CN111544584A - Therapeutic effect of monoclonal antibody on Parkinson's disease - Google Patents
Therapeutic effect of monoclonal antibody on Parkinson's disease Download PDFInfo
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- A61P25/00—Drugs for disorders of the nervous system
- A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
- A61P25/16—Anti-Parkinson drugs
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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Abstract
The invention relates to a therapeutic action of a monoclonal antibody on Parkinson's disease, wherein the monoclonal antibody is a p-alpha-synuclein (p-alpha-syn) monoclonal antibody (C140S) for resisting phosphorylation alpha synuclein at 129 th position of serine, and particularly relates to an action research of the antibody in treating abnormal activation of immune cells.
Description
Technical Field
The invention relates to a therapeutic action of a monoclonal antibody on Parkinson's disease, wherein the monoclonal antibody is a p-alpha-synuclein (p-alpha-syn) monoclonal antibody (C140S) for resisting phosphorylation alpha synuclein at 129 th position of serine, and particularly relates to an action research of the antibody in treating abnormal activation of immune cells.
Background
Parkinson's Disease (PD) is a common neurodegenerative Disease, which is mainly manifested by motor symptoms such as bradykinesia, muscular rigidity, resting tremor, and postural instability, while other neuromodulation and neurotransmitter systems are affected, resulting in various non-motor symptoms, such as decreased cognitive ability, depression, sleep disorders, olfactory disorders, micturition, and gastrointestinal disorders; the pathogenesis of Parkinson's disease is not completely understood, and the main pathological feature is the progressive death of dopaminergic neuron cells in the substantia nigra of the midbrain, and the aggregation of alpha-synuclein (alpha-syn) in the neuron cells to form Lewy bodies, and the main component of the parkinsonism is serine phosphorylated alpha-synuclein (p-alpha-syn) at position 129.
The current treatment method of PD is mainly drug treatment, the most classical drug in the drug treatment is levodopa, but levodopa can only improve the movement symptoms, the drugs have larger and larger toxic and side effects along with the prolonging of the use time and the increasing of the dosage, most PD patients have the phenomena of curative effect decline and 'on-off', the other class of drugs are enzyme inhibitors such as MAO-B inhibitors, the dopamine metabolism can be reduced by specifically inhibiting monoamine oxidase B, but some patients can have the side effects of involuntary movement and the like. Deep Brain Stimulation (DBS) therapy is to stimulate the subthalamic nucleus with electrical signals of a certain frequency for the tremor symptoms of patients, thereby achieving the purpose of relieving dyskinesia. These treatments are aimed at the treatment of motor symptoms, do not really delay the progression of the disease, and have certain limitations on the indications of the disease, so that immunotherapy with a strong specificity for the cause of disease shows its advantages. The antibody can be used as a medicine for identifying target protein causing diseases in a targeted manner, promoting the elimination of the target protein, reducing the aggregation of the target protein and preventing the spread of the target protein, thereby playing a role in treatment.
The target protein for immunotherapy treatment in parkinson's disease is alpha-synuclein. Abnormal aggregation of alpha-synuclein and modification of phosphorylation can cause cytotoxicity and spread among cells, which in turn causes cell death and leads to Parkinson's disease. Currently antibodies against alpha-syn are antibodies against alpha-syn in different aggregation states (anti-monomeric, anti-oligomeric, anti-fibrotic antibodies); alpha-syn is divided into three domains, N-terminal, NAC region and C-terminal, so there are antibodies (anti-N-terminal, anti-C-terminal, anti-NAC region antibodies) directed against different regions of alpha-syn, but none are currently clinically applied. While the toxic action of alpha-synuclein is mainly related to its phosphorylation modification, alpha synuclein phosphorylated at the 129-position of serine has larger damage to cells, is easier to gather and spread, and is more difficult to degrade, thereby causing cytotoxicity and neurodegenerative change. The use of specific anti-serine 129-phosphorylated alpha-synuclein monoclonal antibodies to neutralize toxic proteins and thereby reduce their cytotoxicity is a new therapeutic approach to the etiology.
Anti-serine 129-site phosphorylated alpha synuclein (p-alpha-synuclein, p-alpha-syn) monoclonal antibody (abbreviated as: C140S): the source of the antibody is described in the patent specification, which is disclosed in the Chinese patent ZL 201610913085.1: in order to establish the ELISA detection method, a mouse-derived monoclonal antibody C140S aiming at 129-bit phosphorylated human alpha-syn is specially prepared, and the preparation method of the monoclonal antibody is as follows: p1(M18631-1hz-1-1, Ac-CEAYMP (pS) EGG-NH2, 123-131 peptide fragment of humanized 129-serine phosphorylated alpha-syn) is used for immunizing a balb-C mouse, spleen is taken to prepare a fused myeloma cell line, an indirect ELISA is used for screening out a cell line with the clone number of C140S, the cell line is injected into the abdominal cavity of 4 balb-C/nu mice, the antibody is purified after the specificity of ascites reaction is verified by the indirect ELISA, and the reactivity of the purified antibody is verified by the indirect ELISA (namely, the absorbance value of 490nm wavelength light of a positive group with the recognition of 1000ng/ml by 0.125ug/ml is at least 30 times higher than that of a negative group, and the readings of the background and the negative group are less than 0.2), so that the antibody can be used as a detection antibody in the method, and the final concentration is 4 mg/ml.
(the cell strain is preserved in China general microbiological culture collection center with the number of CGMCC12993, the preservation date is 16 years, 9 months and 26 days, and the cell strain is classified and named as antibody hybridoma cell, address No. 3 Xilu No. 1 of Beijing Korean district, Beicheng).
Disclosure of Invention
The invention provides an application of an anti-serine 129-site phosphorylated alpha synuclein monoclonal antibody (C140S) in preparation of a medicament for treating Parkinson's disease, wherein the application is based on that the antibody can block p-alpha-syn from spreading among cells.
The invention therefore further provides a pharmaceutical preparation comprising an anti-serine 129-phosphorylated alpha-synuclein monoclonal antibody (C140S), wherein said monoclonal antibody is of the type: IgG, this antibody subtype is: IgG2 b. The pharmaceutical formulation, may be administered to a mammal, including a human, in any manner. Parenteral administration is preferred, and injection is particularly preferred.
The application of the invention is that the antibody can block p-alpha-syn from transmitting among cells.
The application of the invention is that the antibody can enter the brain parenchyma through the blood brain barrier within 6 hours.
The application of the invention is that the antibody can improve the proportion inversion of CD4+/CD8+ T cells, can relieve the behavior disorder of a transgenic mouse, increase the rod-rotating retention time of the transgenic mouse, increase the limb strength of the mouse and enhance the coordination ability of the transgenic mouse.
The application of the invention is that the antibody can neutralize toxic protein and reduce the cytotoxicity of the toxic protein.
The application of the invention is that the antibody can reduce the p-alpha-syn content in the brain of a transgenic animal, increase the TH content in the brain and relieve the evasion barrier of a transgenic mouse, which shows that the antibody C140S has neuroprotective effect and therapeutic effect on a PD model.
In order to prove the application of the invention, the invention takes the anti-serine 129-site phosphorylated alpha synuclein monoclonal antibody (C140S) as an experimental drug to research a relevant Parkinson disease model, and the research results are as follows:
the cell model used was a HEK293T cell line overexpressing alpha-syn.
The method comprises the following specific steps:
1) alpha-syn gene is transfected into HEK293T cells, cell supernatant is collected after 24 hours to carry out indirect ELISA experiment, and the result proves that p-alpha-syn is secreted in the cell supernatant.
2) Respectively connecting the N segment and the C segment of the Luciferase with alpha-syn, respectively transfecting into HEK293T cells, collecting supernatant of a transfected alpha-syn-Luciferase-N culture medium after 24h, adding the supernatant into a cell culture medium for transfecting alpha-syn-Luciferase-C, and developing color by using coelenterase after 24h, wherein the result proves that the p-alpha-syn can be transmitted among cells.
3) After C140S was added to the supernatant of the alpha-syn-luciferase-N medium, the intracellular fluorescence intensity of the transfected alpha-syn-luciferase-C plasmid was significantly decreased, indicating that C140S exerted a blocking effect during the transmission of p-alpha-syn.
The invention further provides a method for treating a PD transgenic animal model by using the anti-serine 129-site phosphorylation alpha synuclein monoclonal antibody (C140S).
The PD animal model adopted by the invention is a transgenic animal over-expressing alpha-syn.
The method comprises the following specific steps:
1) after wild type mice are injected with rhodamine-labeled C140S in the abdominal cavity, the fluorescence intensity in the brain of the mice is detected by using live imaging of the mice, and the result proves that the antibody drug C140S with the rhodamine label can enter the brain parenchyma through the blood brain barrier in 6 hours.
2) After 10mg/kg of antibody C140S is injected into the abdominal cavity of the transgenic mice for one week, the CD4 in the plasma of each group of mice is detected by using a flow cytometer+/CD8+T cell ratio, the results demonstrate that the antibody drug C140S can improve CD4+/CD8+The inverted proportion of the T cells can relieve the behavior disorder of the transgenic mouse, increase the rod-rotating retention time of the transgenic mouse, increase the limb strength of the mouse and enhance the coordination ability of the transgenic mouse.
3) The brain of the transgenic mouse is subjected to stereotactic injection of the C140S antibody to a dorsal striatum, and the p-alpha-syn and Tyrosine Hydroxylase (TH) content in the brain of the mouse and the movement behavior of the mouse are detected. The result proves that the antibody drug C140S can reduce the p-alpha-syn content in the brain of the transgenic animal, increase the TH content in the brain, and relieve the evasion disorder of the transgenic mouse, which indicates that the antibody C140S has neuroprotective effect and certain therapeutic effect on a PD model.
The invention has the advantages that the change of abnormal immunity in peripheral blood can be relieved after the PD animal model is injected with the C140S antibody medicine in the abdominal cavity, and the pathological symptoms of the PD model can be relieved and the dyskinesia of a mouse can be relieved after the brain is injected with the C140S antibody in a stereotaxic manner into the striatum.
Description of the drawings:
FIG. 1P- α -syn is used to propagate intercellularAn antibody drug C140S can block the transmission process, α -syn plasmid is transfected into HEK293T cells for 24 hours, cell supernatant is collected and concentrated by 10 times, ELISA detection is carried out, the result shows that α -syn in the supernatant of an over-expressed α -syn group is obviously more than that of a control group, B, luciferase is divided into an N end and a C end, the N end and the C end are respectively connected with α -syn, then the supernatant enters HEK293T cells through plasmid transfection, α -syn + luciferase-N end group is collected, the supernatant of the α -syn + luciferase-N end group is concentrated and then added into a α -syn + luciferase-C end group, coelenterazine is added after 24 hours, the autofluorescence intensity is detected under an enzyme labeling instrument, C, transfected α -syn + luciferase-N end cell protein is collected, a WB experiment is used for detecting that α -syn protein and phosphorylated α -syn protein (17kd) are expressed in the cells, and α -syn protein is also expressed at the same time+Fluorescence detection by enzyme labeling instrument, finding that adding 50 μ l of concentrated α -syn + luciferase-N end group cell supernatant into α -syn + luciferase-C end group cell transfected cells, the fluorescence intensity is obviously increased after adding coelenterazine, adding phosphorylated α -syn antibody C140s into the concentrated supernatant can neutralize p- α -syn + luciferase-N in the transmission process, the fluorescence intensity is obviously reduced, adding other volume of concentrated supernatant and antibody are not obviously different, E, simultaneously transfecting α -syn + luciferase-C and α -syn + luciferase-N into HEK293T cells, the fluorescence intensity is obviously increased after using coelenterazine, and the fluorescence intensity is not influenced by adding C140S antibody into the cell supernatant at the moment, and only the fluorescence emission of luciferase and the fluorescence emission of luciferase end cannot be realized due to the lack of the fluorescence characteristics of the phosphorylated α -syn + luciferase-N (27 kd).
FIG. 2: the half-life of the antibody C140S in mice is 5 days, and the intraperitoneal injection can reduce the content of p-alpha-syn in the peripheral blood of transgenic mice. A. Injecting 10mg/kg of p-alpha-syn antibody drug (C140S) into the abdominal cavity of TG mice, taking peripheral blood on days 1, 3, 5, 7 and 9 respectively, and detecting the amount of C140S and p-alpha-syn in the blood by using an ELISA method; (B, C) the amount of p-alpha-syn in the peripheral blood was significantly reduced after the 9 th day after the administration.
FIG. 3 intraperitoneal injection of antibody C140S can relieve abnormal immune response in transgenic mice.
A, taking peripheral plasma CD4 of transgenic animals+/CD8+The cell proportion is reduced compared with that of a wild mouse; b, C intraperitoneal Injection (INTRA)10mg/kg antibody C140S IPost-week, CD8 in peripheral plasma+A significant reduction in T cell number, CD4+/CD8+The ratio is significantly increased.
FIG. 4 intraperitoneal injection of antibody C140S can cross the blood brain barrier of transgenic mice. Coupling rhodamine serving as a fluorescent label with a C140S antibody, and performing intraperitoneal injection, wherein A, the diffusion degree of C140S in a mouse body is detected in a living body; and B, imaging after 4h, 5h and 6h of mice are subjected to cerebral hemorrhage respectively, and finding that C140S with a rhodamine label has diffused to the brain parenchyma at 4h and the amount of the medicine entering the brain is gradually increased along with the time increase.
FIG. 5 shows that p-alpha-syn in peripheral blood and brain parenchyma of transgenic mice is reduced and TH positive neurons in the brain of the mice are lost after brain localization injection of C140S. A, the number of TH positive neurons on the side of C140S injection is obviously larger than that on the side of control; b, performing stereotaxic injection of a commercially available alpha-syn antibody serving as a control antibody, and performing Western blot experiment by using C140S as an experimental group to extract a soluble protein of the tattoo after antibody drug treatment; c, D: as a result, the TH expression level of the TG group is obviously reduced compared with that of the WT group, the TH protein expression level of the group injected with the C140S antibody drug is obviously higher than that of the TG group, and the TH expression level of the control alpha-syn antibody group is not obviously different from that of the TG group.
FIG. 6 significant reduction of p-alpha-syn in striations of transgenic mice following brain-mapping injection of C140S. Respectively extracting soluble and insoluble protein components, and carrying out Western blot experiment; TG group had significantly less p-alpha-syn than TG group after treatment with C140S, whereas injection of alpha-syn antibody failed to reduce p-alpha-syn.
FIG. 7C 140S treatment ameliorated dyskinesias in transgenic mice
A. A 10-12 month old mouse is used for carrying out a rod rotating fatigue test, accelerated motion with the speed of 10-60r/min is carried out, the motion time is recorded, the rod rotating time of the TG mouse is found to be 136 +/-4.427 seconds and is obviously reduced compared with the rod rotating time of 166.2 +/-6.339 seconds of the WT group, and the rod rotating time of an animal subjected to intraperitoneal injection administration is 152.1 +/-5.123 seconds and is obviously different from that of the TG group, which indicates that the treatment effect is achieved; B. the open field experiment is carried out by using mice of 10-12 months old, normal mice fear a wide place and are willing to walk on the wall, after statistics, the ratio of the residence time of the mice in the TG group in the peripheral area to the total activity time is found to be about 0.81 +/-0.01 and is obviously lower than 0.98 +/-0.02 of the WT group, which shows that the avoidance performance of the mice in the TG group is changed, the avoidance performance is improved after the treatment of the antibody through the stereotaxic injection, the time ratio is 0.96 +/-0.03, and the increase is obvious compared with the increase of the mice in the TG group; C. using a 10-12 month old mouse to carry out a mesh drilling experiment to evaluate the strength of four limbs, scoring according to a score table, successfully drilling the mesh upwards for +3 minutes, grabbing the mesh for 10 seconds +2 minutes by the four limbs, grabbing the mesh for 10 seconds +1 minute by the double forelimbs, dropping the mesh into 0 minute within 10 seconds, and finding that the score of a TG group is 2.333 +/-0.4216 minutes after statistics, which is obviously lower than that of a WT group by 5 +/-0.4472 minutes, and the score of the abdominal cavity administration treatment group is 2.833 +/-0.4773 minutes, which is no difference compared with the TG group, so that the strength of the four limbs is not recovered; D. the coordination ability of the mice of 10-12 months old is evaluated by a pole-climbing experiment, and statistics show that the mice of the TG group take 16.23 +/-2.684 seconds when climbing from top to bottom, which is obviously higher than that of the WT group by 8.39 +/-1.438 seconds, and the mice of the intraperitoneal administration treatment group take 11.62 +/-1.596 seconds and have no difference compared with the TG group.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples. The cell lines used in the following examples are as follows:
thy-1- α -syn transgenic mice: purchased from Jackson Laboratory, usa, animal license number: 3377883HEK293T cell line: human embryo kidney cell (this chamber frozen)
Example 1:
1. indirect ELISA comparison of P-alpha-syn content in culture cell supernatants
(1) Preparation of related reagents
A. Coating liquid: CBS pH 9.6500 ml (Na2CO30.84g, NaHCO 31.45g, H2O500ml) was stored at 4 ℃.
B. Wash and diluent (mother liquor): PBST pH 7.41000 ml (10 ×) (NaH2PO 4.2H 2O 2.964.964 g, Na2HPO 4.12H 2O 28.998g, NaCl29.000g, H2O 1000 ml).
C.20% Tween-20100 ml (Tween-2020 ml, H2O 80ml), autoclaved, and stored at room temperature.
D. Washing and dilution (working solution): 50ml PBST pH 7.4,1ml Tween-20 constant volume 500 ml.
E. Sealing liquid: 5% BSA, 25ml (BSA1.25g, 25ml dilution, ready to use)
F. Substrate solution: OPD (O-phenylenediamine) -H2O2, phosphate-citric acid buffer 0.1M, and mixing 4.86ml with 5.14ml
G. Color developing agent (prepared as used): 10ml of F solution +4mg of OPD + 5. mu. l H were taken out2O2
H. Stopping liquid: concentrated H2SO420ml+H2O 80ml
(2) The experimental steps are as follows:
elisa plate coating: the antigen is sucked and added with the coating solution for dilution, 100 mu l of each hole is added into a 96-hole plate, and the mixture is kept overnight at 4 ℃;
B. washing the plate with 200. mu.l of washing solution for 5 minutes, 3 times, and patting dry;
C. sealing with a sealing liquid plate at 200 μ l/hole at 37 deg.C for 2 hr, and washing the plate for 3 times;
D. adding 100 μ l of diluted C140S antibody;
e.37 ℃ after 2 hours of incubation, wash the plate 3 times;
F. adding 100 μ l HRP-labeled anti-mouse IgG, incubating at 37 ℃ for 1 hour, washing the plate 3 times,
G. adding substrate color developing agent, developing for 30 minutes at 37 ℃; adding a stop solution;
H. absorbance values at 490nm were determined with a microplate reader (Victor 3).
2 extraction of alpha-syn-luciferase-C and alpha-syn-luciferase-N plasmids
(1) Preparing a reagent:
LB liquid culture Medium configuration
Tryptone | 10g |
Bacterial yeast extract | 5g |
NaCl | 10g |
Deionized water | 950ml |
Adjusting pH to 7.0 with 5M NaOH, adding deionized water to desired volume of 1L, sterilizing at 122 deg.C under high pressure for 20min, cooling, adding ampicillin (final concentration of 100 μ g/ml) or kanamycin (final concentration of 50 μ g/ml) in aseptic operation table, mixing, sealing, and storing at room temperature. Solid LB medium configuration and plating
Tryptone | 2g |
Bacterial yeast extract | 1g |
NaCl | 2g |
Agar for bacterial culture | 3g |
Deionized water to 200ml, autoclaving at 122 deg.C for 20min, cooling to 50 deg.C, adding ampicillin (final concentration of 100 μ g/ml) in sterile operating platform, mixing, pouring 5ml into sterile glass culture dish, air drying, and storing at 4 deg.C.
(2) The plasmid is transformed into a highly competent cell.
A. Mu.l of ligation reaction was gently added to DH 5. alpha. -E.coli (100. mu.l, removed from-80 ℃ freezer before use, thawed on ice) and gently mixed. Incubating on ice for 30 min;
B. and (5) placing the mixture in a water bath kettle at 42 ℃ for 60-90 s, and quickly placing the mixture on ice for 2min for standing.
C. Adding 400 μ l LB liquid medium without ampicillin resistance preheated to room temperature, placing into horizontal shaking table, shaking at 200rpm and 37 deg.C, and culturing for 40 min;
D. after shaking, 200ml of bacterial liquid is taken from the reaction tube, smeared on an ampicillin resistance LB culture plate preheated at room temperature and cultured for 12-16 h at 37 ℃.
(3) Plasmid extraction Using Wizard R Plus Midipreps DNApurification System kit
A. The bacterial liquid reserved for the correct sequencing group is plated on a culture dish, cultured for 12 hours at 37 ℃, randomly picked and single colony (independent and not crossed with other colony) is inoculated in LB culture medium which is added with 3ml ampicillin or kanamycin in advance to ensure good ventilation, and shaken for 12 hours at 37 ℃ and 250 rpm.
B. Adding 1ml of the bacterial liquid in the shake tube into 100ml of LB culture medium containing ampicillin or kanamycin, shaking at 37 ℃ and 250rpm for 12-16 h, mixing 1ml of the bacterial liquid in each group with glycerol (mixing according to a ratio of 4: 1), keeping the bacterial strain, and using the rest of the bacterial liquid for plasmid extraction.
C. Pouring the bacteria liquid into a 50ml large centrifuge tube, centrifuging at 4 ℃ and 10000g for 10min to enrich bacteria, discarding supernatant, reversely buckling the centrifuge tube on absorbent paper, and repeating twice to collect bacteria.
D. Adding 3ml of Cell reuse Solution, completely resuspending the bacteria Solution, then adding 3ml of lyssolution, and turning upside down and mixing uniformly for 4 times until the bacteria Solution is clear. Adding 3ml of neutrallization, reversing the upside down, mixing uniformly for 4 times, standing for 5min, centrifuging at 4 ℃ for 15min at 14,000 g for 15min, centrifuging once again under the same conditions if the precipitate is not firmly attached, sucking the supernatant and transferring to another sterile centrifuge tube (avoiding sucking white precipitate). Add 10ml of the fully mixed resin (DNA purification resin), reverse 3 times, stand for 2min, repeat 2-3 times. Transferring the mixture into midicolumn, sucking in vacuum under negative pressure, pouring 15ml of the mixture, continuously adding the mixture until the mixture completely passes, adding 15ml of Column Washing Solution, and Washing once again. The cut midicolumn was centrifuged at 12,000 g for 2min in a sterile EP tube in order to remove the remaining Washing Solution in the midicolumn.
Placed midicolumn in another sterile EP tube, added 150. mu.l of 60 ℃ preheated sterile deionized water, placed in 60 ℃ to incubate for 2min, centrifuged at 12,000 g for 2min, and the first batch of plasmid was collected. Then placing midicolumn in another sterile EP tube, adding 150 μ l of preheated sterile deionized water, placing in 60 ℃ and incubating for 2-3min, centrifuging for 2min at 12,000 g, collecting a second batch of plasmid, and centrifuging for 5min at 12,000 g of each collected tube sample. E. The concentration of the plasmid was determined by Nanodrop and the resulting plasmid was stored at-20 ℃ for cell transfection.
HEK-293T cells transfected with alpha-syn-luciferase-C and alpha-syn-luciferase-N plasmids
A.90nm Petri dish was inoculated with 1X106The number of the individual cells is one,
B. mu.l of OPTI-MEM was added to the transfection tube, and 20. mu.l of the transfection reagent Lipofectamine TM2000 was added thereto while shaking, and allowed to stand at room temperature for 5 min. 375. mu.l of OPTI-MEM was added to the transfection tube, and 12. mu.g of the target plasmid was added thereto, followed by shaking and standing at room temperature for 5 min.
C. The two liquids were mixed and gently shaken and allowed to stand at room temperature for 20 min.
D. The intracellular medium was aspirated, the cells were washed 3 times with OPTI-MEM to remove serum, and then 7.5ml of OPTI-MEM was added to each dish.
E. Adding the mixture of plasmid and transfection reagent into a culture dish, placing at 37 ℃ and 5% CO2And (5) incubating for 6h in the incubator, replacing with a normal culture medium, and treating the cells for a corresponding time.
P-alpha-syn Transmission and blocking experiments
(1) Experimental reagent: LuciferaseN and LuciferaceC terminal plasmids (constructed in the same room), Coelenterazine (BD Co.)
(2) The Luciferase is a protein which generates autofluorescence under the stimulation of coelenterazine, and the N end and the C end of the Luciferase are respectively connected with alpha-syn to construct plasmids which are transfected into HEK293T cells.
(3) After 24h, the cell supernatant of the Luciferase N-alpha-syn transfected cells was collected, concentrated 10-fold using a concentration tube, and then added to the cell culture dish of the Luciferase C-alpha-syn transfected cells, and C140S antibody was added as a blocking drug to the treatment group.
(4) After 24h coelenterazine was added and the autofluorescence intensity was observed in a microplate reader.
Example 2
1. CD4 in plasma of transgenic mice+,CD8+T cell content detection
(1) Blood is taken from inner canthus of the mouse, 3 times volume of erythrocyte lysate is added, 2,000g of erythrocyte lysate is centrifuged for 15min, then the supernatant is discarded, 2 times volume of erythrocyte lysate is added, 2,000g of erythrocyte lysate is centrifuged again for 15min after heavy suspension, and the supernatant is discarded to retain the precipitate.
(2) Live dead stain, add 0.1. mu.L of Zombie Green TM Dye to each tube, shake and mix, incubate 15min at room temperature in the dark, Wash Buffer solution, centrifuge 5min at 1500 g, discard the supernatant.
(3) Blocking Fc receptors: adding 1 mu g/10 of the mixture into each tube6The Anti-Mouse CD16/CD32 antibody of the cells blocks non-specific staining caused by the fluorescent antibody Fc receptor. Incubate at 4 ℃ for 30min with shaking.
(4) The corresponding surface-stained antibody was added to each tube and incubated for 30min at 4 ℃ with shaking in the dark.
(5) Adding 100 μ L of fixing solution, and fixing for 30 min.
(6) Cells were washed with 200. mu.l of permeabilization solution and centrifuged at 600g for 5min at room temperature.
(7) The cells were resuspended by adding 100. mu.l of permeabilization solution, and intracellular protein was stained by adding 1. mu.l of antibody at room temperature for 30 min.
(8) 200 μ l of the permeabilization solution was added, centrifuged at 600g for 5min at room temperature, and the supernatant was discarded.
(9) PBS is washed once, and 1ml PBS is added into the sediment for heavy suspension, and the sediment is detected on a machine.
2. Small animal in vivo imaging experiment
(1) An experimental instrument: kodak small animal imager, depilatory cream (gentamicin).
(2) Rhodamine excitation light is 590nm, connecting rhodamine and C140S.
(3) C140S with a rhodamine tag was intraperitoneally injected into mice, then the mice were anesthetized with 1.5ml of 10% chloral hydrate, abdominal hair loss on the chest avoided affecting imaging, and then the mice were fixed on a photographic plate.
(4) The mouse profile was first saved using the X-ray function of the in vivo imager and then adjusted to the appropriate excitation and emission light for one scan at 1 hour intervals.
(5) In order to avoid the shielding effect of the skull, after administration for 4h, 5h, 6h and 7h, the mice in each group are perfused with physiological saline, brain tissues are taken out for imaging, and the condition that the medicine enters the brain parenchyma through the blood brain barrier is observed. 3, detecting the concentration of C140S in the plasma of the transgenic mice:
A. wrapping a plate: diluting ovalbumin with CBS, adding each 100 μ l of the diluted solution into a 96-well plate, and standing overnight at 4 ℃;
B. washing the plate with 200. mu.l of washing solution for 5 minutes, 3 times, and patting dry;
C. adding 100 ul of diluted C140S serum protein with 100 ul of biotin tag;
d.37 ℃ incubation for 2 hours, plate washing for 3 times;
E. adding alkaline phosphatase-labeled anti-mouse IgG diluted by D solution 1:10000 into the solution, standing the solution at 37 ℃ for incubation for 1 hour in a dark place, wherein each well contains 100 mu l of the solution;
F. washing for 2 times;
G. adding 100 mul of pNPP into each hole, standing at 37 ℃ and developing for 1 hour;
H. the absorbance values at 405nm were read on a PerkinElmer Viktor 3 plate reader set to light for 0.1 second per well.
I. Absorbance values at 490nm were determined with a microplate reader (Victor 3).
4, extracting protein components of transgenic mice
(1) 40mg of fresh brain tissue was minced and placed in a 1.5ml EP tube, and 200. mu.l of RIPA lysate (containing protease inhibitor and phosphatase inhibitor) was added and the whole procedure was performed on ice.
(2) The tissue was ground to a homogenate using a homogenizer and then sonicated under a sonicator.
(3) Lysis was performed on ice for 30 min.
(4) The lysate is centrifuged at 4 ℃ for 30min at a rotation speed of 12,000 g, and the supernatant is collected as a soluble protein fraction and precipitated as an insoluble protein fraction.
(5) After the supernatant was assayed for protein concentration, loading buffer was added and denatured at 95 ℃ for 10min and stored at-20 ℃.
(6) Adding 10M urea into the precipitate, mixing, and crushing under an ultrasonic instrument.
(7) After the insoluble protein is dissolved, directly adding loading buffer to denature for 10min at 95 ℃ and storing at-20 ℃.5 transgenic mice were stereotactically injected with antibody C140S and a commercially available alpha-syn antibody.
(1) The 10-month-old transgenic mice are injected with 0.1ml of 10% chloral hydrate in the abdominal cavity, so that shallow anesthesia is guaranteed, and the survival rate after operation is improved.
(2) The mice were fixed on a stereotaxic apparatus and hair at the top of the mouse head was removed with a razor blade.
(3) Making a longitudinal incision on the top of the mouse head by using a scalpel, exposing bregma, adjusting the position of the microinjector to make a positioning mark by using an oil pen, wherein bregma is +0.2mm and the midline is +2.0 mm.
(4) The surgical instrument was cleaned with medical alcohol and saline and a dental drill was used to drill a hole in the marked site.
(5) The microinjector was lowered to the tunnel position, zeroed and then inserted 2.6mm, the right tattoo. (6) Mu.l of antibody was injected at a rate of 0.1. mu.l per minute, and the needle was left for 10min after injection.
(7) After removing the needle, the wound was cleared and sutured, and the mice were observed for warmth and vital signs.
6: animal behavioural testing
(1) Fatigue test of rotating rod
In order to keep the body balance of the mouse on the rotating rod and prevent the mouse from falling down, the mouse needs to move in the opposite direction of the rotating rod and keep the muscles of the four limbs in coordination, otherwise, the mouse falls off from the rod, and the mouse with dyskinesia or poor coordination can fall down more quickly. Through statistical analysis of quantitative indicators of the fall time of the mice, the dyskinesia or disorder relief condition of the mice can be evaluated. In order to evaluate the endurance of the mouse, the initial speed of the rotarod fatigue tester is set to be 10rpm/min, the acceleration is set to be 9.0, the maximum speed is set to be 60rpm/min, the mouse continuously records test data on the rotarod for 6 times, the test is repeated for 3 days, and then statistics is carried out.
(2) Grip strength test of drilling net
In order to evaluate the grip strength and the coordination ability of the mouse, the caliber of each hole of the used wire gauze is 5cm x 5cm, the mouse is placed under the wire gauze upside down, the falling time of the mouse is recorded, if the mouse drills the wire gauze upwards, the grip strength and the coordination ability are better, and the wire gauze can be used as an adding item during statistics.
(3) Pole climbing experiment
In order to evaluate the four-limb coordination ability of the mouse, the mouse is placed at one end (top end) of a spherical metal rod with the height of 60cm, the time of the mouse climbing from the top end to the ground is recorded, a formal experiment is started after three days of training, and data statistics is carried out.
(4) Open field experiment
The open field experiment is a common mouse ethology experiment, can detect the spontaneous activity behavior and exploration behavior of a mouse, is based on the principle that animals are afraid of the nature of an open field, and the activity of the animals is evasive, and on the other hand, the animals can generate curiosity to explore a new place in the face of new things; in order to evaluate the autonomous movement and avoidance of the experimental animal in a new environment, the animal is placed in the center of the bottom surface in the box, and the shooting and timing are carried out simultaneously. And stopping shooting after observation for 5min, cleaning the inner wall and the bottom surface of the square box to prevent the information remained by the animal at the last time from influencing the next test result, replacing the animal, continuing the experiment, and recording the distance of the mouse edge extending movement and the time of searching the middle part for statistics.
Claims (10)
1. The application of the monoclonal antibody in preparing the medicine for treating the Parkinson's disease is that the monoclonal antibody (C140S) is used for resisting serine 129-site phosphorylated alpha synuclein (p-alpha-synuclein, p-alpha-syn).
2. The use according to claim 1, wherein the monoclonal antibody is of the type: IgG, this antibody subtype is: IgG2 b.
3. The use according to claim 1, wherein the medicament is in any form of an ingestible preparation.
4. The use according to claim 1, wherein the formulation is in a form for parenteral administration.
5. The use according to claim 1, wherein the formulation is in the form of an injectable dosage form.
6. The use according to claim 1, wherein said use is such that said antibody blocks p-a-syn transmission from cells to cells.
7. The use of claim 1, wherein said use is that said antibody can pass the blood-brain barrier into the brain parenchyma at 6 hours.
8. The use of claim 1, wherein the use is that the antibody can improve the ratio inversion of CD4+/CD8+ T cells, relieve the behavior disorder of transgenic mice, increase the rod-rotating residence time of the transgenic mice, increase the limb strength of the mice, and enhance the coordination ability of the transgenic mice.
9. The use according to claim 1, wherein said use is that said antibody neutralizes toxic proteins, reducing their cytotoxicity.
10. The use of claim 1, wherein the use is that the antibody can reduce the p- α -syn content in the brain of the transgenic animal, increase the TH content in the brain, and relieve the evasive disorder of the transgenic mouse, which indicates that the antibody C140S has neuroprotective effect and therapeutic effect on PD model.
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