WO2021136240A1 - 人4IgB7-H3的突变编码基因及其调节免疫的应用 - Google Patents

人4IgB7-H3的突变编码基因及其调节免疫的应用 Download PDF

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WO2021136240A1
WO2021136240A1 PCT/CN2020/140691 CN2020140691W WO2021136240A1 WO 2021136240 A1 WO2021136240 A1 WO 2021136240A1 CN 2020140691 W CN2020140691 W CN 2020140691W WO 2021136240 A1 WO2021136240 A1 WO 2021136240A1
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白素梅
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Definitions

  • the present invention relates to the field of biomedical immunotherapy, in particular to a human 4IgB7-H3 mutation coding gene and its application for regulating immunity.
  • T lymphocytes (referred to as T cells) depends on the positive/negative dual signal, which is necessary to effectively stimulate the specificity of T cell antigens and to stop the immune effect in a timely manner.
  • Immunity regulation is out of balance, regardless of suppression or over-activation, all belong to immune abnormality, which affects the body's immune response, and the diseases caused are various immune diseases, including tumors, inflammations, autoimmune diseases and so on.
  • T cell activation and proliferation depend on dual signals: one is the antigen peptide-human leukocyte antigen (HLA) complex presented by antigen presenting cells (APC), and the other is the costimulatory signal (the second signal).
  • the antigen peptide-human leukocyte antigen (HLA) complex presented by APC is recognized by a special T cell antigen receptor (TCR) and transmits a signal to T cells, which is necessary for T cell activation, but does not cause T cell proliferation And secrete cytokines.
  • TCR T cell antigen receptor
  • Co-stimulatory signals are necessary for the specific activation of T cell antigens. The initiation, maintenance and regulation of the activation cascade determines whether T cells activate proliferation, or turn into anergy or even apoptosis.
  • the two-signal theory (two-signal model) is proposed, explaining how mature peripheral B cells recognize antigens and how T cells obtain antigen peptides presented by APC-major histocompatibility complex (major histocompatibility complex I, MHC) Signal, so costimulatory signal has become a hot research topic.
  • the costimulatory signal is antigen (Ag) non-specific, in addition to affecting the activation and proliferation of T cells, it also directly affects the production of cytokines.
  • Co-stimulatory molecules play an important role in tumor immunity. Studies have found that most tumor cells express MHC-1 molecules and tumor Ag, which are potential APCs, but their immunogenicity is weak.
  • tumors Because they do not express or under-express B7-1, it is difficult to induce tumor-specific T cell activation. It is one of the important reasons why tumors evade immune surveillance. Therefore, the effect of treating tumors can be achieved by increasing or enhancing costimulatory signals.
  • costimulatory molecules also known as costimulatory molecules
  • costimulatory molecules are an important class of regulatory molecules in the activation process of T lymphocytes. Costimulatory molecules and their receptors or ligands act as costimulatory signals for T and B cell activation, helper T cell differentiation, signal transduction, and effector cell activation. Cytokines play an important role in the secretion process.
  • Costimulatory molecules are mainly divided into two super families, B7/CD28 and TNF/TNFR.
  • the classic B7/CD28 signaling pathway mainly regulates the activation of naive T cells in the form of co-stimulation.
  • some newly discovered new members of the B7 family can also negatively regulate the immune response by acting on activated T cells.
  • the B7 family belongs to the class of immunoglobulins, which are transmembrane glycoproteins of 50,000 to 70,000 days.
  • the members of the B7 immune protein family include B7-1, B7-2, B7-H1 (ie PD-L1), B7-H2, B7-H3 And PD-L2.
  • the B7 family plays an important role in the treatment of tumors, organ transplants and autoimmune diseases. In basic research, it can be used for T and B cell differentiation, activation mechanisms, antigen presentation, costimulation mechanisms, immune tolerance, Research on transplant rejection and autoimmunity.
  • B7-H1 and B7-H3 are two important negative regulatory co-stimulatory molecules. Chen Lujun et al. reported for the first time the expression of negative costimulatory molecules B7-H1 and B7-H3 in colorectal cancer and its clinical significance (Chen Lujun, Master's degree thesis of Soochow University, 2009, costimulatory molecules B7-H1 and B7-H3 The clinical significance and regulatory mechanism of expression in colorectal cancer), confirming that they play an important role in the occurrence and development of colorectal cancer and negatively regulate the infiltration of T lymphocytes in tumor tissues. The detection of B7-H1 and B7-H3 molecules can be used as An important biological indicator for the diagnosis and prognosis of bowel cancer.
  • the cytokines IFN- ⁇ and TNF- ⁇ in the tumor microenvironment play an important role in regulating the expression of costimulatory molecules B7-H1 and B7-H3 in tumor cells.
  • the B7-H3 molecule can play a role in the tumor immune escape process of intestinal cancer through two forms of membrane and soluble (also known as SB7-H3).
  • B7-H3 is an immune checkpoint molecule belonging to the B7-CD28 pathway.
  • B7-H3 is a type I transmembrane protein, which is encoded by chromosome 9 in mice and chromosome 15 in humans.
  • B7-H3 mRNA is widely present in non-lymphatic tissues, and is also expressed in many lymphoid organs such as the spleen, lymph nodes, thymus, liver and thymus of infants.
  • tumor cell lines such as melanoma G361, cervical adenoma, HeLa S3, chronic myeloid lymphoma K562, lung cancer A546, and colorectal adenocarcinoma SW480 also have expression of B7-H3 mRNA. Its receptor can be induced to express on the surface of activated T cells. B7-H3 can increase the production of CD4+ and CD8+ cells, and can induce the production of CTL, and can selectively increase the production of IFN- ⁇ , which participates in T, B cell activation and body immunity play an important role in tumor therapy, organ transplantation, and treatment of autoimmune diseases (Suh WK, et al.
  • B7-H3 Prefeerntialy down-regulates T helper type1-mediated immune responses [J]. Nat Immunol, 2003, 4(9): 899-906.). B7-H3 cannot bind to CTLA-4 ICOS and PD-1, indicating that B7-H3 has a receptor or ligand, but it has not been isolated yet.
  • the B7-H3 gene has four Ig-like repeats, which can be spliced alternately to produce a protein containing four Ig-like domains (4Ig) or two Ig-like domains (2Ig), the isoform 4IgB7 -H3 (or B7-H3b) and 2IgB7-H3.
  • 4IgB7-H3 has two copies of IgV-IgC domain, which is the main expression form in most human cells and tissues.
  • the base repetition rate is 98%, which is the result of 2IgB7-H3 gene duplication.
  • Humans 2IgB7-H3 and 4IgB7-H3 proteins have different spatial conformations; 3. Two receptors can be expressed on activated T cells to bind to the two isoforms of B7-H3; human 2IgB7-H3 and mouse B7 -H3 combines with stimulatory receptors to promote the activation of T cells and enhance the function of monocytes; while human 4IgB7-H3 can bind to inhibitory receptors and has an inhibitory effect on the activation of T cells.
  • Human 2IgB7-H3 and mouse B7-H3 genes can promote the proliferation of activated T cells and enhance the effect of monocytes; human 4IgB7-H3 is mainly expressed in antigen-presenting cells and can play a biological function of inhibiting T cell activation.
  • human B7-H3 plays an important role in tumor therapy, organ transplantation, and treatment of autoimmune diseases, and its isoforms 2IgB7-H3 and 4IgB7-H3 are functionally coordinated with each other, increasing the importance of this signaling pathway. Because the B7-H3 receptor or ligand has not yet been discovered, there is still a lot of controversy in the report on its functional research. In-depth research on the two isomers of B7-H3 has important biological significance for revealing the precise regulation of costimulatory molecules in the immune response. The provision of various mutants and functional studies of 2IgB7-H3 and 4IgB7-H3 also provides a new way for the functional research of the above-mentioned B7-H3. However, there are no mutants of 4IgB7-H3 or mutation coding genes of 4IgB7-H3. Report.
  • the present invention provides a new mutant coding gene of human 4IgB7-H3, which has enhanced biological functions than wild-type 4IgB7-H3 gene, and is designed to inhibit excessive or abnormally activated immunity, especially T lymphocyte-mediated cellular immunity and T lymphocytes.
  • the development of lymphocytes or the cooperation with the 2IgB7-H3 mutant with enhanced function provides a new technical means; especially through the new human 4IgB7-H3 mutant coding gene, its encoded protein or its receptor or ligand , Relevant drugs such as RNA molecules or pharmaceutical compositions containing them to interfere with immune escape, thereby regulating or improving the immune response of subjects; at the same time, it provides applications in regulating immunity, such as the preparation of prevention or treatment of malignant tumors and infectious diseases.
  • one of the technical solutions of the present invention is to provide a human 4IgB7-H3 mutant encoding gene, wherein the nucleotide sequence of the human 4IgB7-H3 mutant encoding gene is as SEQ ID NO:1 Shown.
  • DNA is the genetic material of most organisms.
  • the genetic information, DNA replication, DNA repair and other processes carried by DNA play an important role in maintaining chromatin stability and gene expression and regulation; according to the central law, DNA is transcribed into mRNA and translated into Proteins perform corresponding physiological functions.
  • the transcription of mRNA in the cell is finely regulated at multiple levels in time and space; on the other hand, the latest experimental study has discovered another feedback regulation mechanism in the cell, that is, RNA regulates the phase separation of Mediator to perform the transcription process. Feedback regulation.
  • the main function of genes (DNA molecules) is to encode functional protein products or RNA products.
  • protein products can be macromolecular structural proteins, proteases, protein aggregates, nuclear pore complexes (NPC), etc., or amino acids, polypeptides (polypeptide chains), etc.;
  • RNA products can be ribozymes, tRNA, and rRNA , MRNA, etc.
  • protein folding and other proteins above the secondary structure are mainly determined by the amino acid sequence in the primary structure
  • the latest research such as the synonymous mutation study of chloramphenicol acetyltransferase in Escherichia coli has shown that the gene level is important for protein
  • the structure above the secondary structure may also have an impact; on the other hand, the conformation of proteins with the same amino acid sequence in the primary structure may also be different.
  • RNA Similar to the chirality of small molecule compounds, large molecules may also have different spatial conformations and may have different conformations. active.
  • the present invention relates to the genetic level, so it may have an impact on the above-mentioned RNA, protein and other materials and material structure, gene expression, transcription and other attributes, and application aspects of gene medicine in the central law.
  • the mutation coding gene belongs to an exon sequence, and is preferably used to transcribe mRNA.
  • a heterozygous mutation C>T was carried out at position 2142 at the 5'end.
  • Mutations are usually divided into synonymous mutations and missense mutations.
  • Classical theory believes that missense mutations cause an amino acid in a protein molecule to be replaced by another amino acid; while synonymous mutations (using synonymous codons) change the coding sequence of a gene, but retain the encoded amino acid sequence. It affects the correct folding process of the translated protein and the structure and function of the protein, so it is called "silent mutation".
  • synonymous mutations can change the protein level, translation accuracy, post-translational modification, secretion efficiency, and the final folding structure of the protein through a variety of mechanisms, thereby significantly changing the protein in the body and affecting the function of the cell. Therefore, the study of synonymous mutations will have a broad impact on effective protein design and the treatment of diseases related to synonymous mutations.
  • the mutant coding gene of the present invention may have an impact on the secondary and tertiary protein conformation of 4IgB7-H3.
  • 4IgB7-H3 acts as a receptor or a ligand in immune regulation. Therefore, in order to solve the above technical problems, the second technical solution of the present invention is to provide the protein encoded by the mutant gene as described above or its receptor or ligand; wherein, when the protein encoded by the mutant gene of the present invention is a receptor, the same The specific binding is the ligand; and when the protein encoded by the mutant gene of the present invention is the ligand, the specific binding is the receptor.
  • the end of the first C-like domain of the protein contains a PQRSPT motif, which may be an important motif for human 4IgB7-H3 not forming a soluble form.
  • the protein may be a membrane type protein.
  • the third technical solution of the present invention is to provide a recombinant expression vector, wherein the recombinant expression vector contains the human 4IgB7-H3 mutation encoding gene as described above.
  • the recombinant expression vector is a viral expression vector or a prokaryotic expression vector or a eukaryotic expression vector or a retroviral vector or an adenovirus vector or a herpes simplex virus.
  • the viral expression vector is a lentiviral expression vector or an adeno-associated virus expression vector.
  • the fourth technical solution of the present invention is to provide a host cell, wherein the host cell includes the above-mentioned recombinant expression vector.
  • the host cell is a eukaryotic cell, more preferably a mammalian cell.
  • the fifth technical solution of the present invention is to provide an isolated mRNA, wherein the mRNA is transcribed from the nucleotide sequence shown in SEQ ID NO:1.
  • the mRNA is IVT mRNA.
  • the in vitro transcription messenger RNA (IVT mRNA) of the present invention is a kind of mRNA transcribed under in vitro conditions using DNA as a template.
  • This mRNA can guide the synthesis of specific proteins, prevent or change certain Specific diseases. Therefore, IVT mRNA can be used as a potential new drug to transmit genetic information.
  • This IVT mRNA can better control the transcription of genes, transcription process and protein function.
  • IVT mRNA can introduce specific genetic information into the patient’s cells and quickly express proteins, thereby preventing or changing certain diseases.
  • IVT mRNA immunotherapy is mainly used in three areas: immunotherapy for malignant tumors, vaccines for the treatment of infectious diseases, and vaccines for alleviating allergies.
  • IVT mRNA delivery routes There are two delivery routes for IVT mRNA.
  • One is to introduce IVT mRNA into the patient's isolated cells, and then selectively introduce the transfected cells back into the patient's body.
  • This method is suitable for genome engineering, gene recombination, immunotherapy based on T cells and dendritic cells (DC) to treat malignant tumors and infectious diseases, and some protein replacement therapies.
  • Another way is to directly deliver IVT mRNA through a variety of methods, including intramuscular injection, intradermal injection, intranodal injection, intravenous injection, subcutaneous injection, intratracheal instillation, and intrathecal injection.
  • This method is suitable for the treatment of tumors, infectious diseases, immune tolerance regimens for the treatment of allergies, and other protein replacement therapies.
  • the strong immunostimulatory effect and inherent adjuvant properties of IVT mRNA have many advantages. It can cause effective antigen-specific cellular and humoral immune responses.
  • IVT mRNA causes various downstream effects by activating the pattern recognition receptor (PRR), thereby recognizing viral RNA and causing an immune response.
  • PRR pattern recognition receptor
  • TLR3 Toll-like receptor 3
  • TLR7 and TLR8 exist in endosomes.
  • Cells can activate TLR after phagocytosing IVT mRNA and cause the secretion of interferon.
  • TLR3 is used to identify double-stranded RNA (dsRNA)
  • TLR7 and TLR8 are used to identify single-stranded RNA (ssRNA).
  • the most effective interferon inducer, polyuridine acid works through TLR7.
  • most interferons are produced by activating cytoplasmic matrix receptors.
  • the RNA sensor in the cytoplasm can affect the immune stimulation and the translation ability of mRNA.
  • IVT mRNA can inhibit protein translation, degrade RNA, and directly produce antiviral activity.
  • the main part where IVT mRNA exerts its drug effect is the cytoplasm. Unlike natural mRNA, IVT mRNA enters the cytoplasm from outside the cell, and is translated and modified to produce a corresponding protein with pharmacological activity. In immunotherapy, the efficacy of IVT mRNA depends on the processing and processing of the encoded protein. Similar to endogenous proteins, protein molecules encoded by mRNA are degraded into peptide fragments by the proteasome, and then MHC-I molecules present these peptide fragments to CD8+ T cells to induce T cell immune responses.
  • intracellular proteins will not bind to MHC-II molecules to cause helper T (Th) cell responses.
  • the secretion signal is introduced into the antigen coding sequence, the antigen protein can be redirected to the outside of the cell under the action of the secretion signal to bind to MHC-II molecules, thereby simultaneously inducing Th cell response.
  • the sixth technical solution of the present invention is to provide a cell comprising the above-mentioned isolated mRNA and RNA sensor and/or mRNA immune recognition structural elements; preferably, the said Cells include immune cells and non-immune cells; more preferably, the immune cells include dendritic cells and lymphocytes, the lymphocytes include B cells and T cells, and the T cells include helper T cells and suppressor T cells. , Effector T cells, cytotoxic T cells, delayed allergic T cells, natural T cells and memory T cells; among them, helper T cells are divided into Th1, Th2, Th17 or Treg.
  • the seventh technical solution of the present invention is to provide an isolated miRNA, wherein the isolated miRNA targets the nucleotides shown in SEQ ID NO:1 and/or SEQ ID NO:2 .
  • the miRNA drug targets the 2142th position of the nucleotide shown in SEQ ID NO:1 and/or SEQ ID NO:2.
  • the isolated miRNA can be used as a detection marker for a disease associated with the mutant coding gene or its coding protein of the present invention, and can also be applied to prevent and treat related diseases such as tumors.
  • the eighth of the technical solutions of the present invention is to provide a targeted drug that targets the mutant coding gene of the human 4IgB7-H3 of the present invention
  • the targeted drug is an antisense RNA Or siRNA or circular RNA (circRNA) or non-coding RNA (ncRNA) such as lncRNA or shRNA; or target the protein encoded by the mutant coding gene of the present invention or its receptor or ligand, and the targeted drug
  • the targeted drug can inhibit the mutant coding gene of 4IgB7-H3 of the present invention, or its coding protein, or the receptor or ligand of the protein, and interfere with its biological function; or it can be used to detect the above-mentioned substances.
  • the antibody selects and optimizes the biopharmaceutical antibody through internalization and uptake in the cell.
  • RNA also plays an important role in gene expression regulation and gene transcription. RNA splicing, degradation, metabolism, and chemical modification are closely related to cell functions. Each RNA modification All need their specific regulatory factors, including modification enzymes, demodification enzymes and recognition proteins.
  • non-coding RNA ncRNA
  • RNAs with known functions such as circRNA and miRNA, also include RNAs with unknown functions. Among them, circRNA is different from traditional linear RNA.
  • the circRNA molecule has a closed circular structure, which is not affected by RNA exonuclease, and its expression is more stable and not easily degraded.
  • circRNA molecules are rich in miRNA binding sites and act as miRNA sponges in cells, thereby releasing miRNA's inhibitory effect on its target genes and increasing the expression level of target genes .
  • circRNA plays an important regulatory role in diseases. Therefore, circRNA targeting the mutant coding gene of the present invention, its coding protein or its related miRNA is also an important means for diagnosing or preventing and treating immune diseases.
  • the ninth technical solution of the present invention is to provide a pharmaceutical composition, which includes the human 4IgB7-H3 mutation coding gene, the protein or its receptor or ligand, The recombinant vector, the host cell, the isolated mRNA, the cell, the miRNA drug or the targeted drug.
  • the pharmaceutical compound includes a pharmaceutically acceptable carrier or excipient.
  • the tenth technical solution of the present invention is to provide a mutant coding gene of human 4IgB7-H3, the protein or its receptor or ligand, the recombinant vector, and the above-mentioned human 4IgB7-H3
  • the host cell, the isolated mRNA, the cell, the isolated miRNA, the targeted drug or the pharmaceutical composition are prepared to regulate T cell activity and/or regulate T cell proliferation Further, its use in the preparation of drugs for regulating immunity or immunotherapy is provided; preferably, the drugs for regulating immunity or immunotherapy are prevention and/or treatment of tumors, infectious diseases or alleviation of allergies medicine.
  • the drug for regulating immunity or immunotherapy is a vaccine.
  • the tumor is one or more of gastric cancer, bowel cancer, liver cancer, lung cancer, bladder cancer, testicular cancer, prostate cancer, cervical cancer, breast cancer and lymphoma.
  • the "immune regulation” refers to the suppression of an excessively or abnormally activated immune response, and the promotion or activation of an excessively low or suppressed normal immune response.
  • the reagents and raw materials used in the present invention are all commercially available.
  • the HEK293T cells transfected with the 4IgB7-H3 mutant encoding gene of the present invention proliferate faster than the cells transfected with the wild-type gene, indicating that the mutant gene has a stronger biological function than the wild-type gene.
  • the human 4IgB7-H3 mutant encoding gene has an enhanced biological function than the wild-type gene, that is, the binding of the mutant encoding 4IgB7-H3 to an inhibitory receptor or ligand has an enhanced inhibitory effect on the proliferation and activation of T cells.
  • the mutation-encoding gene can also be used as a diagnostic indicator for related diseases, and thus effective disease intervention methods can be designed.
  • the protein or its receptor or ligand encoded by the mutation coding gene, the mRNA and the cells containing the mutation coding gene, miRNA and siRNA, antibodies and other targeted drugs can also be used for tumor treatment, infectious disease treatment, alleviation of allergies, organs Clinical application of transplantation and autoimmune diseases.
  • Figure 1 is a plasmid map of the constructed recombinant expression vector containing the mutant coding gene of human 4IgB7-H3;
  • Figure 2 is a plasmid map of the constructed recombinant expression vector containing the wild-type gene encoding human 4IgB7-H3;
  • Figure 3 shows the results of flow cytometry detection of recombinant expression vectors: A. A recombinant expression vector containing a mutant gene encoding human 4IgB7-H3, B. A recombinant expression vector containing a wild-type gene encoding human 4IgB7-H3;
  • Figure 4 shows the results of flow cytometry detection of HEK293T cells infected by lentivirus: A. Lentivirus containing the mutant gene encoding human 4IgB7-H3, B. Lentivirus containing the wild-type gene encoding human 4IgB7-H3;
  • Figure 5 is a comparison of the effects of wild-type and mutant coding genes on the proliferation of HEK293T cells over time;
  • Figure 6 shows the flow cytometric detection results of jurkat cells infected by lentivirus
  • Figure 7 is a comparison of the effects of wild-type and mutant coding genes on the proliferation of jurkat cells over time.
  • the artificially synthesized wild-type 4IgB7-H3 coding gene (SEQ ID NO: 2, NCBI Reference Sequence: NM_001024736.1) and the 4IgB7-H3 mutant coding gene (SEQ ID NO:1) were constructed into PGMLV-PA7 after PCR. And PGMLV-PA6, and use blasticidin and puro as screening markers, respectively.
  • the vector is transformed into competent cells by conventional techniques in this field, and the competent cells are plated and placed upside down in a 37°C constant temperature incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.) for overnight culture. On the second day, single clones were selected. Two clones of each vector were selected and sent to a sequencing company for sequencing identification.
  • Plasmids containing human 4IgB7-H3 wild-type coding gene (SEQ ID NO: 2) and human 4IgB7-H3 (SEQ ID NO: 1) mutant coding genes were obtained, and they were named CMV-4IgB7-H3(CD276)WT-PGK, respectively -puro (11,459bp, the plasmid map is shown in Figure 2, hereafter referred to as 4IgB7-H3 (CD276)WT plasmid) and CMV-4IgB7-H3 (CD276)MT (c.C2142T)-PGK-Blasticidin (11,267bp, the plasmid map is shown in Figure 1. Hereinafter referred to as 4IgB7-H3 (CD276) MT plasmid).
  • HEK293T DMEM+10% Fetal Bovine Serum (GIBICO); HG transgene reagent (Giman Biotechnology); 0.25% Trypsin (GIBICO); High Purity Plasmid Extraction Kit (QIAGEN); Fluorescence Microscope (Olympus); Propanol; flow cytometry antibody; biological safety cabinet (Thermo); CO 2 incubator (Thermo), BD FACSCalibur flow cytometer.
  • the blank cells in good condition and in the logarithmic growth phase are digested with 0.25% pancreatin (GIBICO) and suspended in complete medium to form a single cell suspension. After cell counting, 5 ⁇ 10 5 cells per well are seeded in 35mm petri dish (Shanghai Qianchen Biology);
  • Example 2 Packaging of lentivirus and determination of virus titer
  • Cell line HEK 293T, lentivirus packaging cells, which are anchorage-dependent epithelial-like cells, and the growth medium is DMEM (containing 10% FBS).
  • Adherent cells grow and proliferate through culture to form a monolayer of cells.
  • Escherichia coli strain DH5 ⁇ Used to amplify lentiviral vectors and auxiliary packaging vector plasmids.
  • Lentiviral packaging system The successfully constructed lentiviral recombinant plasmid and packaging plasmid are extracted with Qiagen's plasmid extraction kit. The obtained plasmid DNA is dissolved in sterile TE, and its concentration and purity are determined by ultraviolet light absorption method to ensure that the A260/A280 of the extracted plasmid DNA is between 1.8 and 2.0.
  • HEK 293T cell partitioning the day before transfection, pass the grown cells into a 10cm culture dish at an appropriate ratio, and prepare for transfection when the cells grow to 70%-80%.
  • Virus collection After changing the medium for 48 hours, pipette the cell supernatant into a 50ml centrifuge tube, centrifuge at 4,500 ⁇ g for 5min at 4°C, filter the supernatant with a 0.45 ⁇ m filter and transfer it to a new centrifuge tube, and finally divide the filtrate into a centrifuge tube. Transfer the batch to a concentration device, 4°C, 4,500 ⁇ g, centrifugation for 10 min, discard the lower layer of liquid in the waste liquid cup containing the disinfectant, the last time 4°C, 4,500 ⁇ g, centrifugation for 20 minutes, then the upper layer of the filter The liquid is the virus concentrate.
  • Virus packaging and storage After packaging the virus, store it at -80°C.
  • HEK 293T cells were cultured to the logarithmic growth phase, and the culture medium for virus dilution was cell culture medium containing 10% FBS.
  • the culture medium for virus dilution was cell culture medium containing 10% FBS.
  • the volume of each component in the PCR system is as follows:
  • the above table shows that the titers of the lentivirus transfected with the wild-type gene and the lentivirus transfected with the mutant coding gene are basically the same, and can be used for subsequent parallel experiments for comparison of in vitro effects.
  • target cells HEK293T purchased from the Cell Bank of Shanghai Chinese Academy of Sciences
  • HEK293T purchased from the Cell Bank of Shanghai Chinese Academy of Sciences
  • the virus stock solution On the second day, before infection, the virus stock solution was taken out of the refrigerator at -80°C and melted in an ice bath.
  • the virus stock solution was diluted with a fresh medium containing 5 ⁇ g/ml Polybrene according to the appropriate MOI value, and the original culture of the control group and the treatment group was aspirated Base, the negative control (NC-) diluent containing the lentivirus was added to the control group cells, and the diluent containing the lentivirus solution was added to the cells of the treatment group.
  • NC- negative control
  • the culture medium was changed, and the culture medium containing the lentivirus was replaced with normal culture medium after 16 hours of infection.
  • the cells are basically stable (4IgB7-H3(CD276) WT stable transgenic puro, total lethal concentration 1.5 ⁇ g/ml, maintenance concentration 0.75 ⁇ g/ml; 4IgB7 -H3(CD276)MT stable transformation strain blast total lethal concentration 8 ⁇ g/ml, maintenance concentration 4 ⁇ g/ml).
  • HEK293T cells culture medium; serum (GIBICO); antibiotics: penicillin and streptomycin (Penicillin, Streptomycin); trypsin: 0.25% Trypsin (GIBICO); high-purity plasmid extraction kit (QIAGEN); flow cytometry Antibody;
  • Virus infection The overexpression lentivirus and lentivirus negative control are packaged to infect the target cells, and the overexpression effect of the overexpression vector is detected by the flow antibody staining method.
  • the cells are directly collected in the flow tube, the cells are sucked off the upper medium, and 500 ⁇ l PBS (1%BSA) is added to wash the cells, 1000rpm, 5min, the liquid is directly poured out, leaving about 100 ⁇ l PBS (1%BSA), If there is no 100 ⁇ l directly less than 100 ⁇ l, resuspend the cells;
  • HEK293T DMEM medium + 10% fetal bovine serum (GIBICO); 0.25% Trypsin (GIBICO); fluorescence microscope (Olympus); biological safety cabinet (Thermo); CO 2 incubator (Thermo); centrifuge (Thermo) ; CCK-8 (Fluka); Automated Enzyme Immunoassay Analyzer (Beijing Tianshi Medical Products Manufacturing Co., Ltd.).
  • CON represents empty cells
  • NC represents cell lines infected with wild-type control lentivirus and mutant control lentivirus
  • WT represents cell lines infected with 4IgB7-H3 (CD276) WT wild-type lentivirus
  • MT represents 4IgB7-H3( CD276) MT mutant lentivirus cell line
  • WT+MT represents a cell line infected with 4IgB7-H3 (CD276) WT wild-type lentivirus and 4IgB7-H3 (CD276) MT mutant lentivirus at the same time.
  • SS represents the sum of squared deviations from the mean
  • df represents the degrees of freedom.
  • MS stands for mean square
  • F stands for F statistic
  • P-value stands for p value
  • F crit stands for F critical value.
  • Figure 5 further shows the cell proliferation trend in the table above.
  • the above cell lines were cultured for 72 hours and verified by CCK8.
  • the 4IgB7-H3(CD276)MT mutant cell line expressing the lentivirus has enhanced cell proliferation
  • HEK293T cells transfected with the human 4IgB7-H3 mutant coding gene proliferate faster than the cells transfected with the 4IgB7-H3 wild-type gene, indicating that the mutant coding gene has stronger effects in HEK293T cells than the wild-type gene. Promote the biological function of proliferation.
  • the target cells infected by the lentivirus are jurkat cells (purchased from the cell bank of the Shanghai Chinese Academy of Sciences), and the infection method is the same as in Example 3.
  • the cells are basically stable.
  • 4IgB7-H3(CD276)WT stable transgenic strain puro has a total lethal concentration of 1.5 ⁇ g/ml, maintaining a concentration of 0.75 ⁇ g/ml
  • 4IgB7-H3(CD276)MT stable transgenic strain blast has a total lethal concentration of 7 ⁇ g/ml, maintaining The concentration is 3.5 ⁇ g/ml.
  • Example 3 When verifying the effect of gene overexpression by flow cytometry, the other conditions were the same as in Example 3 except that jurkat cells were used as the cells.
  • 4IgB7-H3(CD276)WT Infect 4IgB7-H3(CD276)WT wild-type jurkat cells expressing lentivirus alone;
  • 4IgB7-H3(CD276)WT+MT Simultaneously infect jurkat cells expressing 4IgB7-H3(CD276)WT wild-type lentivirus and 4IgB7-H3(CD276)MT mutant expressing lentivirus;
  • WT-IgG Isotype control.
  • jurkat refers to empty cells not transfected with virus
  • NC refers to jurkat cell lines infected with wild-type control lentivirus and mutant control lentivirus
  • WT refers to jurkat infected with 4IgB7-H3 (CD276)WT wild-type lentivirus expressing lentivirus Cell line
  • MT is a jurkat cell line infected with 4IgB7-H3 (CD276) MT mutant expressing lentivirus
  • WT+MT is simultaneously infected with 4IgB7-H3 (CD276) WT wild-type expressing lentivirus and 4IgB7 -H3(CD276)MT mutant jurkat cell line expressing lentivirus.
  • SS represents the sum of squared deviations from the mean
  • df represents the degrees of freedom.
  • MS stands for mean square
  • F stands for F statistic
  • P-value stands for p value
  • F crit stands for F critical value.
  • Jurkat cells belong to a human acute T lymphocyte leukemia cell line, which can mimic the function of T lymphocytes and are widely used in in vitro studies of T lymphocyte signal transduction, cytokine and receptor expression.
  • the in vitro experimental results reveal that the mutation code 4IgB7-H3 The inhibitory effect of binding to inhibitory receptors on the proliferation of T lymphocytes is enhanced.
  • the new human 4IgB7-H3 mutation coding gene shown in SEQ ID NO:1 of the present invention is used in T and B cell proliferation, activation, helper T cell differentiation, signal transduction and effector cell cytokines It plays an important role in the secretion process, and plays a role in solving and treating the main reason why it is difficult to induce tumor-specific T cell activation in tumor evading immune surveillance. It has outstanding and broad application prospects in the preparation of genome engineering, genetic recombination, T cell and dendritic cell-based immunotherapy drugs, vaccines, protein replacement therapy, alleviation of allergies, etc. for the treatment of malignant tumors and infectious diseases.

Abstract

提供了一种新的人4IgB7-H3的突变编码基因及其调节免疫的应用，其核苷酸序列如SEQ ID NO:1所示。该人4IgB7-H3的突变编码基因比野生型基因的生物学功能增强，即突变编码4IgB7-H3与抑制性受体结合对T细胞的增殖、活化的抑制作用增强。对4IgB7-H3作为共刺激分子在T、B细胞活化、辅助T细胞分化，信号转导及效应细胞的细胞因子分泌过程中起重要作用。

Description

人4IgB7-H3的突变编码基因及其调节免疫的应用
本申请要求申请日为2019/12/30的中国专利申请201911399571.6，以及申请日为2020/9/10的中国专利申请202010949660.X的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物医药免疫疗法领域，尤其涉及一种人4IgB7-H3的突变编码基因及其调节免疫的应用。
背景技术
正性/负性协同刺激信号(又称共刺激信号，Co-stimulatory signals)之间的有序调节达到的动态平衡在机体抵抗外来抗原的入侵以及防止自身免疫性疾病的发生中起着重要的作用。T淋巴细胞(简称T细胞)的活化或增殖依赖于正性/负性双重信号，其对T细胞抗原特异性的有效激发以及免疫效应适时中止是必须的。免疫调节失去平衡、不管抑制或者过度激活，都属于免疫异常，从而影响机体的免疫应答，引起的疾病即为各种免疫性疾病，包括肿瘤、炎症、自身免疫性疾病等。另一方面，T细胞活化与增殖依赖于双信号：一是抗原呈递细胞(APC)呈递的抗原肽-人类白细胞抗原(HLA)复合物，二是共刺激信号(第2信号)。APC呈递的抗原肽-人类白细胞抗原(HLA)复合物由特殊T细胞抗原受体(TCR)识别，并将信号传递给T细胞，该信号对T细胞活化是必需的，但不引起T细胞增生和分泌细胞因子。共刺激信号为T细胞抗原特异性激活所必需，启动、维持并调节活化级联反应决定了T细胞是活化增殖、抑或转变为无反应状态(anergy)甚至凋亡(apoptosis)。双信号学说(two-signal model)的提出，解释了成熟外周B细胞如何识别抗原，以及T细胞如何获得来自于APC呈递的抗原肽-主要组织相容性复合物(major histocompatibility complex I，MHC)信号，因此共刺激信号已经成为人们研究的热点。共刺激信号为抗原(Ag)非特异性，除影响T细胞的活化与增殖外，还直接影响细胞因子的产生。共刺激分子在肿瘤免疫中起重要作用。研究发现大多数肿瘤细胞表达MHC-1类分子和肿瘤Ag，是潜在的APC，但免疫原性较弱，因为其不表达或低表达B7-1，故难以诱导肿瘤特异性T细胞激活，这是肿瘤逃避免疫监视的重要原因之一。因此，可以通过增加或增强共刺激信号，达到治疗肿瘤的作用。
由此可见，肿瘤免疫逃逸是肿瘤发生、侵袭及转移的重要机制。由于肿瘤细胞通常 不能提供有效的抗原信号，自身具备抵抗免疫效应细胞攻击的能力，或者机体存在免疫应答缺陷及免疫抑制，这些情况均导致了肿瘤细胞逃避机体的免疫监控。在这一过程中，协同刺激分子(又称共刺激分子)及其调节网络发挥着重要作用。协同刺激分子是T淋巴细胞活化过程中一类重要的调节分子，共刺激分子与其受体或配体作为共刺激信号，在T、B细胞活化、辅助T细胞分化、信号转导及效应细胞的细胞因子分泌过程中起重要作用。协同刺激分子主要分为B7/CD28和TNF/TNFR两个超家族。经典的B7/CD28信号途径主要以协同刺激的形式来调节初始T细胞的活化。然而，新近发现的一些B7家族新成员亦可通过作用于活化的T细胞，对免疫应答起到负性调控作用。B7家族属免疫球蛋白类，为50000～70000d的跨膜糖蛋白，B7免疫蛋白家族的成员包括B7-1、B7-2、B7-H1(即PD-L1)、B7-H2、B7-H3和PD-L2。它们结构相似均可在机体免疫过程中作为共刺激信号，参与T、B细胞的活化和机体免疫。因此，B7家族在肿瘤治疗、器官移植和自体免疫病的治疗方面有重要作用，在基础研究方面则可以用于T、B细胞分化、活化机制、抗原提呈、共刺激机制、免疫耐受、移植排斥反应和自体免疫等的研究。
B7-H1、B7-H3就是其中两个重要的负性调控协同刺激分子。陈陆俊等首次报导了负性协同刺激分子B7-H1、B7-H3在结直肠癌中的表达及其临床意义(陈陆俊，苏州大学硕士学位论文，2009，协同刺激分子B7-H1和B7-H3在结直肠癌中表达的临床意义及调控机制)，证实它们在肠癌的发生发展中发挥重要作用并负性调控T淋巴细胞在肿瘤组织中的浸润，检测B7-H1、B7-H3分子可作为肠癌诊断和预后判断的重要生物学指标。同时，肿瘤微环境中的细胞因子IFN-γ和TNF-α对肿瘤细胞表达协同刺激分子B7-H1、B7-H3起到重要的调控作用。B7-H3分子可通过膜型和可溶性(又称SB7-H3)两种形式在肠癌的肿瘤免疫逃逸过程中发挥作用。
人B7-H3，也称为CD276，是属于B7-CD28途径的免疫检查点分子。B7-H3是一种I型跨膜蛋白，在小鼠中由9号染色体编码，在人类中由15号染色体编码。B7-H3作为B7家族的最新成员，mRNA广泛存在于非淋巴组织，在许多淋巴器官如脾、淋巴结、胸腺、婴儿的肝和胸腺也有表达。此外，部分肿瘤细胞株如黑色素瘤G361、子宫颈腺瘤、HeLa S3、慢性髓淋巴瘤K562、肺癌A546和结直肠腺癌SW480中，B7-H3的mRNA也有表达。其受体在活化的T细胞表面可被诱导表达，B7-H3对CD4+和CD8+细胞的生成均具有增加作用，并能诱导CTL产生，并可选择性地增加IFN-γ的生成，参与T、B细胞的活化和机体免疫，在肿瘤治疗、器官移植和自身免疫病的治疗方面具有重要的作用(Suh WK,et al.The B7 family member B7-H3 Prefeerntialy down-regulates T helper type1-mediated immune responses[J].Nat Immunol,2003,4(9):899-906.)。B7-H3不能与CTLA-4  ICOS和PD-1结合，表明B7-H3有受体或配体，但目前还没有分离到。
在人类中，B7-H3基因具有四个Ig样重复，这些重复可以交替剪接以产生包含四个Ig样结构域(4Ig)或两个Ig样结构域(2Ig)的蛋白质，即异构体4IgB7-H3(或B7-H3b)和2IgB7-H3。4IgB7-H3带有两个拷贝的IgV-IgC结构域，为人大部分细胞和组织中的主要表达形式，其胞外段两对VC之间碱基重复率为98％，是2IgB7-H3基因复制的结果。孙静等(孙静，苏州大学博士学位论文，2010，协同刺激分子B7-H3两种异构体的基因演化及其生物学功能差异的研究)根据B7-H3分子2种异构体在不同物种中的分布及其基因复制模式的研究、人2IgB7-H3和4IgB7-H3蛋白质的空间结构模拟及其受体表达分析、及人2IgB7-H3和4IgB7-H3生物学功能差异研究等发现：1、4IgB7-H3是2IgB7-H3基因复制的结果，除表达于人类外还可表达于豚鼠、家犬、非洲象、牛、熊猫、蝙蝠及高级灵长类动物如猩猩、猴体内；2、人2IgB7-H3和4IgB7-H3两种蛋白质具备不同的空间构象；3、活化的T细胞上可表达两种受体与B7-H3的2种异构体相结合；人2IgB7-H3与小鼠B7-H3与刺激性受体相结合，发挥促进T细胞的活化与增强单核细胞的功能；而人4IgB7-H3可与抑制性受体结合，对T细胞的活化具有抑制作用。
在人4IgB7-H3分子“PQRSPT”基序的生物学特性的研究发现在外显子复制过程中产生的保守性氨基酸“PQRSPT”可成为人4IgB7-H3不能形成可溶性形式及对活化T细胞产生抑制作用的重要基序。蛋白质建模发现氨基酸高度重复的2种异构体具备不同的空间构象，融合蛋白结合实验推测其可结合不同的受体发挥生物学功能。体外实验对活化T细胞和单核细胞的作用证实了之前的实验结果。人2IgB7-H3与小鼠B7-H3基因可促进活化T细胞的增殖，增强单核细胞的作用；人4IgB7-H3主要表达于抗原递呈细胞中可发挥抑制T细胞活化的生物学功能。
综上，人B7-H3在肿瘤治疗、器官移植和自身免疫病的治疗方面具有重要的作用，且其异构体2IgB7-H3与4IgB7-H3在功能上相互配合，增加了该信号通路的重要性和复杂性，且由于B7-H3受体或者配体尚未被发现，因此关于其功能学研究的报道还存在很大的争议。围绕B7-H3两个异构体开展深入研究对揭示共刺激分子在免疫应答中的精确调控具有重要的生物学意义。而提供2IgB7-H3与4IgB7-H3的各种突变体及功能研究，也为上述B7-H3的功能研究提供新途径，但目前尚未有4IgB7-H3的突变体或4IgB7-H3的突变编码基因的报道。
发明内容
本发明提供了一种新的人4IgB7-H3的突变编码基因，比野生型4IgB7-H3基因具有 增强的生物功能，为抑制过度或异常激活的免疫特别是T淋巴细胞介导的细胞免疫及T淋巴细胞的发育、或与功能增强的2IgB7-H3突变体的配合提供一种新的技术手段；尤其是通过新的人4IgB7-H3的突变编码基因、其编码的蛋白或其受体或配体、相关的例如RNA分子等药物或含其的药物组合物来干预免疫逃逸，从而调节或改善受试者的免疫响应；同时提供在调节免疫的应用例如其在制备预防或治疗恶性肿瘤和传染性疾病的免疫治疗药物、疫苗以及缓解过敏药物等中的应用。
为解决上述技术问题，本发明的技术方案之一为：提供一种人4IgB7-H3的突变编码基因，其中，所述人4IgB7-H3的突变编码基因的核苷酸序列如SEQ ID NO:1所示。
众所周知，DNA是绝大多数生物的遗传物质，DNA携带的遗传信息、DNA复制、DNA修复等过程对维持染色质稳定和基因表达、调控具有重要作用；按照中心法则DNA转录为mRNA，并翻译为蛋白质，执行相应的生理功能。一方面，细胞内mRNA的转录在时间和空间上受到了多层次的精细调控；另一方面，最新实验研究发现了细胞内另一反馈调节机制，即RNA通过调控Mediator的相分离对转录过程进行反馈调节。基因(DNA分子)最主要的功能是编码功能性蛋白质产物或RNA产物。其中，蛋白质产物可以是大分子的结构蛋白、蛋白酶、蛋白质聚合体、核孔复合物(NPC)等，也可以是氨基酸、多肽(多肽链)等；RNA产物则可以是核酶和tRNA、rRNA、mRNA等。虽然之前技术显示蛋白质折叠等蛋白质二级以上结构主要由一级结构中的氨基酸序列决定，但最新的研究例如大肠杆菌中的氯霉素乙酰基转移酶的同义突变研究则显示基因层面对于蛋白质二级以上结构可能也有影响；另一方面，一级结构氨基酸序列相同的蛋白质构象也有可能不一样，类似于小分子化合物有手性，大分子也会有不同的空间构象，且可能具有不同的活性。而本发明涉及基因层面，故可能对于中心法则中的上述RNA、蛋白质等物质及物质结构，基因表达、转录等属性，以及基因药物等应用方面均能产生影响。
根据本发明，所述的突变编码基因属于外显子序列，较佳地用于转录mRNA，于5，端第2142位点进行了杂合突变C>T。突变通常分为同义突变和错义突变。经典理论认为，错义突变导致蛋白质分子中某一氨基酸为另一氨基酸所替代；而同义突变(使用同义密码子)改变了基因编码序列，却保留了编码的氨基酸序列，由此不会影响到其所翻译的蛋白质的正确折叠过程以及蛋白质的结构和功能，因此被称为“沉默突变”。然而，近期的研究证实，罕见的同义密码子往往比普通密码子翻译得慢，其改变了核糖体合成蛋白质的速度，由此不仅会扰乱蛋白质折叠，导致更容易降解的结构形成，造成细胞生长缺陷。越来越多的研究表明，同义突变可以通过多种机制改变蛋白质水平、翻译准确性、翻译后修饰、分泌效率和蛋白质的最终折叠结构，从而显著改变体内蛋白质进而影 响细胞的功能。因此，同义突变的研究对有效的蛋白质设计和与同义突变相关疾病的治疗将产生广泛的影响。
如上所述，本发明的突变编码基因可能对于4IgB7-H3的二级、三级蛋白质构象产生影响，而目前4IgB7-H3在免疫调节中作为受体还是配体作用尚未有定论。故为解决上述技术问题，本发明的技术方案之二为：提供如上所述的突变基因编码的蛋白或其受体或配体；其中，当本发明突变基因编码的蛋白为受体时，与其特异性结合的为配体；而当本发明突变基因编码的蛋白为配体时，与其特异性结合的为受体。优选地，所述蛋白的第一个C样结构域末端包含PQRSPT基序，该基序可能是人4IgB7-H3不能形成可溶性形式的一个重要基序。所述蛋白可为膜型蛋白。为解决上述技术问题，本发明的技术方案之三为：提供一种重组表达载体，其中，所述重组表达载体包含如上所述的人4IgB7-H3突变编码基因。
优选地，所述重组表达载体为病毒表达载体或原核表达载体或真核表达载体或逆转录病毒载体或腺病毒载体或单纯孢疹病毒。
更优选地，所述病毒表达载体为慢病毒表达载体或腺相关病毒表达载体。
为解决上述技术问题，本发明的技术方案之四为：提供一种宿主细胞，其中，所述宿主细胞包括如上所述的重组表达载体。
优选地，所述宿主细胞为真核细胞，更优选哺乳动物细胞。
为解决上述技术问题，本发明的技术方案之五为：提供一种分离的mRNA，其中，所述mRNA由如SEQ ID NO:1所示的核苷酸序列转录而得。优选地，所述mRNA为IVT mRNA。
本发明所述的体外转录信使RNA(in vitro transcription messenger RNA,IVT mRNA)是在体外条件下以DNA为模板转录得到的一种mRNA，这种mRNA可以指导特定蛋白质的合成，阻止或改变某种特定的疾病。因此，IVT mRNA可以作为一种传递遗传信息的潜在新药。这种IVT mRNA能够更好地控制转录基因，转录过程与蛋白功能等。IVT mRNA可以将特定的遗传信息导入患者的细胞内，快速表达出蛋白质，从而阻止或改变某种疾病。目前，IVT mRNA免疫疗法主要应用于三个领域：恶性肿瘤的免疫治疗、治疗传染性疾病的疫苗、缓解过敏的疫苗。
IVT mRNA有2条递送途径。一条是将IVT mRNA导入患者离体细胞，再将转染的细胞有选择性地导回患者体内。这种方法适用于基因组工程、基因重组、以T细胞和树突状细胞(dendritic cells，DC)为基础治疗恶性肿瘤和传染性疾病的免疫疗法以及一些蛋白替补疗法等。另一条途径是通过多种方式直接递送IVT mRNA，包括肌肉注射、真皮 内注射、结内注射、静脉注射、皮下注射、气管内滴入和鞘内注射等。这种方法适用于肿瘤、传染性疾病的治疗、治疗过敏的免疫耐受养生法以及其他蛋白替补疗法等。对于疫苗而言，IVT mRNA具有的强烈免疫刺激作用和固有的佐剂特性有很多优势，它能够引起有效的抗原特异性细胞和体液免疫应答。
细胞内RNA感应器的识别机制和mRNA免疫识别结构元件：
IVT mRNA通过激活模式识别受体(pattern recognition receptor，PRR)引起各种下游效应，从而识别病毒RNA，引起免疫应答。
在免疫细胞中，Toll样受体3(Toll-like receptor 3，TLR3)、TLR7和TLR8存在于内体之中。细胞吞噬IVT mRNA后能够激活TLR，并且引起干扰素分泌。其中，TLR3用于识别双链RNA(double-stranded RNA，dsRNA)，TLR7和TLR8用于识别单链RNA(single-stranded RNA，ssRNA)。最有效的干扰素诱导剂聚尿苷酸通过TLR7发挥作用。而在非免疫细胞中，大多数干扰素是通过激活细胞质基质受体产生的。另外，细胞质中的RNA感应器能够影响免疫刺激作用和mRNA的翻译能力。它可以抑制蛋白质翻译，降解RNA，直接产生抗病毒活性。IVT mRNA发挥药效的部位主要是细胞质。与天然mRNA不同，IVT mRNA由细胞外进入细胞质，翻译并修饰后产生相应的具有药理活性的蛋白质。在免疫治疗中，IVT mRNA的药效取决于编码蛋白的处理加工过程。与内源性蛋白相似，mRNA编码产生的蛋白分子会被蛋白酶体降解为肽片段，然后MHC-I类分子将这些肽片段呈递给CD8+T细胞，诱导T细胞免疫应答。一般来说，胞内蛋白不会与MHC-II类分子结合引起辅助T(helper T，Th)细胞应答。但如果将分泌信号导入抗原编码序列，抗原蛋白在分泌信号作用下就可以重新导向细胞外，与MHC-II类分子结合，从而同时诱导Th细胞应答。
由此，为解决上述技术问题，本发明的技术方案之六为：提供一种细胞，包含如上所述的分离的mRNA和RNA感应器和/或mRNA免疫识别结构元件；较佳地，所述细胞包括免疫细胞和非免疫细胞；更佳地所述免疫细胞包括树突状细胞和淋巴细胞，所述淋巴细胞包括B细胞和T细胞，所述T细胞包括辅助性T细胞、抑制性T细胞、效应T细胞、细胞毒T细胞、迟发性变态反应T细胞、天然T细胞和记忆T细胞；其中，辅助性T细胞分为Th1、Th2、Th17或Treg。
为解决上述技术问题，本发明的技术方案之七为：提供一种分离的miRNA，其中，所述分离的miRNA靶向SEQ ID NO:1和/或SEQ ID NO:2所示的核苷酸。优选地，所述miRNA药物靶向SEQ ID NO:1和/或SEQ ID NO:2所示的核苷酸的第2142位点。所述分离的miRNA可以作为本发明突变编码基因或其编码蛋白关联疾病的检测标志物，也 可以应用于防治相关疾病例如肿瘤。
为解决上述技术问题，本发明的技术方案之八为：提供一种靶向药物，所述靶向药物靶向本发明上述人4IgB7-H3的突变编码基因，所述靶向药物为反义RNA或siRNA或环状RNA(circRNA)或lncRNA或shRNA等不编码RNA(Non-coding RNA，ncRNA)；或者靶向本发明突变编码基因编码的蛋白或其受体或配体、所述靶向药物例如抗体。所述靶向药物可抑制本发明人4IgB7-H3的突变编码基因、或其编码蛋白、或该蛋白的受体或配体，干预其生物功能；或者用于检测上述物质。
其中，所述抗体通过在细胞内的内化和摄取情况来进行生物药物抗体的选择和优化。
目前的分子生物学研究显示，在基因表达调控和基因转录等过程中，除了DNA，RNA也发挥着重要的作用，RNA剪接、降解、代谢和化学修饰等都与细胞功能息息相关，每种RNA修饰都需要其特异的调控因子，包括修饰酶、去修饰酶和识别蛋白。特别是非编码RNA(non-coding RNA，ncRNA)在细胞各项生物过程中发挥着不可忽视的作用，被喻为生命的“暗物质”，其包括但不仅限于rRNA、tRNA、snRNA、IncRNA、snoRNA、circRNA和miRNA等多种已知功能的RNA，还包括未知功能的RNA。其中，circRNA与传统的线性RNA不同，circRNA分子呈封闭环状结构，不受RNA外切酶影响，表达更稳定，不易降解。在功能上，近年的研究表明，circRNA分子富含miRNA结合位点，在细胞中起到miRNA海绵(miRNA sponge)的作用，进而解除miRNA对其靶基因的抑制作用，升高靶基因的表达水平。通过与疾病关联的miRNA相互作用，circRNA在疾病中发挥着重要的调控作用。由此，靶向本发明突变编码基因、其编码蛋白或其相关miRNA的circRNA也是诊断或防治免疫性疾病的重要手段。
为解决上述技术问题，本发明的技术方案之九为：提供一种药物组合物，其中，其包括如上所述的人4IgB7-H3突变编码基因、所述的蛋白或其受体或配体、所述的重组载体、所述的宿主细胞、所述的分离的mRNA、所述的细胞、所述的miRNA药物或靶向药物。优选地，所述药物化合物包括药学上可接受的载体或赋形剂。
为解决上述技术问题，本发明的技术方案之十为：提供一种如上所述的人4IgB7-H3的突变编码基因、所述的蛋白或其受体或配体、所述的重组载体、所述的宿主细胞、所述的分离的mRNA、所述的细胞、所述的分离的miRNA、所述的靶向药物或所述的药物组合物在制备调节T细胞活性和/或调节T细胞增殖的药物中的用途；进一步地，提供其在制备调节免疫或免疫治疗的药物中的用途；优选地，所述调节免疫或免疫治疗的药物为预防和/或治疗肿瘤、传染性疾病或缓解过敏的药物。
其中，所述调节免疫或免疫治疗的药物为疫苗。
所述肿瘤为胃癌、肠癌、肝癌、肺癌、膀胱癌、睾丸癌、前列腺癌、宫颈癌、乳房癌和淋巴癌中的一种或多种。
根据本发明，所述的“免疫调节”是指对过度或异常激活的免疫反应进行抑制，而对过低或被抑制的正常免疫反应进行促进或激活。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
转染了本发明人4IgB7-H3突变编码基因的HEK293T细胞比转染了野生型基因的细胞增殖快，说明突变基因比野生型基因具有更强的生物学功能。换言之，该人4IgB7-H3的突变编码基因比野生型基因的生物学功能增强，即突变编码4IgB7-H3与抑制性受体或配体结合对T细胞的增殖、活化的抑制作用增强。从而可用于抑制过度或异常激活的免疫、或与2IgB7-H3及其突变体的配合调节免疫，在肿瘤治疗、器官移植和自身免疫病的治疗方面有广泛的应用前景。此外，该突变编码基因还可以作为相关疾病的诊断指标，并由此设计有效的疾病干预手段。突变编码基因编码的蛋白或其受体或配体、针对突变编码基因的mRNA及含其的细胞、miRNA和siRNA、抗体等靶向药物也可以用于肿瘤治疗、传染病治疗、缓解过敏、器官移植和自身免疫病的临床应用。
附图说明
图1为构建的含人4IgB7-H3的突变编码基因的重组表达载体的质粒图谱；
图2为构建的含编码人4IgB7-H3的野生型基因的重组表达载体的质粒图谱；
图3为重组表达载体的流式细胞检测结果：A.含人4IgB7-H3的突变编码基因的重组表达载体，B.含编码人4IgB7-H3的野生型基因的重组表达载体；
图4为慢病毒感染HEK293T细胞的流式细胞检测结果：A.含人4IgB7-H3的突变编码基因的慢病毒，B.含编码人4IgB7-H3的野生型基因的慢病毒；
图5为野生型和突变编码基因对HEK293T细胞增殖效果随时间变化影响的比较；
图6为慢病毒感染jurkat细胞的流式细胞检测结果；
图7为野生型和突变编码基因对jurkat细胞增殖效果随时间变化影响的比较。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
实施例1含4IgB7-H3野生型编码基因和4IgB7-H3突变编码基因的质粒的制备
1、材料和方法
将人工合成的野生型4IgB7-H3编码基因(SEQ ID NO:2，NCBI Reference Sequence:NM_001024736.1)和4IgB7-H3突变编码基因(SEQ ID NO:1)进行PCR后，分别构建到PGMLV-PA7和PGMLV-PA6，并分别以Blasticidin和puro为筛选标记。载体以本领域常规技术转化到感受态细胞中，将感受态细胞涂板，倒置于37℃恒温培养箱(上海一恒科学仪器有限公司)内过夜培养。第二天挑选单克隆，每一种载体克隆挑选两个送测序公司进行测序鉴定，经过比对，重组克隆中插入片段序列与目的片段序列完全一致，因此质粒构建成功。获得人4IgB7-H3野生型编码基因(SEQ ID NO:2)及人4IgB7-H3(SEQ ID NO:1)的突变编码基因。流式细胞实验验证目的基因的表达水平。具体的转化、测序和流式细胞实验验证目的基因的表达水平过程如下。
(1)转化
A.将感受态细胞置于冰上(4℃)待其自然解冻后，取10μl连接产物加入感受态细胞中于冰上(4℃)放置30min。
B.之后于42℃水浴中热击90S。然后迅速置于冰上(4℃)放置2-3min。
C.加入500μL不含抗生素的SOC培养基于37℃，225rpm振荡培养45min。
D.3000转离心2min，弃掉900μL的上清液，将管底的菌液吹打散开，加入到含有载体上对应抗性(氨苄或卡那等)的培养平板中，用灭菌的涂布器涂匀，倒置于37℃恒温培养箱内过夜培养。
(2)测序
每个克隆挑选两个送测序公司进行测序鉴定，经过比对，重组克隆中插入片段序列与目的片段序列完全一致，因此质粒构建成功。获得含人4IgB7-H3野生型编码基因(SEQ ID NO:2)及人4IgB7-H3(SEQ ID NO:1)的突变编码基因的质粒，分别命名为CMV-4IgB7-H3(CD276)WT-PGK-puro(11,459bp，质粒图谱见图2，以下简称4IgB7-H3(CD276)WT质粒)和CMV-4IgB7-H3(CD276)MT(c.C2142T)-PGK-Blasticidin(11,267bp，质粒图谱见图1，以下简称4IgB7-H3(CD276)MT质粒)。
(3)流式细胞实验验证目的基因的表达水平
细胞：HEK293T；DMEM+10％胎牛血清(GIBICO)；HG transgene reagent(吉满生物科技)；0.25％Trypsin(GIBICO)；高纯度质粒抽提试剂盒(QIAGEN)；荧光显微镜(Olympus)；异丙醇；流式抗体；生物安全柜(Thermo)；CO 2培养箱(Thermo)，BD FACSCalibur流式细胞仪。
A.过表达质粒瞬时转染靶细胞
a.将状态良好，处于对数生长期的空白细胞用0.25％胰酶(GIBICO)消化，用完全培养基悬浮成单细胞悬液，细胞计数后，按照每孔5×10 5个细胞接种于35mm培养皿(上海前尘生物)中；
b.培养过夜，观察细胞生长状态。在细胞覆盖率80％左右时，进行转染实验；
c.转染2h前换液为新鲜的培养基。
d.转染
取无菌的1.5ml EP管，转染体系按下表：
Figure PCTCN2020140691-appb-000001
混匀后，室温放置15min-20min后，均匀滴加到提前换过液的培养皿中，后置于CO 2培养箱中培养。
B.通过流式抗体细胞染色检测目的基因的表达
a.转染72h后，吸除上层培养基，适当PBS清洗细胞，后加入适当胰酶消化、终止。使用血球计数板计数，调整细胞密度至1×10 6cells/ml，以每管100μl的细胞液分装到流式管中；
b.加入适当体积荧光标记的一抗到已经准备好的细胞悬液里，4℃避光孵育20min；
c.加入1ml的PBS清洗细胞，300×g离心5min，小心吸除上清，重复此步骤1次；
d.用300μl PBS重悬细胞；
e.流式细胞仪分析。
结果：如图3所示，经流式验证，4IgB7-H3(CD276)WT质粒和4IgB7-H3(CD276)MT质粒与对照组相比，表达量升高，启动子正常启动，有过表达效果，由此说明4IgB7-H3(CD276)WT表达质粒和4IgB7-H3(CD276)MT表达质粒构建成功，可以进行后续的病毒包装。
实施例2慢病毒的包装和病毒滴度的测定
细胞株：HEK 293T，慢病毒的包装细胞，为贴壁依赖型成上皮样细胞，生长培养基为DMEM(含10％FBS)。贴壁细胞经培养生长增殖形成单层细胞。
菌株：大肠杆菌菌株DH5α。用于扩增慢病毒载体和辅助包装载体质粒。
慢病毒包装系统：将构建成功的慢病毒重组质粒和包装质粒采用Qiagen公司的质粒抽提试剂盒提取。所得的质粒DNA溶于无菌的TE中，以紫外光吸收法测定其浓度及纯度，保证所提质粒DNA的A260/A280在1.8～2.0之间。
一、慢病毒包装
1)HEK 293T细胞分盘：转染前一天，将已经长好的细胞以合适的比例传代到10cm培养皿中，当细胞长到70％～80％时准备转染。
2)转染前换液：转染前1～2h将需要转染的细胞换新鲜的培养基，12ml/10cm皿。
3)转染：取无菌的1.5ml EP管或15ml离心管，转染体系按下表：
Figure PCTCN2020140691-appb-000002
混匀后，室温放置15min-20min后，均匀滴加到提前换过液的培养皿中，后置于CO 2培养箱中培养。
4)加Enhancing buffer：转染10～12h后，均匀滴加100×Enhancing buffer促进转染，120μl/皿。
5)换液：转染18～20h后，小心吸掉细胞培养液弃于盛有消毒液的废液杯中，然后加15ml新鲜的细胞培养基继续培养。
6)病毒收集：换液48h后，吸取细胞上清液于50ml离心管，4℃，4,500×g离心5min，上清液用0.45μm滤器过滤后转移到新的离心管中，最后将滤液分批转移到浓缩装置中，4℃，4,500×g，离心10min，弃下层的液体于盛有消毒液的废液杯中，最后一次4℃，4,500×g，离心20min，此时滤器上层中的液体即为病毒浓缩液。
7)病毒分装与保存：将病毒分装后保存于-80℃。
二、Lentivirus滴度测定
样品制备：将HEK 293T细胞培养至对数生长期，病毒稀释用培养液为含10％FBS的细胞培养基。第一天，细胞胰酶消化计数后，按照每孔2×10 5细胞接种12孔板，37度培养过夜，感染时细胞长至20～40％的融合密度；感染前换液，感染前1～2h将需要感染的细胞换新鲜的培养基，0.91ml每孔，Polybrene浓度为1μg/ml。第二天，对12孔板当天转染时，将保存于-80度冰箱中病毒液冰浴融化，用含有10％FBS的细胞培养液 进行梯度稀释。选取所需的细胞孔，轻轻混匀各管慢病毒稀释液，取90μl加入每孔细胞中，放入37度的细胞培养箱中过夜培养；已知滴度的对照病毒加入1-5号稀释液，待测样品加入1号和2号稀释液。第三天，去除含慢病毒的培养基，加入1ml的完全培养基；4天后，抽提RNA准备做RT-qPCR。总RNA根据本领域常规手段进行提取。
反转录cDNA
a)取一灭菌的无RNA酶的eppendorf管，每个样本加入如下组分得到Mix 1。
Figure PCTCN2020140691-appb-000003
b)轻轻混匀后，25℃孵育10min。
c)42℃孵育60min。
d)85℃孵育5min终止反应。所得到的cDNA保存在-20℃备用。
Real-time PCR检测：目的基因的检测用引物序列(5’-3’)：
Figure PCTCN2020140691-appb-000004
内参基因的检测用引物序列(5’-3’)：
Figure PCTCN2020140691-appb-000005
PCR体系中各组分的体积如下：
Figure PCTCN2020140691-appb-000006
Figure PCTCN2020140691-appb-000007
PCR反应条件：
Figure PCTCN2020140691-appb-000008
结果：
Figure PCTCN2020140691-appb-000009
上表说明，转染野生型基因的慢病毒和转染突变编码基因的慢病毒滴度基本相同，可以用于后续的体外效果比较的平行试验。
实施例3突变体和野生型HEK293T细胞体外效果的比较
一、慢病毒感染靶细胞
第一天，以合适的比例接种靶细胞HEK293T(购自上海中科院细胞库)于培养皿中。
第二天，感染前，病毒原液从-80℃冰箱取出后冰浴融化，用含5μg/ml Polybrene的新鲜培养基按合适的MOI值稀释病毒原液，吸除对照组和处理组原有的培养基，含有慢病毒阴性对照(NC-)稀释液加到对照组细胞中，含有慢病毒液稀释液加到处理组细胞中。
第三天，更换培养液，在感染16h后将含有慢病毒的培养液更换成正常培养液。
第五天，选择合适的真核抗性筛选细胞，药筛两轮，细胞基本稳定(4IgB7-H3(CD276)WT稳转株puro全致死浓度1.5μg/ml，维持浓度0.75μg/ml；4IgB7-H3(CD276)MT稳转株blast全致死浓度8μg/ml，维持浓度4μg/ml)。
二、流式染色验证基因过表达效果
实验材料：HEK293T细胞；培养基；血清(GIBICO)；抗生素：青霉素和链霉素(Penicillin，Streptomycin)；胰酶：0.25％Trypsin(GIBICO)；高纯度质粒抽提试剂盒(QIAGEN)；流式抗体；
实验仪器：CO 2培养箱(Thermo)；荧光显微镜(Olympus)；生物安全柜(Thermo)，BD FACSCalibur流式细胞仪。
1、病毒感染：包装好过表达慢病毒和慢病毒阴性对照感染目的细胞，通过流式抗体染色方法检测过表达载体的过表达效果。
2、通过流式抗体细胞染色检测目的基因的表达
A.细胞直接收在流式管里，细胞吸除上层培养基，加入500μl PBS(1％BSA)清洗细胞，1000rpm，5min，将液体直接倒出，留100μl左右的PBS(1％BSA)，没有100μl直接不足100μl，重悬细胞；
B.加入合适体积的荧光标记一抗到已经准备好的细胞悬液里，混匀(不要用枪头混匀，推动混匀)，4℃避光孵育30-50min；
C.加入1ml PBS(1％BSA)清洗细胞，1000rpm，5min，将液体直接倒出，留300μl左右的PBS(1％BSA)，没有300μl直接补足300μl，重悬细胞。
D.流式细胞仪分析。
HEK293T细胞结果如图4所示，经流式验证，4IgB7-H3(CD276)WT稳转株和4IgB7-H3(CD276)MT稳转株与对照组相比较，表达量升高，有过表达效果，由此说明4IgB7-H3(CD276)WT和4IgB7-H3(CD276)MT稳转株细胞构建成功。
三、细胞增殖测定
细胞：HEK293T，DMEM培养基+10％胎牛血清(GIBICO)；0.25％Trypsin(GIBICO)；荧光显微镜(Olympus)；生物安全柜(Thermo)；CO 2培养箱(Thermo)；离心机(Thermo)；CCK-8(Fluka)；自动化酶免分析仪(北京天石医疗用品制作所)。
1、第一天，准备4个96孔板，在96孔板中接种细胞悬液(2000细胞每孔)，每组细胞3个复孔。将培养板放在培养箱中预培养(37℃,5％CO 2)。
2、瞬转后，取其中一个96孔板，向每孔加入5μL CCK8溶液(注意不要在孔中生成气泡，它们会影响OD值的读数)。其他3块96孔板在瞬转24h、48h、72h小时后进行同样的操作。
3、将培养板在培养箱内孵育3小时。
4、用酶标仪测定在450nm处的吸光度。
HEK293T细胞实验结果和数据
Figure PCTCN2020140691-appb-000010
Figure PCTCN2020140691-appb-000011
其中，CON代表空细胞，NC代表感染野生型对照慢病毒和突变型对照慢病毒的细胞系，WT代表感染4IgB7-H3(CD276)WT野生型慢病毒的细胞系，MT代表感染4IgB7-H3(CD276)MT突变型慢病毒的细胞系，WT+MT代表同时感染4IgB7-H3(CD276)WT野生型慢病毒和4IgB7-H3(CD276)MT突变型慢病毒的细胞系。
上表数据的统计学分析结果如下：
WT和MT方差分析
Figure PCTCN2020140691-appb-000012
上表中，SS代表离均差平方和，df代表自由度。MS代表均方，F代表F统计量，P-value代表p值，F crit代表F临界值。
图5进一步显示了上表的细胞增殖趋势。
结论：上述细胞系培养72h后经CCK8检测验证，4IgB7-H3(CD276)WT稳转株、4IgB7-H3(CD276)MT稳转株和同时感染4IgB7-H3(CD276)WT野生型表达慢病毒和4IgB7-H3(CD276)MT突变型表达慢病毒的细胞系与对照细胞相比，细胞增殖能力增强；4IgB7-H3(CD276)WT稳转株与4IgB7-H3(CD276)MT稳转株相比，p＝2.37E-8<0.01有显著统计学意义。由此可见，转染了人4IgB7-H3的突变编码基因的HEK293T细胞比转染了4IgB7-H3野生型基因的细胞增殖快，说明突变编码基因在HEK293T细胞中比野生型 基因具有更强的例如促进增殖的生物学功能。
实施例4突变体和野生型jurkat细胞体外效果的比较
一、慢病毒感染靶细胞
本实施例中，慢病毒感染的靶细胞为jurkat细胞(购自上海中科院细胞库)，感染方法同实施例3。至第五天选择合适的真核抗性筛选细胞，药筛两轮，细胞基本稳定。在jurkat细胞中，4IgB7-H3(CD276)WT稳转株puro全致死浓度1.5μg/ml，维持浓度0.75μg/ml；4IgB7-H3(CD276)MT稳转株blast全致死浓度7μg/ml，维持浓度3.5μg/ml。
二、流式染色验证基因过表达效果
流式染色验证基因过表达效果时，除了细胞采用jurkat细胞以外，其它条件同实施例3。
jurkat细胞中结果如图6所示，单独感染4IgB7-H3(CD276)WT稳转株和4IgB7-H3(CD276)MT稳转株与对照组相比较，表达量升高，有过表达效果。同时感染4IgB7-H3(CD276)WT野生型表达慢病毒和4IgB7-H3(CD276)MT突变型表达慢病毒的细胞系(4IgB7-H3(CD276)WT+MT)和对照组相比较，表达量升高，也有过表达效果，由此说明4IgB7-H3(CD276)WT、4IgB7-H3(CD276)MT稳转株以及4IgB7-H3(CD276)WT+MT稳转株细胞构建成功。
以下为图6中的缩略语说明：
A，jurkat不染：jurkat空细胞，无荧光标记；
B，jurkat：jurkat空细胞；
C，NC染：PGK-Puro+PGK-Blasticidin，感染野生型对照慢病毒和突变型对照慢病毒的jurkat细胞；
D，4IgB7-H3(CD276)WT：单独感染4IgB7-H3(CD276)WT野生型表达慢病毒的jurkat细胞；
E，4IgB7-H3(CD276)MT：单独感染4IgB7-H3(CD276)MT突变型表达慢病毒的jurkat细胞；
F，4IgB7-H3(CD276)WT+MT：同时感染4IgB7-H3(CD276)WT野生型表达慢病毒和4IgB7-H3(CD276)MT突变型表达慢病毒的jurkat细胞；
jurkat IgG：同型对照；
WT-IgG：同型对照。
三、细胞增殖测定
同样地，除了细胞采用jurkat细胞以外，其它条件同实施例3对应的实验。
jurkat细胞实验结果和数据见下表及图7：
Figure PCTCN2020140691-appb-000013
图7中，jurkat指未转染病毒的空细胞，NC指感染野生型对照慢病毒和突变型对照慢病毒的jurkat细胞系，WT为感染4IgB7-H3(CD276)WT野生型表达慢病毒的jurkat细胞系，MT为感染4IgB7-H3(CD276)MT突变型表达慢病毒的jurkat细胞系，WT+MT(或称杂合型)为同时感染4IgB7-H3(CD276)WT野生型表达慢病毒和4IgB7-H3(CD276)MT突变型表达慢病毒的jurkat细胞系。
上表数据的统计学分析结果如下：
WT和MT方差分析
Figure PCTCN2020140691-appb-000014
上表中，SS代表离均差平方和，df代表自由度。MS代表均方，F代表F统计量，P-value代表p值，F crit代表F临界值。
图7的结果表明，上述细胞系培养72h后经CCK8检测验证，WT+MT(即杂合型) 稳转株与jurkat细胞以及WT细胞相比较细胞增殖减慢，由此可以表明杂合型对T细胞增殖的抑制性增强；WT+MT稳转株与WT稳转株相比，p＝0.000236<0.01有显著统计学意义。
jurkat细胞属于人急性T淋巴细胞白血病细胞系，可模拟T淋巴细胞功能，在T淋巴细胞信号转导、细胞因子和受体表达的体外研究中应用广泛，其体外实验结果揭示突变编码4IgB7-H3与抑制性受体结合对T淋巴细胞的增殖的抑制作用增强。由此可推知，本发明的如SEQ ID NO:1所示的新的人4IgB7-H3的突变编码基因在T、B细胞增殖、活化、辅助T细胞分化，信号转导及效应细胞的细胞因子分泌过程中起重要作用，对肿瘤逃避免疫监视中难以诱导肿瘤特异性T细胞激活的主要原因起到了解决及治疗作用。其在制备基因组工程、基因重组、以T细胞和树突状细胞为基础的治疗恶性肿瘤和传染性疾病的免疫治疗药物、疫苗以及蛋白替补疗法、缓解过敏等中有突出而广泛的应用前景。

Claims (10)

  1. 一种人4IgB7-H3的突变编码基因，其特征在于，所述人4IgB7-H3的突变编码基因的核苷酸序列如SEQ ID NO:1所示。
  2. 如权利要求1所述的突变编码基因编码的蛋白或其受体或配体；优选地，所述蛋白的第一个C样结构域末端包含PQRSPT基序。
  3. 一种重组表达载体，其特征在于，所述重组表达载体包含如权利要求1所述的人4IgB7-H3突变编码基因；优选地，所述重组表达载体为病毒表达载体或原核表达载体或真核表达载体；更优选地，所述病毒表达载体为慢病毒表达载体或腺相关病毒表达载体或逆转录病毒载体或腺病毒载体或单纯孢疹病毒。
  4. 一种宿主细胞，其特征在于，所述宿主细胞包括如权利要求3所述的重组表达载体；优选地，所述宿主细胞为真核细胞，更优选哺乳动物细胞。
  5. 一种分离的mRNA，其特征在于，所述mRNA由如SEQ ID NO:1所示的核苷酸序列转录而得；优选地，所述mRNA为IVT mRNA。
  6. 一种细胞，包含如权利要求5所述的分离的mRNA和RNA感应器和/或mRNA免疫识别结构元件；较佳地，所述细胞包括免疫细胞和非免疫细胞；更佳地所述免疫细胞包括树突状细胞和淋巴细胞，所述淋巴细胞包括B细胞和T细胞，所述T细胞包括辅助性T细胞、抑制性T细胞、效应T细胞、细胞毒T细胞、迟发性变态反应T细胞、天然T细胞和记忆T细胞；其中，辅助性T细胞分为Th1、Th2、Th17或Treg。
  7. 一种分离的miRNA，其特征在于，所述分离的miRNA靶向SEQ ID NO:1和/或SEQ ID NO:2所示的核苷酸；优选地，所述分离的miRNA靶向SEQ ID NO:1和/或SEQ ID NO:2所示的核苷酸的第2142个位点。
  8. 一种靶向药物，其特征在于，所述靶向药物靶向如权利要求1所述的人4IgB7-H3的突变编码基因，所述靶向药物为反义RNA、siRNA、环状RNA、lncRNA或shRNA；或者靶向如权利要求2所述的蛋白或其受体或配体，所述靶向药物例如为抗体。
  9. 一种药物组合物，其特征在于，其包括如权利要求1所述的人4IgB7-H3突变编码基因、权利要求2所述的蛋白或其受体或配体、权利要求3所述的重组载体、权利要求4所述的宿主细胞、权利要求5所述的分离的mRNA、权利要求6所述的细胞、权利要求7所述的分离的miRNA或权利要求8所述的靶向药物；优选地，所述药物化合物包括药学上可接受的载体或赋形剂。
  10. 一种如权利要求1所述的人4IgB7-H3的突变编码基因、权利要求2所述的蛋白 或其受体或配体、权利要求3所述的重组载体、权利要求4所述的宿主细胞、权利要求5所述的分离的mRNA、权利要求6所述的细胞、权利要求7所述的分离的miRNA、权利要求8所述的靶向药物或权利要求9所述的药物组合物在制备调节T细胞活化和/或调节T细胞增殖的药物中的用途；
    进一步地，在制备调节免疫或免疫治疗的药物中的用途；优选地，所述调节免疫或免疫治疗的药物为预防和/或治疗肿瘤、传染性疾病或缓解过敏的药物；更优选地，所述肿瘤优选为胃癌、肠癌、肝癌、肺癌、膀胱癌、睾丸癌、前列腺癌、宫颈癌、乳房癌和淋巴癌中的一种或多种；
    所述药物优选为疫苗。
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