WO2019047921A1 - Short peptide atr001, monoclonal antibody prepared by short peptide and having function of biased regulation of at1r, and application thereof - Google Patents

Short peptide atr001, monoclonal antibody prepared by short peptide and having function of biased regulation of at1r, and application thereof Download PDF

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WO2019047921A1
WO2019047921A1 PCT/CN2018/104608 CN2018104608W WO2019047921A1 WO 2019047921 A1 WO2019047921 A1 WO 2019047921A1 CN 2018104608 W CN2018104608 W CN 2018104608W WO 2019047921 A1 WO2019047921 A1 WO 2019047921A1
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at1r
monoclonal antibody
short peptide
hybridoma cell
atr001
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陈霄
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武汉华纪元生物技术开发有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/14—Angiotensins: Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to short peptides and monoclonal antibodies, in particular to a short peptide ATR001 and to the preparation of monoclonal antibodies and applications for the regulation of AT1R by a short peptide.
  • Renin is a proteolytic enzyme produced and secreted by the para-balloon cells of the kidney, which hydrolyzes angiotensinogen (AGT) to produce angiotensin I (AngI).
  • AGT angiotensinogen
  • AngI angiotensin I
  • Angiotensin-converting enzyme (ACE) widely distributed in tissues such as heart, blood vessels, kidneys, and lungs, can degrade AngI to angiotensin II (Ang II).
  • AngI can also be used in cathepsins, elastase, and chymase.
  • angiotensin-converting enzyme 2 ACE2 can also act on AngII to produce active small molecules such as angiotensin 1-7 (Ang1-7).
  • RAS works in two ways: one is the renin secreted by the renal paracellular cells and the AGT released by the liver enters the blood circulation, and a small amount of active small molecule fragments are generated by a series of enzyme hydrolysis to exert physiological effects. The other is to produce an active molecule such as AngII locally in a paracrine manner. It is currently believed that AngII is the most important bioactive peptide in RAS and binds to the corresponding receptor to exert biological effects. The AT1 receptor (AT1R) is the most important AngII receptor.
  • AT1R As a G-protein coupled receptor (GPCR), AT1R has a typical seven-transmembrane alpha helix structure, which is mainly distributed in tissues and organs such as blood vessels, heart, kidney, and fat. The combination of AngII and AT1R plays the following roles:
  • AT1R plays an important role in the regulation of blood pressure in human body and plays an important role in the occurrence and development of hypertension.
  • AngII can also stimulate the release of endothelin, which ultimately leads to the proliferation and contraction of vascular smooth muscle, endothelial damage, and remodeling of blood vessels, which leads to the development and progression of hypertension.
  • AT1R is also involved in the development and progression of atherosclerosis.
  • Wassmann et al also confirmed that apoE and AT1R double knock mice can effectively block vascular oxidative stress, endothelial dysfunction, atherosclerotic lesion formation caused by high cholesterol diet, and independent of blood pressure and blood lipid levels.
  • AngII activates NA2-/NADPH oxidase on the cell membrane of vascular endothelial cells through AT1R, which produces O 2- , which in turn activates NF ⁇ B, initiates MCP-1 expression, and promotes the accumulation of monocytes/macrophages in the blood vessel wall. It migrates to the endothelium, then changes to the inflammatory phenotype, and participates in early lesions of atherosclerosis such as foam cell formation.
  • AT1R abdominal aortic aneurysm
  • apoE -/- or Ldlr -/- pumped high concentrations of AngII showed tube diameter dilatation, thrombosis, vascular smooth muscle apoptosis and loss, elastic fiber rupture, extracellular matrix deposition, a large number of inflammatory cell infiltration of the adventitia, Similar changes in human abdominal aortic aneurysm lesions such as MMPs/TIMPs imbalance, and the above changes were not related to blood pressure and blood lipid levels.
  • ACEI angiotensin-converting enzyme inhibitors
  • ARB angiotensin 1 receptor antagonist
  • GPCRs can be coupled with a variety of downstream G proteins, even non-G proteins (such as ⁇ -arrestin), and ligands can selectively make GPCRs when interacting with GPCRs.
  • Certain G proteins or ⁇ -arrestins are coupled to certain signaling pathways, referred to as ligand bias.
  • ligand bias Taking AT1R as an example, after binding of AngII, PKC can be activated by coupling Gq to increase intracellular calcium, and then the downstream phosphorylase activation pathway is initiated.
  • Gq activation can phosphorylate the intracellular portion of AT1R by GPCR-associated protein kinase 5 (GRK5), and ⁇ -arrestin2 binds to it, and is degraded or recycled to the cell surface by ⁇ -arrestin2-mediated desensitization.
  • GPCR-associated protein kinase 5 GRK5
  • ⁇ -arrestin2 binds to it, and is degraded or recycled to the cell surface by ⁇ -arrestin2-mediated desensitization.
  • ⁇ -arrestin activation can also initiate downstream signal transduction, such as ERK activation and is not affected by the Gi protein inhibitor PTX.
  • the binding of Ang II and AT1R can also be coupled to Gi, Gi as an inhibitory regulation of GPCR, and also considered to be involved in receptor internalization desensitization and intracellular phosphorylase activation. way.
  • the biasing regulation of receptor action is more precise and can play a role that needs to be exerted, while avoiding the occurrence of adverse effects caused by total blockage.
  • the regulation of receptor bias is a hot spot in drug development, but there is no bias to regulate AT1R drug development.
  • the extracellular second loop of AT1R is critical for receptor function, and early studies have shown that autoantibodies against the extracellular second loop of AT1R can be detected in the serum of patients with pre-eclampsia and refractory hypertension, which can aggravate vascular damage. To promote the development of hypertension.
  • drugs that can block the activation of AT1R and bias the regulation of AT1R we screened the extracellular epitope of AT1R and screened the antibodies against the selected AT1R target sequences. The effects were confirmed in animal models of hypertension, atherosclerosis and aneurysms.
  • the present invention provides a short peptide ATR001 and a monoclonal antibody and application for preparing a biased regulatory AT1R function from a short peptide.
  • the invention adopts the monoclonal antibody technology to prepare a monoclonal antibody against AT1R, and the antibody can bias the AT1R function, and exerts a therapeutic effect in vascular remodeling diseases such as hypertension, atherosclerosis and aneurysm.
  • the present invention protects a short peptide ATR001 which produces hybridoma cell CQ8-A2D9, the amino acid sequence of which is AFHYESQ.
  • the present invention also protects a hybridoma cell CQ8-A2D9, which has the accession number: CCTCC NO: C2017108.
  • the above hybridoma cell CQ8-A2D9 was deposited with the China Center for Type Culture Collection (CCTCC) on November 16, 2016; the deposit number is CCTCC NO: C2017108; the deposit location is: China, Wuhan, Wuhan University.
  • CTCC China Center for Type Culture Collection
  • the invention also protects a preparation method of the above hybridoma cell CQ8-A2D9, comprising the following steps:
  • the short peptide ATR001 is coupled to the carrier protein KLH as an immuno-coupled antigen
  • the immunoconjugated antigen is mixed with Freund's adjuvant to immunize the mouse, and then the spleen cells of the immunized mouse are fused with the myeloma cell SP2/0, and the hybridoma cell CQ8-A2D9 is obtained by screening.
  • the amino acid sequence of the short peptide ATR001 is AFHYESQ
  • the present invention also protects the above hybridoma cell CQ8-A2D9 for the preparation of the monoclonal antibody anti-AT1R, the hybridoma cell CQ8-A2D9 has the accession number: CCTCC NO: C2017108.
  • the principle of monoclonal antibody antibody technology is that B lymphocytes can produce antibodies, but incapable of infinite division in vitro; while tumor cells can be infinitely passaged in vitro, but can not produce antibodies.
  • the hybridoma cells obtained by fusing these two cells have the characteristics of two parental cells. It is capable of generating highly specific and high affinity antibodies against a single antigenic epitope.
  • the above method prepares a monoclonal antibody anti-AT1R having a bias-regulating AT1R function, and the amino acid sequence of the monoclonal antibody anti-AT1R includes a heavy chain or a light chain, the sequences of which are SEQ ID No. 1 and SEQ ID No. 2, respectively. .
  • the present invention also contemplates the use of the monoclonal antibody anti-AT1R prepared by the above-described preparation to have a bias-regulating AT1R function for the treatment of hypertension, atherosclerosis and aneurysms.
  • the beneficial effects of the present invention are that the monoclonal antibody anti-AT1R prepared by ATR001 can preferentially regulate AT1R, thereby exerting a protective effect in vascular remodeling diseases (hypertension, atherosclerosis, aneurysm).
  • vascular remodeling diseases hypertension, atherosclerosis, aneurysm
  • Figure 1 shows the effect of anti-AT1R antibody on AngII-induced ERK phosphorylation
  • Figure 2 shows the effect of anti-AT1R antibody on AngII-induced JNK phosphorylation
  • FIG. 3 shows the effect of anti-AT1R antibody on the change of intracellular calcium concentration induced by AngII.
  • AT1R-antibody is anti-AT1R;
  • Figure 4 is a graph showing the serum antibody titer of mice in the monoclonal antibody group on the second day of monoclonal antibody injection;
  • Figure 5 is a graph showing changes in blood pressure of mice during the experiment.
  • FIG. 6 is a comparison of the monoclonal antibody anti-AT1R aorta gross atherosclerotic plaque.
  • mAb refers to anti-AT1R antibody
  • Val refers to valsartan
  • Con is a control group
  • FIG. 6A is a general view of vascular lesions
  • FIG. 6B is a statistical diagram of the area of vascular gross lesions
  • Figure 7 shows the area of the aortic valve plaque of the monoclonal antibody anti-AT1R compared with the control group.
  • mAb refers to anti-AT1R antibody
  • Val refers to valsartan
  • Con is a control group
  • Figure 7A is a sectional view of aortic annulus
  • Figure 7B is a statistical diagram of aortic annulus lesions
  • Figure 8 is a comparison of monoclonal antibody anti-AT1R on AAA tumor formation rate and tumor size, C is control, S is AngII perfusion group, A is AngII perfusion plus anti-AT1R antibody;
  • Fig. 8A is a general view of an aneurysm
  • Fig. 8B is a statistical diagram of the incidence of an aneurysm
  • Figure 8C is a statistical diagram of the diameter of the aneurysm
  • Figure 8D is a statistical classification of the aneurysm pathology
  • Figure 9 is a schematic diagram showing the integrity of the monoclonal antibody anti-AT1R to protect the vascular wall, C is the control, S is the AngII perfusion group, and A is the AngII perfusion plus anti-AT1R antibody group.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from a conventional biochemical reagent store.
  • the quantitative tests in the following examples were set up with three replicate experiments, and the results were averaged, unless otherwise specified.
  • the conventional pharmaceutical preparations in the examples are as follows:
  • the cell culture medium used in the following experiments mainly consisted of two basic media, RPMI-1640 or DMEM. After being prepared, the cells were sterilized by filtration (0.22 um), and stored at 4 ° C.
  • the incomplete RPMI-1640 medium formulation is:
  • Complete RPMI-1640 or DMEM medium incomplete RPMI-1640 or DMEM medium 80ml + calf serum 15-20ml;
  • HT medium formula complete RPMI-1640 or DMEM medium 99ml + HT stock solution 1ml;
  • HAT medium formula complete RPMI-1640 or DMEM medium 98ml + HT stock solution 1ml + A stock solution 1ml
  • aminoguanidine (Aminopterin MW 440.4) was weighed and dissolved in 90 ml of ultrapure water or four distilled water, neutralized by adding 1 mol/L NaOH 0.5 ml, and then added with ultrapure water or four distilled water to 100 ml. Filter and sterilize, dispense vials (2ml/bottle), and store at -20 °C.
  • F.7.5% NaHCO 3 solution Weigh analytically pure NaHCO 3 7.5g, dissolve in 100ml ultrapure water or four distilled water, filter and sterilize, dispense vial (4-5ml/bottle), close the stopper, 4°C save.
  • HEPES N-2-Hydroxyethylpiperazine-N,-2-ethanesulfonic acid, N-2-hydroxyethylpiperazine-N,-2-ethylsulfonic acid, MW 238.3 in 100ml of ultrapure water or In four distilled water, filter and sterilize, dispense vials (4-5ml/bottle), and store at 4°C.
  • the short peptide ATR001 with a purity of ⁇ 85% of the peptide synthesis is carried out for the sequence AFHYESQ, and then the short peptide ATR001 is coupled with the carrier protein KLH as an immunizing antigen;
  • mice 6-week-old Balb/c mice (live mice) were immunized with immunizing antigens, regular cycle: 3 weeks - 2 weeks - 2 weeks... Intensive cycle: 2 weeks - 2 weeks - 1 week...
  • Mouse antiserum ELISA titer ⁇ 1:80000 the specific steps are as follows:
  • the dose is 50ug, intraperitoneal injection
  • mice spleen fusion was performed, and mouse spleen cells with titer ⁇ 1:80000 were taken.
  • the preparation method of myeloma cell suspension is as follows:
  • Bone marrow cells are expanded and cultured 48-36 hours before the fusion (generally, about 2-3 bottles of 100 ml culture flasks are prepared by a 96-well fusion test);
  • the cells are gently blown from the bottle wall tumor with a curved pipette and collected in a 50 ml centrifuge tube or a fusion tube;
  • mice that had been immunized were taken, the eyeballs were removed, and the serum was separated as a positive control serum at the time of antibody detection.
  • the mice were sacrificed by cervical dislocation, soaked in 75% alcohol for 5 minutes, fixed on the culture dish, and then the left abdomen skin was opened. The spleen was visible, and the eye scissors were cut. The scissors were opened in a clean bench. The peritoneum was taken out and placed in a dish containing 10 ml of incomplete medium, gently washed, and the surrounding connective tissue was carefully peeled off.
  • the plate was placed in a stainless steel sieve, and the cell suspension was ground with a syringe needle and counted to allow the spleen cells to enter the incomplete medium in the plate. Sip several times with a pipette to make a single cell suspension. Usually 1 x 10 8 - 2.5 x 10 8 splenocytes per mouse.
  • mice After the mice were sacrificed, the surface was disinfected and fixed, the abdominal skin was lifted from the posterior abdomen with a sterile scissors to expose the peritoneum. Wipe the peritoneum with an alcohol cotton ball. Inject 10 ml of incomplete medium into the abdominal cavity with a syringe, taking care to avoid penetrating the intestine. Fix the syringe in the right hand, leave the needle in the abdominal cavity, gently massage the abdomen with an alcohol cotton ball for 1 minute, then aspirate the injected culture solution. Centrifuge at 1000 r/min for 5-10 minutes and discard the supernatant.
  • the precipitated cells were first suspended in 5 ml of HAT medium, and HAT medium was added according to the cell count to make the cell concentration 2 ⁇ 10 5 /ml, and was used.
  • HAT medium for macrophages, a 96-well plate requires 2 x 10 4 cells per well, and a 24-well plate requires 105 cells per well.
  • Each mouse can obtain 3-5 ⁇ 10 6 cells, so one mouse can be used for feeding cells of two 96-well plates.
  • the feeder cells can also be prepared and cultured 1-2 days before cell fusion, such that the bottom of the culture plate is first covered with a layer of feeder cells.
  • the above cell suspension was added to a 96-well plate at 0.1 ml per well, and then cultured in an incubator at 37 ° C in a 6% CO 2 atmosphere.
  • the selection medium was added, 50ul per well, and after 3 days, the medium was replaced with a half medium;
  • 1 antigen is diluted to 10ug/ml with a coating solution
  • each hybridoma cell is divided into 96-well plates, each well is 100ul;
  • the cells were cultured at 537 ° C, 5% CO 2 for 6 days, and the clones were detected by macroscopic detection; under the inverted microscope, only the wells of single clone growth were marked, and the supernatant was taken for antibody detection.
  • Hybridoma cells diluted with PBS or serum-free medium were inoculated intraperitoneally after 27-10 days, 5 ⁇ 10 5 /0.2 ml per mouse.
  • mice After 5 days, observe the ascites production in mice every day. If the abdomen is obviously enlarged, when the skin is touched by hand, the skin can be used to collect ascites with a 16-gauge needle. Generally, it can be taken continuously for 2-3 times, usually for each mouse. Can take 5-10ml ascites;
  • the ascites was centrifuged (2000 r/min for 5 minutes) to remove the cellular components and other precipitates, and the supernatant was collected, and the antibody titer was determined, and the mixture was stored at -70 ° C for storage or stored by lyophilization.
  • the monoclonal antibody anti-AT1R is sequenced, and the amino acid sequence of the monoclonal antibody anti-AT1R includes a heavy chain or a light chain, and the sequences thereof are SEQ ID No. 1 and SEQ ID No. 2, respectively.
  • AT1R-CHOK1 cell line (PerkinElmer): DMEM/F12 complete medium containing 10% FBS, cultured in a 5% CO 2 cell incubator;
  • AT1R-CHOK1 was inoculated into a six-well plate at a density of about 80% and cultured overnight. Then, HBS was starved for 2 h, AngII concentration gradient stimulation and time gradient stimulation were performed, and the expression of p-ERK/ERK and p-JNK/JNK was detected by Western Blot.
  • Fig. 1 AngII-induced ERK
  • Fig. 2 JNK phosphorylation
  • Fig. 3 the monoclonal antibody anti-AT1R had no effect on the increase of intracellular calcium concentration induced by AngII (Fig. 3), namely the monoclonal antibody anti-AT1R.
  • a blood pressure model was constructed by instilling AngII into the Balb/c mouse subcutaneously.
  • the monoclonal antibody group (group A) was injected with anti-AT1R monoclonal antibody 100 ⁇ g/only in the tail vein of the buried pump, and the hydralazine group (group J) was used.
  • gavage was administered at 3 mg/kg/d.
  • a Western diet-induced atherosclerosis (AS) mouse model was established, in which apoE -/- mice were fed with high fat for 17 weeks, and anti-AT1R monoclonal antibody (mAb) was 20ug and 100ug per week.
  • mAb monoclonal antibody
  • the apoE -/- mouse aorta was roughly stained with oil red after 17 weeks, and the area of atherosclerotic plaque was significantly decreased (Fig. 6).
  • the aortic annulus was stained with oil-red stained in frozen sections, and the anti-AT1R monoclonal antibody was found to significantly reduce atherosclerotic plaques (Fig. 7).
  • an AngII-induced abdominal aortic aneurysm (AAA) mouse model was constructed, in which apoE -/- mice were subcutaneously implanted with a micro-osmotic pump and AngII was administered at 1000 ng/kg/min for 28 days.
  • the monoclonal antibody anti-AT1R was found to be significant. Reduce the incidence of AAA in apoE -/- mice and reduce the severity of the tumor ( Figure 8). Cross-sectional pathology of the tumor vessels revealed a significant expansion of the control vessels with thrombosis, while the monoclonal antibody anti-AT1R protected the vascular morphology and morphology (Fig. 9).

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Abstract

A short peptide ATR001, a monoclonal antibody that is prepared by a short peptide and that has a function of the biased regulation of AT1R, and an application thereof; the short peptide ATR001 has an amino acid sequence of AFHYESQ, a hybridoma cell CQ8-A2D9, and a storage number thereof is: CCTCC NO: C2017108. Monoclonal antibody technology is used to prepare a monoclonal antibody targeting AT1R, and the antibody may have a function of the biased regulation of AT1R, having a therapeutic effect in vascular remodeling diseases such as hypertension, atherosclerosis and aneurysms.

Description

短肽ATR001以及由短肽制备具有偏向性调节AT1R功能的单克隆抗体和应用Short peptide ATR001 and monoclonal antibodies prepared by short peptides with biased regulation of AT1R function and applications 技术领域Technical field
本发明涉及短肽和单克隆抗体，具体地指一种短肽ATR001以及由短肽制备具有偏向性调节AT1R功能的单克隆抗体和应用。The present invention relates to short peptides and monoclonal antibodies, in particular to a short peptide ATR001 and to the preparation of monoclonal antibodies and applications for the regulation of AT1R by a short peptide.
背景技术Background technique
肾素-血管紧张素***(RAS)是调控人体生理功能的重要***。肾素是由肾脏球旁细胞产生和分泌的一种蛋白水解酶，可水解血管张素原(AGT)生成血管紧张素Ⅰ(AngⅠ)。心脏、血管、肾、肺等组织中广泛分布的血管紧张素转化酶(ACE)，可将AngⅠ降解为血管紧张素Ⅱ(AngⅡ)，此外，AngⅠ也可在组织蛋白酶、弹性蛋白酶、糜酶(chymase)等作用下生成AngⅡ，或由内肽酶直接降解为血管紧张素1-7(Ang1-7)等一些列小分子活性物质。同时血管紧张素转化酶2(ACE2)也可作用于AngⅡ生成血管紧张素1-7(Ang1-7)等活性小分子。The renin-angiotensin system (RAS) is an important system for regulating physiological functions of the human body. Renin is a proteolytic enzyme produced and secreted by the para-balloon cells of the kidney, which hydrolyzes angiotensinogen (AGT) to produce angiotensin I (AngI). Angiotensin-converting enzyme (ACE), widely distributed in tissues such as heart, blood vessels, kidneys, and lungs, can degrade AngI to angiotensin II (Ang II). In addition, AngI can also be used in cathepsins, elastase, and chymase. Under the action of chymase), AngII is produced, or directly degraded by endopeptidase into angiotensin 1-7 (Ang1-7) and other small molecule active substances. At the same time, angiotensin-converting enzyme 2 (ACE2) can also act on AngII to produce active small molecules such as angiotensin 1-7 (Ang1-7).
目前研究表明，RAS通过两种方式发挥作用：一种是肾球旁细胞分泌的肾素及肝脏释放的AGT进入血液循环，通过一系列酶的水解作用生成活性小分子片段发挥生理作用。另一种是以旁分泌方式在局部生成AngII等活性分子发挥作用。目前认为，AngⅡ是RAS中最主要的生物活性肽，与相应受体结合发挥生物学效应，其中AT1受体(AT1R)是最关键的一种AngⅡ受体。作为一种G蛋白耦联受体(GPCR)，AT1R有典型的七次跨膜α螺旋结构，主要分布在血管、心脏、肾脏、脂肪等组织和器官。AngⅡ和AT1R结合后发挥以下作用：At present, studies have shown that RAS works in two ways: one is the renin secreted by the renal paracellular cells and the AGT released by the liver enters the blood circulation, and a small amount of active small molecule fragments are generated by a series of enzyme hydrolysis to exert physiological effects. The other is to produce an active molecule such as AngII locally in a paracrine manner. It is currently believed that AngII is the most important bioactive peptide in RAS and binds to the corresponding receptor to exert biological effects. The AT1 receptor (AT1R) is the most important AngII receptor. As a G-protein coupled receptor (GPCR), AT1R has a typical seven-transmembrane alpha helix structure, which is mainly distributed in tissues and organs such as blood vessels, heart, kidney, and fat. The combination of AngII and AT1R plays the following roles:
1、缩血管，升血压；1, shrinking blood vessels, raising blood pressure;
2、促进细胞增生和肥大；2. Promote cell proliferation and hypertrophy;
3、促进间质纤维化；3. Promote interstitial fibrosis;
4、参与细胞凋亡；4. Participate in apoptosis;
5、调节细胞代谢；5. Regulate cellular metabolism;
6、促凝血作用；6, procoagulant effect;
7、促进醛固酮生成；7. Promote aldosterone production;
8、作用于中枢神经***，调节水盐代谢，影响交感神经张力。8, acting on the central nervous system, regulating water and salt metabolism, affecting sympathetic tone.
AT1R在人体血压调节中起重要作用，对高血压疾病的发生发展发挥着举足轻重的作用。有研究表明AngⅡ和AT1R作用，可激活血管内皮细胞膜上的NADH/NADPH氧化酶，诱导产生超氧阴离子(O 2-)，O 2-一方面可直接收缩血管，另一方面可与NO相互作用，抑制NO的舒血管作用，并可促进H 2O 2、NO 2、PGF2等缩血管物质的产生。此外，AngⅡ还可刺激内皮素的释放，最终导致血管平滑肌增殖和收缩、内皮损伤、血管壁重构而引起高血压病的发生和发展。 AT1R plays an important role in the regulation of blood pressure in human body and plays an important role in the occurrence and development of hypertension. Studies have shown that AngⅡ AT1R and effect can be activated NADH / NADPH oxidase in the endothelial cell membrane, inducing superoxide anion (O 2-), O 2- on the one hand directly to constrict blood vessels, on the other hand interact with NO It inhibits the vasodilating action of NO and promotes the production of vasoconstrictors such as H 2 O 2 , NO 2 and PGF2. In addition, AngII can also stimulate the release of endothelin, which ultimately leads to the proliferation and contraction of vascular smooth muscle, endothelial damage, and remodeling of blood vessels, which leads to the development and progression of hypertension.
AT1R也参与了动脉粥样硬化的发生与发展过程。研究发现，相比Ldlr -/-小鼠，AT1R与Ldlr基因双敲的小鼠能明显减轻高胆固醇血症诱导的动脉粥样硬化程度。Wassmann等还研究证实apoE与AT1R基因双敲小鼠能有效阻断高胆固醇饮食所致的血管的氧化应激、内皮功能失调、动脉粥样硬化病变形成，且独立于血压和血脂水平。进一步研究表明，AngⅡ通过AT1R激活血管内皮细胞细胞膜上的NADP/NADPH氧化酶，产生的O 2-，进而活化NFκB，启动MCP-1表达，促使血中单核/巨噬细胞向血管壁聚集并迁移至内皮下，随后向炎症表型变化，参与泡沫细胞的形成等动脉粥样硬化早期病变。 AT1R is also involved in the development and progression of atherosclerosis. The study found that compared with Ldlr -/- mice, mice with double knockdown of AT1R and Ldlr gene can significantly reduce the degree of atherosclerosis induced by hypercholesterolemia. Wassmann et al also confirmed that apoE and AT1R double knock mice can effectively block vascular oxidative stress, endothelial dysfunction, atherosclerotic lesion formation caused by high cholesterol diet, and independent of blood pressure and blood lipid levels. Further studies have shown that AngII activates NA2-/NADPH oxidase on the cell membrane of vascular endothelial cells through AT1R, which produces O 2- , which in turn activates NFκB, initiates MCP-1 expression, and promotes the accumulation of monocytes/macrophages in the blood vessel wall. It migrates to the endothelium, then changes to the inflammatory phenotype, and participates in early lesions of atherosclerosis such as foam cell formation.
多项动物实验表明，AT1R与腹主动脉瘤(AAA)的发生发展关系密切。利用apoE -/-小鼠皮下持续泵注AngⅡ已成为研究腹主动脉瘤最常用的动物模型之一。apoE -/-或Ldlr -/-泵注高浓度的AngⅡ后表现出管径扩张、血栓形成、血管中层平滑肌凋亡和缺失、弹力纤维断裂、细胞外基质沉积、血管外膜大量炎细胞浸润、MMPs/TIMPs失衡等人腹主动脉瘤病变相似的变化，且上述变化与血压和血脂水平无关。有研究表明，血管紧张素转化酶抑制剂(ACEI)和血管紧张素1受体拮抗剂(ARB)能抑制实验动物动脉瘤的发生发展、降低动脉瘤患者死亡率。 A number of animal experiments have shown that AT1R is closely related to the development of abdominal aortic aneurysm (AAA). Continuous subcutaneous injection of AngII in apoE -/- mice has become one of the most commonly used animal models for the study of abdominal aortic aneurysms. apoE -/- or Ldlr -/- pumped high concentrations of AngII showed tube diameter dilatation, thrombosis, vascular smooth muscle apoptosis and loss, elastic fiber rupture, extracellular matrix deposition, a large number of inflammatory cell infiltration of the adventitia, Similar changes in human abdominal aortic aneurysm lesions such as MMPs/TIMPs imbalance, and the above changes were not related to blood pressure and blood lipid levels. Studies have shown that angiotensin-converting enzyme inhibitors (ACEI) and angiotensin 1 receptor antagonist (ARB) can inhibit the development of aneurysms in experimental animals and reduce mortality in patients with aneurysms.
尽管ARB在临床上已经广泛运用，但是近年来发现GPCR可以和下游多种G蛋白，甚至非G蛋白(如β-arrestin)耦联，配体在和GPCR相互作用时，可以选择性使GPCR与某些G蛋白或β-arrestin耦联，偏向某些信号途径，称之为配体的偏向性。以AT1R为例，AngⅡ与之结合后，可以通过耦联Gq激活PKC使得胞内钙增加，继而开始下游的磷酸化酶激活途径。Gq活化同时，可以通过GPCR相关蛋白激酶5(GRK5)使得AT1R胞内部分磷酸化，β-arrestin2与之结合，通过β-arrestin2介导的脱敏作用被定向降解或再循环到细胞表面。最近研究表明，β-arrestin活化后亦可开启下游信号转导，如ERK的活化且不受Gi蛋白抑制剂PTX影响。除了上述Gq和β-arrestin2的参与外，理论上Ang Ⅱ和AT1R的结合还可以耦联Gi，Gi作为GPCR的抑制性调控，也认为参与受体的内化脱敏和胞内磷酸化酶激活途径。受体作用的偏向性调节更具精确靶向作用，可以发挥需要发挥的作用，同时避免因全部阻断而导致的不良效应的产生。对受体的偏向性调节是目前药物研发的热点，但是尚无偏向性调节AT1R的药物研制成功。Although ARB has been widely used in clinical practice, it has been found in recent years that GPCRs can be coupled with a variety of downstream G proteins, even non-G proteins (such as β-arrestin), and ligands can selectively make GPCRs when interacting with GPCRs. Certain G proteins or β-arrestins are coupled to certain signaling pathways, referred to as ligand bias. Taking AT1R as an example, after binding of AngII, PKC can be activated by coupling Gq to increase intracellular calcium, and then the downstream phosphorylase activation pathway is initiated. At the same time, Gq activation can phosphorylate the intracellular portion of AT1R by GPCR-associated protein kinase 5 (GRK5), and β-arrestin2 binds to it, and is degraded or recycled to the cell surface by β-arrestin2-mediated desensitization. Recent studies have shown that β-arrestin activation can also initiate downstream signal transduction, such as ERK activation and is not affected by the Gi protein inhibitor PTX. In addition to the above-mentioned participation of Gq and β-arrestin2, in theory, the binding of Ang II and AT1R can also be coupled to Gi, Gi as an inhibitory regulation of GPCR, and also considered to be involved in receptor internalization desensitization and intracellular phosphorylase activation. way. The biasing regulation of receptor action is more precise and can play a role that needs to be exerted, while avoiding the occurrence of adverse effects caused by total blockage. The regulation of receptor bias is a hot spot in drug development, but there is no bias to regulate AT1R drug development.
AT1R胞外第二环对受体功能至关重要，早期研究显示在先兆子痫和难治性高血压患者血清中可以检测到针对AT1R胞外第二环的自身抗体，该抗体可以加重血管损害，促进高血压发生发展。为了寻找既能阻断AT1R激活效应，又能偏向性调节AT1R的药物，我们对AT1R胞外表位序列进行了筛选验证，同时针对所筛选的AT1R靶点序列的抗体进行了单克隆化筛选，并在高血压、动脉粥样硬化及动脉瘤动物模型上确证了其效应。The extracellular second loop of AT1R is critical for receptor function, and early studies have shown that autoantibodies against the extracellular second loop of AT1R can be detected in the serum of patients with pre-eclampsia and refractory hypertension, which can aggravate vascular damage. To promote the development of hypertension. In order to find drugs that can block the activation of AT1R and bias the regulation of AT1R, we screened the extracellular epitope of AT1R and screened the antibodies against the selected AT1R target sequences. The effects were confirmed in animal models of hypertension, atherosclerosis and aneurysms.
发明内容Summary of the invention
本发明提供了一种短肽ATR001以及由短肽制备具有偏向性调节AT1R功能的单克隆抗体和应用。本发明运用单克隆抗体技术制备针对AT1R单克隆抗体，且该抗体可以偏向性调节AT1R功能，在血管重构类疾病，如高血压、动脉粥样硬化、动脉瘤中发挥治疗作用。The present invention provides a short peptide ATR001 and a monoclonal antibody and application for preparing a biased regulatory AT1R function from a short peptide. The invention adopts the monoclonal antibody technology to prepare a monoclonal antibody against AT1R, and the antibody can bias the AT1R function, and exerts a therapeutic effect in vascular remodeling diseases such as hypertension, atherosclerosis and aneurysm.
为实现上述目的，本发明保护了一种制备杂交瘤细胞CQ8-A2D9的短肽ATR001，其氨基酸序列为AFHYESQ。To achieve the above object, the present invention protects a short peptide ATR001 which produces hybridoma cell CQ8-A2D9, the amino acid sequence of which is AFHYESQ.
本发明还保护了一种杂交瘤细胞CQ8-A2D9，其保藏编号为：CCTCC NO：C2017108。The present invention also protects a hybridoma cell CQ8-A2D9, which has the accession number: CCTCC NO: C2017108.
上述杂交瘤细胞CQ8-A2D9已于2016年11月16日保藏于中国典型培养物保藏中心(简称CCTCC)；保藏登记号为CCTCC NO：C2017108；保藏地点为：中国、武汉、武汉大学。The above hybridoma cell CQ8-A2D9 was deposited with the China Center for Type Culture Collection (CCTCC) on November 16, 2016; the deposit number is CCTCC NO: C2017108; the deposit location is: China, Wuhan, Wuhan University.
本发明还保护了一种上述杂交瘤细胞CQ8-A2D9的制备方法，包括以下步骤：The invention also protects a preparation method of the above hybridoma cell CQ8-A2D9, comprising the following steps:
1)短肽ATR001与载体蛋白KLH进行耦联作为免疫耦联抗原；1) The short peptide ATR001 is coupled to the carrier protein KLH as an immuno-coupled antigen;
2)免疫耦联抗原与弗氏佐剂混合免疫小鼠，然后去免疫小鼠的脾细胞与骨髓瘤细胞SP2/0进行融合，检测筛选获得杂交瘤细胞CQ8-A2D9。2) The immunoconjugated antigen is mixed with Freund's adjuvant to immunize the mouse, and then the spleen cells of the immunized mouse are fused with the myeloma cell SP2/0, and the hybridoma cell CQ8-A2D9 is obtained by screening.
进一步地，所述步骤1)中，短肽ATR001的氨基酸序列为AFHYESQ；Further, in the step 1), the amino acid sequence of the short peptide ATR001 is AFHYESQ;
本发明还保护了上述杂交瘤细胞CQ8-A2D9制备单克隆抗体anti-AT1R的方法，所述杂交瘤细胞CQ8-A2D9保藏编号为：CCTCC NO：C2017108。The present invention also protects the above hybridoma cell CQ8-A2D9 for the preparation of the monoclonal antibody anti-AT1R, the hybridoma cell CQ8-A2D9 has the accession number: CCTCC NO: C2017108.
上述制备具有偏向性调节AT1R功能的单克隆抗体anti-AT1R的方法，具体方法如下：The above method for preparing the monoclonal antibody anti-AT1R having the function of bias regulating AT1R is as follows:
1)将杂交瘤细胞CQ8-A2D9制备腹水；1) Preparation of ascites by hybridoma cell CQ8-A2D9;
2)利用腹水制备纯化，得到单克隆抗体anti-AT1R。2) Purification by ascites to obtain monoclonal antibody anti-AT1R.
单抗隆抗体技术原理是：B淋巴细胞能够产生抗体，但在体外不能进行无限***；而瘤细胞虽然可以在体外进行无限传代，但不能产生抗体。将这两种细胞融合后得到的杂交瘤细胞具有两种亲本细胞的特性。能够生成针对单一抗原表位的，具有高度特异性和高亲和力的抗体。The principle of monoclonal antibody antibody technology is that B lymphocytes can produce antibodies, but incapable of infinite division in vitro; while tumor cells can be infinitely passaged in vitro, but can not produce antibodies. The hybridoma cells obtained by fusing these two cells have the characteristics of two parental cells. It is capable of generating highly specific and high affinity antibodies against a single antigenic epitope.
上述方法制备具有偏向性调节AT1R功能的单克隆抗体anti-AT1R，所述单克隆抗体anti-AT1R的氨基酸序列包括重链或轻链，其序列分别为SEQ ID No.1和SEQ ID No.2。The above method prepares a monoclonal antibody anti-AT1R having a bias-regulating AT1R function, and the amino acid sequence of the monoclonal antibody anti-AT1R includes a heavy chain or a light chain, the sequences of which are SEQ ID No. 1 and SEQ ID No. 2, respectively. .
本发明还保护了一种利用上述制备得到具有偏向性调节AT1R功能的单克隆抗体anti-AT1R在治疗高血压、动脉粥样硬化和动脉瘤的药品中的应用。The present invention also contemplates the use of the monoclonal antibody anti-AT1R prepared by the above-described preparation to have a bias-regulating AT1R function for the treatment of hypertension, atherosclerosis and aneurysms.
本发明的有益效果在于：由ATR001制备的单克隆抗体anti-AT1R能偏向性调节AT1R，从而发挥在血管重构类疾病(高血压、动脉粥样硬化、动脉瘤)中的保护作用。The beneficial effects of the present invention are that the monoclonal antibody anti-AT1R prepared by ATR001 can preferentially regulate AT1R, thereby exerting a protective effect in vascular remodeling diseases (hypertension, atherosclerosis, aneurysm).
附图说明DRAWINGS
图1为anti-AT1R抗体对AngⅡ诱导ERK磷酸化影响；Figure 1 shows the effect of anti-AT1R antibody on AngII-induced ERK phosphorylation;
图2为anti-AT1R抗体对AngⅡ诱导JNK磷酸化影响；Figure 2 shows the effect of anti-AT1R antibody on AngII-induced JNK phosphorylation;
图3为anti-AT1R抗体对AngⅡ诱导细胞内钙离子浓度变化影响，图中AT1R-antibody即为anti-AT1R；Figure 3 shows the effect of anti-AT1R antibody on the change of intracellular calcium concentration induced by AngII. In the figure, AT1R-antibody is anti-AT1R;
图4为单抗注射第二天单抗组小鼠血清抗体滴度情况图；Figure 4 is a graph showing the serum antibody titer of mice in the monoclonal antibody group on the second day of monoclonal antibody injection;
图5为实验过程中小鼠血压变化情况图；Figure 5 is a graph showing changes in blood pressure of mice during the experiment;
图6为单克隆抗体anti-AT1R主动脉大体动脉粥样硬化斑块较对照图，图中mAb是指anti-AT1R抗体，Val是指缬沙坦，Con是对照组；Figure 6 is a comparison of the monoclonal antibody anti-AT1R aorta gross atherosclerotic plaque. In the figure, mAb refers to anti-AT1R antibody, Val refers to valsartan, and Con is a control group;
图中，图6A为血管病变大体图；图6B为血管大体病变面积统计图；In the figure, FIG. 6A is a general view of vascular lesions; FIG. 6B is a statistical diagram of the area of vascular gross lesions;
图7为单克隆抗体anti-AT1R主动脉瓣斑块面积较对照组图，图中mAb是指anti-AT1R抗体，Val是指缬沙坦，Con是对照组；Figure 7 shows the area of the aortic valve plaque of the monoclonal antibody anti-AT1R compared with the control group. In the figure, mAb refers to anti-AT1R antibody, Val refers to valsartan, and Con is a control group;
图7A为主动脉瓣环切片图；图7B为主动脉瓣环病变统计图；Figure 7A is a sectional view of aortic annulus; Figure 7B is a statistical diagram of aortic annulus lesions;
图8为单克隆抗体anti-AT1R对AAA成瘤率及瘤体大小对比图，C是对照，S是 AngⅡ灌注组，A是AngⅡ灌注加anti-AT1R抗体；Figure 8 is a comparison of monoclonal antibody anti-AT1R on AAA tumor formation rate and tumor size, C is control, S is AngII perfusion group, A is AngII perfusion plus anti-AT1R antibody;
图中，图8A为动脉瘤大体图；图8B为动脉瘤发生率统计图；In the figure, Fig. 8A is a general view of an aneurysm; Fig. 8B is a statistical diagram of the incidence of an aneurysm;
图8C为动脉瘤直径大小统计图；图8D为动脉瘤病理分类统计图；Figure 8C is a statistical diagram of the diameter of the aneurysm; Figure 8D is a statistical classification of the aneurysm pathology;
图9为单克隆抗体anti-AT1R保护血管壁完整性示意图，C是对照，S是AngⅡ灌注组，A是AngⅡ灌注加anti-AT1R抗体组。Figure 9 is a schematic diagram showing the integrity of the monoclonal antibody anti-AT1R to protect the vascular wall, C is the control, S is the AngII perfusion group, and A is the AngII perfusion plus anti-AT1R antibody group.
具体实施方式Detailed ways
为了更好地解释本发明，以下结合具体实施例进一步阐明本发明的主要内容，但本发明的内容不仅仅局限于以下实施例。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为常规生化试剂商店购买得到的，以下实施例中的定量试验，均设置有三次重复实验，结果取平均值，如无特殊说明，实施例中的常规药品制备如下：In order to better explain the present invention, the main contents of the present invention will be further clarified below with reference to specific embodiments, but the content of the present invention is not limited to the following embodiments. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, were purchased from a conventional biochemical reagent store. The quantitative tests in the following examples were set up with three replicate experiments, and the results were averaged, unless otherwise specified. The conventional pharmaceutical preparations in the examples are as follows:
a.下述实验中使用的细胞培养基主要有RPMI-1640或DMEM两种基础培养基，配好后过滤除菌(0.22um)，分装，4℃保存。a. The cell culture medium used in the following experiments mainly consisted of two basic media, RPMI-1640 or DMEM. After being prepared, the cells were sterilized by filtration (0.22 um), and stored at 4 ° C.
不完全RPMI-1640培养基配方为：The incomplete RPMI-1640 medium formulation is:
Figure PCTCN2018104608-appb-000001
Figure PCTCN2018104608-appb-000001
不完全DMEM培养基(用1N HCl调试pH至7.2-7.4，过滤除菌，分装4℃保存)配方：Incomplete DMEM medium (pH adjusted to 7.2-7.4 with 1N HCl, filter sterilized, stored at 4 ° C) formula:
Figure PCTCN2018104608-appb-000002
Figure PCTCN2018104608-appb-000002
完全RPMI-1640或DMEM培养基：不完全RPMI-1640或DMEM培养基80ml+小牛血清15-20ml；Complete RPMI-1640 or DMEM medium: incomplete RPMI-1640 or DMEM medium 80ml + calf serum 15-20ml;
HT培养基配方：完全RPMI-1640或DMEM培养基99ml+HT贮存液1ml；HT medium formula: complete RPMI-1640 or DMEM medium 99ml + HT stock solution 1ml;
HAT培养基配方：完全RPMI-1640或DMEM培养基98ml+HT贮存液1ml+A贮存液1mlHAT medium formula: complete RPMI-1640 or DMEM medium 98ml + HT stock solution 1ml + A stock solution 1ml
b.氨基喋呤(A)贮存液(100×，4×10-5mol/L)：b. Aminoguanidine (A) stock solution (100×, 4×10-5 mol/L):
称取1.76mg氨基喋呤(Aminopterin MW 440.4)，溶于90ml超纯水或四蒸水中，滴加1mol/L NaOH 0.5ml中和，再补加超纯水或四蒸水至100ml。过滤除菌，分装小瓶(2ml/瓶)，-20℃保存。1.76 mg of aminoguanidine (Aminopterin MW 440.4) was weighed and dissolved in 90 ml of ultrapure water or four distilled water, neutralized by adding 1 mol/L NaOH 0.5 ml, and then added with ultrapure water or four distilled water to 100 ml. Filter and sterilize, dispense vials (2ml/bottle), and store at -20 °C.
c.次黄嘌呤和胸腺嘧啶核苷(HT)贮存液(100×,H:10 -2mol/L，T：1.6×10 -3mol/L)： c. Hypoxanthine and thymidine (HT) stock solution (100×, H: 10 -2 mol/L, T: 1.6×10 -3 mol/L):
称取136.1mg次黄嘌呤(Hypoxanthine,MW 136.1)和38.8mg胸腺嘧啶核苷(Thymidine,MW 242.2)，加超纯水或四蒸水至100ml，-20℃冻存。136.1 mg of hypoxanthine (Hypoxanthine, MW 136.1) and 38.8 mg of thymidine (Thymidine, MW 242.2) were weighed, and ultrapure water or four distilled water was added to 100 ml, and frozen at -20 °C.
d.L-谷氨酰胺(L.G.)溶液(100×，0.2mol/L)：d. L-Glutamine (L.G.) solution (100×, 0.2 mol/L):
称取2.92g L-谷氨酰胺(L-glutamine，MW 146.15)，用100ml不完全培养液或超纯水(或四蒸水)溶解，过滤除菌，分装小瓶(4-5ml/瓶)，-20℃冻存。Weigh 2.92 g of L-glutamine (MW 146.15), dissolve it with 100 ml of incomplete medium or ultrapure water (or four distilled water), filter and sterilize, and dispense vials (4-5ml/bottle). Store at -20 °C.
e.青、链霉素(双抗)溶液(100×)：e. Cyan and streptomycin (double antibody) solution (100×):
取青霉素G(钠盐)100万单位和链霉素(硫酸盐)1g，溶于100ml灭菌超纯水或四蒸水中，分装小瓶(4-5ml/瓶)，-20℃冻存。Take 1 million units of penicillin G (sodium salt) and 1 g of streptomycin (sulfate), dissolve in 100 ml of sterilized ultrapure water or four distilled water, dispense vials (4-5 ml/bottle), and store at -20 °C.
f.7.5％NaHCO 3溶液：称取分析纯NaHCO 3 7.5g，溶于100ml超纯水或四蒸水中，过滤除菌，分装小瓶(4-5ml/瓶)，盖紧瓶塞，4℃保存。 F.7.5% NaHCO 3 solution: Weigh analytically pure NaHCO 3 7.5g, dissolve in 100ml ultrapure water or four distilled water, filter and sterilize, dispense vial (4-5ml/bottle), close the stopper, 4°C save.
g.HEPES溶液(1mol/L)：g. HEPES solution (1mol/L):
称取23.83g HEPES(N-2-Hydroxyethylpiperazine-N，-2-ethanesulfonic acid，N-2-羟乙基哌嗪-N，-2-乙基磺酸，MW 238.3)溶于100ml超纯水或四蒸水中，过滤除菌，分装小瓶(4-5ml/瓶)，4℃保存。Weigh 23.83g HEPES (N-2-Hydroxyethylpiperazine-N,-2-ethanesulfonic acid, N-2-hydroxyethylpiperazine-N,-2-ethylsulfonic acid, MW 238.3) in 100ml of ultrapure water or In four distilled water, filter and sterilize, dispense vials (4-5ml/bottle), and store at 4°C.
h.8-氮鸟嘌呤贮存液(100×)：H.8-Azaguanine stock solution (100×):
称取200mg 8-氮鸟嘌呤(8-azaguanine，MW152.1)，加入4mol/L NaOH 1ml，待其溶解后，加入超纯水或四蒸水99ml，过滤除菌；分装小瓶，-20℃冻存。使用时按1％浓度加入到培养液中(即终浓度为20ug/ml)。Weigh 200mg 8-azaguanine (MW152.1), add 1ml / L NaOH 1ml, after it is dissolved, add ultra-pure water or four distilled water 99ml, filter sterilization; sub-package, -20 Store at °C. When used, it was added to the culture solution at a concentration of 1% (ie, the final concentration was 20 ug/ml).
i.50％PEG：I.50% PEG:
称取PEG1000或4000 20-50g于三角瓶中，盖紧，60-80℃水浴融化，0.6ml分装于 青霉素小瓶中，盖紧，8磅高压蒸汽15分钟，-20℃存放备用。临用前加热融化，加等量不完全培养基，用少许7.5％NaHCO 3调pH至8.0。 Weigh PEG1000 or 4000 20-50g in a triangular flask, tightly cap, melt in a 60-80 ° C water bath, 0.6 ml in a penicillin vial, tightly capped, 8 lbs of high pressure steam for 15 minutes, and store at -20 ° C for later use. Heat and melt before use, add an equal amount of incomplete medium, and adjust the pH to 8.0 with a little 7.5% NaHCO 3 .
实施例1 杂交瘤细胞CQ8-A2D9的制备Example 1 Preparation of hybridoma cell CQ8-A2D9
1、小鼠脾细胞的获得1. Acquisition of mouse spleen cells
1)免疫抗原获得1) Immune antigen acquisition
针对序列AFHYESQ进行多肽合成纯度≥85％的短肽ATR001，然后短肽ATR001与载体蛋白KLH进行耦联作为免疫抗原；The short peptide ATR001 with a purity of ≥85% of the peptide synthesis is carried out for the sequence AFHYESQ, and then the short peptide ATR001 is coupled with the carrier protein KLH as an immunizing antigen;
2)动物免疫2) Animal immunity
用免疫抗原免疫4-6只6周龄的Balb/c小鼠(活体小鼠)，常规周期：3周-2周-2周……强化周期：2周-2周-1周……使小鼠抗血清ELISA效价≥1:80000，具体步骤如下：4-6 6-week-old Balb/c mice (live mice) were immunized with immunizing antigens, regular cycle: 3 weeks - 2 weeks - 2 weeks... Intensive cycle: 2 weeks - 2 weeks - 1 week... Mouse antiserum ELISA titer ≥ 1:80000, the specific steps are as follows:
(1)初次免疫，免疫抗原使用量：50ug/只，加弗氏完全佐剂皮下多点注射，间隔3周；(1) First immunization, the amount of immunized antigen: 50ug/only, plus Freund's complete adjuvant subcutaneous injection, separated by 3 weeks;
(2)第21天第二次免疫，剂量途径同上，加弗氏不完全佐剂，间隔2周；(2) The second immunization on the 21st day, the dose route is the same as above, and the Freund's incomplete adjuvant is separated by 2 weeks;
(3)第35天第三次免疫，剂量同上，加弗氏不完全佐剂或不加佐剂，腹腔注射，7-10天后采血进行ELISA检测效价，检测免疫效果，间隔2周；(3) The third immunization on the 35th day, the dose is the same as above, Jiafu's incomplete adjuvant or no adjuvant, intraperitoneal injection, 7-10 days after the blood collection for ELISA detection titer, detection of immune effect, interval 2 weeks;
(4)加强免疫，剂量50ug，腹腔注射；(4) boosting the immunization, the dose is 50ug, intraperitoneal injection;
(5)在最后一次加强免疫后第3天，取脾融合，取效价≥1:80000的小鼠脾细胞。(5) On the third day after the last booster immunization, spleen fusion was performed, and mouse spleen cells with titer ≥ 1:80000 were taken.
2、细胞融合及杂交瘤筛选2. Cell fusion and hybridoma screening
1)骨髓瘤细胞悬液的制备方法如下：1) The preparation method of myeloma cell suspension is as follows:
a.于融合前48-36小时，将骨髓细胞扩大培养(一般按一块96孔板的融合试验约需2-3瓶100ml培养瓶培养的细胞进行准备)；a. Bone marrow cells are expanded and cultured 48-36 hours before the fusion (generally, about 2-3 bottles of 100 ml culture flasks are prepared by a 96-well fusion test);
b.融合当天，用弯头滴管将细胞从瓶壁瘤轻轻吹下，收集于50ml离心管或融合管内；b. On the day of fusion, the cells are gently blown from the bottle wall tumor with a curved pipette and collected in a 50 ml centrifuge tube or a fusion tube;
c.1000r/min离心5-10分钟，弃去上清；C. 1000r / min centrifugation for 5-10 minutes, discard the supernatant;
d.加入30ml不完全培养基，同法离心洗涤一次。然后将细胞重悬浮于10ml不完全培养基，混匀。d. Add 30 ml of incomplete medium and wash once with the same method. The cells were then resuspended in 10 ml of incomplete medium and mixed.
e.取骨髓瘤细胞悬液，加0.4％台盼蓝染液作活细胞计数后备用。细胞计数时，取细胞悬液0.1ml加入0.9ml台盼蓝染液中，混匀，用血球计数板计数。计算细胞数目的公式为：每毫升细胞数＝4个大方格细胞数×105/4；或每毫升细胞数＝5个中方格细胞数 ×106/2e. Take the myeloma cell suspension, add 0.4% trypan blue dye solution for live cell counting and use. When the cells were counted, 0.1 ml of the cell suspension was added to 0.9 ml of trypan blue dye solution, mixed, and counted using a hemocytometer. The formula for calculating the number of cells is: number of cells per ml = 4 large square cells × 105 / 4; or number of cells per ml = 5 square cells × 106 / 2
2)脾淋巴细胞的准备：2) Preparation of spleen lymphocytes:
取已经免疫的BALB/c小鼠，摘除眼球采血，并分离血清作为抗体检测时的阳性对照血清。同时通过颈脱位致死小鼠，浸泡于75％酒精中5分钟，于培养皿上固定后掀开左侧腹部皮肤，可看到脾脏，换眼科剪镊，在超净台中用无菌手术剪开腹膜，取出脾脏置于已盛有10ml不完全培养基的平皿中，轻轻洗涤，并细心剥去周围***。置平皿中不锈钢筛网上，用注射器针芯研磨成细胞悬液后计数，使脾细胞进入平皿中的不完全培养基。用吸管吹打数次，制成单细胞悬液。通常每只小鼠1×10 8-2.5×10 8个脾细胞。 The BALB/c mice that had been immunized were taken, the eyeballs were removed, and the serum was separated as a positive control serum at the time of antibody detection. At the same time, the mice were sacrificed by cervical dislocation, soaked in 75% alcohol for 5 minutes, fixed on the culture dish, and then the left abdomen skin was opened. The spleen was visible, and the eye scissors were cut. The scissors were opened in a clean bench. The peritoneum was taken out and placed in a dish containing 10 ml of incomplete medium, gently washed, and the surrounding connective tissue was carefully peeled off. The plate was placed in a stainless steel sieve, and the cell suspension was ground with a syringe needle and counted to allow the spleen cells to enter the incomplete medium in the plate. Sip several times with a pipette to make a single cell suspension. Usually 1 x 10 8 - 2.5 x 10 8 splenocytes per mouse.
3)饲养细胞(小鼠腹腔巨噬细胞)的制备：3) Preparation of feeder cells (mouse peritoneal macrophages):
将小鼠处死、体表消毒和固定后，用消毒剪镊从后腹掀起腹部皮肤，暴露腹膜。用酒精棉球擦拭腹膜消毒。用注射器注射10ml不完全培养基至腹腔，注意避免穿入肠管。右手固定注射器，使针头留置在腹腔内，左手持酒精棉球轻轻按摩腹部1分钟，随后吸出注入的培养液。1000r/min离心5-10分钟，弃上清。先用5ml HAT培养基将沉淀细胞悬浮，根据细胞计数结果，补加HAT培养基，使细胞浓度为2×10 5/ml，备用。通常对巨噬细胞来说，96孔培养板每孔需2×10 4个细胞，24孔板每孔需105细胞。每只小鼠可得3-5×10 6个细胞，因此一只小鼠可供两块96孔板的饲养细胞。也可在细胞融合前1-2天制备并培养饲养细胞，这样使培养板孔底先铺上一层饲养细胞层。做法是，将上述细胞悬液加入96孔板，每孔0.1ml，然后置37℃6％CO 2的培养箱中培养。 After the mice were sacrificed, the surface was disinfected and fixed, the abdominal skin was lifted from the posterior abdomen with a sterile scissors to expose the peritoneum. Wipe the peritoneum with an alcohol cotton ball. Inject 10 ml of incomplete medium into the abdominal cavity with a syringe, taking care to avoid penetrating the intestine. Fix the syringe in the right hand, leave the needle in the abdominal cavity, gently massage the abdomen with an alcohol cotton ball for 1 minute, then aspirate the injected culture solution. Centrifuge at 1000 r/min for 5-10 minutes and discard the supernatant. The precipitated cells were first suspended in 5 ml of HAT medium, and HAT medium was added according to the cell count to make the cell concentration 2 × 10 5 /ml, and was used. Generally, for macrophages, a 96-well plate requires 2 x 10 4 cells per well, and a 24-well plate requires 105 cells per well. Each mouse can obtain 3-5 × 10 6 cells, so one mouse can be used for feeding cells of two 96-well plates. The feeder cells can also be prepared and cultured 1-2 days before cell fusion, such that the bottom of the culture plate is first covered with a layer of feeder cells. Alternatively, the above cell suspension was added to a 96-well plate at 0.1 ml per well, and then cultured in an incubator at 37 ° C in a 6% CO 2 atmosphere.
4)细胞融合4) Cell fusion
①将1×10 8脾细胞与1×10 7骨髓瘤细胞SP2/0混合于一支50ml融合管中，补加不完全培养基至30ml，充分混匀； 1 Mix 1×10 8 splenocytes and 1×10 7 myeloma cells SP2/0 in a 50 ml fusion tube, add incomplete medium to 30 ml, and mix well;
②1000r/min离心5-10分钟，将上清尽量吸净；Centrifuge at 21000r/min for 5-10 minutes, and absorb the supernatant as much as possible;
③在手掌上轻击融合管底，使沉淀细胞松散均匀；3 tap the bottom of the fusion tube on the palm to make the precipitated cells loose and even;
④用1ml吸管在30s内加入预热的50％PEG1ml，边加边轻轻搅拌；4 Add 1 ml of preheated 50% PEG 1 ml with a 1 ml pipette, and gently stir while adding;
⑤吸入吸管静置1min；5 suction pipe is allowed to stand for 1 min;
⑥加入预热的不完全培养液，终止PEG作用，连续每2min内分别加入1ml、2ml、3ml、4ml、5ml、10ml；6 adding pre-heated incomplete culture solution, stop the action of PEG, continuously add 1ml, 2ml, 3ml, 4ml, 5ml, 10ml every 2min;
⑦800rpm,5分钟，弃去上清；7800 rpm, 5 minutes, discard the supernatant;
⑧加入5ml完全培养基，轻轻吹吸沉淀细胞，使其悬浮并混匀，然后补加完全培养基至40-50ml。分装96孔细胞培养板，每孔100ul,然后将培养板置37℃，5％CO 2培养箱内培养； 8 Add 5 ml of complete medium, gently pipette the pelleted cells, suspend and mix, then add complete medium to 40-50 ml. The 96-well cell culture plate was divided into 100 ul per well, and then the culture plate was placed at 37 ° C in a 5% CO 2 incubator;
⑨6h后补加选择培养基，每孔50ul，3天后用选择培养基半换液；After 96h, the selection medium was added, 50ul per well, and after 3 days, the medium was replaced with a half medium;
⑩经常观察杂交瘤细胞生长情况，待其长至孔底面积1/10以上时吸出上清供抗体检测；10 often observe the growth of hybridoma cells, when the length of the bottom of the pores is more than 1/10, the supernatant is aspirated for antibody detection;
5)杂交瘤细胞的筛选5) Screening of hybridoma cells
①抗原用包被液稀释至10ug/ml；1 antigen is diluted to 10ug/ml with a coating solution;
②以100ul/孔量加入酶标板孔中，置4℃过夜或37℃吸附2小时；2 was added to the wells of the microtiter plate at a dose of 100 ul/well, and allowed to stand at 4 ° C overnight or at 37 ° C for 2 hours;
③弃去孔内的液体，同时用洗涤液洗3次，每次3分钟，拍干；3 Discard the liquid in the well and wash it 3 times with the washing solution for 3 minutes each time, pat dry;
④每孔加100ul封闭液37℃封闭1小时；4 Each well was sealed with 100 ul of blocking solution at 37 ° C for 1 hour;
⑤洗涤液洗3次；5 washing liquid wash 3 times;
⑥每孔加100ul待检杂交瘤细胞培养上清，同时设立阳性、阴性对照和空白对照；37℃孵育1小时；洗涤，拍干；6 Add 100 ul of hybridoma cell culture supernatant to be per well, and set up positive, negative control and blank control; incubate for 1 hour at 37 ° C; wash and pat dry;
⑦加酶标二抗，每孔100ul，37℃孵育1小时，洗涤，拍干；7 plus enzyme-labeled secondary antibody, 100ul per well, incubate for 1 hour at 37 ° C, wash, pat dry;
⑧加底物液，每孔加新鲜配制的底物使用液100ul，37℃20分钟；8 Add substrate solution, add 100ul of freshly prepared substrate per well, and incubate at 37 °C for 20 minutes;
⑨以2mol/L H 2SO 4终止反应，在酶联免疫阅读仪上读取OD值； 9 The reaction was stopped with 2 mol/L H 2 SO 4 and the OD value was read on an enzyme-linked immunosorber;
⑩结果判定：以P/N≧2.1，或P≧N+3SD为阳性。若阴性对照孔无色或接近无色，阳性对照孔明确显色，则可直接用肉眼观察结果。即得到杂交瘤细胞CQ8-A2D9，并将杂交瘤细胞CQ8-A2D9于2016年11月16日保藏于中国典型培养物保藏中心(简称CCTCC)；保藏登记号为CCTCC NO：C2017108；保藏地点为：中国、武汉、武汉大学。10 Results: P/N≧2.1, or P≧N+3SD was positive. If the negative control well is colorless or nearly colorless, and the positive control well is clearly colored, the results can be observed directly with the naked eye. The hybridoma cell CQ8-A2D9 was obtained, and the hybridoma cell CQ8-A2D9 was deposited with the China Center for Type Culture Collection (CCTCC) on November 16, 2016; the deposit number is CCTCC NO: C2017108; the deposit location is: China, Wuhan, Wuhan University.
实施例2Example 2
1、杂交瘤亚克隆及建株1. Hybridoma subcloning and construction
①制备小鼠脾细胞为饲养细胞；1 preparing mouse spleen cells as feeder cells;
②制备待克隆的杂交瘤细胞悬液，用含20％血清的HT培养基稀释至每毫升含5、10和20个细胞3种不同的稀释度；2 preparing a suspension of hybridoma cells to be cloned, and diluting with HT medium containing 20% serum to 3 different dilutions of 5, 10 and 20 cells per ml;
③按每毫升加入5×10 4-1×10 5细胞的比例，在上述杂交瘤细胞悬液中分别加入腹腔巨噬细胞； 3 adding peritoneal macrophages to the above hybridoma cell suspension in a ratio of 5×10 4 -1×10 5 cells per ml;
④每种杂交瘤细胞分装96孔板一块，每孔量为100ul；4 each hybridoma cell is divided into 96-well plates, each well is 100ul;
⑤37℃、5％CO 2培养6天，出现肉眼可见的克隆即可检测抗体；在倒置显微镜下观察，标出只有单个克隆生长的孔，取上清作抗体检测。 The cells were cultured at 537 ° C, 5% CO 2 for 6 days, and the clones were detected by macroscopic detection; under the inverted microscope, only the wells of single clone growth were marked, and the supernatant was taken for antibody detection.
⑥取抗体检测阳性孔的细胞扩大培养，并冻存，亚克隆2-3次，得到单克隆细胞2-5株。即为杂交瘤细胞CQ8-A2D96 The cells in which the positive cells of the antibody were detected were expanded and frozen, and subcloned 2-3 times to obtain 2-5 monoclonal cells. Hybridoma cell CQ8-A2D9
2、单抗生产2, monoclonal antibody production
①腹腔接种降植烷或液体石蜡，每只小鼠0.3-0.5ml。1 Intraperitoneal inoculation of norparin or liquid paraffin, 0.3-0.5 ml per mouse.
②7-10天后腹腔接种用PBS或无血清培养基稀释的杂交瘤细胞，每只小鼠5×10 5/0.2ml。 Hybridoma cells diluted with PBS or serum-free medium were inoculated intraperitoneally after 27-10 days, 5 × 10 5 /0.2 ml per mouse.
③间隔5天后，每天观察小鼠腹水产生情况，如腹部明显膨大，以手触摸时，皮肤有紧张感，即可用16号针头采集腹水，一般可连续采2-3次，通常每只小鼠可采5-10ml腹水；3 After 5 days, observe the ascites production in mice every day. If the abdomen is obviously enlarged, when the skin is touched by hand, the skin can be used to collect ascites with a 16-gauge needle. Generally, it can be taken continuously for 2-3 times, usually for each mouse. Can take 5-10ml ascites;
④将腹水离心(2000r/min 5分钟)，除去细胞成分和其他的沉淀物，收集上清，测定抗体效价，分装，-70℃冻存备用，或冻干保存。4 The ascites was centrifuged (2000 r/min for 5 minutes) to remove the cellular components and other precipitates, and the supernatant was collected, and the antibody titer was determined, and the mixture was stored at -70 ° C for storage or stored by lyophilization.
3、单克隆抗体的纯化3. Purification of monoclonal antibodies
取1份预处理过的腹水加2份0.06mol/L pH5.0醋酸缓冲液，用1mol/L HCl调pH至4.8；按每毫升稀释腹水加11ul辛酸的比例，室温搅拌下逐滴加入辛酸，于30分钟内加完，4℃静置2小时，取出15000g离心30分钟，弃沉淀；上清经尼龙筛过滤(125um)，加入1/10体积的0.01mol/L PBS，用1mol/L NaOH调pH至7.2；在4℃下加入饱和硫酸铵至45％饱和度，作用30分钟，静置1小时；10000g离心30分钟，弃上清；沉淀溶于适量PBS(含137mmol/L NaCl，2.6mol/L KCl，0.2mmol/L EDTA)中，对50-100倍体积的PBS透析，4℃过夜，其间换水3次以上；取出10000g离心30分钟，除去不溶性沉渣，测定蛋白质含量后，即为单克隆抗体anti-AT1R：分装，冻存备用。Take 1 part of pretreated ascites and add 2 parts of 0.06mol/L pH 5.0 acetate buffer, adjust the pH to 4.8 with 1mol/L HCl; add 11ul octanoic acid per ml of diluted ascites, add octanoic acid dropwise at room temperature Add in 30 minutes, let stand at 4 °C for 2 hours, remove 15000g for 30 minutes, discard the precipitate; the supernatant is filtered through a nylon sieve (125um), add 1/10 volume of 0.01mol/L PBS, use 1mol/L Adjust the pH to 7.2 with NaOH; add saturated ammonium sulfate to 45% saturation at 4 ° C for 30 minutes, let stand for 1 hour; centrifuge at 10000 g for 30 minutes, discard the supernatant; the precipitate is dissolved in an appropriate amount of PBS (containing 137 mmol / L NaCl, In 2.6mol/L KCl, 0.2mmol/L EDTA), dialyze against 50-100 volumes of PBS, overnight at 4°C, and change water for more than 3 times; remove 10000g for 30 minutes, remove insoluble sediment, and determine protein content. That is, the monoclonal antibody anti-AT1R: packed, frozen and stored.
将单克隆抗体anti-AT1R进行测序，该单克隆抗体anti-AT1R的氨基酸序列包括重链或轻链，其序列分别为SEQ ID No.1和SEQ ID No.2。The monoclonal antibody anti-AT1R is sequenced, and the amino acid sequence of the monoclonal antibody anti-AT1R includes a heavy chain or a light chain, and the sequences thereof are SEQ ID No. 1 and SEQ ID No. 2, respectively.
结果：选取两个克隆株，运用ELISA方法，我们检测制备单克隆抗体滴度，结果表明，抗体效价≥1:80000RESULTS: Two clones were selected and the monoclonal antibody titer was determined by ELISA. The results showed that the antibody titer was ≥1:80000.
Clone IDClone ID BlankBlank 1:10001:1000 1:30001:3000 1:90001:9000 1:270001:27000 1:810001:81000 1:2430001:243000 1:7290001:729000
CQ8-A2D9CQ8-A2D9 0.0760.076 2.4192.419 2.1452.145 1.8041.804 1.1721.172 0.5630.563 0.2940.294 0.1650.165
CQ8-C3B7CQ8-C3B7 0.0760.076 2.6512.651 2.4342.434 2.1162.116 1.471.47 0.8320.832 0.4140.414 0.2040.204
实施例3 单克隆抗体anti-AT1R对稳定表达AT1R CHO细胞ERK和JNK磷酸化的影响Example 3 Effect of monoclonal antibody anti-AT1R on ERK and JNK phosphorylation in stably expressing AT1R CHO cells
1.培养AT1R-CHOK1细胞系(PerkinElmer)：含10％FBS的DMEM/F12完全培养基，5％CO 2细胞培养箱培养； 1. Cultured AT1R-CHOK1 cell line (PerkinElmer): DMEM/F12 complete medium containing 10% FBS, cultured in a 5% CO 2 cell incubator;
2.AT1R-CHOK1中检测AngⅡ诱导的ERK和JNK的磷酸化2. Detection of AngII-induced phosphorylation of ERK and JNK in AT1R-CHOK1
将AT1R-CHOK1接种于六孔板中，密度80％左右，过夜培养。然后以HBS饥饿2h，做AngⅡ浓度梯度刺激和时间梯度刺激，Western Blot检测p-ERK/ERK和p-JNK/JNK的表达。AT1R-CHOK1 was inoculated into a six-well plate at a density of about 80% and cultured overnight. Then, HBS was starved for 2 h, AngII concentration gradient stimulation and time gradient stimulation were performed, and the expression of p-ERK/ERK and p-JNK/JNK was detected by Western Blot.
3.检测单克隆抗体anti-AT1R对AT1R激活引起ERK/JNK磷酸化的影响3. Detection of the effect of monoclonal antibody anti-AT1R on ERK/JNK phosphorylation induced by AT1R activation
细胞培养、种板见步骤1、2。HBS饥饿2h后ARB组和单抗组分别以洛沙坦和单克隆抗体anti-AT1R预孵育1h，而后各组(除control组外)均以AngII刺激，Western Blot检测单克隆抗体anti-AT1R对AT1R激活引起ERK和JNK磷酸化的影响，结果如下：For cell culture and seed plating, see steps 1 and 2. After 2 hours of HBS starvation, ARB group and monoclonal antibody group were pre-incubated with losartan and monoclonal antibody anti-AT1R for 1 h, respectively. Then each group (except control group) was stimulated with AngII, and Western blot was used to detect anti-AT1R monoclonal antibody. AT1R activation causes the effects of ERK and JNK phosphorylation, and the results are as follows:
结果表明单克隆抗体anti-AT1R可以有效抑制AngII诱导的ERK(图1)及JNK磷酸化(图2)。我们也检测了稳定表达AT1R的CHO细胞内钙离子浓度的变化，结果表明单克隆抗体anti-AT1R对AngII诱导的细胞内钙离子浓度增加无影响(图3)，即单克隆抗体anti-AT1R并不完全通过经典的AT1R-Gq-PKC途径起作用，单克隆抗体anti-AT1R偏向性调节AT1R的功能。The results showed that the monoclonal antibody anti-AT1R can effectively inhibit AngII-induced ERK (Fig. 1) and JNK phosphorylation (Fig. 2). We also examined changes in intracellular calcium concentration in CHO cells stably expressing AT1R, and the results showed that the monoclonal antibody anti-AT1R had no effect on the increase of intracellular calcium concentration induced by AngII (Fig. 3), namely the monoclonal antibody anti-AT1R. Not fully functioning through the classical AT1R-Gq-PKC pathway, the monoclonal antibody anti-AT1R bias regulates the function of AT1R.
实施例4 单克隆抗体anti-AT1R在高血压中的降压作用Example 4 Antihypertensive effect of monoclonal antibody anti-AT1R in hypertension
以Balb/c小鼠皮下埋泵灌注AngⅡ构建高血压模型，单抗组(A组)于埋泵当日鼠尾静脉注射anti-AT1R单克隆抗体100μg/只，肼屈嗪组(J组)于埋泵前一周开始以肼屈嗪3mg/kg/d灌胃。A blood pressure model was constructed by instilling AngII into the Balb/c mouse subcutaneously. The monoclonal antibody group (group A) was injected with anti-AT1R monoclonal antibody 100 μg/only in the tail vein of the buried pump, and the hydralazine group (group J) was used. One week before the pump was buried, gavage was administered at 3 mg/kg/d.
分别监测各组埋泵前、埋泵后2周和4周血压变化，发现anti-AT1R抗体能显著降低AngⅡ模型小鼠血压(如图4～5)。The blood pressure changes of the groups before and after the pump were monitored, and the anti-AT1R antibody was found to significantly reduce the blood pressure of the AngII model mice (Fig. 4-5).
实施例5 单克隆抗体anti-AT1R在抗动脉粥样硬化中的作用Example 5 The role of monoclonal antibody anti-AT1R in anti-atherosclerosis
首先构建西方饮食诱导的动脉粥样硬化(AS)小鼠模型，即给apoE -/-小鼠高脂喂养17周，每周把anti-AT1R单抗(mAb)20ug和100ug分别鼠尾静脉回输apoE -/-小鼠，17周后对apoE -/-小鼠主动脉大体进行油红染色，发现粥样硬化斑块面积显著下降(图6)。主动脉瓣瓣环进行冰冻切片油红染色，发现anti-AT1R单抗显著减小粥样硬化斑块(图7)。 First, a Western diet-induced atherosclerosis (AS) mouse model was established, in which apoE -/- mice were fed with high fat for 17 weeks, and anti-AT1R monoclonal antibody (mAb) was 20ug and 100ug per week. After apoE -/- mice were transfused, the apoE -/- mouse aorta was roughly stained with oil red after 17 weeks, and the area of atherosclerotic plaque was significantly decreased (Fig. 6). The aortic annulus was stained with oil-red stained in frozen sections, and the anti-AT1R monoclonal antibody was found to significantly reduce atherosclerotic plaques (Fig. 7).
实施例6 单克隆抗体anti-AT1R在抗动脉瘤中的作用Example 6 The role of monoclonal antibody anti-AT1R in anti-aneurysms
首先构建AngⅡ诱导的腹主动脉瘤(AAA)小鼠模型，即给apoE -/-小鼠皮下埋入微型渗透泵持续给予AngII 1000ng/kg/min 28天，发现单克隆抗体anti-AT1R能显著降低apoE -/-小鼠AAA的发生率，减轻瘤体严重程度(图8)。瘤体血管横截面病理检测发现对照组血管显著扩张，并伴血栓形成，而单克隆抗体anti-AT1R保护血管的形态结构相对完整(图9)。 Firstly, an AngII-induced abdominal aortic aneurysm (AAA) mouse model was constructed, in which apoE -/- mice were subcutaneously implanted with a micro-osmotic pump and AngII was administered at 1000 ng/kg/min for 28 days. The monoclonal antibody anti-AT1R was found to be significant. Reduce the incidence of AAA in apoE -/- mice and reduce the severity of the tumor (Figure 8). Cross-sectional pathology of the tumor vessels revealed a significant expansion of the control vessels with thrombosis, while the monoclonal antibody anti-AT1R protected the vascular morphology and morphology (Fig. 9).
其它未详细说明的部分均为现有技术。尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。Other parts not described in detail are prior art. While the above-described embodiments have been described in detail, the present invention is only a part of the embodiments of the present invention, but not all of the embodiments, and other embodiments may be obtained without inventiveness according to the embodiments. All belong to the scope of protection of the present invention.

Claims (8)

  1. 一种制备杂交瘤细胞CQ8-A2D9的短肽ATR001，其氨基酸序列为AFHYESQ。A short peptide ATR001 for preparing hybridoma cell CQ8-A2D9, the amino acid sequence of which is AFHYESQ.
  2. 一种杂交瘤细胞CQ8-A2D9，其保藏编号为：CCTCC NO：C2017108。A hybridoma cell CQ8-A2D9, the preservation number is: CCTCC NO: C2017108.
  3. 一种权利要求2所述杂交瘤细胞CQ8-A2D9的制备方法，其特征在于：包括以下步骤：A method for preparing the hybridoma cell CQ8-A2D9 according to claim 2, comprising the steps of:
    1)短肽ATR001与载体蛋白KLH进行耦联作为免疫耦联抗原；1) The short peptide ATR001 is coupled to the carrier protein KLH as an immuno-coupled antigen;
    2)免疫耦联抗原与弗氏佐剂混合免疫小鼠，然后去免疫小鼠的脾细胞与骨髓瘤细胞SP2/0进行融合，检测筛选获得杂交瘤细胞CQ8-A2D9。2) The immunoconjugated antigen is mixed with Freund's adjuvant to immunize the mouse, and then the spleen cells of the immunized mouse are fused with the myeloma cell SP2/0, and the hybridoma cell CQ8-A2D9 is obtained by screening.
  4. 根据权利要求3所述杂交瘤细胞CQ8-A2D9的制备方法，其特征在于：所述步骤1)中，短肽ATR001的氨基酸序列为AFHYESQ；The method for preparing the hybridoma cell CQ8-A2D9 according to claim 3, wherein in the step 1), the amino acid sequence of the short peptide ATR001 is AFHYESQ;
  5. 由杂交瘤细胞CQ8-A2D9制备单克隆抗体anti-AT1R的方法，其特征在于：所述杂交瘤细胞CQ8-A2D9保藏编号为：CCTCC NO：C2017108。A method for producing the monoclonal antibody anti-AT1R from the hybridoma cell CQ8-A2D9, characterized in that the hybridoma cell CQ8-A2D9 has the accession number: CCTCC NO: C2017108.
  6. 根据权利要求5所述制备单克隆抗体anti-AT1R的方法，其特征在于：包括以下步骤：The method for preparing a monoclonal antibody anti-AT1R according to claim 5, comprising the steps of:
    1)将杂交瘤细胞CQ8-A2D9制备腹水；1) Preparation of ascites by hybridoma cell CQ8-A2D9;
    2)利用腹水制备纯化，得到单克隆抗体anti-AT1R。2) Purification by ascites to obtain monoclonal antibody anti-AT1R.
  7. 一种权利要求5和6所述方法制备具有偏向性调节AT1R功能的单克隆抗体anti-AT1R，其特征在于：所述单克隆抗体anti-AT1R的氨基酸序列包括重链或轻链，其序列分别为SEQ ID No.1和SEQ ID No.2。A monoclonal antibody anti-AT1R having a bias-regulating AT1R function according to the method of claims 5 and 6, wherein the amino acid sequence of the monoclonal antibody anti-AT1R comprises a heavy chain or a light chain, the sequences of which are respectively Is SEQ ID No. 1 and SEQ ID No. 2.
  8. 利用权利要求7制备得到具有偏向性调节AT1R功能的单克隆抗体anti-AT1R在制备治疗高血压、动脉粥样硬化和动脉瘤的药品中的应用。The use of the monoclonal antibody anti-AT1R having the function of bias-regulating AT1R for the preparation of a medicament for the treatment of hypertension, atherosclerosis and aneurysm is prepared by the method of claim 7.
PCT/CN2018/104608 2017-09-08 2018-09-07 Short peptide atr001, monoclonal antibody prepared by short peptide and having function of biased regulation of at1r, and application thereof WO2019047921A1 (en)

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