Anti-human CD47 monoclonal antibody as well as preparation method and application thereof

Abstract

The invention discloses an anti-human CD47 monoclonal antibody which comprises a heavy chain variable region and a light chain variable region, and a complementarity determining region of the heavy chain variable region comprises CDRH1 with an amino acid sequence as shown in SEQ ID No.1; the complementarity determining region of the heavy chain variable region comprises CDRH2 of which the amino acid sequence is shown as SEQ ID No.2; the complementarity determining region of the heavy chain variable region comprises CDRH3 of which the amino acid sequence is shown as SEQ ID No.3; the complementarity determining region of the light chain variable region comprises CDRL1 of which the amino acid sequence is as shown in SEQ ID No.4; the complementarity determining region of the light chain variable region comprises an amino acid sequence CDRL2 which is KVS; the complementarity determining region of the light chain variable region comprises CDRL3 of which the amino acid sequence is as shown in SEQ ID No.5. The invention also discloses a preparation method of the anti-human CD47 monoclonal antibody and an application of the anti-human CD47 monoclonal antibody in CD47 protein immunodetection.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and in particular to an anti-human CD47 monoclonal antibody and a preparation method and application thereof. Background Art

[0002] CD47 protein is a 47kDa transmembrane immunoglobulin, also known as integrin-associated protein (IAP) because it can bind to integrin αvβ3. It was originally found on the erythrocyte membrane, and later its role in phagocytosis was found in CD47 knockout mice.

[0003] The structure of CD47 protein can be roughly divided into three parts: intracellular region, transmembrane region and extracellular region. The extracellular region is the most important, which is the protein recognition region. The N-terminus is an IgV domain with five N-glycosylation sites; the transmembrane region is five transmembrane helical domains; the intracellular C-terminus is the cytoplasmic tail region. Due to the different amino acid content of the intracellular tail, CD47 is expressed in four different subtypes. Among them, subtype II containing 16 amino acids is highly expressed in the body, such as hematopoietic cells, vascular endothelial cells and epithelial cells. Because CD47 protein expresses different subtypes in different cells and mediates a series of processes such as immune homeostasis, cell proliferation, migration, phagocytosis and apoptosis by binding to different ligands. This protein is often highly expressed in normal cells and lowly expressed in senescent cells, resulting in the recognition and elimination of senescent cells. So far, it has been found that CD47 protein is highly expressed in almost all tumor cells. The expression of CD47 protein increases in many tumors such as cervical cancer, ovarian cancer, acute myeloid leukemia, lymphoma, etc., which is much greater than the expression in normal cells.

[0004] CD47 protein plays an important role in the mechanism of tumor immune escape. In the human body, macrophages distinguish "self" from "non-self" by the expression content of CD47 protein. CD47 protein is expressed on the surface of non-malignant cells and various types of cancer cells, and can bind to SIRPα transmembrane protein on macrophages to form CD47-SIRPα signaling complex. The extracellular region of SIRPα has three immunoglobulin superfamily-like regions, among which the amino terminal structure can bind to CD47; the intracellular region includes four tyrosine phosphorylation sites and two immunoreceptor tyrosine kinase inhibitor motifs. SIRPα regulates cell migration and phagocytic activities, as well as immune homeostasis and the formation of neural networks. The binding of CD47 to SIRPα can cause phosphorylation of SIRPα tyrosine on the intracellular ITIM motif, and activate the binding of two tyrosine phosphatases that can inhibit the accumulation of myosin ⅡA in phagocytic cell synapses, prompting macrophages to release "Don't eat me" signals, inhibit phagocytosis, and protect normal cells from damage by the immune system. In contrast, in senescent cells, the CD47-SIRPα signaling pathway is weakened, allowing macrophages to approach and remove these cells. Compared with non-malignant cells, cancer cells release the "Don't eat me" signal by expressing higher levels of CD47 on their surface, thereby escaping the pursuit and phagocytosis of macrophages. In response to this phenomenon, scientists regard CD47-SIRPα as an important target for tumor immunotherapy.

[0005] In recent years, drug development targeting the tumor escape mechanism mediated by CD47-SIRPα has become a hot topic in tumor immunotherapy after PD-1 / PD-L1, and the development of CD47 antibodies has now entered the preclinical stage. However, there are still some problems with the antibodies currently developed, such as unclear antigen epitopes. The market is currently in urgent need of a CD47 monoclonal antibody drug that guarantees affinity while reducing side effects (such as strong binding ability to red blood cells). Summary of the invention

[0006] Purpose of the invention: In order to solve the problem of unclear antigen epitope of current anti-CD47 monoclonal antibodies, the present invention provides an anti-human CD47 monoclonal antibody and a preparation method and application thereof.

[0007] Technical solution: An anti-human CD47 monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, wherein the anti-CD47 antibody has the following technical features:

[0008] The complementarity determining region of the heavy chain variable region includes CDRH1 with the amino acid sequence shown in SEQ ID No. 1;

[0009] The complementarity determining region of the heavy chain variable region includes CDRH2 with the amino acid sequence shown in SEQ ID No. 2;

[0010] The complementarity determining region of the heavy chain variable region includes CDRH3 with the amino acid sequence shown in SEQ ID No. 3;

[0011] The complementarity determining region of the light chain variable region includes CDRL1 with the amino acid sequence shown in SEQ ID No. 4;

[0012] The complementarity determining region of the light chain variable region includes the amino acid sequence CDRL2, which is KVS;

[0013] The complementarity determining region of the light chain variable region includes CDRL3 whose amino acid sequence is shown in SEQ ID No.5.

[0014] CDR (complementarity determining region) usually refers to the region in an antibody that can form complementarity with an antigenic determinant in spatial structure. The variability in an antibody is usually not evenly distributed throughout the variable region of the antibody. The heavy chain variable region and light chain variable region of a monoclonal antibody usually have three hypervariable regions (HVRs), which can usually form complementarity with an antigenic determinant in spatial structure, so the hypervariable region is also called the complementarity determining region (CDR), that is, the heavy chain variable region usually includes three complementarity determining regions, i.e., CDRH1, CDRH2 and CDRH3, and the light chain variable region usually includes three complementarity determining regions, i.e., CDRL1, CDRL2 and CDRL3.

[0015] The heavy chain variable region and the light chain variable region of the anti-human CD47 monoclonal antibody may further include a framework region, and the framework region may be located between the complementarity determining regions or at both ends of the complementarity determining regions.

[0016] Furthermore, the amino acid sequence of the heavy chain variable region of the anti-human CD47 monoclonal antibody is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region of the anti-human CD47 monoclonal antibody is shown in SEQ ID NO.7.

[0017] Furthermore, the anti-human CD47 monoclonal antibody is a mouse IgG1κ subtype monoclonal antibody.

[0018] The anti-human CD47 monoclonal antibody is produced by the hybridoma cell line HD-47 with the deposit number CCTCC NO: C2023238. The Latin name of the hybridoma cell line is Hybridoma cell line HD-47. It is deposited in the China Center for Type Culture Collection and the deposit date is August 10, 2023.

[0019] A nucleotide molecule encoding any one of the above anti-human CD47 monoclonal antibodies.

[0020] An expression vector contains the above-mentioned nucleotide molecule.

[0021] A host cell contains the above expression vector or is integrated with the above exogenous nucleotide molecule.

[0022] The method for preparing anti-human CD47 monoclonal antibodies comprises selecting a suitable CD47 protein, adding a cysteine ​​to the carboxyl terminus thereof, coupling it with a carrier protein KLH as an immunogen, and then immunizing mice, and obtaining anti-human CD47 monoclonal antibodies through cell fusion, screening and subcloning, wherein:

[0023] The amino acid sequence of positions 24-63 of the selected CD47 protein is shown in SEQ ID NO.8.

[0024] Application of the above anti-human CD47 monoclonal antibody in CD47 protein immunoassay.

[0025] Furthermore, the CD47 protein immunoassay includes CD47 protein blotting immunoassay and CD47 enzyme-linked immunosorbent assay.

[0026] Beneficial effects: The anti-human CD47 monoclonal antibody disclosed in the present invention and its preparation method and application have the following beneficial technical effects:

[0027] The above technical scheme selects the 24-63 amino acids of CD47 as the antigenic peptide, and the immunogen obtained after coupling with KLH is used to immunize mice. After cell fusion, screening and subcloning, a hybridoma cell line HD-47 that efficiently secretes anti-CD47 protein monoclonal antibodies and anti-CD47 protein monoclonal antibodies secreted by the cell line are obtained.

[0028] The antibody obtained in this scheme has high specificity and sensitivity, can specifically recognize cells expressing CD47 protein, and is suitable for immunological detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 and Figure 2 This is the protein expression result when IPTG is used as the inducer.

[0030] Figure 3 and Figure 4 This is the protein expression result when lactose is used as the inducer.

[0031] Figure 5 and Figure 6 The figure shows the elution results under different concentrations of imidazole.

[0032] Figure 7 This is the Western Blot result of recombinant CD47 protein before and after renaturation.

[0033] Figure 8 The figure is a Western Blot result diagram for detecting the recognition specificity of the monoclonal antibody of the present invention. DETAILED DESCRIPTION

[0034] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0035] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0036] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0037] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0038] Experimental Materials:

[0039] 6-8 week old Balb / c female mice, provided by Beijing Weitonglihua Animal Co., Ltd.;

[0040] Cell line: SP2 / 0 (mouse myeloma cell line), using RPMI1640 medium, which contains essential nutrients for cell growth, fetal bovine serum (10%) and a certain amount of double antibody (1%, penicillin and streptomycin solution);

[0041] Bacterial suspension: 0.073 g EDTA·2Na, 1.463 g NaCl, 3.025 g Tris, 25 mL glycerol, weigh and add a small amount of double distilled water to dissolve, make up to 500 mL, add pH adjuster to adjust pH to about 7.9, filter, and store at room temperature.

[0042] TGE Buffer: 50 mM Tris, 0.5 mM EDTA, 50 mM NaCl, 5% glycerol, 1% glycine, 0.5 mM GSH, 0.05 mM GSSH, add pH adjuster to adjust the pH to about 7.9.

[0043] Example 1: Preparation of immunogen

[0044] 1. Preparation of Immunogen

[0045] According to the sequence and secondary structure analysis of the protein sequence with accession number Q08722 in Uniprot, the amino acid sequence SEQ ID NO.8 at positions 24-63 was selected as the antigen peptide for chemical synthesis of the peptide, and KLH was added to the carboxyl end of the peptide to obtain the immunogen. Among them:

[0046] The peptide synthesis work was entrusted to Jiangsu Jitai Peptide Co., Ltd.

[0047] 2. Preparation of CD47 extracellular domain protein for detection

[0048] (1) Construction of expression vector

[0049] Entrusted Sangon Biotech (Shanghai) Co., Ltd. to synthesize the CD47 extracellular region gene onto the T vector;

[0050] TaKaRa online primer design tool was used to design primers for polymerase chain reaction (PCR) to amplify the extracellular domain protein of human CD47. The purity and size of the product were analyzed by nucleic acid electrophoresis; the pET28a(+) vector was linearized by PCR reaction, and the PCR products of the target gene and vector were quickly recovered and purified using the Agarose Gel DNA Extraction Kit (TaKaRa, 9762) purification kit. Then, the vector and the target gene were connected using the In-Fusion HD Cloning Kit (TaKaRa, 639648) based on the principle of homologous recombination, followed by transformation. After overnight culture, 16 single colonies were picked for colony PCR detection, and then streaked onto LB plates with corresponding resistance grid numbers for culture. The clones with positive results were picked and cultured in a 37℃ bacterial shaker overnight. The plasmid was extracted the next day, and the positive clones were verified by restriction enzyme digestion and sequencing. The electrophoresis bands after digestion were consistent with the size of the target fragment, and the sequencing results corresponded to the original sequence, indicating that the plasmid was successfully constructed.

[0051] (2) Optimization of CD47 protein expression conditions

[0052] After verification, the plasmid was transformed into BL21 competent cells. The colony of the transformed bacteria p was inoculated into 10 ml LB medium containing 50 ug / ml kanamycin and cultured overnight at 37°C, 200 rpm to obtain seed liquid. Measure 30 mL of LB medium, 0.3 mL of seed liquid and 30 μL of kanamycin in a 100 mL conical flask to prepare the target protein culture system.

[0053] The target protein culture system was placed in a shaker at 37°C and 200 rpm for constant temperature incubation. The absorbance of the protein was measured every 0.5-1 h. When OD600 reached 0.6-0.8, which was about 3 h, the colony reached the logarithmic growth phase, and a certain amount of inducer was added at this time.

[0054] Then the best inducer type and concentration were explored and incubated according to the conditions in Table 1. Collect bacteria, centrifuge the cultured bacterial solution at 8500rpm and 4℃ for 10min, and remove the supernatant. Add the precipitate to the bacterial suspension at a ratio of 1:10 and resuspend it. Ultrasound the resuspended bacterial solution in an ice bath. Take 40μL of the ultrasonic bacterial solution to prepare the whole bacterial sample, and take another 40μL of the bacterial solution and centrifuge it at 12000rpm for 5min. Collect the supernatant and precipitate respectively, and add 40μL of redistilled water to the precipitate for sample preparation. Add 10μL of 5×Loading buffer to the above whole bacterial solution, supernatant and precipitate to prepare the sample. Heat the sample at 100℃ and cook for 10min.

[0055] Table 1 Protein expression conditions with different inducer types and concentrations

[0056]

[0057] Take 20 μL of the mixture for 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and stain with Coomassie Brilliant Blue Fast Staining Solution for 20 min to evaluate the optimal expression conditions of the target protein. The protein expression results when IPTG was used as the inducer are shown in Figure 1 and Figure 2 .exist Figure 1 and Figure 2 In the figure, M is a marker, 1-3 are the whole cells, supernatant and precipitate when no inducer is added; 4-6 are the whole cells, supernatant and precipitate when 0.1 mM IPTG is added; 7-9 are the whole cells, supernatant and precipitate when 0.3 mM IPTG is added; 10-12 are the whole cells, supernatant and precipitate when 0.5 mM IPTG is added; 13-15 are the whole cells, supernatant and precipitate when 0.8 mM IPTG is added; 16-18 are the whole cells, supernatant and precipitate when 1.0 mM IPTG is added.

[0058] When lactose was used as an inducer, the protein expression results were shown in Figure 3 and Figure 4 .exist Figure 3 and Figure 4 In the figure, M is a marker, 1-3 is the protein expression when the lactose concentration is 0.5 mM; 4-6 is the protein expression when 1 mM lactose is used as an inducer; 7-9 is the protein expression when 2 mM lactose is used as an inducer; 10-12 is the protein expression when 5 mM lactose is used as an inducer; 13-15 is the protein expression when 10 mM lactose is used as an inducer.

[0059] After analysis by Image J software, the protein expression is shown in Table 2. The target protein was expressed at a high level when induced with 0.5 mM IPTG at 37°C, and most of it was not in the supernatant. The target protein needed to be purified from the inclusion body.

[0060] Table 2 Protein expression under different inducers and different induction concentrations

[0061]

[0062]

[0063] (3) Large-scale expression and purification of CD47 protein

[0064] Inoculate 3 mL of seed liquid into 300 ml of fresh culture medium, seal with a breathable membrane, and incubate at 37°C, 200 rpm on a shaker until OD600 is about 0.6-0.8, which takes about 3 hours, and induce with 0.5 mM IPTG at 37°C for 5 hours.

[0065] Centrifuge at 8500rpm, 4℃ for 10min, remove the supernatant, and keep the light yellow precipitate. Add a certain volume of bacterial suspension at a ratio of 1:10 to the precipitate mass and resuspend. The resuspended bacterial solution is ultrasonicated in an ice bath, and the bacterial solution is milky white after ultrasonication.

[0066] The bacterial solution after ultrasound was centrifuged at 8500 rpm and 4°C for 10 min, and the supernatant was discarded.

[0067] At this point, the protein is expressed in the inclusion body precipitate. Use 10 mL of washing solution to resuspend the washed protein precipitate. After washing, centrifuge at 8500 rpm and 4°C for 10 minutes and wash the precipitate twice. Resuspend with 10 mL of lysis solution. The protein inclusion body will appear as yellow filaments when resuspended. Shake rapidly on a horizontal shaker at room temperature for 1 hour to promote lysis.

[0068] According to the above conditions, ice bath ultrasound was used to dissolve and denature the inclusion bodies. Then, centrifugation was performed at 8500 rpm and 4°C for 10 min, and the supernatant was collected in a clean centrifuge tube and filtered through a 0.22 μM filter membrane for later use.

[0069] Use Ni-NTA nickel column to purify CD47 protein. Prepare 20mM, 50mM, 80mM, 100mM, 150mM, and 250mM imidazole eluents. Rinse the nickel column with 50mL of water, and then balance the column material with 20mM imidazole solution. Take 1mL of loading solution for sample preparation, and inject the remaining loading solution into the nickel column. Then add 20mM, 50mM, 80mM, 100mM, 150mM, 250mM, and 500mM imidazole eluents for elution and collection. Take 40μL of eluent in each tube, add 10μL of 5× Loading buffer, and prepare samples. Perform SDS-PAGE detection, and the results are shown in Figure 5 and Figure 6 .exist Figure 5 and Figure 6 In the figure, ①②③ are three tubes of eluent collected in sequence of different concentrations, M is marker, 1 is the loading solution, 2 is the flow-through solution, 3-5 are ①②③ of 20 mM imidazole, 6-8 are ①②③ of 50 mM imidazole, 9-11 are ①②③ of 80 mM imidazole, 12-14 are ①②③ of 100 mM imidazole, 15-17 are ①②③ of 150 mM imidazole, 18-20 are ①②③ of 250 mM imidazole, 21-23 are ①②③ of 500 mM imidazole.

[0070] from Figure 5and Figure 6 It can be seen that 80mM imidazole solution can elute a large amount of CD47 protein with high purity.

[0071] (4) Renaturation and identification of CD47 protein

[0072] Combine the relatively pure eluted samples and adjust them to a concentration of 0.5 mg / ml using a BCA assay kit. Place the protein in a dialysis bag and place the dialyzed sample (about 10 mL) in 500 mL TGE Buffer. Place it in a 4°C refrigerator and change the dialysate every 7-8 hours for a total dialysis time of 48 hours. Use a 0.22 μM filter membrane to filter the purified protein, and then use SDS-PAGE to determine the size and purity of the protein. Use a BCA kit to determine the protein concentration after renaturation, and use Western Blot to test whether the recombinant protein after renaturation is the target protein. The results are as follows: Figure 7 As shown, the target band appeared at 15KD, which was the target protein, and its concentration was higher than that before renaturation. ELISA experiment verified that the recombinant protein could be recognized by the antibody, and the results were shown in Table 3. The recombinant protein was then stored in a -80℃ refrigerator.

[0073] Table 3 ELISA experimental results

[0074] Group <![CDATA[OD 450nm ]]> KLH (negative control) 0.266 CD47 (positive control) 3.44 Before Renaturation 4.02 After renaturation 4.26

[0075] Example 2: Establishment of hybridoma cell lines

[0076] 1. Immunity

[0077] The cross-linked polypeptide in Example 1 was emulsified with a rapid immune adjuvant (QuickAntibody-Mouse 5W, Suzhou Biolong Company), and Balb / c mice (6-8 weeks, female mice, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were immunized. The water-soluble adjuvant QuickAntibody-Mouse5W and the antigen were quickly mixed upside down at a volume ratio of 1:1 under sterile conditions, and the mice were injected into the hind calf muscles. Each mouse was injected with 50 μg of antigen, 100 μL volume. Three weeks later, the mice were immunized twice according to the same amount of antigen. After an interval of two weeks, 100-200 μL of tail tip blood was taken from the immunized mice, and the polyantibody titer against the immunogen in the mouse serum was detected by indirect ELISA (wavelength 450nm). The mice with the highest titer were immunized by intraperitoneal injection, and the antigen was mixed with physiological saline at a dose of 50 μg / mouse.

[0078] 2. Cell Fusion

[0079] (1) Preparation of spleen cells from immune mice

[0080] The immunized mice were killed by dislocating the cervical vertebrae and disinfected by soaking in 75% alcohol for 5 minutes. A small incision was made on the lower left side of the mouse chest with high-pressure sterilized surgical scissors in the clean bench, and the spleen was removed with a scissor and placed in a dish containing 3-5 ml of incomplete culture medium. The surrounding connective tissue was removed, and the spleen was transferred to a moistened sieve and gently ground with a grinding rod. An appropriate amount of pre-cooled incomplete culture medium was added to make a cell suspension, and the suspension was centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in an appropriate amount of incomplete culture medium. The cells were centrifuged at 1200 rpm for 5 minutes, and the cells were washed 2-3 times, and the cells were resuspended in an appropriate amount of incomplete culture medium. The cells were counted and set aside.

[0081] (2) Integration

[0082] Prepare 25 ml of incomplete culture medium as fusion termination solution and preheat the prepared PEG solution in a 37°C cell culture incubator. Before fusion, blow SP2 / 0 cells from the bottom of the culture dish with a gun and suspend them, collect them in a 50 ml centrifuge tube, centrifuge at 1200 rpm for 5 minutes, wash the cells 2 to 3 times, add 10 ml of incomplete culture medium to resuspend the cells, take 0.1 ml of the prepared immune spleen cell solution and the same volume of red blood cell lysis solution, mix them thoroughly, and count the cells. Beat the SP2 / 0 cells evenly and count the cells. Set aside after counting.

[0083] Mix with mouse myeloma cells SP2 / 0 (Wuhan Pronocell Life Science Co., Ltd., CL-0445) at a ratio of 5:1 and centrifuge at 1500rpm for 5min. After discarding the supernatant, place the centrifuge tube in a 37℃ water bath, keep the tube mouth downward, use a pipette to suck out the cell supernatant remaining in the tube wall, and gently tap the cell sediment at the bottom of the tube with your fingertips to loosen the cells. Slowly add 1 ml of preheated PEG1450 (Sigma, P7181) within 1 minute, and continue to stir slowly with a pipette tip for 1-2 minutes; then add 1 ml of incomplete culture medium preheated at 37°C at a uniform speed within 1 minute, and gently stir with a pipette tip during the period; add 3 ml of incomplete culture medium preheated at 37°C within 3 minutes, and gently stir with a pipette tip during the period; finally, add 10 ml of incomplete culture medium preheated at 37°C, incubate at 37°C for 5 minutes, centrifuge at 200-400g for 5 minutes to remove the cells, add 10 ml of complete culture medium, that is, 1640 culture medium containing 20% ​​fetal bovine serum and 10% Hybridoma Feeder supplement factor, gently resuspend the cells and dilute to 100 ml; take 100 μL and add it to a 96-well plate, 37°C, 5% CO 2 The cells were cultured in a saturated humidity incubator.

[0084] 24 hours after fusion, add selective rehydration medium containing HAT, i.e. HAT culture medium (containing 20% ​​fetal bovine serum and 10% Hybridoma Feeder supplement factor) containing hypoxanthine (H), aminopterin (A) and thymidine nucleoside (T), 50 μL / well. Two days after rehydration, perform the first half-rehydration, aspirate 50 μL from each well. The HAT content in the culture medium is halved in the first half-rehydration, 50 μL / well. Two days later, perform the second half-rehydration, aspirate 50 μL from each well and replace it with culture medium containing HT, 50 μL / well.

[0085] 3. ELISA screening of positive hybridoma cells and cloning

[0086] One week after fusion, 100 μL of cell supernatant was taken for double antibody sandwich ELISA to detect the antibody concentration in the supernatant. The serum after mouse booster immunization was used as a positive control, and the fresh culture medium was used as a negative control to detect the production of antibodies in the supernatant. KLH was coated as a reverse screening to remove hybridoma cells that specifically bind to KLH. According to the results of ELISA, the positive cell wells were picked out, and combined with the round and transparent single cell cluster morphology under the microscope, it was preliminarily determined to be the required hybridoma cells, and subcloning was performed when a certain density was reached. HT culture medium (containing 20% ​​fetal bovine serum, 10% Hybridoma Feeder supplement factor, HT) was used in the subcloning stage.

[0087] The selected cells were gently blown off and counted, and gradient dilutions were performed in a centrifuge tube with HT medium. Each concentration gradient of cell suspension was seeded into 3 columns of cell wells, with 100 μL in each well; 37°C, 5% CO 2 Cultured in a saturated humidity cell culture incubator, observed the cell growth status after 6 days, added culture medium appropriately, circled the cell wells with single / few hybridoma cell clusters under a microscope, and used them as the objects of subsequent ELISA detection; when the hybridoma cells in the marked wells covered more than 1 / 10 of the bottom area, the antibody content in the cell supernatant was detected by indirect ELISA. In order to ensure the stable growth of hybridoma cells during the first cloning, the cell density was diluted to 10, 5, and 1 cells. In order to ensure that a single cell line can be obtained in subsequent cloning, the density gradient was adjusted to 2, 1, and 0.5 cells. After 3 rounds of limiting dilution and monoclonal culture, monoclonal hybridoma cells were obtained, the obtained monoclonal cell lines were expanded and cultured, and frozen in liquid nitrogen. The culture medium selected for expansion culture and subsequent recovery was complete 1640 culture medium containing 5% Hybridoma Feeder supplement factor and 10% FBS.

[0088] Example 3: Preparation and purification of ascites antibodies

[0089] (1) Preparation of ascites antibodies

[0090] The ascites-specific adjuvant (provided by Suzhou Biolong Company) was injected into the peritoneal cavity of Balb / c mice (6-8 weeks, female mice, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), with 500 μL injected into each mouse. After about 7-15 days, the screened hybridoma cell line HD-47 was expanded and cultured and inoculated into the peritoneal cavity of Balb / c mice; 7 days after the injection of hybridoma cells, a bulge appeared in the abdomen of the mouse, and the ascites was collected and centrifuged at 4°C, 4000rpm, 5min. The colorless and transparent middle layer was ascites, which was collected in a centrifuge tube and stored at -20°C.

[0091] (2) Purification of Ascites Antibodies

[0092] After the above peritoneal fluid was diluted with Binding Buffer at a ratio of 1:10, it was filtered and sterilized with a 0.45μm filter membrane and loaded onto a gravity column preloaded with 1ml Protein G agarose gel (Sangon Biotechnology Co., Ltd., C600951) for purification; 5 column volumes of binding / washing buffer were added for equilibrium, and after the equilibrium solution in the resin was completely drained, the sample to be purified was slowly added to the resin, and the effluent was collected; non-specifically adsorbed impurities were eluted with 10 to 15 column volumes of binding / washing buffer, and the effluent was collected; the antibodies were then eluted with 5 to 10 column volumes of elution buffer, and the effluent, i.e., the target protein component, was collected; the eluted target protein component needed to be immediately adjusted to neutrality to prevent the low pH value from denaturing and inactivating the purified protein. It is generally recommended to use 1 / 10 of the volume of the elution component with 1M Neutralize with Tris-HCL; take 40 μL of each collected solution and add it to 10 μL of 5× loading buffer, boil the sample at 100℃ for 10 min; perform SDS-PAGE to analyze the size and purity of the target protein in the eluate; combine the relatively pure eluted samples, then transfer them to a dialysis bag and dialyze them with 1L of 1×PBS, change the solution after 12 hours and continue dialysis once. Collect the dialyzed samples, determine the protein concentration with a BCA kit, and store them in aliquots at -80℃.

[0093] Example 4: Identification of subtypes of mouse anti-human CD47 monoclonal antibodies

[0094] The protein expressed by E. coli in Example 1 was coated onto an ELISA plate, about 100 ng (1 μg / μL) per well, and incubated at 4°C overnight; the liquid in the plate was shaken off and the residual liquid was wiped off, 200 μL of washing solution was added to each well, washed 3 times and the residual liquid was wiped off; 200 μL of 5% BSA dilution / blocking solution was added to each well; the reaction was carried out at 37°C for 1 hour, the liquid in the plate was shaken off and the residual liquid was wiped off, 200 μL of washing solution was added to each well, washed 3 times and the residual liquid was wiped off; 100 μL of primary antibody (antibody to be detected) was added to each well, and the reaction was carried out at 37°C. React for 1 hour, shake off the liquid in the plate and tap off the residual liquid; add 200 μL of 1× washing solution to each well, shake off the liquid in the plate and tap off the residual liquid, repeat 5 times; add 100 μL of different subtypes of enzyme-labeled secondary antibodies (provided by Luoyang Bioton Company, C060102-L) to each well, dilute with PBS, incubate at 37°C for 1 hour, shake off the liquid in the plate and tap off the residual liquid, wash the plate 5 times with washing solution; add 100 μL of substrate to each well, react in the dark at room temperature for 15 minutes; 2. Add 50 μL of stop solution to each well; measure OD450 nm value with an enzyme reader.

[0095] The results showed that the monoclonal antibody of the present invention is an IgG1k type mouse monoclonal antibody.

[0096] Example 5: Reaction Specificity and Application Effect of Mouse Anti-Human CD47 Monoclonal Antibody

[0097] Take the protein expressed by E. coli in Example 1, and use the Western-blotting method to detect the recognition specificity of the monoclonal antibody of the present invention. Perform 15% SDS-PAGE electrophoresis, transfer the membrane after running the gel, and block it with TBST containing 5% skim milk powder after the transfer. Then take 5 ml of the purified antibody diluted to 4 ng / μL as the primary antibody for incubation, and react on a shaker at 4°C overnight; the next day, take out the membrane and wash it 5 times with 1×TBST, each time for 5 minutes; after washing the membrane, incubate it with HRP-labeled goat anti-mouse IgG secondary antibody (provided by Beijing Solebow Technology Co., Ltd.) at room temperature for 1 hour; after washing the membrane with TBST, add ECL ultrasensitive colorimetric solution (provided by Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.) for development, and the results are shown in Figure 8 , a specific band appeared at 15KD, which not only verified the recognition specificity of the monoclonal antibody of the present invention, but also showed that the monoclonal antibody produced by the present invention can be applied to Western-blotting experiments.

[0098] Example 6: Determination of variable region sequence of mouse anti-human CD47 monoclonal antibody

[0099] Take the freshly cultured hybridoma cell line HD-47 and collect more than 106 hybridoma cells by centrifugation. Extract total cell RNA by Trizol method, and detect its concentration using Nanodrop ultra-micro UV analyzer; then take 2μg RNA and mix it evenly with an appropriate amount of loading buffer, perform electrophoresis in 2% agarose gel, and observe RNA integrity. Use SMARTerRACE 5` / 3`Kit (TAKARA, 634858)an according to the instructions for reverse transcription and variable region gene sequence PCR amplification, where:

[0100] The primer sequences used for the heavy chain were: GGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGC;

[0101] The primer sequence used for the light chain was: GGATACAGTTGGTGCAGCATC.

[0102] After obtaining the PCR product, 2% DNA agarose gel electrophoresis was performed to confirm the product size and purity. The length of the light chain (kappa light chain) was between 320-340bp, and the length of the heavy chain was between 340-370bp. The electrophoresis product in this area was cut and recovered, purified using a kit, and sent for sequencing. After comparison by NCBI, 3 antigen-determining complementary regions and 4 framework regions were determined.

[0103] SEQ ID No:

[0104] SEQ ID No.1

[0105] GFDFSRYW

[0106] SEQ ID No.2

[0107] INPDSSSI

[0108] SEQ ID No.3

[0109] TRPGLYGNFDY

[0110] SEQ ID No.4

[0111] QSLVHSNGYTY

[0112] SEQ ID No.5

[0113] SQNTHFPLT

[0114] SEQ ID No.6

[0115] EVKLLESGGGLVQPGGSLKISCAASGFDFSRYWMSWVRQAPGKGLEYIGEINPDSSSIIYTPSLK DKFIISRDNAKKTLYLQMSKVRSEDTALYYCTRPGLYGNFDYWGQRAYSRQC

[0116] SEQ ID No.7

[0117] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGYTYFHWYLQKPGQSPDLLIYKVSNRFSGVPD RFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTHFPLTFGAGTRLELK

[0118] SEQ ID No.8

[0119] FVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDF

[0120] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of this application, this does not limit the scope of patent protection of this application. All technical solutions generated by replacing or modifying equivalent structures or equivalent processes based on the essential concept of this application using the contents recorded in the specification and drawings of this application, as well as directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, are included in the scope of patent protection of this application.

Claims

1. An anti-human CD47 monoclonal antibody, characterized in that: Comprising a heavy chain variable region and a light chain variable region, the anti-CD47 antibody has the following technical features: The complementarity determining region of the heavy chain variable region includes CDRH1 with the amino acid sequence shown in SEQ ID No. 1; The complementarity determining region of the heavy chain variable region includes CDRH2 with the amino acid sequence shown in SEQ ID No. 2; The complementarity determining region of the heavy chain variable region includes CDRH3 with the amino acid sequence shown in SEQ ID No. 3; The complementarity determining region of the light chain variable region includes CDRL1 with the amino acid sequence shown in SEQ ID No. 4; The complementarity determining region of the light chain variable region includes the amino acid sequence CDRL2, which is KVS; The complementarity determining region of the light chain variable region includes CDRL3 whose amino acid sequence is shown in SEQ ID No.

5.

2. The anti-human CD47 monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-human CD47 monoclonal antibody is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region of the anti-human CD47 monoclonal antibody is shown in SEQ ID NO.

7.

3. The anti-human CD47 monoclonal antibody according to claim 1, characterized in that The anti-human CD47 monoclonal antibody is a mouse IgG1κ subtype monoclonal antibody.

4. An anti-human CD47 monoclonal antibody, characterized in that: It is produced by the hybridoma cell line HD-47 with the deposit number CCTCC NO: C2023238 and is deposited in the China Center for Type Culture Collection.

5. A nucleotide molecule, characterized in that Encodes the anti-human CD47 monoclonal antibody according to any one of claims 1 to 4.

6. An expression vector, characterized in that: Containing the nucleotide molecule according to claim 5.

7. A host cell, characterized in that Contains the expression vector of claim 6 or has integrated the exogenous nucleotide molecule of claim 5.

8. The method for preparing the anti-human CD47 monoclonal antibody according to any one of claims 1 to 4, characterized in that: Select a suitable CD47 protein, add a cysteine ​​to its carboxyl terminus, and couple it with the carrier protein KLH as an immunogen. Then immunize mice, and obtain anti-human CD47 monoclonal antibodies through cell fusion, screening and subcloning. The amino acid sequence of positions 24-63 of the selected CD47 protein is shown in SEQ ID NO.

8.

9. Use of the anti-human CD47 monoclonal antibody according to any one of claims 1 to 4 in immunodetection of CD47 protein.

10. The use according to claim 9, characterized in that The CD47 protein immunoassay includes CD47 protein blotting immunoassay and CD47 enzyme-linked immunosorbent assay.

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