CN117187190A - Anti-cyclophilin A monoclonal antibody and application thereof in treatment of rheumatoid arthritis - Google Patents
Anti-cyclophilin A monoclonal antibody and application thereof in treatment of rheumatoid arthritis Download PDFInfo
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Abstract
The invention discloses an anti-cyclophilin A monoclonal antibody and application thereof in treatment of rheumatoid arthritis. The invention provides a hybridoma cell BQHA7 secreting anti-CypA monoclonal antibody, the preservation number of which is CGMCC No. 45101, and the anti-CypA monoclonal antibody secreted by the hybridoma cell BQHA7. The experiments of the invention prove that the invention provides a hybridoma cell strain capable of secreting anti-CypA monoclonal antibody, the hybridoma cell strain can secrete the monoclonal antibody BQHA7, the monoclonal antibody BQHA7 can specifically recognize CypA, and the hybridoma cell strain has good therapeutic effect on type II collagen-induced mouse rheumatoid arthritis (rheumatoid arthritis, RA).
Description
Technical Field
The invention belongs to the field of biological medicine, and relates to an anti-cyclophilin A monoclonal antibody and application thereof in the treatment of rheumatoid arthritis.
Background
Cyclopylin A (CypA, cyclophilin A) has peptidyl prolyl cis/trans isomerase activity and is a protein which is widely existing in the biological world and is highly conserved. CypA is mainly distributed in cytoplasm and is the main receptor of immunosuppressive drug cycloporin a (CsA, cyclosporin a) in cells. Intracellular CypA is also involved in a variety of biological processes such as apoptosis, immune regulation, bacterial infection, viral replication, etc. CypA can also be secreted extracellularly when stimulated by mechanical injury, reactive oxygen species, pathogenic microbial infections, and the like. Extracellular CypA (eCypA) plays an important role in the development of a variety of inflammatory diseases. For example, in the occurrence of autoimmune diseases related to inflammation such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus, etc., the relationship between eCypA and inflammation is observed.
Because the anti-CypA monoclonal antibody only acts on the eCypA outside the cell, the normal function of the intracellular CypA is not affected. Therefore, the feasibility of the anti-CypA monoclonal antibody serving as a medicament for treating inflammatory diseases is far higher than that of other medicaments, the specificity of the medicaments is greatly enhanced, and the generation of side effects is reduced. Chen Zhina et al obtained an anti-CypA camelid heavy chain VHH antibody (patent CN 102660551B) and an anti-human cyclophilin A monoclonal antibody (CN 108893449A); liu Wenjun and the like obtain a monoclonal antibody (CN 113248617B) for resisting cyclopylin A, and has good treatment effect on acute pneumonia induced by viruses. The antibodies provide tools for diagnosis and treatment of inflammation related to eCrpA, but related experimental data about the application of the CypA antibodies in the treatment of autoimmune diseases such as rheumatoid arthritis are not reported at present.
Disclosure of Invention
The invention aims to provide anti-cyclophilin A monoclonal antibody and application thereof in treatment of rheumatoid arthritis.
In a first aspect, the invention provides a hybridoma cell strain BQHA7 secreting an anti-CypA monoclonal antibody (abbreviated as CypA monoclonal antibody or anti-CypA monoclonal antibody), and the preservation number of the hybridoma cell strain BQHA7 is CGMCC No. 45101.
In a second aspect, the invention provides an anti-CypA monoclonal antibody secreted by the hybridoma cell line BQHA7 of the first aspect.
In a third aspect, the invention provides the use of an anti-CypA monoclonal antibody according to the second aspect in any one of the following;
1) Preparing a product for treating rheumatoid arthritis;
2) Preparing a product that reduces the extent of injury or destruction of rheumatoid arthritis joints;
3) Preparing a product for delaying the disease process of rheumatoid arthritis;
4) Preparing a product for alleviating inflammatory response of rheumatoid arthritis;
5) Preparing a product for improving the degree of arthrocele and/or bone erosion of rheumatoid arthritis;
6) Preparing a product for reducing the infiltration quantity of inflammatory cells;
7) Preparing a product for reducing the expression level of factors related to inflammatory reaction;
8) The product for detecting the cyclophilin A is prepared.
Factors associated with inflammatory responses in the above text are in particular IL-17A, TNF-alpha and/or IL-6 in serum.
In the above, detecting the cyclophilin A is detecting whether the sample to be detected contains the cyclophilin A; the sample to be tested is a cell or a tissue.
In a fourth aspect, the invention provides a product comprising an anti-CypA monoclonal antibody according to the second aspect.
The product described above has at least one of the following functions:
1) Treating rheumatoid arthritis;
2) Reducing the degree of injury or destruction of rheumatoid arthritis joints;
3) Delay the disease course of rheumatoid arthritis;
4) Relieving inflammatory response of rheumatoid arthritis;
5) Improving the arthrocele and/or bone erosion of rheumatoid arthritis;
6) Reducing inflammatory cell infiltration quantity;
7) Reducing the expression level of factors associated with inflammatory response;
8) Detecting the cyclophilin A.
The above rheumatoid arthritis is exemplified by type II collagen-induced rheumatoid arthritis.
In the above, the product is a kit.
In a fifth aspect, the present invention provides a method for detecting cyclophilin a comprising the steps of: and detecting whether the sample to be detected contains the cyclophilin A by taking the anti-CypA monoclonal antibody of the second aspect as an antibody.
Experiments prove that the invention provides a hybridoma cell strain capable of secreting anti-CypA monoclonal antibody, the hybridoma cell strain can secrete monoclonal antibody BQHA7, the monoclonal antibody BQHA7 can specifically recognize CypA, and the hybridoma cell strain has good therapeutic effect on type II collagen-induced mouse rheumatoid arthritis (rheumatoid arthritis, RA).
Preservation description
Strain name: BQHA7
Classification naming: mouse hybridoma cells
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
Preservation date: 2022, 2 and 24 days
Accession numbers of the preservation center: CGMCC No. 45101
Drawings
FIG. 1 shows ELISA for detecting the titers of BQHA7 mab.
FIG. 2 is a graph showing the detection of specific recognition of CypA in cell lines and tissues by BQHA7.
FIG. 3 is a graph showing the effect of detecting BQHA7 on the score of joint inflammation in mice.
FIG. 4 is a graph showing the effect of BQHA7 on micro-CT imaging of joint inflammation in mice.
FIG. 5 shows the therapeutic effect of BQHA7 on pathological lesions of the ankle joints in mice.
FIG. 6 is a graph showing the therapeutic effect of BQHA7 on cartilage injury in ankle joints of mice.
FIG. 7 is a graph showing the effect of BQHA7 on mouse serum cytokines.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified.
The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same.
The quantitative tests in the following examples were all set up in triplicate and the results averaged.
Male DBA/1 mice of 7-8 weeks old were purchased from Peking Vitolith laboratory animal Limited.
The CypA mab QH01 in the example is QH01 in CN 113248617B (grant date 2021.10.01; application number 202110682376.5), and the preservation number of the hybridoma cell strain secreting the CypA mab QH01 is CGMCC No.21909.
PBS (pH 7.2-7.4) in the examples below was purchased from Beijing Soy Bao technology Co., ltd., product number P1020.
The CypA protein in the examples below was purchased from peri coast protein technologies Inc., under the designation CR15.
The reagents used for the ELISA method were as follows:
coating solution (ph=9.6) was purchased from beijing solibao technologies limited under the trade designation C1050.
Washing solution (pH 7.2-7.4) was purchased from Beijing Soy Bao technology Co., ltd., product number P1031.
The sealing liquid is a washing liquid containing (mass volume percentage, g: ml) 2% of skimmed milk powder;
the color development liquid is obtained by mixing 1%A volume percent liquid and 10% B volume percent liquid, wherein the A volume percent liquid is: 1% TMB in DMSO; and (2) liquid B: containing 0.1% CH 4 N 2 O·H 2 O 2 Is added to the solution.
The stop solution was a 0.5M aqueous sulfuric acid solution.
Example 1 preparation of anti-CypA mab
1. Acquisition of hybridoma cell line secreting anti-CypA monoclonal antibody
1. Immunized mice
6 SPF-class BALB/c female mice are immunized with CypA protein, and the initial immunization is performed by intramuscular injection, wherein the immunization amount is 20 mug protein per mouse; subsequent 6 booster immunizations were then performed in the same manner and dose, each one at one week intervals; mice were immunized by intraperitoneal impact with 50 μg CypA protein one week after the boost was completed, and after one week the mice were bled from their orbits and serum antibody titers were determined.
2. Screening BQHA7
(1) ELISA detects serum titers of 6 immunized mice, and mice with higher titers are screened for hybridoma fusion.
(2) Cell fusion experiments: mouse spleen cells were fused with SP2/0 cells by PEG method. The fused cells were subjected to selection culture using a semisolid medium (containing HAT).
(3) Picking a monoclonal: 10 plates×93 cells were selected and cultured in 96-well cell culture plates (previously plated with thymocytes, 100 μl/well).
(4) Primary screening of monoclonal cells: the monoclonal cell supernatant from the 96-well plates was discarded in total, 200. Mu.L/well, and 20% fresh bovine IMDM medium (containing HT) was added for the first screening.
(5) Secondary screening of monoclonal cells: and (3) coating the recombinant CypA protein, and performing secondary screening on the selected clone by adopting an ELISA method to obtain a positive hybridoma cell strain.
(6) Three screens of monoclonal cells: and (3) respectively coating the positive cell strain obtained by the secondary screening with recombinant CypA protein and "tag protein", and performing the third screening by adopting an ELISA method to finally obtain the hybridoma cell strain BQHA7 secreting the CypA monoclonal antibody (BQHA 7).
The hybridoma cell strain BQHA7 secreting BQHA7 is preserved in China general microbiological culture collection center (CGMCC, address: north Chen Xiylu No. 1, 3, postal code 100101) of China general microbiological culture Collection center (China) for 2 months and 24 days in 2022, the preservation number is CGMCC No. 45101, and the classification is named as mouse hybridoma cells.
2. Preparation of anti-CypA monoclonal antibody by ascites
Injecting the obtained BQHA 7-secreting hybridoma cell strain BQHA7 into the abdominal cavity of a BALB/c mouse, wherein the cell concentration is 1.5X10 6 And/mouse. And collecting ascites after 10-14 days.
The antibody in ascites was purified using a Protein G-Sepharose4B adsorption chromatography column to give anti-CypA mab BQHA7, which was collected in 0.1M glycine-hcl buffer (ph=2.7), then replaced in PBS and stored for subsequent experiments.
1. Diluting the CypA protein with a coating solution to a final concentration of 2ug/ml,100 ul/well, and washing with a washing solution for 3 times at 4 ℃ overnight;
2. sealing the sealing liquid, incubating for 2 hours at 37 ℃ at 200 ul/hole, and then washing 3 times by using a washing liquid;
3. adding primary antibody (anti-CypA monoclonal antibody BQHA7 or CypA monoclonal antibody QH 01) or PBS (Blank), performing 4-fold gradient dilution on the primary hole 10 mug/mL, performing 8-point and end hole Blank on the primary hole, performing 100 mug/well incubation for 1h at 25 ℃, and then washing 3 times by using a washing solution;
4. adding HRP-labeled goat anti-mouse IgG secondary antibody (available from Aibotag biotechnology Co., ltd., catalog number: AS 003) diluted 10000 times with PBS, incubating at 25deg.C for 1 hr, taking out, and washing with washing solution for 3 times;
5. adding 100 mu L/hole of color development liquid, and the color development time is about 10 min;
6. adding 50 mu L of stop solution into each hole for stopping;
7. measuring absorbance values at two wavelengths (450 nm,630 nm), and recording and storing data;
as shown in FIG. 1, the half-effective concentration (EC 50) of anti-CypA mab BQHA7 was 0.2629. Mu.g/mL and the EC50 of QH01 was 2.415. Mu.g/mL.
Example 2 identification of anti-CypA mab BQHA7 subtype
The subtype of anti-CypA mab BQHA7 was identified using a mouse monoclonal antibody subtype identification kit (available from proteontech under the accession number PK 20002). Reference is made to the description of the steps.
The analysis results are shown in Table 1, wherein the heavy chain of anti-CypA monoclonal antibody BQHA7 is IgG1 and the light chain is kappa.
Example 3 detection of specific recognition of CypA in cells and tissues by anti-CypA mab BQHA7
The human monocytic leukemia cell line (THP 1, available from ATCC, TIB-202) was lysed using Lysis Buffer (150 mM NaCl,20 mM HEPES,1 mM EDTA,1% Triton100, 10% glycerol) containing Protease Inhibitor Cocktail (Roche, #5871, applied in amounts referred to the instructions), respectively, and the lysates were collected from 7-8 week old male DBA/1 mice (available from Vetong ritus) from tissues such as lung, spleen and lymph nodes.
An appropriate amount of 5 XSDS-PAGE protein loading buffer was added to each lysate and heated at 98℃for 15 min. Conventional SDS-PAGE electrophoresis and Western blot analysis were performed using anti-CypA monoclonal antibody BQHA7 and anti-GAPDH antibody (Santa Cruz Co., cat. No. sc-47724) from ascites as primary antibodies and HRP-labeled goat anti-mouse monoclonal antibody (Jackson, cat. No. 115035003) as secondary antibodies, prepared in example 1.
The specific recognition results of the anti-CypA monoclonal antibody BQHA7 on the CypA in each tissue of the cells and the mice are shown in FIG. 2, 2A is the specific recognition result of the anti-CypA monoclonal antibody BQHA7 on the CypA in the cells, 2B is the specific recognition result of the anti-CypA monoclonal antibody BQHA7 on the CypA in each tissue of the mice, and it can be seen that the anti-CypA monoclonal antibody BQHA7 can specifically recognize the CypA protein (with the molecular weight of about 18 kDa) in the cell line and in different tissues of the mice.
EXAMPLE 4 therapeutic Effect of anti-CypA mab BQHA7 on type II collagen-induced DBA/1 mice (hereinafter referred to as mice) on rheumatoid arthritis
1. Mouse grouping and construction of mouse rheumatoid arthritis model
(1) Grouping mice
Male mice of 7-8 weeks of age were randomly assigned to groups of 6 animals per group, including a blank group (PBS, purchased from Dalian Meen, mass. 0008), untreated group, BQHA 7-treated group, QH 01-treated group, and methotrexate (MTX, product of American-type-Chemie).
(2) Construction of a treatment model for rheumatoid arthritis in mice
Primary immunization: equal volumes of complete Freund's adjuvant (brand: chondrex cat# 7008) and bovine type II collagen (brand: chondrex cat# 20022) were emulsified by a homogenizer for 2-3 min, with no dispersion of the emulsion in water being considered successful. The roots of mice in the untreated group, the BQHA7 treated group, the QH01 treated group and the methotrexate treated group were subcutaneously injected with 100. Mu.L of the emulsion using a 500. Mu.L insulin syringe, and the blank group was injected with 100. Mu.L PBS.
Boosting: boosting was performed 21 days after the first immunization, and equal volumes of incomplete Freund's adjuvant (brand: chondrex cat# 7002) and bovine type II collagen were emulsified in an ice bath by a homogenizer for 2-3 min. The roots of mice in the untreated group, BQHA 7-treated group, QH 01-treated group and methotrexate-treated group were subcutaneously injected with 100. Mu.L of the emulsion at the roots of the mice, and the blank group was injected with 100. Mu.L of PBS. The appearance of obvious red swelling in the foot claw part of the DBA/1 mouse within 1 week after the second-day immunization indicates that the model establishment is successful.
Drug administration treatment: the dosing treatment was started on the day of the second day, specifically as follows:
blank group: no treatment is carried out;
untreated group: mice were intraperitoneally injected with 200 μl PBS;
BQHA7 treatment group: mice were intraperitoneally injected with 100 μg of anti-CypA mab BQHA7 (5 mg antibody/kg body weight, dissolved in 200 μl PBS) once every 7 days;
QH01 treatment group: mice were intraperitoneally injected with 100 μg of anti-CypA mab QH01 (5 mg antibody/kg body weight, dissolved in 200 μl PBS) once every 7 days;
methotrexate (MTX) treatment group: mice were injected i.v. with 30 μg MTX (1.5 mg MTX/kg body weight in 50 μl PBS) at 3 day intervals.
Each group of mice was sacrificed on day 42 after the first immunization, and samples were collected and tested.
2. Evaluation of inflammation of phalangeal and ankle joints in mice
(1) Paw arthritis score in DBA/1 mice
The thickness of the same hind paw was measured every 3 days from day 24 after the first immunization using an electronic vernier caliper and scored for ocular redness. The swelling degree and clinical scoring of the mouse claw refer to European anti-rheumatism alliance and American society of rheumatology (EULAR/ACR) standard, each claw of the mouse is 0-4, 0 is no red swelling, 1 is slight red swelling of ankle joint and finger tip, 2 is moderate red swelling of ankle joint and finger tip, 3 is severe red swelling of ankle joint and finger tip, and 4 is maximum red swelling of limbs.
The scores of the hind paws of the mice in the different groups are shown in FIG. 3, the scores of the thickness, eye redness and swelling and the like of the paws of the mice in the BQHA7 treatment group are obviously lower than those of the untreated group (P < 0.01), the treatment effect of the mice in the untreated group is equivalent to that of MTX, and the BQHA7 has better treatment effect (P < 0.05) than that of QH 01.
(2) DBA/1 mouse claw micro-CT imaging analysis
micro-CT imaging analysis was performed as soon as possible after mice were sacrificed. The isolated mouse hind paws were kept in paraformaldehyde solution and subsequently scanned as soon as possible in a high resolution micro-CT system. After scanning, 2D picture analysis and 3D image reconstruction were performed using a 3D sler. The degree of joint damage caused by inflammation was analyzed based on micro-CT images. In the micro-CT image, the bone of the normal joint tissue is uniform and smooth in boundary, and the bone tissue near the joint of the rheumatoid arthritis mouse is seriously damaged, and the characteristics of local deformation swelling, erosion of partial bone, rough boundary and the like are particularly shown.
micro-CT images of hind paws of different groups of mice are shown in FIG. 4, and the joint injury degree of the hind paws of the mice in the BQHA7 treatment group is obviously reduced. Compared with untreated mice, the BQHA7 treated mice have significantly improved arthrocele and bone erosion, and smoother joint bone boundaries. BQHA7 shows a therapeutic effect superior to MTX. QH01 did not show good therapeutic effect.
(3) Ankle synovial tissue H & E staining
Mice are sacrificed, 4% paraformaldehyde is put into the hind limbs of the mice for fixation for 24H, then 10% EDTA decalcification treatment is carried out for 21 days, conventional paraffin embedding and slicing manufacture are carried out after decalcification, and indexes such as inflammatory cell infiltration, synovial hyperplasia, bone destruction degree and the like at the ankle joint are detected by H & E staining, so that the inflammatory reaction and the inflammatory injury degree of the ankle joint are comprehensively analyzed. Normal synovial tissue is smooth and uniform, synovial cells are generally in a single layer, are orderly arranged, have little or no inflammatory cell infiltration, have no vascular proliferation, and have no papillary proliferation.
The results of H & E staining of the ankle joints of mice in the different groups are shown in fig. 5, and compared with untreated groups, the number of inflammatory cell infiltration of the ankle joints of mice in the BQHA7 treated group is obviously reduced, and the mice basically have no synovial hyperplasia and papillary hyperplasia, and the overall joint destruction degree is obviously lower than that of untreated groups, and even shows a better treatment effect than that of MTX. QH01 did not show good therapeutic effect.
(4) Ankle safranine fast green staining analysis
Mice were sacrificed, hind limbs of the mice were fixed in 4% paraformaldehyde for 24 h, then decalcified with 10% EDTA for 21 days, and after decalcification, conventional paraffin embedding and slicing were performed, and the cartilage injury degree at the ankle joint was detected by safranine fast green staining, wherein the cartilage tissue was safranine colored and the bone tissue was green colored. The normal cartilage tissue is distributed uniformly, and the boundary is clear and has no hyperplasia.
The results of the red-solid green staining of the ankle joint of the mice in the different groups are shown in fig. 6, and the ankle joint cartilage of the mice in the BQHA7 treatment group has neat edges and clear boundaries (red), has no obvious hyperplasia, has obviously reduced damage degree compared with the untreated group, and shows a treatment effect superior to that of MTX. Although QH01 shows a certain effect, the therapeutic effect is inferior to BQHA7.
(5) Detection of cytokines by enzyme-linked immunosorbent assay (ELISA)
Mice were sacrificed after the experimental endpoint was reached, blood was collected from the mice and serum was prepared. The serum was assayed for IL-17A (catalog number EK217-01, hangzhou Bidentia Biotechnology Co., ltd.), TNF-alpha (catalog number EK282-01, hangzhou Bidentia Biotechnology Co., ltd.) and IL-6 (catalog number EK206-01, hangzhou Bidentia Biotechnology Co., ltd.) by ELISA. The content of IL-17A, TNF-alpha and IL-6 in serum is an important index for the reaction of the disease course of rheumatoid arthritis and systemic inflammatory response. ELISA procedure reference is made to the instructions.
The levels of IL-17A, TNF-alpha and IL-6 in the serum of mice from different groups are shown in FIG. 7, and the levels of IL-17A, TNF-alpha and IL-6 in the serum of mice from BQHA7 treated groups are significantly lower than those in untreated groups, and exhibit therapeutic effects comparable to MTX, indicating that BQHA7 has good effects in alleviating inflammatory responses associated with rheumatoid arthritis. BQHA7 shows better therapeutic effect than QH 01.
Claims (8)
1. Hybridoma cell strain BQHA7 secreting anti-CypA monoclonal antibody, and the preservation number is CGMCC No. 45101.
2. An anti-CypA monoclonal antibody secreted by the hybridoma cell line BQHA7 of claim 1.
3. Use of the anti-CypA monoclonal antibody of claim 2 in any one of the following;
1) Preparing a product for treating rheumatoid arthritis;
2) Preparing a product that reduces the extent of injury or destruction of rheumatoid arthritis joints;
3) Preparing a product for delaying the disease process of rheumatoid arthritis;
4) Preparing a product for alleviating inflammatory response of rheumatoid arthritis;
5) Preparing a product for improving the degree of arthrocele and/or bone erosion of rheumatoid arthritis;
6) Preparing a product for reducing the infiltration quantity of inflammatory cells;
7) Preparing a product for reducing the expression level of factors related to inflammatory reaction;
8) The product for detecting the cyclophilin A is prepared.
4. A use according to claim 3, characterized in that: the product is a kit.
5. A product comprising the anti-CypA monoclonal antibody of claim 2.
6. The product according to claim 5, wherein:
the product has at least one of the following functions:
1) Treating rheumatoid arthritis;
2) Reducing the degree of injury or destruction of rheumatoid arthritis joints;
3) Delay the disease course of rheumatoid arthritis;
4) Relieving inflammatory response of rheumatoid arthritis;
5) Improving the arthrocele and/or bone erosion of rheumatoid arthritis;
6) Reducing inflammatory cell infiltration quantity;
7) Reducing the expression level of factors associated with inflammatory response;
8) Detecting the cyclophilin A.
7. The product according to claim 5 or 6, characterized in that: the product is a kit.
8. A method of detecting cyclophilin a comprising the steps of: the anti-CypA monoclonal antibody according to claim 2 is used as an antibody to detect whether the sample to be detected contains cyclophilin a.
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Citations (3)
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WO2002013763A2 (en) * | 2000-08-10 | 2002-02-21 | The Picower Institute For Medical Research | Treatment of hiv-1 infection and inflammatory disease using cyclophilin receptor antagonists |
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CN113248617A (en) * | 2021-06-21 | 2021-08-13 | 中国科学院微生物研究所 | Monoclonal antibody against Cyclophilin A and its use in treating inflammation |
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WO2002013763A2 (en) * | 2000-08-10 | 2002-02-21 | The Picower Institute For Medical Research | Treatment of hiv-1 infection and inflammatory disease using cyclophilin receptor antagonists |
CN108893449A (en) * | 2018-06-07 | 2018-11-27 | 中国人民解放军第四军医大学 | The monoclonal antibody and its application of hybridoma, anti-human cyclophilin albumin A |
CN113248617A (en) * | 2021-06-21 | 2021-08-13 | 中国科学院微生物研究所 | Monoclonal antibody against Cyclophilin A and its use in treating inflammation |
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