRNA.Respectively will After 4 couples of sgRNA and Cas9 albumen are incubated for, mixed liquor is injected in 0.5 day fertilized eggs, after culture to blastocyst stage, The identification for carrying out the cutting efficiency of mouse TIGIT gene, screens high sgRNA pairs of cleavage activity with this.

SgRNA cuts identification method: carrying out nested PCR amplification to the blastaea of collection, (PCR primer and amplification scheme are as follows Shown in table), amplified band is subjected to the sequencing of two generations, is compared with wt band, the probability (qualification result that statistics mutates or deletes See the table below), final choice TIGIT-5S1 and TIGIT-3S1.

SgRNA detection scheme

SgRNA qualification result:

SgRNA title	Cutting efficiency	Remarks
			TIGIT-5S1	81%	It selects
TIGIT-5S2	80%
			TIGIT-3S1	72.2%	It selects
TIGIT-3S2	78.6%

3, carrier and transplanting product building

1) C57BL/6 genome amplification TIGIT-5arm (end Exon1 homology arm) is used, (end Exon3 is same by TIGIT-3arm Source arm).Using the BAC of Human TIGIT as template, amplification source of people replaces segment both ends HuTIGIT-H1 H2, first by TIGIT- 5arm, TIGIT-3arm, HuTIGIT-H1 H2 fusion, then SLIC is connected on PMD18T carrier, apply Amp plate.Successful connection Carrier be named as TIGIT-RV.Pac1 linearizes TIGIT-RV, and CIAP processing is spare.

2) it identifies that correct Human TIGIT BAC electricity is gone in EL350, applies chl plate, electricity turns the TIGIT-RV of linearisation Carrier extracts Human BAC into the host strain of Human TIGIT BAC, applies Amp plate.Successful carrier is extracted to be named as TIGIT-dsDNA-5S1-3S1。

3) final carrier is identified in PCR and digestion, uses digestion scheme preparation injection sample after correct.ASC1 digestion: 7238 3829, recycle the band of 7238bp for injecting.

PCR amplification scheme

4, transplanting injection obtains positive mouse

Product, purpose sgRNA, cas9 protein injection to fertilized eggs will be transplanted, pseudopregnant mouse is migrated to.

It obtains positive F0: genotype identification being carried out to the raw newborn mouse of false pregnancy, identification primer see the table below, and screening is successively inserted into just The positive mouse F0 mouse of true source of people segment, qualification result are shown in Fig. 3.

F0 identifies primer:

PCR reaction condition

Fig. 3 and Fig. 4 shows F0 for qualification result, and wherein Fig. 3 shows that 5 end TIGIT-KI-target and 3 ends are identified As a result, Fig. 4 shows the wild band identification of TIGIT-KI-.Wherein negative control is B6 genomic DNA；N is blank control, nothing The control of template；P is positive control；TRANS 2K PLUS II band: 8000bp 5000bp 3000bp 2000bp 1000bp\750bp\500bp\250bp\100bp.Only the both ends 5# PCR is consistent with positive control, and sequencing passes through.

F0 and background mouse match numerous acquisition F1, carry out identified for genes to F1 generation rat-tail, F1 generation mouse PCR experiment result is shown in Fig. 5 And Fig. 6, it is as follows to obtain mouse number: 90#, 96#, 99#, 100#, 102#, 104#, 106#, 107#.F1 largely expands numerous rear progress Interworking obtains homozygote.We are numerous by matching for 2-3 generation with PD1 humanization mouse by the pure and mild mouse of TIGIT humanization simultaneously, Obtain TIGIT and the bis- humanization mouse of PD1, abbreviation B6-hPD1/hTIGIT.

Fig. 4 shows that 5 end F1 generation TIGIT-KI-target and 3 ends identify that electrophoretogram, Fig. 5 show F1 generation TIGIT- The wild band identification of KI-.Wherein negative control is B6 genomic DNA；N is blank control, the control of no template；P is positive right According to；TRANS 2K PLUS II band: 8000bp 5000bp 3000bp 2000bp 1000bp 750bp 500bp 250bp 100bp.Positive mice both ends PCR result is consistent with positive control.

Source of people TIGIT expression and immune system verifying in 2 B6-hTIGIT mouse of embodiment

Pass through the expression of flow cytometry TIGIT Mice homozygous and immunocyte monoid is checked, passes through Analysis can successful expression humanization gene, and the mouse for not causing immune system obvious abnormal is for tumour effect experiment.

1. protein expression detects

The main expression tissue thymus gland of TIGIT or spleen are chosen, the expression of TIGIT albumen in tissue is detected.

TIGIT albumen streaming detection method:

A) it draws materials: to B6-hTIGIT Mice homozygous and C57BL/6 background mouse (see mouse information list) clip spleen, claiming Weight, is placed in c-type pipe.

B) digest: peripheral blood room temperature, which is protected from light, to be split red, is washed 1 time with FACS buffer；Spleen and thymus gland use enzymic digestion liquid (PBS contains Ca, Mg+2%CS+10mM HEPES+30ugDNase+1.75mg clostridiopetidase A D), digests 30min by 37 DEG C.It will digest At spleen cell add 300ul 0.1M EDTA terminate digestion.1mL membrane filtration is taken, removing does not digest complete tissue Block, every pipe adds in 2mL FACS buffer and EDTA.8 DEG C, 400g, be centrifuged 5min, remove supernatant, the every pipe of spleen add 3ml 1 × RBC is mixed, and room temperature is protected from light splitting erythrocyte 5min, 8 DEG C, 400g, is centrifuged 5min, removes supernatant, adds 1mL FACS buffer weight It is outstanding, filtering；8 DEG C, 400g, it is centrifuged 5min, removes supernatant, FACS buffer is added to be resuspended, adjusts cell concentration 1 × 10⁷/ mL, is assigned to 100uL in streaming pipe prepares to be incubated for antibody.

C) it closes: according to experiment needs, by 100uL (about 10⁶It is a) cell assigns in different streaming pipes, add Fc block (CD16/32 antibody) every pipe adds 1ul CD16/32 antibody (1:100 dilution) to mix, and is incubated for 5min on ice.

D) antibody incubation: antibody mixed liquor (hTIGIT, mTIGIT, CD3 antibody) is configured according to sample cell number, according to anti- The addition of body optimum amount, each sample add 50uL antibody mixed liquor, are vortexed and mix；Single dye manages every pipe and adds 0.5ul antibody, is vortexed mixed It is even；It is protected from light on ice and is incubated for 1h；

E) clean: FACS buffer, upper machine testing is added in FACS buffer washing.5min is added before loading Sytoxblue (final concentration 1:10000 dilution) distinguishes life or death cell.

Mouse information and spleen weight

Testing result: the anti-TIGIT antibody of source of people and the anti-TIGIT antibody of source of mouse are used, flow cytomery B6- is passed through Source of people and source of mouse TIGIT T-cell situation are expressed in hTIGIT Mice homozygous and B6 background mouse, and people is not detected in B6 background mouse Source TIGIT expression, and be only capable of detecting that source of people TIGIT is expressed in B6-hTIGIT Mice homozygous.And source of people in B6-hTIGIT TIGIT protein expression abundance is similar to source of mouse TIGIT protein expression (see Fig. 7).

2. immune system is verified

Choose the Main Immune Organs thymus gland or spleen of mouse, clip B6-hTIGIT Mice homozygous and C57BL/6 background mouse Thymus gland or spleen (mouse information such as following table), are digested to individual cells for tissue grinder, with source of mouse T B NK surface antibody to group It knits cell extracellular protein to be dyed, after cleaning cell with PBS, carries out FCM analysis T (CD4+, CD8+), B, NK cell Quantity.

Immunocyte flow cytometer detection method:

A) draw materials: clip B6-hTIGIT Mice homozygous and the weighing of C57BL/6 background mouse spleen are placed in c-type pipe.

B) digest: using enzymic digestion liquid, (PBS contains Ca, Mg+2%CS+10mM HEPES+30ugDNase+1.75mg to spleen Clostridiopetidase A D), 37 DEG C, digest 30min.The EDTA of 300ul 0.1M is added to terminate digestion the spleen cell that digestion is completed.Take 1mL With membrane filtration, removing does not digest complete tissue block, and every pipe adds in 2mL FACS buffer and EDTA.8 DEG C, 400g, centrifugation 5min removes supernatant, and the every pipe of spleen adds 1 × RBC of 3ml to mix, and room temperature is protected from light splitting erythrocyte 5min, 8 DEG C, 400g, is centrifuged 5min removes supernatant, and 1mL FACS buffer is added to be resuspended, filtering；8 DEG C, 400g, it is centrifuged 5min, supernatant is removed, adds FACS buffer It is resuspended, adjusts cell concentration 1 × 10⁷/ mL assigns to 100uL in streaming pipe, prepares to be incubated for antibody.

C) it closes: according to experiment needs, by 100uL (about 10⁶It is a) cell assigns in different streaming pipes, add Fc block (CD16/32 antibody) every pipe adds 1ul CD16/32 antibody (1:100 dilution) to mix, and is incubated for 5min on ice.

D) antibody incubation: configuring antibody mixed liquor (CD19, CD4, CD8, CD335, CD3 antibody) according to sample cell number, It is added according to antibody optimum amount, each sample adds 50uL antibody mixed liquor, is vortexed and mixes；Single dye manages every pipe and adds 0.5ul antibody, whirlpool Rotation mixes；It is protected from light on ice and is incubated for 1h；

E) clean: FACS buffer, upper machine testing is added in FACS buffer washing.5min is added before loading Sytoxblue (final concentration 1:10000 dilution) distinguishes life or death cell.

Testing result: as shown in fig. 7, each T, B, NK immunocyte quantity of B6-hTIGIT Mice homozygous and C57BL/6 background The basic indifference of mouse.Above data shows that source of people TIGIT has participated in the immune response process of mouse, and B6-hTIGIT mouse is exempted from Epidemic disease system is normal, compares indifference with generic background mouse, can be used for the evaluation of TIGIT targeted drug.

Source of people TIGIT and PD1 expression and functional verification in embodiment 3B6-hPD1/hTIGIT mouse

1, the pure and mild humanization mouse of PD1 is mated with TIGIT humanization mouse and obtains PD1 heterozygosis TIGIT heterozygosis humanization Mouse, male mouse and the pure and mild humanization mouse of PD1 carry out IVF, the pure and mild TIGIT heterozygosis humanization mouse of a large amount of PD1 are obtained, to PD1 Pure and mild TIGIT heterozygosis humanization mouse obtains PD1 and the bis- pure and mild humanization mouse of TIGIT to the adaptation age, by its brother and sister's interworking, Qualification program is as follows with qualification result, the bis- pure and mild humanization mouse numbers of the PD1 and TIGIT of acquisition be 148,155,161,168, 172、177-180、182、194#。

PCR primer information

Fig. 8-Figure 11 shows the electrophoretic identification in B6-hPD1/hTIGIT mouse preparation process, and wherein Fig. 9 is shown 5 end TIGIT-KI-target and 3 end qualification results, Figure 10 show the wild band identification of TIGIT-KI-.Figure 11 is shown Electrophoretogram is identified at 5 end PDCD1-KI-target and 3 ends；Figure 12 shows PDCD1-KI- wild type identification electrophoretogram.It is wherein negative Property control be B6 genomic DNA；P is positive control, TIGIT or PD1 humanization mouse rat-tail；N is blank control, no template Control；TRANS 2K PLUS II band: 8000bp 5000bp 3000bp 2000bp 1000bp 750bp 500bp 250bp\100bp.Using positive control as reference, stripe size and the consistent as positive mice of positive control, both ends PCR and open country Raw type is identified while having band to show that the mouse is heterozygote KI/WT, if only both ends PCR has band to show that the murine genes type can It can be KI/KI or KI/KO.

2, expression and functional verification

Pass through the expression of flow cytometry TIGIT&PD1 Mice homozygous and immunocyte monoid is examined Look into, through analysis can successful expression humanization gene, and the mouse for not causing immune system obvious abnormal is real for tumour drug effect It tests.

The detection of 2.1 protein expressions

TIGIT and the main expression tissue thymus gland of PD1 or spleen are chosen, the expression of TIGIT and PD1 in tissue is detected.Small Mouse internal injection Anti-CD3e antibody can activate the intracorporal T cell of mouse, thus using Anti-CD3e Antibody on Mouse into It assassinates after swashing, the expression of TIGIT and PD1 after taking same tissue detection to stimulate.

Stimulation process method: 7.5ug is injected intraperitoneally in B6-hPD1/hTIGIT Mice homozygous and C57BL/6 background mouse The antibody (Sigma, SAB4700553) of Anti-CD3e, draws materials after 24 hours, does flow cytometer detection.

Immunocyte flow cytometer detection method:

F) it draws materials: to B6-hPD1/hTIGIT Mice homozygous and C57BL/6 background mouse (see mouse information list) clip spleen Dirty and thymic tissue, weighing, is placed in c-type pipe.

G) digest: peripheral blood room temperature, which is protected from light, to be split red, is washed 1 time with FACS buffer；Spleen and thymus gland use enzymic digestion liquid (PBS contains Ca, Mg+2%CS+10mM HEPES+30ugDNase+1.75mg clostridiopetidase A D), digests 30min by 37 DEG C.It will digest At spleen cell add 300ul 0.1M EDTA terminate digestion.1mL membrane filtration is taken, removing does not digest complete tissue Block, every pipe adds in 2mL FACS buffer and EDTA.8 DEG C, 400g, be centrifuged 5min, remove supernatant, the every pipe of spleen add 3ml 1 × RBC is mixed, and room temperature is protected from light splitting erythrocyte 5min, 8 DEG C, 400g, is centrifuged 5min, removes supernatant, adds 1mL FACS buffer weight It is outstanding, filtering；8 DEG C, 400g, it is centrifuged 5min, removes supernatant, FACS buffer is added to be resuspended, adjusts cell concentration 1 × 10⁷/ mL, is assigned to 100uL in streaming pipe prepares to be incubated for antibody.

H) it closes: according to experiment needs, by 100uL (about 10⁶It is a) cell assigns in different streaming pipes, add Fc block (CD16/32 antibody) every pipe adds 1ul CD16/32 antibody (1:100 dilution) to mix, and is incubated for 5min on ice.

I) antibody mixed liquor (hPD-1, mPD-1, hTIGIT, mTIGIT, CD3 antibody incubation: are configured according to sample cell number Antibody), it is added according to antibody optimum amount, each sample adds 50uL antibody mixed liquor, is vortexed and mixes；Single dye manages every pipe and adds 0.5ul Antibody is vortexed and mixes；It is protected from light on ice and is incubated for 1h；

J) clean: FACS buffer, upper machine testing is added in FACS buffer washing.5min is added before loading Sytoxblue (final concentration 1:10000 dilution) distinguishes life or death cell.

Mouse information and spleen weight

Testing result: using the anti-PD1 of source of people and TIGIT antibody and the anti-PD1 of source of mouse and TIGIT antibody, pass through fluidic cell Instrument detection B6-hPD1/hTIGIT Mice homozygous and B6 background mouse express source of people and source of mouse PD1 and TIGIT in stimulating and not stimulating Source of people PD1 and TIGIT expression are not detected in B6 background mouse for T-cell situation, and in B6-hPD1/hTIGIT Mice homozygous only It can detect that source of people PD1 and TIGIT are expressed.And after the stimulation of CD3e antibody, source of people PD1 and TIGIT expression with it is significant It improves, it is similar to TIGIT promotion amplitude to source of mouse PD1 to promote amplitude.The above result shows that the PD1 that is prepared of this method with TIGIT gene modification humanization mouse can successful expression PD1 and TIGIT albumen, and wherein TIGIT expression reach with it is common small Mouse is on close level (see Figure 13).

The verifying of 2.2 immune systems

Choose the Main Immune Organs thymus gland or spleen of mouse, clip B6-hPD1/hTIGIT Mice homozygous and C57BL/6 Background mouse thymus gland or spleen (mouse information is as follows), are digested to individual cells for tissue grinder, with source of mouse T B NK surface antibody Histocyte extracellular protein is dyed, after cleaning cell with PBS, carries out FCM analysis T (CD4+, CD8+), B, NK Cell quantity.

Immunocyte flow cytometer detection method:

F) draw materials: clip B6-hPD1/hTIGIT Mice homozygous and the weighing of C57BL/6 background mouse spleen are placed in c-type pipe.

G) digest: peripheral blood room temperature, which is protected from light, to be split red, is washed 1 time with FACS buffer；Spleen and thymus gland use enzymic digestion liquid (PBS contains Ca, Mg+2%CS+10mM HEPES+30ugDNase+1.75mg clostridiopetidase A D), digests 30min by 37 DEG C.It will digest At spleen cell add 300ul 0.1M EDTA terminate digestion.1mL membrane filtration is taken, removing does not digest complete tissue Block, every pipe adds in 2mL FACS buffer and EDTA.8 DEG C, 400g, be centrifuged 5min, remove supernatant, the every pipe of spleen add 3ml 1 × RBC is mixed, and room temperature is protected from light splitting erythrocyte 5min, 8 DEG C, 400g, is centrifuged 5min, removes supernatant, adds 1mL FACS buffer weight It is outstanding, filtering；8 DEG C, 400g, it is centrifuged 5min, removes supernatant, FACS buffer is added to be resuspended, adjusts cell concentration 1 × 10⁷/ mL, is assigned to 100uL in streaming pipe prepares to be incubated for antibody.

H) it closes: according to experiment needs, by 100uL (about 10⁶It is a) cell assigns in different streaming pipes, add Fc block (CD16/32 antibody) every pipe adds 1ul CD16/32 antibody (1:100 dilution) to mix, and is incubated for 5min on ice.

I) antibody incubation: configuring antibody mixed liquor (CD19, CD4, CD8, CD335, CD3 antibody) according to sample cell number, It is added according to antibody optimum amount, each sample adds 50uL antibody mixed liquor, is vortexed and mixes；Single dye manages every pipe and adds 0.5ul antibody, whirlpool Rotation mixes；It is protected from light on ice and is incubated for 1h；

J) clean: FACS buffer, upper machine testing is added in FACS buffer washing.5min is added before loading Sytoxblue (final concentration 1:10000 dilution) distinguishes life or death cell.

Mouse information and spleen weight

Testing result: as shown in figure 14, each immunocyte quantity of B6-hPD1/hTIGIT Mice homozygous each T, B, NK with The basic indifference of C57BL/6 background mouse.Above data shows that source of people PD1 and TIGIT have participated in the immune response process of mouse, B6-hPD1/hTIGIT mouse immune system is normal, compares indifference with generic background mouse, can be used for need to by immune system into The evaluating drug effect system of row drug evaluation.

Flow cytometer detection statistical form

Compared with wt mouse, total T cell in the dual-gene humanization mouse spleen of hTIGIT/hPD1, B cell, NK cell Ratio does not have notable difference.

Embodiment 4PD1 and TIGIT humanization rat evaluation PD1 and TIGIT inhibitor antineoplastic effect are demonstrate,proved

Vitro data have verified that PD1 and TIGIT humanization mouse can normal expression PD1 and TIGIT albumen, and the albumen exists The intracorporal immune response of mouse is shown normally.But whether the mouse, which can be used to evaluate PD1 and TIGIT anticarcinogen actual efficacy, still has It is to be verified, therefore the tumor cell line of our Mice Inoculateds on PD1 and TIGIT humanization mouse, use PD1 and TIGIT people Source antibody drug carries out cancer resistant effect evaluation.

4.1 PD1 and TIGIT inhibitor evaluate MC38-hPD1/hTIGIT mouse model

It chooses 6-7w B6-hPD1/hTIGIT Mice homozygous (female), inoculates mouse colonic cell MC38, to swollen Knurl accumulates about 100 ± 50mm³Be divided into 4 groups immediately afterwards, respectively the mono- medicine group of control group, TIGIT, the mono- medicine group of PD1 and TIGIT and PD1 co-administered group.The anti-human TIGIT antibody of the mono- medicine group intraperitoneal injection 10mg/kg of TIGIT, the mono- medicine group control group abdominal cavity note of PD1 Penetrate the anti-human PD1 antibody of 3mg/kg, co-administered group inject simultaneously 10mg/kg anti-human TIGIT antibody and 3mg/kg it is anti-human PD1 antibody, control group inject 10mg/kg humanized IgG Isotype control；Dosage rate is successive administration 3 weeks 2 times a week.(administration Scheme see the table below) administration terminal, mouse is put to death.

Dosage regimen

Experimental result

1) mouse weight changes

Figure 15 shows the treatment group of tumor-bearing mice and the changes of weight situation of control group.It can be seen that mouse weight is integrally slow Increase, treatment group mouse is similar to control group changes of weight trend.

2) tumor growth curve

Figure 16 shows the treatment group of tumor-bearing mice and the tumor growth curve of control group.

As the result is shown:

Compared with the control group, anti-TIGIT antibody, anti-PD-1 antibody have significant suppression cancer effect after being administered alone 10 days Fruit has apparent tumor killing effect.And inhibition tumour growth is administered simultaneously using anti-TIGIT antibody and anti-PD-1 antibody Effect is more significant.The result shows that B6-hPD1/hTIGIT mouse can be not only used for the evaluation of PD1 human antibody and TIGIT human antibody The evaluation of independent target drug is evaluated, while can be used for the evaluation of PD1 inhibitor and TIGIT inhibitor administering drug combinations mode.

Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed With.It can be applied to various suitable the field of the invention completely.It for those skilled in the art, can be easily Realize other modification.Therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited In specific details and legend shown and described herein.

Sequence table

<110>Jiangsu treasury Yao Kang Biotechnology Co., Ltd

<120>a kind of construction method of TIGIT humanized mouse model and its application

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Pro Leu Ala Ser Gly Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn

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Ile Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser

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Ser Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln

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Leu Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser

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Phe Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln

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Ser Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr

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Tyr Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu

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Ser Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Leu Gly Gly

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Thr Met Ala Ala Val Leu Gly Leu Ile Cys Leu Met Val Thr Gly Val

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cttttcacca gcttggtttc accttacagc tgcagtgagc cagtttcagt tggaggagag 60

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ctgggcccag gggctgaggc aggctcccct cgcctcaggt aaggcctgaa acccagcaga 180

gcagcaggga ggaaaaacaa ggctaagctt ggttggagac ttgggtgctt gtgtagggaa 240

gactccaaga gcatgccaca gctgcttgag agaaagaaat tgcaactttt agagtcctgt 300

gtactcttat ttaagaaagg acaatctctg agaatgaggt ggggttctgt ttttggtggc 360

agatgaagga ctttgcagac ttccggggtt ggaggagcct gactggccag cacaagaagg 420

agcaggagaa gaagaagggc tgggcaggag gcattgccag tttgctttta gaagctcatt 480

cctgctccag cttcttttct gttgcctttc aggaactttc caggaaaaca ttataagttc 540

tctgtgaatt gaaaatgtct ttttcacaga ttccagaggt tctaccacta gagactgatt 600

ttttgttaaa tttatttctt ctatattaaa aatcagagtc aaatatttct gagggagggg 660

aacacaaata tttaaaaact gtggtataaa ttagttttta tgttgccttc tgaactatct 720

gaggatggag atgtgcccag ggttgctggg gagaggttaa gtgctgagag ggtggttatg 780

cctgggagaa gcagggtttc ctgctaagct tcctttgcta ttggatttct tggctttcct 840

gaaggttttt tcaggcactg caaatgacaa tcaaccaaac agcaaggttt tatttcaaag 900

gataatgcat ccagcataga actaggccgc aggaagaaac ataaaaacaa aacaaaataa 960

aaacagaaga ggtggtatca gctgacaagg gcttatattc taagtgggta gacaaaatag 1020

gcctacgaaa ataacaagct ggccatgaag ctacatagca gaatatgcga gggggcaacg 1080

tgctaagttg tgtagtcata agccaggcat ttagtaaggt gctaattata caatacaggc 1140

agtgaatgtg aaaggaaaag ggaggttgag atgggctgag gtcttctagg aggcggggag 1200

ggagtgcagc cttgacaagc ctccctgtgg gcgaggtgta aagaggggca gaagtcagct 1260

ctggaagcat agggcagtgg gtggggagga gatgggctgg gctgggctgg agtagagatg 1320

tgtggagaag ggtgagaaga ctggaaagac aacctgaatg ggggactggg agccttgaat 1380

aacaggcatg gagaggagcg tctcttgaaa tggaagaaac aggaaataat tacagcctct 1440

atggaggagc aacaggatgg actggagaaa ctatcattcc aaaatccagt tggggcctca 1500

aaggccctta gaatttttct aggaaggttg aaggccagct gctgacccag gactcacatg 1560

tgcttcgtcc tcttccctag gaatgatgac aggcacaata gaaacaacgg ggaacatttc 1620

tgcagagaaa ggtggctcta tcatcttaca atgtcacctc tcctccacca cggcacaagt 1680

gacccaggtc aactgggagc agcaggacca gcttctggcc atttgtaatg ctgacttggg 1740

gtggcacatc tccccatcct tcaaggatcg agtggcccca ggtcccggcc tgggcctcac 1800

cctccagtcg ctgaccgtga acgatacagg ggagtacttc tgcatctatc acacctaccc 1860

tgatgggacg tacactggga gaatcttcct ggaggtccta gaaagctcag gtattcctgc 1920

tggagcaagt tggtggataa acctctccct ctagcataga aaatgcaatc ctgaaacact 1980

gcacagcagg gcttctcaat tcgggatcac atttgaatca cctgaggaga ttttaaatca 2040

tactgatgcc gaggcctcac ccagaccaat tcaatcagaa tccctaatag cagagctaaa 2100

caagggtaag gtctaaaagc atttccaggt gattctaatg ggcagccaat actgagaacc 2160

actgttctta tgtaagaagc acatcttacc tatatttcct aggaagacca gttgatgagg 2220

tcatatgcaa aagttcccat ttattggttt agtataattg tgcaaattag aattaacccc 2280

taagtgtata aagagtagag ctggttaaaa acatagcctg tgctaagttt aattgtacag 2340

taatttacat ttgtgtggta cttccaagtt tcccaaatgc cctcatattt gttatccacc 2400

tgtacattta aaccaattcc cagaggttag aaaaggattc ttatcttatc tgagagacag 2460

tatagcacca tggttaaaag ctcagactct gacaccagac agtctgggtg caaatcctgg 2520

ctctgctgtt gccctcagac aggatttctt agcttctctg tgtatcagtt ttctcatctg 2580

tgaaaggagg atgaatataa gaccttatgc gatgctaatg taggattaaa ttccctacta 2640

caagaaaatg gtcttagaac aatgcctgga acatggtaag tgctgtataa gtgttaacta 2700

ttagtatgat tcccatttgt ttgatgcagg cagaagctgg agcttggagt tgctcagagt 2760

ggtagagctg attttaacac agatgttttt gttctactaa actgcatcct cagaactcct 2820

atttactgct gtcagttatg ttgaagatta agttatatgg ttgcaaaata tgtgtaaact 2880

gagaagataa aaggttacct taaagtttcc tttcaaatgt tacttctttt tttttttttt 2940

tttttttgag acggagtttc gctcttgttg cccaggctgg agtgcaatgg cgcaatctca 3000

gctcactgca acttccgcct cctgagttca agcgattctc tcctgcctca gcctcctgag 3060

tatctgggat tacaggcacc caccaccatg cccggctaat tttgtatttt tagtagagac 3120

agggtttctt cacgttggtc aggctggtct caaactcccg acctccagtg atctgcccgc 3180

ctcagcctcc caaagtgtta cttcttaact gatttcacat aggccaaaca aagactggtt 3240

aagagttgag gctctggaat tgcactgtct ggggttgact attgcagttg accccacgaa 3300

gtctgggtaa attatgtact ttttttctca gttttcttgt cgataaaatg tggcaaataa 3360

tttcatgagc tgatacaagt aaggtcttac aacagtgcta gataggtaat aagcttaact 3420

tcataagggc tagtttttat gctatttatt ggttcatggt tagaggaggc ctgggacaat 3480

aagcagggca ggggacctag attctggcaa agtaaccaag tgatggcctt gaccattctg 3540

ggctctttac cccagtgtca tatgccagag gacccatcac cagacggcag gaccgggaac 3600

agggagggag ttcatcaggc agaaggcccc tgtggagtgg tagtaccagc aactgaagac 3660

caaatgggag ctatgtctgg ttgctaatct ccaggccagg ccattcatgg agatcggtgg 3720

tcctgacatg cacactcctc cccaagacca cagtggcacg gctatttccc agcctgaacc 3780

tggggaaatg aaggtgcaaa tttcttctct gtggctcatg gacccctggg gagagagatc 3840

agaagatgtt ctattggacc cctgtgcagc ctccccacaa agcctgtatg tttccaccct 3900

tttgccattg cagactccag ggaacaccat tggtcactcg gctcttccct cttgtttccc 3960

aagtggtcag ttgcttatgt ggaatgcaaa ctctccttaa ttacttgcat tagcacaaac 4020

cctggcttct ttttgaacct aatctcaaac cttagtccaa gtccctatcc taatactatc 4080

tgtaatattt aagctattcc taaccttaat gctaaaattg tacttaaccc caaagtcaac 4140

ctggttaata ggcagaagtt ttaagtaacc cttatacaga gtctgcccct tccccaccct 4200

gcatgtagtg atgtcatgct tactcagggc tgcttctgtt tcaaagacct tgatatcttt 4260

gttctatgac cataactgtg aatggagaca ttggtgcttt tatttcaaac caaaaattcc 4320

aaatgaaatt gatacatcta atatcccaag ttccactctg gtcccaattg tatctcgaac 4380

ttatccttaa acctaaccaa actcattatc ataatcttcc ctaaacctca acatgctttt 4440

cactccatct atcttcatct gtccccaagg tgtaagagaa cccaggccta accctgcctc 4500

caatctggct ctgtccccaa actgatcata tatcctttcc tggttttatg gatagccctc 4560

tgcagcccct cttacctgct gcagtgtgtt ctctatgacc cctgtgcttt ccatctgtcc 4620

tctttactcc agagctaact gtatggctat ggctaaccag aagccgtcct ggccaactgt 4680

gactgagtcc ctcaaggcag gaggaacatg ttctggggaa tttgaattgt gcaggcacca 4740

cccttgtctt atggattgga ttcctaagcc accttttagc aacccagacc cttaggtaga 4800

gggatgagat gtgaggcttc tgctgcatcc tttgaagact cccctggata cctgagccct 4860

acctgagctg ctggggcaga ctggaaacct acctacagcg cagcatgcat gggggtgatg 4920

gtgttctttt tctcacattc ttcttgggtc agatcttact catctcttca aaactcccct 4980

cctgcatctt cttgcttcca cttacatgtt atctcttaat gaaatgcact agttcatgat 5040

ttcacttatg ccacaaatat ttatatttac tgagcattcg ctatgtgtca gatgctgaga 5100

aaacagcaaa ggataagagt ccagagcctc atgccctcca gggtttcaca gcctagctgg 5160

aaataagtgt tataggctta agggaaaagg ttacatactt tttttcattt ttgtaattta 5220

atgttaactt tattttaatt ttgaattaaa tatttttatt gatatgattt catatttgaa 5280

gttcaataaa agaaaatatt cactttctga gtttttttct ggaactacta tttgtttcat 5340

ttcatgatta ccaaaaatga ttttgtcaaa tagaagaaac gaatgttaaa atgacctgct 5400

ctggtatcaa atatacaagg tacaccactg ctgaaaatat cagtcattga tttcatgatg 5460

ggccagatgg atactctgct gttcaaagtg ttgggcttaa aggctccagt cccatggtta 5520

cacaaagggc ttggccaatc ctcccctctc tgccgtgaag ctcaggctgc cctggggaaa 5580

tcagcttcac ttctggcttc tgccactcat ctctgttttg tcctccctct agtggctgag 5640

cacggtgcca ggttccagat tccacttgga ggaaccatgg ctgctgtgct gggactcatt 5700

tgcttaatgg tcacaggagt gactgtactg gctagaaag 5739

<210> 3

<211> 17

<212> DNA

<213> homo sapiens

<400> 3

cagcctgtat cagcccc 17

<210> 4

<211> 16

<212> DNA

<213> homo sapiens

<400> 4

gaactgagcc actaca 16

<210> 5

<211> 40

<212> DNA

<213> homo sapiens

<400> 5

tgggatgggc agggcgcgcc ggcatgaggc tttggatgtc 40

<210> 6

<211> 23

<212> DNA

<213> homo sapiens

<400> 6

gcttctaacc agaggcctac agc 23

<210> 7

<211> 20

<212> DNA

<213> homo sapiens

<400> 7

cttggaggaa ccatggctgc 20

<210> 8

<211> 30

<212> DNA

<213> homo sapiens

<400> 8

ggcgcgccaa agggccgagg aaaccagacc 30

<210> 9

<211> 40

<212> DNA

<213> homo sapiens

<400> 9

gtaggcctct ggttagaagc atgcgctggt gtctcctcct 40

<210> 10

<211> 28

<212> DNA

<213> homo sapiens

<400> 10

ttaattaacc tggaaagttc ctgaaagg 28

<210> 11

<211> 40

<212> DNA

<213> homo sapiens

<400> 11

gaactttcca ggttaattaa ccagggtttc acagcctagc 40

<210> 12

<211> 41

<212> DNA

<213> homo sapiens

<400> 12

gcagccatgg ttcctccaag tggaatctgg aacctggcac c 41

Claims (11)

1. a kind of method for preparing TIGIT humanized mouse model using CRISPR/Cas9 technology, which is characterized in that the people In the mouse of source, whole extracellular regions of mouse TIGIT gene are replaced by the homologous segment of the gene of source of people TIGIT, retain Mouse TIGIT transmembrane region and intracellular region.

2. the method as described in claim 1 comprising following steps:

(1) plasmid of the building expression for the sgRNA of mouse TIGIT gene；

(2) expression vector of humanization TIGIT gene is constructed；

(3) step (1) plasmid is transcribed in vitro to the carrier and Cas9mRNA or Cas9 of the sgRNA and step (2) that obtain Protein injection is implanted into receptor female rat production TIGIT gene modification humanization mouse mould into mouse fertilized egg cell matter or nucleus Type；

The extracellular region of source of mouse TIGIT gene can be replaced with the extracellular region of source of people TIGIT gene by the carrier, remain mouse The transmembrane region and intracellular region of TIGIT gene.

3. method according to claim 2, wherein the humanization TIGIT gene includes nucleic acid sequence SEQ ID NO:1.

4. method according to claim 2, wherein the chimeric protein sequence of humanization TIGIT gene coding is SEQ ID NO:2.

5. it is method according to claim 1 or 2, wherein the sequence such as SEQ ID NO of the sgRNA for source of mouse TIGIT gene: Shown in 3 and SEQ ID NO:4.

6. wherein the process of carrier construction is as follows in step (2) such as the described in any item methods of claim 2-5: using mouse C57BL/6 genome expands the homology arm segment at 1 end of mouse TIGIT gene extron and the homologous arm pieces at exon 3 end respectively Section, using the BAC of Human TIGIT as template, above-mentioned segment composition is then attached to PMD18T by amplification source of people replacement segment On carrier, the carrier of humanization TIGIT gene is constructed.

7. the primer that the homology arm for method as claimed in claim 6, expanding 1 end of mouse TIGIT gene extron uses is SEQ ID NO:5 and SEQ ID NO:6, the primer that the homology arm at amplification 3 end of mouse TIGIT gene extron uses is SEQ ID NO: 7 and SEQ ID NO:8, the primer that uses of amplification source of people replacement segment be SEQ ID NO:9 and SEQ ID NO:10 primer pair with And SEQ ID NO:11 and SEQ ID NO:12 primer pair.

8. further comprising the method for claim 7, the step of identifying the genotype of the animal model using primer.

9. a kind of method for preparing the bis- humanization mouse models of TIGIT/PD1, includes the following steps:

(a) TIGIT gene humanization mouse is obtained using any one of claim 1-8 the method；

(b) the humanization mouse that step (a) obtains is mated and is screened with PD1 humanization mouse, it is small to obtain double humanizations Mouse model.

10. drug effectiveness, TIGIT targeted drug sieve of the described in any item methods of claim 1-9 in assessment targeting TIGIT In the toxicological study of choosing exploitation, the antitumor evaluating drug effect of TIGIT targeted drug joint other drugs or TIGIT targeted drug Using.

11. the sgRNA of selectively targeted mouse TIGIT gene, sequence is as shown in SEQ ID NO:3 and SEQID NO:4.