Recombinant chimeric membrane protein based on Luciferase reporter gene system and its application

Abstract

The present invention relates to a recombinant chimeric membrane protein based on a Luciferase reporter gene system and an application thereof. The recombinant chimeric membrane protein can be used to screen specific molecular drugs for CD47 or SIRPα and detect the activity of blocking the CD47-SIRPα signaling pathway. The protein has the characteristics of convenient and rapid detection, good stability, high target specificity, etc.

Description

Technical Field

[0001] The invention relates to the field of molecular biotechnology, in particular to a recombinant chimeric membrane protein based on a Luciferase reporter gene system and an application thereof. Background Art

[0002] The signal regulatory proteins (SIRPs) family is a transmembrane glycoprotein belonging to the immunoglobulin superfamily (Ig-SF). According to the different structures of the transmembrane and intracellular regions, they are generally divided into SIRPα, SIRPβ and SIRPγ. The extracellular regions of the three are highly homologous and are all composed of three Ig-like domains. The intracellular regions of SIRPβ and SIRPγ are very short, with only six and four amino acids respectively, and neither has a signal motif that interacts with phosphatases. The four tyrosine residues in the cytoplasmic region of SIRPα form two typical immunoreceptor tyrosine inhibitory motifs (ITIMs). SIRPα, also known as CD172α, is a typical inhibitory receptor in the signal regulatory protein family. It can interact with the transmembrane glycoprotein CD47 that is widely present in the body to transmit inhibitory signals. SIRPα activates tyrosine phosphorylation in the cytoplasmic region, and then recruits and activates tyrosine phosphatases with SH2 domains (SHP-1, SHP-2). SIRPα can dephosphorylate as a substrate protein of SHP-1 / SHP-2, thereby inhibiting protein tyrosine kinase (PIK)-dependent cell activation and immune cell activation. SIRPα requires ligands to bind to it in the process of inhibiting macrophage phagocytosis. Currently, the known ligands of SIRPα are CD47, alveolar surfactant protein A (SP-A), and alveolar surfactant protein D (SP-D). Among them, SIRPα / CD47 binding is of great significance in the process of macrophage phagocytosis.

[0003] CD47 is a widely expressed member of the immunoglobulin superfamily and a ligand of SIRPα. It has long been considered a marker of red blood cells themselves. Normal red blood cells bind to SIRPα on the surface of macrophages through CD47, generating inhibitory signals, thereby inhibiting the phagocytosis of red blood cells. Red blood cells lacking CD47 are rapidly cleared by macrophages in the spleen, leading to hemolysis. In addition to non-opsonized phagocytosis of red blood cells, the CD47-SIRPα signaling pathway also plays a role in many other physiological and pathological processes, such as tumor cell immune escape, and can be used as a therapeutic target. Since CD47 is widely expressed in a variety of tumor cells, tumor cells can escape the immune surveillance of macrophages through the CD47-SIRPα signaling pathway. Therefore, blocking the binding of CD47 and SIRPα through CD47 antibodies or receptors can activate the phagocytosis of macrophages on tumors.

[0004] In recent years, the development of CD47-targeted drugs has received more and more attention in the field of pharmaceutical research and development, and its biological activity analysis at the cellular level is essential. The traditional analysis method uses lymphocyte separation fluid to separate peripheral blood mononuclear cells (PBMC) from human peripheral blood, further uses magnetic bead sorting to obtain monocytes, and then differentiates them into macrophages through long-term stimulation and cultivation, so that they can be used to study the ability of CD47-targeted drugs to promote the phagocytosis of tumor cells. This method has low operability, long cycle, high cost, low throughput, and cannot be used for drug release testing. Summary of the invention

[0005] The invention provides a recombinant chimeric membrane protein based on a Luciferase reporter gene system which can be used for screening drugs for blocking a CD47-SIRPα signaling pathway.

[0006] The present invention provides a recombinant chimeric membrane protein, which comprises a SIRPα extracellular domain, a transmembrane domain, and an intracellular domain.

[0007] In some embodiments, the amino acid sequence of the extracellular domain of SIRPα comprises the sequence shown in amino acid position 20 to amino acid position 359 in SEQ ID NO:1.

[0008] In some embodiments, the transmembrane domain is a CD28 transmembrane domain.

[0009] In some embodiments, a hinge region is connected between the SIRPα extracellular domain and the transmembrane domain.

[0010] In some embodiments, the intracellular domain is a CD28 signaling domain and a CD3 zeta signaling domain.

[0011] In some embodiments, the amino acid sequence of the CD28 transmembrane domain comprises the sequence shown in amino acid position 405 to amino acid position 431 in SEQ ID NO: 1; and / or

[0012] The amino acid sequence of the hinge region comprises the sequence shown in amino acid position 360 to amino acid position 404 in SEQ ID NO: 1; and / or

[0013] The amino acid sequence of the intracellular domain comprises the sequence shown from amino acid 432 to amino acid 584 in SEQ ID NO:1.

[0014] In some embodiments, the recombinant chimeric membrane protein is an isolated recombinant chimeric membrane protein.

[0015] A recombinant chimeric membrane protein, whose amino acid sequence is shown in SEQ ID NO: 1.

[0016] The present invention also provides a nucleic acid molecule encoding the recombinant chimeric membrane protein as described above. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

[0017] In one embodiment, the nucleotide sequence is as shown in SEQ ID NO:2.

[0018] The present invention also provides a recombinant vector, the recombinant vector comprising the nucleic acid molecule as described above. In some embodiments, the recombinant vector is an isolated recombinant vector.

[0019] In one embodiment, the recombinant vector is a plasmid, a recombinant lentiviral vector or an adenoviral vector.

[0020] The present invention also provides a host cell, the genome of which contains the nucleic acid molecule described above. In some embodiments, the host cell is an isolated host cell.

[0021] In one embodiment, the genome of the host cell further contains a NFAT luciferase reporter gene vector.

[0022] In one embodiment, the host cell is a Jurkat-NFAT cell, a NK cell or a T cell.

[0023] The present invention also provides the use of the above-mentioned recombinant chimeric membrane protein, nucleic acid molecule, recombinant vector or host cell in screening drugs that block the CD47-SIRPα signaling pathway.

[0024] The present invention also provides a method for screening drugs that block the CD47-SIRPα signaling pathway, comprising the following steps: mixing and culturing cells expressing the recombinant chimeric membrane protein as described above and containing a NFAT luciferase reporter gene vector, cells expressing CD47, and a drug to be screened, and then detecting whether the drug has the activity of blocking the CD47-SIRPα signaling pathway by detecting the expression of the NFAT luciferase reporter gene.

[0025] Host cells can specifically bind to CD47 protein, and within a certain period of time after binding, signal transduction can be initiated and the expression of intracellular luciferase reporter gene can be activated. The expression of reporter gene is positively correlated with the binding of CD47-SIRPα. When specific molecular drugs for CD47 or SIRPα (monoclonal antibodies, recombinant proteins, targeted small molecule drugs, etc.) are added, the binding of CD47-SIRPα is blocked, resulting in a decrease in the expression of downstream reporter genes. The method is simple to operate, and the entire experiment only takes 4 to 6 hours, which greatly shortens the experimental time. In addition, the use of reporter genes for detection enhances the sensitivity and accuracy of the detection method. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic diagram of the structure of the recombinant chimeric membrane protein;

[0027] Figure 2 This is the result of the detection of CD47 protein expression in Jurkat-NFAT-CD47KO monoclonal cell line;

[0028] Figure 3 The results of the detection of CSR molecule expression in Jurkat-NFAT-CSR monoclonal cell line;

[0029] Figure 4 This is the detection result of CD47 antibody blocking CD47-induced Jurkat-NFAT-CSR luciferase reporter gene expression. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0032] Explanation of terms

[0033] The term "and / or" includes any and all combinations of one or more of the associated listed items.

[0034] "Vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomas (such as SV40).

[0035] "Host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or other cells.

[0036] "CD47" is a transmembrane glycoprotein that belongs to the immunoglobulin superfamily and is expressed on the surface of almost all cells, including red blood cells. The ligands of CD47 include adhesion factors (integrins), thrombospondin-1, and signal regulatory proteins (SIRPs). CD47 has a variety of biological functions, including cell migration, T cell and dendritic cell activation, axon development, etc. In addition, CD47 can inhibit the phagocytic activity of macrophages by interacting with SIRPα. In this way, CD47 transmits a so-called "Don't eat me" signal, which can protect normal cells such as blood cells from being phagocytosed by macrophages. In addition to normal tissue cells expressing CD47, many tumor cells overexpress CD47 and prevent macrophages from phagocytosing tumor cells by binding to SIRPα on the surface of macrophages, which is regarded as a mechanism for tumors to escape the body's immune surveillance. Tumor cells that highly express CD47 include acute myeloid leukemia cells (AML), chronic myeloid leukemia cells (CML), acute lymphoblastic leukemia cells (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, breast cancer, pancreatic cancer, etc.

[0037] In one or more embodiments, the amino acid sequence of the recombinant chimeric membrane protein is as shown in SEQ ID NO:1.

[0038] Through extensive and in-depth research, the inventors obtained the above-mentioned recombinant chimeric membrane protein, which includes a signal peptide, an extracellular domain, a hinge region, a transmembrane domain and an intracellular domain. The extracellular domain includes the extracellular end of SIRPα, and the intracellular domain includes the signal transduction region of CD3ζ. The recombinant chimeric membrane protein can be used to screen specific molecular drugs for CD47 or SIRPα, detect its activity in blocking the CD47-SIRPα signaling pathway, and has the characteristics of convenient and rapid detection, good stability, and high target specificity. .

[0039] It is understood that the analogs of the above-mentioned recombinant chimeric membrane protein are also within the scope of the present invention. The difference between these analogs and the polypeptide shown in SEQ ID NO: 1 may be a difference in the form of modification that does not affect the sequence, or may be an analog with residues different from natural L-amino acids (such as D-amino acids), as well as with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). Modifications (usually without changing the primary structure) include chemical derivatization forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modification during the synthesis and processing of the polypeptide or in further processing steps. This modification can be accomplished by exposing the polypeptide to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylation enzyme). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine). Modified forms also include polypeptides that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0040] The intracellular signaling domain of the recombinant chimeric membrane protein is responsible for the activation of at least one normal effector function of the immune cell that has expressed the recombinant chimeric membrane protein. "Effector function" refers to a specific function of a cell. For example, the effector function of a T cell may be a cytolytic activity or an auxiliary activity including cytokine secretion. "Intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specific function. Although the entire intracellular signaling domain can generally be used, in many examples, the entire chain does not have to be used. Insofar as a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used in place of the complete chain as long as it transduces an effector function signal. Therefore, an intracellular signaling domain refers to any truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal.

[0041] In one or more embodiments, the nucleic acid molecule encodes a recombinant chimeric membrane protein as described above.

[0042] In one or more embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 2. It is understood that due to the degeneracy of codons, nucleic acid sequences capable of expressing the same protein have multiple forms, and the above is a codon-optimized nucleic acid sequence, but the sequence form is not limited thereto.

[0043] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by isolating directly from cells and tissues containing the gene using standard techniques, or they can be produced synthetically.

[0044] In one or more embodiments, the recombinant vector contains a nucleic acid molecule as described above.

[0045] In one or more embodiments, the recombinant vector can be a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid, etc. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be separated and delivered to the subject cell in vivo or in vitro. In some embodiments, an adenoviral vector is used. In some embodiments, a slow virus vector is used. Vectors derived from retroviruses such as slow viruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Slow virus vectors have the advantage of being more than vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes, and they also have the advantage of low immunogenicity.

[0046] In one or more embodiments, the vector may further comprise regulatory elements commonly used in genetic engineering, such as enhancers, promoters, and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0047] In one or more embodiments, the genome of the host cell contains a nucleic acid molecule as described above.

[0048] The host cells of the present invention can specifically bind to the CD47 protein, and can initiate signal transduction and activate the expression of the intracellular luciferase reporter gene within a certain period of time after binding. The addition of anti-CD47 antibodies or SIRPα recombinant proteins can inhibit the activation of CD47 on host cells and reduce the expression of luciferase reporter genes. The host cells can be used to detect the cell biological activity function of CD47 / SIRPα blockers and are used for the screening of CD47 or SIRPα specific drugs (monoclonal antibodies, gene recombinant proteins, targeted small molecule drugs, etc.).

[0049] In one or more embodiments, the genome of the host cell also contains a NFAT luciferase reporter gene vector. In one or more embodiments, the host cell is a mammalian immune cell. In one or more embodiments, the host cell is a Jurkat-NFAT cell, a NK cell or a T cell. In one or more embodiments, the host cell does not express CD47, and not expressing CD47 means that the expression of CD47 in the host cell is reduced by more than 80% compared with the wild-type cell strain. In one or more embodiments, the expression of CD47 in the host cell is reduced by more than 90%. In one or more embodiments, the expression of CD47 in the host cell is reduced by 100%.

[0050] The method of introducing gene into cell and expressing gene into cell is known in the art, and vector can be easily introduced into host cell such as mammal, bacterium, yeast or insect cell by any method in the art. For example, vector can be transferred into host cell by physical, chemical or biological means.

[0051] The physical method of introducing polynucleotide into host cell comprises calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation etc., and in one or more embodiments, the physical method of introducing host cell is calcium phosphate transfection. The biological method of introducing polynucleotide into host cell comprises the use of DNA and RNA vector. Viral vector, particularly retroviral vector, has become the most widely used method of inserting gene into mammal such as CHO cell, and other viral vectors can be derived from slow virus, pox virus, herpes simplex virus I, adenovirus and adeno-associated virus etc. The chemical means of introducing polynucleotide into host cell comprises colloidal dispersion system such as macromolecular complex, nanocapsule, microsphere, lipid-based system such as water-in-oil emulsion, micelle, mixed micelle and liposome. In the case of using non-viral delivery system, exemplary delivery tool is liposome.

[0052] In one or more embodiments, the method for preparing the Jurkat-NFAT-CSR engineered cell line comprises the following steps: (1) packaging a recombinant chimeric membrane protein (i.e., a recombinant chimeric SIRPα receptor membrane protein, CSR) overexpressing lentivirus; (2) knocking out the CD47 gene of Jurkat-NFAT cells to obtain Jurkat-NFAT-CD47KO cells; (3) infecting Jurkat-NFAT-CD47KO cells with CSR lentivirus to screen Jurkat-NFAT-CSR cells; and (4) identifying the function of Jurkat-NFAT-CSR cells.

[0053] In one or more embodiments, a method for screening drugs that block the CD47-SIRPα signaling pathway comprises the following steps: adding a drug to be screened to cells expressing the recombinant chimeric membrane protein as described above and containing a NFAT luciferase reporter gene vector and cells expressing CD47, and then detecting whether the above-mentioned drug has the activity of blocking the CD47-SIRPα signaling pathway by detecting the expression of the NFAT luciferase reporter gene.

[0054] If the drug to be screened can bind to CD47 or SIRPα and block the binding site between CD47 and SIRPα, then when the drug is added to the mixed cell culture, the expression of the luciferase reporter gene activated by CD47 can be inhibited; if the drug to be screened cannot bind to CD47 or SIRPα and block the binding site between CD47 and SIRPα, the expression of the luciferase reporter gene activated by CD47 cannot be inhibited. In this way, drugs that block the CD47-SIRPα signaling pathway can be screened based on the expression of the NFAT luciferase reporter gene.

[0055] In one or more embodiments, the drug that blocks the CD47-SIRPα signaling pathway may be an anti-CD47 antibody or an anti-SIRPα antibody.

[0056] In one or more embodiments, the screening method further comprises the step of setting up a positive control group and / or a negative control group.

[0057] In one or more embodiments, the composition comprises a pharmaceutically acceptable carrier and the recombinant chimeric membrane protein, nucleic acid molecule, recombinant vector or host cell as described above. In one or more embodiments, the recombinant chimeric membrane protein can competitively bind to CD47, thereby blocking the CD47-SIRPα signaling pathway.

[0058] In one or more embodiments, the kit includes the recombinant chimeric membrane protein, nucleic acid molecule, recombinant vector or host cell as described above. In one or more embodiments, the kit also includes instructions.

[0059] The embodiments of the present invention will be described in detail below in conjunction with specific examples. The experimental methods in the following examples where detailed conditions are not specified are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0060] Example 1 Construction of CSR expression vector

[0061] 1.1 Synthesis of recombinant chimeric SIRPα receptor membrane protein (CSR) gene

[0062] The DNA coding sequence of CSR was synthesized by General Biosystems (Anhui) Co., Ltd., and the synthesized sequence was subcloned into the pUC57 vector by this company.

[0063] The design structure of CSR is as follows Figure 1 As shown in SEQ ID NO: 2, it is composed of the extracellular domain of SIRPα (SIRPα-ECD), the hinge region of CD8alpha (Hinge), the transmembrane domain and signal transduction domain of CD28 (CD28TMD / ICD), and the signal transduction domain of CD3ζ (CD3ζ). The gene coding sequence of CSR consists of 1755 nucleotides, as shown in SEQ ID NO: 2 (including the stop codon), wherein the signal peptide coding sequence is 57 nucleotides (1-57), SIRPαECD is 1020 nucleotides (58-1077), CD8alpha hinge region is 135 nucleotides (1078-1212), CD28 transmembrane domain and signal transduction domain is 204 nucleotides (1213-1416), and CD3ζ signal transduction domain is 336 nucleotides (1417-1752). The corresponding amino acid sequence is shown in SEQ ID NO: 1.

[0064] 1.2 CSR overexpression lentiviral packaging

[0065] (1) 293T cells within 10 generations after recovery were cultured at 6×10 6 The total amount of cells was seeded in a 10 cm culture dish with 10% FBS and antibiotic-free DMEM medium.

[0066] (2) Preheat serum-free DMEM medium at 37°C to prepare the transfection system, prepare PEI transfection reagent and plasmid system for packaging virus. Take a 10 cm culture dish as an example, use a total of 20 μg plasmid, pLVX:psPAX2:pMD2G is 2:2:1 ratio (8 μg, 8 μg, 4 μg). PEI:plasmid ratio is 3:1. Dissolve the plasmid or PEI in 250 μL serum-free DMEM, mix gently and place at room temperature for 5 minutes, then add the PEI mixture dropwise to the plasmid mixture, mix, continue to incubate at room temperature for 15 minutes, and then add to the cells.

[0067] (3) After culturing 293T cells at 37°C for 4-8 hours, the medium was replaced with DMEM medium containing 10% FBS and double antibiotics (penicillin-streptomycin) and cultured at 37°C for another 48 hours.

[0068] (4) Collect the 293T culture supernatant containing virus particles into a 50 mL centrifuge tube, centrifuge at 1100 rpm / 200 g for 5 min, and remove the cell pellet.

[0069] (5) Prepare 4×PEG-NaCl lentiviral precipitation solution in advance. Formula: 80g PEG-6000, 14.0g NaCl dissolved in 1×PBS, final volume 200mL. Autoclave. Store at 4℃. Add the corresponding volume of 4×PEG-NaCl solution to the supernatant in step 4 to make the final concentration 1×, shake gently to mix, and place at 4℃ for 1 to 2 hours. Use a 4℃ centrifuge and centrifuge at 10,000g for 30 minutes.

[0070] (6) In a cell hood, pour off the supernatant.

[0071] (7) Resuspend the lentiviral particles in the corresponding culture medium at a concentration ratio of more than 100 times. Aliquot and store at -80°C.

[0072] Example 2 Construction of Jurkat-NFAT-CD47KO monoclonal cell line and verification of its CD47 protein level

[0073] Using CRISPR-Cas9 technology, primers targeting the CD47 coding frame sequence were designed to construct a CD47 knockout plasmid (LentiCRISPRV2-CD47). The plasmid was introduced into Jurkat-NFAT cells (Cell Bank of the Chinese Academy of Sciences, Cat#TCHU123) by lentiviral infection. After the cell viability was restored, CD47 expression was detected by flow cytometry (Fluorescence Activating Cell Sorter, FACS).

[0074] The heavy chain of the anti-CD47 antibody Hu5F9-G4 (the variable region of the antibody is shown in patent US13 / 675274) is: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQGLEWIGTIYPGNDDTSYNQKFKDKATLTADKSTSTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVD HKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:3)

[0075] The light chain of antibody Hu5F9-G4 is:

[0076] DVVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYHCFQGSHVPYTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:4)

[0077] The detection process is as follows: the antibody Hu5F9-G4 1nM was incubated with Jurkat-NFAT cells at 4°C for 30 minutes, washed once with PBS, incubated with PE anti-Human IgG antibody (Bioss, Bs-0297G-PE) at 4°C for 20 minutes, washed twice with PBS, and then transferred to flow cytometer for detection. CD47-negative Jurkat-NFAT monoclonal cells were sorted using flow cytometry, and the results showed (e.g. Figure 2) CD47 protein on the cell membrane of cells numbered E1, E4, and E11 could no longer be detected. Jurkat-NFAT-CD47 KO-E1 was selected as a monoclonal cell for amplification and preservation and for subsequent CSR molecule overexpression.

[0078] Example 3 Construction of Jurkat-NFAT-CD47KO-CSR (abbreviated as Jurkat-NFAT-CSR) monoclonal cell line and its functional analysis

[0079] 3.1 Construction of Jurkat-NFAT-CSR stable cell line

[0080] 3.1.1 Infection of Jurkat-NFAT-CD47KO cells with CSR-overexpressing lentivirus

[0081] The lentiviral infection process is as follows:

[0082] (1)2×10 6 Jurkat-NFAT-CD47KO cells were seeded into 6-well plates one day in advance.

[0083] (2) The virus solution stored at -80°C was slowly thawed on ice.

[0084] (3) Add 20 μL of 100-fold concentrated virus solution to 1 mL of culture medium, add polybrene at a final concentration of 8-10 μg / mL, and culture the cells at 37°C overnight.

[0085] (4) On the next day, the virus solution was completely removed and replaced with fresh RPMI-1640 complete medium and cultured for another 72 hours.

[0086] (5) Set up a blank cell control well that is not infected with the virus and add zeocin at a concentration of 100 μg / mL. Continue culturing and change the medium every 2 to 3 days. The blank cells should be completely dead in about 4 to 5 days.

[0087] (6) After zeocin selection, the stably transfected cells were expanded in a medium containing 50 μg / mL zeocin. 6 cell / mL, and the number of cell generations should be counted. Before P4, cells should be expanded in large quantities and frozen for storage.

[0088] (7) Stably transfected cells were cultured in a medium containing 50 μg / mL zeocin and the corresponding experiments were started.

[0089] 3.1.2 Screening of Jurkat-NFAT-CSR monoclonal cell lines

[0090] The heavy chain of the Anti-SIRPα antibody (the variable regions of the antibody are shown in Patent WO2015138600A2) is: EVQLQQSGAELVKPGASVKLSCTASGFNIKDYYIHWVQQRTEQGLEWIGRIDPEDGETKYAPKFQDKATITADTSSNTAYLHLSSLTSEDTAVYYCARWGAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:5)

[0091] The light chain of the Anti-SIRPα antibody is:

[0092] QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPRTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO:6)

[0093] Monoclonal cells with high expression of CSR molecules were sorted by flow cytometry. The sorting process was as follows: Anti-SIRPα antibody 1nM was incubated with Jurkat-NFAT-CSR cells at 4°C for 30min, washed once with PBS, and incubated with PE anti-Human IgG antibody (Bioss, Bs-0297G-PE) at 4°C for 20min, washed twice with PBS, and transferred to flow cytometer for screening. According to 1 cell per well, 96-well plates were plated for a total of 10 plates. When the monoclonal clone grew to a certain size, a portion of the cells were taken out for FACS detection of CSR molecule expression. Cell lines were selected according to expression abundance and uniformity, and they were amplified, cultured, frozen, and subjected to subsequent cell experiments.

[0094] 3.2 Functional analysis of Jurkat-NFAT-CD47KO-CSR cell line

[0095] 3.2.1 Detection of CD47 molecule expression in Jurkat-NFAT-CSR cells

[0096] The CD47 molecule on the surface of Jurkat-NFAT-CSR cells was detected by FACS technology. The detection process was as follows: the antibody Hu5F9-G4 1nM was incubated with Jurkat-NFAT cells at 4°C for 30 minutes, washed once with PBS, and incubated with PE anti-Human IgG antibody (Bioss, Bs-0297G-PE) at 4°C for 20 minutes, washed twice with PBS, and sent to the flow cytometer for detection.

[0097] 3.2.2 Detection of CSR molecule expression in Jurkat-NFAT-CSR cells

[0098] The CSR molecules on the surface of Jurkat-NFAT-CSR cells were detected by FACS technology. The detection process was as follows: 1 nM Anti-SIRPα antibody was incubated with Jurkat-NFAT-CSR cells at 4°C for 30 min, washed once with PBS, incubated with PE anti-Human IgG antibody (Bioss, Bs-0297G-PE) at 4°C for 20 min, washed twice with PBS, and sent to flow cytometer for detection.

[0099] The results showed that the CSR expression abundance on the cell membranes numbered B7, F5, and G5 was very high. The corresponding test results were as follows Figure 3 G5 was selected as the cell line for subsequent cell function experiments.

[0100] Example 4 Analysis of the activity of Jurkat-NFAT-CSR luciferase reporter gene expression induced by CD47 antibody blocking CD47

[0101] Antibody Hu5F9-G4 and IgG isotype antibody (control antibody) were diluted 2-fold starting from 25 nM, with a total of 10 gradients. The diluted antibodies were added to the cells (6×10 Jurkat cells per well). 4 Jurkat-NFAT-CSR cells were expressed at 1×10 per well. 5 ), gently mix with a spray gun, and incubate in a 37°C incubator for 6 hours. Add 100 μL of luciferase substrate to each well, incubate in the dark for 5 minutes, and detect the fluorescence value on a microplate reader.

[0102] The results are as follows Figure 4 As shown, the method shows that the anti-CD47 antibody Hu5F9-G4 can inhibit the expression of Jurkat-NFAT-CSR luciferase reporter gene induced by CD47 by blocking the CD47-SIRPα signaling pathway, and has a significant concentration-dose dependence.

[0103] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0104] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. Sequence Listing <110> Bio-Thera Pharmaceuticals Co., Ltd. <120> Recombinant chimeric membrane protein based on Luciferase reporter gene system and its application <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 584 <212> PRT <213> Artificial Sequence <400> 1 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val 20 25 30 Leu Val Ala Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser 35 40 45 Leu Ile Pro Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly 50 55 60 Arg Glu Leu Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr 65 70 75 80 Thr Val Ser Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg 85 90 95 Ile Gly Asn Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys 100 105 110 Phe Arg Lys Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly 115 120 125 Thr Glu Leu Ser Val Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly 130 135 140 Pro Ala Ala Arg Ala Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu 145 150 155 160 Ser His Gly Phe Ser Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn 165 170 175 Gly Asn Glu Leu Ser Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu 180 185 190 Ser Val Ser Tyr Ser Ile His Ser Thr Ala Lys Val Val Leu Thr Arg 195 200 205 Glu Asp Val His Ser Gln Val Ile Cys Glu Val Ala His Val Thr Leu 210 215 220 Gln Gly Asp Pro Leu Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg 225 230 235 240 Val Pro Pro Thr Leu Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn 245 250 255 Gln Val Asn Val Thr Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu 260 265 270 Gln Leu Thr Trp Leu Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala 275 280 285 Ser Thr Val Thr Glu Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp 290 295 300 Leu Leu Val Asn Val Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys 305 310 315 320 Gln Val Glu His Asp Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu 325 330 335 Lys Val Ser Ala His Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu 340 345 350 Asn Thr Gly Ser Asn Glu Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 355 360 365 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 370 375 380 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 385 390 395 400 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 405 410 415 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 420 425 430 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 435 440 445 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 450 455 460 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 465 470 475 480 Glu Pro Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 485 490 495 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 500 505 510 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 515 520 525 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 530 535 540 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 545 550 555 560 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 565 570 575 His Met Gln Ala Leu Pro Pro Arg 580 <210> 2 <211> 1755 <212> DNA <213> Artificial Sequence <400> 2 atgggctggt cctgtatcat cctgtttctg gtggccaccg ctaccggcgt gcactccgag 60 gaggagctgc aggtcattca gcccgataag tccgtgctgg tggctgctgg cgagaccgct 120 accctgaggt gtaccgccac cagcctgatc cctgtgggcc ccatccagtg gtttcggggc 180 gccggacccg gcagagagct gatctataat cagaaggagg gccacttccc cagggtgacc 240 accgtgagcg acctgaccaa gcggaataac atggatttca gcatcaggat cggcaatatc 300 acccctgctg acgctggcac ctactactgc gtgaagttcc ggaagggcag ccctgacgac 360 gtggagttca agagcggcgc tggcaccgag ctgtccgtga gggctaagcc ttccgctccc 420 gtggtgtccg gccccgctgc tagagctacc cctcagcaca ccgtgagctt cacctgtgag 480 tcccacggct tttcccctag ggatatcacc ctgaagtggt tcaagaacgg caacgagctg 540 tccgacttcc agaccaatgt ggaccctgtg ggcgagtccg tgtcctatag catccacagc 600 accgctaagg tggtgctgac ccgggaggat gtgcactccc aggtcatttg tgaggtggct 660 cacgtgaccc tgcagggcga ccccctgagg ggaaccgcta atctgtccga gaccatccgg 720 gtgcccccca ccctggaggt gacccagcag cctgtgcggg ctgagaacca ggtgaatgtg 780 acctgtcagg tgaggaagtt ttaccctcag cggctgcagc tgacctggct ggagaatggc 840 aacgtgagcc ggaccgagac cgccagcacc gtgaccgaga ataaggacgg cacctataac 900 tggatgtcct ggctgctggt gaacgtgagc gctcaccggg acgacgtgaa gctgacctgt 960 caggtcgagc acgatggcca gcccgctgtg tccaagagcc acgatctgaa ggtgtccgcc 1020 caccccaagg agcagggcag caataccgct gctgagaata ccggcagcaa cgagcggacc 1080 accacccccg ctcccaggcc tcctacccca gctccaacca tcgcctccca gcctctgagc 1140 ctgaggcctg aggcctgtag gcccgctgct ggcggagctg tgcacaccag aggcctggac 1200 tttgcttgcg atttctgggt gctggtggtg gtgggcggcg tgctggcttg ctatagcctg 1260 ctggtgaccg tggcctttat catcttctgg gtgcggtcca agcggagccg gctgctgcac 1320 tccgactaca tgaatatgac ccctaggagg cccggcccta cccggaagca ctaccagcct 1380 tacgctcctc ctcgggactt tgctgcctac cggagccggg tgaagttctc ccggagcgcc 1440 gagcctcccg cctatcagca gggccagaac cagctgtaca atgagctgaa cctgggcagg 1500 cgggaggagt acgacgtgct ggataagcgg cggggccggg accctgagat gggaggaaag 1560 ccccggcgga agaatcccca ggagggcctg tataacgagc tgcagaagga caagatggct 1620 gaggcctata gcgagatcgg catgaagggc gagcggcgga ggggcaaggg ccatgatggc 1680 ctgtatcagg gcctgtccac cgccaccaag gatacctatg atgccctgca catgcaggcc 1740 ctgcctccca ggtga 1755 <210> 3 <211> 444 <212> PRT <213> Artificial Sequence <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Tyr Pro Gly Asn Asp Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gly Tyr Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 <210> 4 <211> 219 <212> PRT <213> Artificial Sequence <400> 4 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Tyr Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Gly Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr His Cys Phe Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 5 <211> 443 <212> PRT <213> Artificial Sequence <400> 5 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr 20 25 30 Tyr Ile His Trp Val Gln Gln Arg Thr Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys Tyr Ala Pro Lys Phe 50 55 60 Gln Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Gly Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 115 120 125 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 180 185 190 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 210 215 220 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 275 280 285 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 290 295 300 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 340 345 350 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 <210> 6 <211> 215 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 6 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Leu Thr Cys Ser Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu 65 70 75 80 Ala Glu Asp Ala Ala Ser Tyr Phe Cys His Gln Trp Ser Ser Tyr Pro 85 90 95 Arg Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. A method for screening drugs that block the CD47-SIRPα signaling pathway, characterized in that: The method comprises the following steps: mixing and culturing cells expressing a recombinant chimeric membrane protein and containing a NFAT luciferase reporter gene vector, cells expressing CD47 and a drug to be screened, and then detecting whether the drug has the activity of blocking the CD47-SIRPα signaling pathway by detecting the expression of the NFAT luciferase reporter gene; the amino acid sequence of the recombinant chimeric membrane protein is shown in SEQ ID NO: 1; If the drug to be screened binds to CD47 or SIRPα and blocks the binding site between CD47 and SIRPα, then when the drug is added to the mixed cell culture, the expression of the luciferase reporter gene activated by CD47 can be inhibited; if the drug to be screened does not bind to CD47 or SIRPα and blocks the binding site between CD47 and SIRPα, the expression of the luciferase reporter gene activated by CD47 cannot be inhibited.

2. The screening method according to claim 1, characterized in that The cells expressing the recombinant chimeric membrane protein and containing the NFATluciferase reporter gene vector are Jurkat-NFAT cells.

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