Application of PD-1IR2 in immunodetection or tumor treatment medicine

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of PD-1IR2 in immunodetection or tumor treatment drugs. A second intron of the PD-1 isomer is reserved, the PD-1 isomer contains a signal peptide, an IgV structural domain and a new short cytoplasm tail, and the protein sequence of the PD-1 isomer is SEQ ID NO.1. According to the invention, a brand new PD-1 isomer is clarified, and the expression profile and immune function of the PD-1IR2 protein in T lymphocytes are explored. In-vivo and in-vitro functions prove that the compound inhibits the function of CD8 + T cells, so that tumor progression is promoted, drug resistance of PD-L1 treatment is mediated, a new mechanism is provided for drug resistance of a PD-1 / PD-L1 inhibitor, and a new target is provided for immunotherapy of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and specifically relates to PD-1 IR2 Application in immunoassay or tumor therapeutic drugs. Background Art

[0002] Cancer is a general term for a class of diseases in which cells grow out of control and have the ability to metastasize to distant sites. Cancer cells are characterized by continuous proliferation, escape from growth inhibition, antagonism of cell death, maintenance of permanent replication, induction of new angiogenesis, activation of invasion and metastasis, reprogramming of energy metabolism, and escape from immune surveillance. Although the 5-year survival rate of patients has made phased progress after cancer intervention, cancer remains one of the leading causes of death worldwide.

[0003] Over the past decade, therapies targeting the immune system have revolutionized cancer treatment. Immune checkpoint inhibitors (ICIs) work by releasing the T cell "brakes," leading to robust activation of the immune system and anti-tumor immune responses that have resulted in durable remissions in patients with a variety of different tumor types. Despite these successes, immunotherapy remains ineffective for most cancer patients, and among patients who initially respond, some develop resistance and experience tumor recurrence at a later time.

[0004] Programmed death receptor-1 (PD-1, also known as CD279), encoded by the PDCD1 gene, is a member of the CD28 receptor superfamily and is a representative immune checkpoint molecule. PD-1 regulates T cell function by interacting with two natural ligands, PD-L1 and PD-L2. When PD-1 binds to its ligand, it induces phosphorylation of tyrosine residues in the ITSM and ITIM domains of the cytoplasmic tail of PD-1, thereby inhibiting T cell activation and differentiation. Under physiological conditions, PD-1 inhibits overactivation of the immune system, maintains immune tolerance, and protects one's own tissues from damage. However, PD-L1 expressed by tumor cells can also bind to PD-1 on the surface of T cells, mediating T cell dysfunction, leading to tumor immune escape. Targeting PD-1 can restore CD8 +T cells function and kill cancer cells. In recent years, antibodies targeting PD-1 or PD-L1 have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of cancer, opening a new chapter in cross-era tumor immunotherapy. Preliminary clinical trials of PD-1 antibodies nivolumab and pembrolizumab have demonstrated potent and durable antitumor activity and limited immunotoxicity in a wide range of cancer types, including melanoma, non-small cell lung cancer, renal cell carcinoma, and colorectal cancer. However, some issues remain to be resolved, including a large number of non-responders and immune-related adverse events, indicating that the mechanism of tumor immune escape is still insufficiently elucidated. Therefore, identifying new immune checkpoints, exploring their mechanisms of action, and conducting new drug research using them as potential therapeutic targets may provide new means for anti-tumor immunotherapy. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention can be achieved through the following technical solutions:

[0006] A PD-1 isoform, wherein the second intron of the PD-1 isoform is retained, the PD-1 isoform contains a signal peptide, an IgV domain and a new short cytoplasmic tail, and the protein sequence thereof is SEQ ID NO.1.

[0007] A use of a reagent for detecting PD-1 isomers in preparing a kit for detecting tumors.

[0008] Furthermore, the reagent for detecting PD-1 isoforms contains an antibody that specifically binds to PD-1 isoforms, specifically Mouse IgG2b.K.

[0009] A use of a reagent containing a knockdown PD-1 isoform in the preparation of a drug for treating tumors.

[0010] Furthermore, the reagent for knocking down PD-1 isoforms contains specific primers that bind to PD-1 isoforms, and the specific primers include Sh-F1, Sh-R1, Sh-F2 and Sh-R2, the sequence of Sh-F1 is SEQ ID NO.2, the sequence of Sh-R1 is SEQ ID NO.3, the sequence of Sh-F2 is SEQ ID NO.4, and the sequence of Sh-R2 is SEQID NO.5.

[0011] A method for constructing a PD-1 isoform knock-in mouse model, the construction method comprising: inserting a complete DNA sequence unit, including a promoter, PDCD1 IR2CDS-Flag region and PolyA tail; specifically, Cre recombinase is used to act on the Loxp fragment, and the gene sequence between the Loxp fragments is deleted or recombined through the Cre recombinase recognition of specific sites; Cd4cre tool mice insert the Cre gene into the CD4 cell promoter, and then cross with Loxp mice to obtain mice that only knock in or knock out specific genes in T cells; PDCD1 is used IR2 The flox / flox mice were crossed with Cd4cre mice, and PDCD was specifically knocked in T cells under the action of Cre recombinase. 1IR2 of mice.

[0012] Furthermore, the gene sequence of Loxp is SEQ ID NO.6.

[0013] A PD-1 isoform knock-in mouse model is constructed by the PD-1 isoform knock-in mouse model construction method described above.

[0014] The PD-1 isoform knock-in mouse model described above is used to study tumor diseases.

[0015] Beneficial effects of the present invention: The present invention clarifies the new PD-1 isomers and explores the IR2 The protein's expression profile and immune function in T lymphocytes. In vitro and in vivo functions have proven that it inhibits CD8 + The function of T cells promotes tumor progression and mediates resistance to PD-L1 treatment, providing a new mechanism for resistance to PD-1 / PD-L1 inhibitors and a new target for tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 For the identification of PD-1 isomers in the present invention;

[0018] Figure 2 PD-1 in the present invention IR2 Preparation of monoclonal antibodies and detection of endogenous proteins;

[0019] Figure 3 PD-1 in the present invention IR2 Inducible expression of

[0020] Figure 4 PD-1 in the present invention IR2Expression in T cell subsets and in tumor patients;

[0021] Figure 5 PD-1 in the present invention IR2 Knockdown promotes immune cell function;

[0022] Figure 6 PD-1 in the present invention IR2 Overexpression inhibits the function of immune cells;

[0023] Figure 7 PD-1 in the present invention IR2 Construction of knock-in mice;

[0024] Figure 8 PD-1 in the present invention IR2 Gene knock-in results in mouse CD8 + Impaired anti-tumor capacity of T cells;

[0025] Fig. 9 PD-1 in the present invention IR2 Gene knock-in leads to resistance to PD-L1 antibody therapy;

[0026] Fig.10 PD-1 in the present invention IR2 The anti-tumor ability of humanized mice was weakened. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example 1

[0029] Peripheral blood mononuclear cells (PBMCs) were flow sorted, and PDCD1 was amplified using reverse transcription PCR (RT-PCR) in PBMCs, T cells, B cells, and monocytes. A larger band was found above the wild-type PDCD1. Based on Sanger sequencing, it was found that compared with the wild-type PDCD1, this band retained the second intron, a total of 267 bp ( Figure 1 AB), which we named PD-1 IR2 , its gene schematic diagram is as follows ( Figure 1 C) Because the retained intron contains a premature termination codon, PD-1 IR2Translated as truncated protein. PD-1 IR2 It contains a signal peptide, an IgV domain and a new short cytoplasmic tail. Its protein sequence is SEQ ID NO.1. Its protein structure diagram is shown in ( Figure 1 D), PD-1 IR2 The protein sequence is as follows ( Figure 1 .E). To further validate PD-1 IR2 Expression profile of PD-1 across the second intron IR2 Specific primers ( Figure 1 F), using quantitative RT-PCR (qRT-PCR) to detect its expression in multiple human leukemia cell lines, including Jurkat (T cell leukemia cell line), Raji and Jeko-1 (B cell lymphoma cell lines), U937 (monocytic leukemia cell line), K562 (myeloid leukemia cell line), and HL-60 (human promyelocytic leukemia cell line). The analysis results showed that PD-1 IR2 It is expressed in all these human leukemia cell lines, especially in Jurkat and K562 cell lines, with relatively high expression levels ( Figure 1 G).

[0030] Example 2

[0031] To verify endogenous PD-1 IR2 The real existence of protein level, using PD-1 IR2 The 10 amino acid fragments specific to the cytoplasmic tail were used as antigens to immunize mice to prepare monoclonal antibodies. The antibody used was Mouse lgG2b.K. First, the antibody was verified to be able to specifically bind to PD-1. IR2 Overexpression of PD-1 in HEK293T cells IR2 -GFP, respectively, with IgG, PD-1 and prepared PD-1 IR2 Monoclonal antibodies were incubated with cells and PD-1 was detected by flow cytometry (FACS) IR2 The flow cytometry results showed that PD-1 IR2 Monoclonal antibodies can specifically recognize this isomer, with a detection efficiency of about 20% ( Figure 2 A), where IgG and PD-1 antibodies were used as negative and positive controls, respectively.

[0032] Wild-type Jurkat cells and the PDCD1 knockout (KO) Jurkat cell line established in the laboratory were used. After 48 h of stimulation with 5 μg / ml PHA, PD-1 IR2The monoclonal antibody was incubated with cells, and FACS was used to detect the endogenous expression of the isoform. The results showed that PD-1 could be detected in activated Jurkat cells compared with Jurkat-KO strains. IR2 The expression of protein level ( Figure 2 B) Due to PD-1 IR2 The lack of a transmembrane segment suggests that it may be localized in the cytoplasm or secreted out of the cell. To verify the hypothesis, PD-1 was overexpressed in Jurkat KO cells. IR2 At the same time, the cell lysate and cell supernatant were collected and subjected to immunoblotting. The experimental results showed that PD-1 IR2 Mainly located in cells, and almost undetectable in the supernatant ( Figure 2 C).

[0033] Example 3

[0034] Jurkat cells were activated using three classical methods. CD3 and CD28 (5 μg / ml) stimulated Jurkat cells for 72 h, PMA (50 ng / ml) and ionomycin (1 μM) stimulated cells for 5 h, and PHA (5 μg / ml) stimulated cells for 48 h. After stimulation, qRT-PCR was used to detect PDD-1 in Jurkat cells. IR2 mRNA level, FACS detection of PD-1 IR2 The results showed that PD-1 in activated Jurkat cells IR2 Both the mRNA and protein levels of Figure 3 AF).

[0035] Example 4

[0036] To evaluate PD-1 IR2 The expression of PD-1 in T cell subsets of healthy people and tumor patients was analyzed by downloading RNA-seq data from the GEO database. The analysis results were presented in TPM (Transcripts Per Million), which refers to the ratio of the measured RNA to 1 million RNA. IR2 CD4 + and CD8 + T cells are expressed, and PD-1 IR2 In CD8 + The expression level in T cells is higher than that in CD4 + T cells ( Figure 4 A). In addition, PD-1 IR2 The expression of PD-1 is correlated with Figure 4 B) Analysis of CD8+T cell subsets in this database showed that PD-1IR2 In CD8 + All T cell subsets express it, but the expression level is lower than that of PD-1 ( Figure 4 CD). Compared with naive T cells, PD-1 IR2 In central memory (CM) T cells, type 1 effector memory (EM1) T cells and PD-1 + CD39 + Increased expression in T cells ( Figure 4 C), which is consistent with PD-1 in CD8 + Expression in T cell subsets is partially consistent ( Figure 4 D) Compared with naive T cells, PD-1 IR2 The expression of Tfh in follicular helper T cells ( Figure 4 E), which is consistent with the expression of PD-1 in CD4 + The expression in T subgroups is similar ( Figure 4 F), the above analysis results suggest that PD-1 IR2 In addition, another set of sequencing data analyzed the expression of PD-1 in healthy people and patients with cutaneous T-cell lymphoma (CTCL). IR2 The expression of PD-1 IR2 The expression level in CTCL patients is higher than that in healthy people, and PD-1 IR2 The expression level of PD-1 is highly correlated with tumor stage, that is, as the stage increases, IR2 The expression level of Figure 4 G), indicating PD-1 IR2 This may be associated with the poor prognosis of CTCL patients. IR2 The researchers analyzed T cell RNA sequencing data from 364 patient samples covering 10 different solid tumor types. The results showed that PDCD1 IR2 and PDCD1 were expressed in all these tumor types ( Figure 4 H). Tumor-infiltrating lymphocytes (TILs) were further isolated from clinical lung cancer samples, and the experimental results showed that PDCD1 IR2 Both PDCD1 and PDCD2 were expressed in TILs, but there was no difference in their expression ( Figure 4 I).

[0037] Example 5

[0038] To explore PD-1 IR2 CFSE staining and FACS technology were used to detect the potential molecular functions of PD-1 IR2 Effects on the proliferation of Jurkat cells. IR2 The reagent for knocking down PD-1 isoforms contains specific primers that bind to PD-1 isoforms, and the specific primers include Sh-F1, Sh-R1, Sh-F2 and Sh-R2, the sequence of Sh-F1 is SEQ ID NO.2, the sequence of Sh-R1 is SEQ ID NO.3, the sequence of Sh-F2 is SEQ ID NO.4, and the sequence of Sh-R2 is SEQ ID NO.5. The results showed that compared with the control group, PDCD1 IR2 Knock down (KD) of Figure 5 A). Next, the secretion level of IL2 was detected using an ELISA kit, and the results showed that PDCD1 IR2 KD promoted the production of IL-2 ( Figure 5 B). Furthermore, immune cells were co-cultured with human non-small cell lung cancer (NCI-H1299) tumor cells, and PDCD1 expression was evaluated using an apoptosis detection kit and FACS technology. IR2 The knockdown of immune cells against tumor cells showed that compared with the control group, PDCD1 IR2 Knockdown Jurkat cells showed enhanced killing ability against NCI-H1299 tumor cells ( Figure 5 C).

[0039] Sh-F1: 5-GATCCGcatctctgtcctctagctctTTCAAGAGAagagctagaggacagagatgCTTTTTTG-3;

[0040] Sh-R1: 5-AATTCAAAAAAGcatctctgtcctctagctctTCTCTTGAAagagctagaggacagagatgCG-3;

[0041] Sh-F2: 5-GATCCCctttgtgcccttccagagagTTCAAGAGActctctggaagggcacaaagGTTTTTTG-3;

[0042] Sh-R2: 5-AATTCAAAAAACctttgtgcccttccagagagTCTCTTGAActctctggaagggcacaaagGG-3;

[0043] Example 6

[0044] Building PD-1 and PD-1 IR2 Overexpression plasmids were used to overexpress PD-1 and PD-1 in two KO strains of PD-1. IR2 CFSE staining and FACS were used to detect PD-1 and PD-1 IR2 The results showed that compared with the control group, PD-1 IR2 Overexpression of Figure 6 A and D). The secretion level of IL2 was detected by ELISA kit, indicating that PD-1 IR2 Overexpression of Figure 6 B and E). Similarly, immune cells were co-cultured with NCI-H1299 tumor cells, and PD-1 expression was assessed using an apoptosis detection kit and FACS technology. IR2 The results showed that compared with the control group, PD-1 IR2 Overexpression leads to a weakened killing ability of Jurkat cells against NCI-H1299 ( Figure 6 C and F).

[0045] Example 7

[0046] Three amino acid sites (K78, I126, E136) of PD-1 are located in the IgV domain, which are crucial for the binding of PD-1 to PD-L1. IR2 The IgV domain that binds to PD-L1 is retained, so it may exhibit inhibitory function by interacting with PD-L1 like PD-1. IR2 Similar mouse PD-1 isoforms, but because mouse PD-l1 has a similar molecular structure to human PD-L1, we speculate that the knocked-in PD-1 IR2 The gene can bind to the mouse Pd-l1 to exert biological effects.

[0047] To further study PD-1 IR2 We prepared PDCD1 IR2flox / flox mice. Insert the complete DNA sequence unit into the safe site of ROSA26 gene in C57BL / 6J mice, including promoter, PDCD1 IR2 CDS-Flag region and PolyA tail. Cre recombinase is often used in conjunction with loxP sites to form a Cre / loxP recombination system. This system is widely used in gene knockout, gene knock-in, and spatiotemporal control of gene expression. loxP sequence: a DNA fragment with a specific order that the Cre recombinase can recognize and cut. The gene sequence of the Loxp is SEQ ID NO.6. Function of Cre recombinase: The Cre enzyme can mediate DNA recombination by recognizing the sequence between loxP sites. For example, it can cause deletion, flipping, or insertion of the DNA sequence between loxP sites. After the Cd4cre tool mouse inserts the Cre gene into the CD4 cell promoter, when it is hybridized with the Loxp mouse, mice that only knock in or knock out specific genes in T cells can be obtained. We used PDCD1 IR2 When flox / flox mice are crossed with Cd4cre mice, the stop unit is deleted under the action of Cre recombinase, and the target gene begins to be expressed, thereby obtaining PDCD1 knock-in specifically in T cells. IR2 mice, namely PDCD1 IR2 Conditional knock-in (PDCD1 IR2 CKI) mice. In the absence of Cd4cre gene, the target gene is not expressed. The genotype of mice was identified by tail PCR, and PD-1 IR2+ / + Cd4cre + / - (PD-1 IR2 Homozygous and Cre) as the experimental group, PD-1 IR2+ / + Cd4cre - / - (PD-1 IR2 Homozygous) served as the control group ( Figure 7 A) Using EasySep TM Mouse CD8 + T cell isolation kit to isolate mouse spleen T cells, PD-1 IR2 After incubation with specific antibodies, mouse PD-1 was detected by FACS IR2 Expression, the results showed that PDCD1 IR2CKI Mouse isoform gene knock-in was successful ( Figure 7 B).

[0048] Example 8

[0049] Using the MC38 colorectal cancer model to explore PD-1 IR2 Function in tumors. IR2+ / +Cd4cre + / - PD-1 in the experimental group and littermates IR2+ / + Cd4cre - / - There were 5 male mice in the control group, and 1×106 MC38 tumor cells were injected subcutaneously on the back of the mice. Figure 8 A) The mice were killed on day 15 and the tumor tissues were removed and weighed. The measurement results showed that PDCD1 IR2 CKI The tumor growth rate of mice was faster than that of WT mice, as shown by the tumor volume ( Figure 8 B) and tumor weight ( Figure 8 The removed tumor tissue was minced and ground, and mouse TILs were isolated by density gradient centrifugation. Anti-CD45, CD3, CD4, CD8, CD25, CD44, FOXP3, Ki-67, Gzmb and IFN-γ staining were performed, and the immune cell phenotype was analyzed by FACS. IR2 CKI and WT mice tumor-infiltrating CD8 + The proportion of CD44 in T cells was similar. CD44 is a T cell activation marker, indicating that the two groups had similar antigen presentation processes ( Figure 8 E) PDCD1 IR2 The knock-in of the gene did not affect the CD4 + T cells, CD8 + The percentage of T cells, CD4 + With CD8 + There was no effect on the ratio of T cells, Treg levels, and Ki-67 expression ( Figure 8 FH). However, compared with WT mice, PDCD1 IR2 CKI GzmB and IFN-γ in mouse TILs were reduced ( Figure 8 IJ), indicating CD8 + These results suggest that PD-1 IR2 Impairment of cytotoxic CD8 in the tumor microenvironment + T cells function, thereby promoting tumor growth.

[0050] Example 9

[0051] Transplantation of MC38 tumor cells into WT and PDCD1 IR2 CKI The results showed that compared with IgG treatment, PD-L1 antibody treatment significantly delayed tumor growth in WT mice, as shown by reduced tumor volume and weight and increased GZMB, while PDCD1 IR2 CKI Mice were resistant to anti-PD-L1 therapy ( Fig. 9AD). In fact, we still observed that anti-PD-L1 therapy IR2 CKI The tumors in mice showed a certain effect, which was manifested by a decrease in tumor volume and weight, and an increase in GZMB ( Fig. 9 AD, CKI+anti-PD-L1 vs CKI+IgG). However, PDCD1 IR2 CKI The tumor volume of mice was still larger than that of WT mice ( Fig. 9 A, CKI+anti-PD-L1 vs WT+anti-PD-L1), which indicates that PD-1 IR2 After anti-PD-L1 treatment, WT and PDCD1 IR2 CKI The number of Treg cells in mice decreased ( Fig. 9 E), which is consistent with previous reports using checkpoint blockade therapy.

[0052] Example 10

[0053] Models in which human genes, cells, tissues, organs or immune systems are transplanted into mice are called humanized mice. Mice with humanized immune systems are mice that have reconstructed human immune systems. By transplanting human hematopoietic stem cells, immune cells or tissues into immunodeficient mice, mice are endowed with human immune functions. Common immune system humanizations include PBMC humanization and hematopoietic stem cell (HSC) humanization. In order to further confirm that PD-1 IR2 In order to investigate the intrinsic role of PBMC in anti-tumor, a humanized PBMC-NOG model (huPBMC-NOG) was constructed using NOG (NOD.Cg-PrkdcsidIL2rgtm1Sug / Jaccard) mice. NOG mice were subcutaneously inoculated with NCI-H1299 tumor cells on their backs. One week later, human PBMCs were injected into the tail vein, where PBMCs overexpressed empty control (Vector) and PD-1. IR2 ( Fig.10 A). Tumor size was measured regularly and tumor growth curve was drawn. Mice were killed on day 23, and tumor tissues were removed and weighed. IR2 The ability of huPBMC-NOG mice to inhibit tumor growth was impaired, as evidenced by increased tumor volume and weight ( Fig.10 BD). The tumor tissue was minced and ground, TILs were isolated, and the results were analyzed by FACS after fluorescent antibody staining. There was no significant difference in the CD44 activation level in tumor-infiltrating CD8+ T cells compared with control mice, indicating that the same antigen presentation was experienced ( Fig.10 E) In addition, no CD4+ T cells and CD8 + The frequency of T cells changed significantly ( Fig.10 F), the expression of Ki-67 in TIL was also not affected ( Fig.10 G). However, we found that CD8 + The immune function of T cells is impaired, as shown by the decreased production of cytokines GmzB and IFN-γ ( Fig.10 H), this conclusion is consistent with PDCD1 IR2 CKI Mice consistent.

[0054] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0055] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A PD-1 isomer, characterized in that: The second intron of the PD-1 isoform is retained, and the PD-1 isoform contains a signal peptide, an IgV domain and a new short cytoplasmic tail, and its protein sequence is SEQ ID NO.

1.

2. Use of a reagent for detecting PD-1 isomers in the preparation of a kit for detecting tumors.

3. The use according to claim 2, characterized in that: The reagent for detecting PD-1 isoforms contains an antibody that specifically binds to PD-1 isoforms, specifically Mouse IgG2b.K.

4. Use of a reagent containing a knockdown PD-1 isoform in the preparation of a drug for treating tumors.

5. The use according to claim 4, characterized in that: The reagent for knocking down PD-1 isoforms contains specific primers that bind to PD-1 isoforms, and the specific primers include Sh-F1, Sh-R1, Sh-F2 and Sh-R2. The sequence of Sh-F1 is SEQ ID NO.2, the sequence of Sh-R1 is SEQ ID NO.3, the sequence of Sh-F2 is SEQ ID NO.4, and the sequence of Sh-R2 is SEQ ID NO.

5.

6. A method for constructing a PD-1 isoform knock-in mouse model, characterized in that: The construction method is: inserting a complete DNA sequence unit, including a promoter, PDCD1 IR2 CDS-Flag region and PolyA tail; specifically, Cre recombinase is used to act on the Loxp fragment, and the gene sequence between the Loxp fragments is deleted or recombined through the Cre recombinase recognition of specific sites; Cd4cre tool mice insert the Cre gene into the CD4 cell promoter, and then cross with Loxp mice to obtain mice that only knock in or knock out specific genes in T cells; PDCD1 is used IR2 The flox / flox mice were crossed with Cd4cre mice, and PDCD was specifically knocked in T cells under the action of Cre recombinase. 1IR2 of mice.

7. The method for constructing a PD-1 isoform knock-in mouse according to claim 6, characterized in that: The gene sequence of Loxp is SEQ ID NO.

6.

8. A PD-1 isoform knock-in mouse model is constructed by the method for constructing a PD-1 isoform knock-in mouse model according to claim 6 or 7.

9. The PD-1 isoform knock-in mouse model as described in claim 8 is used for the study of tumor diseases.

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