CN110028583A - Anti- Tau antibody and its application in treatment Alzheimer disease, traumatic brain injury - Google Patents
Anti- Tau antibody and its application in treatment Alzheimer disease, traumatic brain injury Download PDFInfo
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Abstract
The object of the present invention is to provide anti-Tau antibody and its application in treatment Alzheimer disease, traumatic brain injury, the antibody can specifically bind Protein tau, can especially specifically bind the Protein tau of Hyperphosphorylationof；By giving the antibody, the expression of abnormally phosphorylated tau in Alzheimer disease mouse model can be delayed, prevention can be played or treat the effect of the relevant neurodegenerative diseases of Protein taus such as Alzheimer disease, traumatic brain injury.
Description
Technical field
The invention belongs to antibody art, in particular to anti-Tau antibody and its treatment Alzheimer disease, traumatic brain damage
Application in wound.
Background technique
Tau albumen is a kind of microtubule associated protein, can be divided into four regions, i.e. N-terminal prominent area, Pro-rich
Area, micro-pipe combined area and C-terminal, in the case of Tau protein normal in conjunction with micro-pipe, but by glycogen synthase kinase 3 β
(glycogen synthase kinase-3 β, GSK-3 β) catalysis can fall off after phosphorylation occurs from micro-pipe, cause micro-pipe steady
Qualitative decline or depolymerization；Balance between Tau protein phosphorylation and dephosphorylation is to maintain neuronal microtubules stability, guarantee carefully
Born of the same parents complete the premise of organelles normal transports in axoplasm such as mitochondria.
Traumatic brain injury (traumatic brain injury, TBI) is by accidentality or non-accidentality damage institute
Caused duration cerebral injury.Clinical manifestation includes the loss of consciousness, epileptic attack, specific positions, syncope, hemiplegia etc..In wound
Property cerebral injury research in discovery Protein tau Hyperphosphorylationof can cause microtubule depolymerization, Distribution of mitochondria exception and mitochondria
Dysfunction has eventually led to neuron transformation or apoptosis；Cis- Phosphorylated tau is nerve retrograde affection after cerebral injury
An early stage driven factor, can promote Protein tau pathology.
Mostly onset, onset secret, the course of disease are slow in one's old age for Alzheimer disease (Alzheimer disease, AD)
Time is long and irreversible, is one group of current cause of disease and indefinite brain primary retrograde degeneration's disease, and basic pathology is special
Point shows as that A β deposits the senile plaque to be formed, phosphorylated Tau protein deposits the neurofibrillary tangles to be formed and a large amount of cholinergics
The loss etc. of neuron.At present some researches show that can treat or assist in the treatment of Alzheimer disease by anti-Protein tau antibody,
It can play the role of alleviating progression of disease.
Based on above-mentioned cognition, it is considered as using the specifically extremely relevant disease of anti-Protein tau Antybody therapy Protein tau
One feasible measure.
Summary of the invention
The object of the present invention is to provide a kind of anti-Protein tau antibody, the antibody can specifically bind phosphorylation tau egg
White, the light-chain amino acid sequence of the antibody is as shown in SEQ ID NO:1, and heavy chain amino acid sequence is as shown in SEQ ID NO:2.
Further, the present invention provides a kind of coding nucleotide, can encode anti-Protein tau antibody.
Further, the present invention also provides a kind of recombinant expression carrier, the carrier includes above-mentioned coding nucleotide.
Further, the present invention also provides a kind of hybridoma, the hybridoma can secrete anti-Protein tau
Antibody.
The present invention also provides anti-Protein tau antibody in the relevant nervus retrogression of preparation treatment Hyperphosphorylationof Protein tau
Purposes in the drug of disease.
Further, the light-chain amino acid sequence of the anti-Protein tau antibody is as shown in SEQ ID NO:1, heavy chain amino
Acid sequence is as shown in SEQ ID NO:2.
Further, the neurodegenerative disease is Alzheimer disease, traumatic brain injury.
Further, the drug further includes pharmaceutically acceptable carrier.
The carrier is preferably excipient, stabilizer.
Beneficial effect
Anti- Protein tau antibody of the invention can specifically bind the Protein tau of phosphorylation, can significantly improve A Er ratio
The symptom of the relevant neurodegenerative diseases of Hyperphosphorylationofs Protein tau such as the silent disease in sea.
The present invention is the relevant nervus retrogressions of Hyperphosphorylationofs Protein tau such as A Erbihaimo disease, traumatic brain injury
The prevention and/or treatment of disease provide a kind of new effective way.
Detailed description of the invention
Mice serum titre after Fig. 1: PHF-tau protein immunization.
Fig. 2: anti-tau antibody specificity western blot testing result, wherein PHF-tau is Phosphorylated tau,
Tau is non-phosphorylating Protein tau.
Fig. 3: the affinity of antibody E2 and PHF-tau albumen.
Fig. 4: the monoclonal antibody E2 therapeutic effect to Alzheimer disease mouse.
Specific embodiment
The present invention is described below in more detail to facilitate the understanding of the present invention.
It should be understood that the term or word used in the specification and in the claims is not construed as having
The meaning limited in dictionary, and be interpreted as having on the basis of following principle and its meaning one in the context of the present invention
The meaning of cause: the concept of term can suitably limit best illustration of the invention by inventor.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition such as Sambrook et al.,
Molecular cloning: condition described in laboratory manual, or according to the normal condition proposed by manufacturer.
Embodiment 1: anti-Protein tau antibody hybridoma preparation
Disease occurs in the nervous system diseases such as traumatic brain injury, Alzheimer disease in order to obtain to specifically bind
The anti-Protein tau antibody of the Protein tau of change selects to prepare anti-tau egg as antigen with the closely related PHF-tau of morbid state
Bai Kangti.
The PHF-tau albumen is prepared by known method and is purified, i.e. the skin of selection patients with Alzheimer disease after death
Matter tissue, using tissue homogenizer with 1000rpm by 5mg volume cortex 8-10 times of volume cold buffer liquid H (10nM Tris,
800mM NaCl, ImM EGTA and 10% sucrose, pH 7.4) in homogenization, the material of homogenization in supercentrifuge with
27000g is centrifuged 20 minutes.It discards agglomerate and adjusts supernatant to 1% (W/V) N- Hamposyl L and 1% (V/V) 2- mercapto
The ultimate density of base ethyl alcohol, and incubated 2 hours at 37 DEG C.Then by supernatant with 108000g in Ultracentrifuge
It being centrifuged 35 minutes at 20 DEG C, agglomerate is carefully washed in PBS and is suspended in PBS, supernatant is centrifuged second as mentioned,
And final agglomerate is dissolved and obtained the PHF-tau albumen of purifying, equal part and is freezed at -80 DEG C.
Mouse immune
6-8 weeks female BAl BIc/c mouse 6, picric acid dye marker are taken, docking takes blood to prepare basal serum.
First immunisation:
PHF-tau protein solution (0.4mg/ml) is mixed with isometric Freund's complete adjuvant, piping and druming is mixed to complete repeatedly
Full milk, by the dosage of every 200 μ l of mouse, intraperitoneal injection.
Booster immunization:
Every 2 weeks after initial immunization, PHF-tau protein solution is mixed with isometric incomplete Freund's adjuvant, is blown repeatedly
It beats and mixes to complete emulsification, by the dosage of every 100 μ l of mouse, intraperitoneal injection.
Impact is immune:
Add up after being immunized 8 times, with the mode of intrasplenic injection, carries out booster immunization with PHF-tau albumen, dosage is 25 μ g/
Only.Method is: being first anesthetized with ether mouse, is placed on the right side of table top and crouches, the left flank, that is, part ridge of exposure disinfects in alcohol
Afterwards, sterile working abdominal cut skin exposure peritonaeum, finds spleen through peritonaeum, with 1ml syringe by PHF-tau protein solution
It is slowly injected into spleen across peritonaeum extending shaft inserting needle, injection finishes rear enclosed notch, and spleen cell is taken to carry out cell after three days
Fusion.
Hybridoma preparation:
Myeloma cell (SP2/0) is passed on and expands culture, when fusion, is gently blown down cell from bottle wall with dropper, is received
It combining in centrifuge tube, 1000rpm is centrifuged 5-10min, discards supernatant, and 30ml1640 culture medium is added, and it is primary with method centrifuge washing,
Then Cell resuspension is mixed in 1640 culture medium of 10ml, takes myeloma cell's suspension, it is spare after cell count.
The BALB/c mouse being immunized is taken, extracts eyeball bloodletting, and separate positive control of the serum as antibody test when
Serum takes mouse spleen by routine operation and peels off surrounding connective tissue, and spleen is placed in and fills the flat of incomplete culture medium
In ware, spleen is gently squeezed on nylon wire and obtains Single-cell suspensions.Splenocyte suspension is harvested, 1000rpm is centrifuged 5-
10min cannots be used up complete 1640 culture medium centrifuge washing 2 times, cell is then resuspended in 10ml incomplete culture medium and is mixed, is taken
State suspension make it is spare after viable count.
It prepares and cultivates feeder cells within 1 day before cell fusion.
By 1 × 108Splenocyte and 2 × 107Myeloma cell SP2/0 is mixed in a centrifuge tube, adds 1640 culture mediums
It to 30ml, mixes well, 1000rpm is centrifuged 5-10min, and supernatant is exhausted as far as possible, and tap fusion pipe bottom keeps sedimentation cell loose
It dissipating uniformly, sets in water-bath and preheat, be added dropwise in 1min with 1ml suction pipe and be preheated to 37 DEG C of PEG 1ml, side edged shakes gently,
1-2min is stood, 20-30ml is slowly added dropwise in 2-3min with 1Oml suction pipe and is preheated to 37 DEG C of incomplete 1640 culture medium extremely
Terminate reaction.1000rpm is centrifuged 5min, discards supernatant, and 2 × HAT of 40ml culture medium is added, gently pressure-vaccum sedimentation cell, makes it
It suspends and mixes, have been added in 96 well culture plates of feeder cells and cultivate on the day before being inoculated in, every hole 0.1ml then will training
Feeding plate is placed in 37 DEG C, 5%CO2Culture in incubator is swapped out 1/2 culture medium after 5 days with 2 × HAT culture medium, and later every 3 days with 2
× HAT culture medium swaps out 1/2 culture medium, is swapped out HAT culture medium after 14 days with HT culture medium, common culture completely can be used after 21 days
Base.
Positive colony screening and subclone
Growth of Hybridoma Cell situation is observed, culture supernatant is sucked out when its length to 1/5 or more hole floor space and passes through directly
ELISA method detects positive colony, i.e., the rabbit-anti being conjugated with coated 96 orifice plate of PHF-tau albumen and horseradish peroxidase is small
The positive colony that screening obtains is subcloned by mouse IgG screening positive clone by limiting dilution assay.
The preparation and purification of monoclonal antibody
It is prepared using conventional monoclonal antibody preparation and purification method with BALB/c mouse tail experimental animal and obtains Dan Ke
Grand antibody.
Utilize the specificity of Western blot measurement monoclonal antibody.
With PHF-tau protein competition binding antibody measure affinity constant: monoclonal antibody first with various concentration
PHF-tau albumen is incubated for, and the initial concentration of monoclonal antibody is 400ng/ml (2.6 × 10 in reaction system-12Mol/l), PHF-
The initial concentration of Protein tau forms 7 reaction systems, is added to reaction solution after incubation from 100 μ g/ml doubling dilution down
It has been coated in the ELISA Plate of PHF-tau antigen, every 100 μ l of hole, board-washing 3 times after incubation, the rabbit of 100 μ l HRP label is added in every hole
Anti- mouse that health solution, 37 DEG C of reaction 1h, board-washing 3 times, is added terminate liquid after tmb substrate colour developing 10min is added, measures OD450 value
The antigen binding rate for calculating each reaction system calculates affinity constant.
As the result is shown:
Immune mouse is injected intraperitoneally in PHF-tau albumen, the serum antibody titer after initial immunity can reach 1:
4000, antibody titer can reach 1: 8000 after being immunized three times, and nine times immune rear IgG antibody titre can reach 1: 528000,
Show to obtain good immune effect, the result is shown in Figure 1 using PHF-tau protein immunization mouse.
After positive colony screens, total screening obtains 5 plants can be with the hybridoma of the anti-PHF-tau protein antibodies of stably excreting
Cell strain is respectively designated as A2, A4, B8, C6 and E2, above-mentioned 5 strain of hybridoma strain equal energy after continuous passage culture 2 months
Enough stably excreting antibody, and culture supernatant potency is all larger than 1: 1000.
Western blot detection shows in the monoclonal antibody of above-mentioned 5 strain of hybridoma strain secretion that E2, B8 and A2 have
There is preferable PHF-tau protein binding specificity, and the ability of C6 and A4 specific binding PHF-tau albumen is poor, as a result sees
Fig. 2.
Affinity costant measurement is carried out to antibody E2, B8 and A2, its affinity costant is in 2-8 × 10 as the result is shown-8Mol/1 it
Between, wherein the affinity constant of monoclonal antibody E2 is 7.89 × 10-8Mol/l (R2=0.9938), demonstrating the need for antigen excess could be right
Antibody forms Reverse transcriptase, as a result sees Fig. 3.
Embodiment 2: the building of Alzheimer disease model
In order to study effect of the anti-Protein tau antibody to Hyperphosphorylationof Protein tau under physiological status, A Erci is constructed
The silent sick mouse model in sea.
After BALB/c mouse 2% yellow Jackets (40mg/kg) intraperitoneal injection of anesthesia, it is fixed on stereotaxic apparatus
On, calvarium midsection is exposed to skull, positions stereo spectrum according to mouse cranium brain, to substrate nuclear location, is opened with dental burr brill
Dry bones, with being slowly injected into A β in the miniature sample injector injection needle 5min of 1 μ l25-35It (is purchased in sigma with Iibotenicacid (IBO)
Company) 1 μ l of mixed liquor, the 10min that gives that let the acupuncture needle remain at a certain point, with local soft tissue close pinhole after injection, skin suture is postoperative to give
50,000 u intramuscular injection of penicillin sodium salt is given, 1 time daily, continuous injection 3 days.Construct successfully mouse Alzheimer disease model.Separately
Outside, blank group gives mouse basal nuclei under positioning and disposably injects 1 μ l sterile saline.
After mouse model constructs successfully, 3 mouse are chosen respectively by immunohistochemistry to Protein tau total in brain and phosphorylation
The accumulation optical density and optical density of Protein tau are measured and analyze.
As the result is shown: the accumulation optical density of the total Protein tau of model group and Phosphorylated tau is respectively 0.385 ±
0.015,0.339 ± 0.018, total Protein tau of blank group and the accumulation optical density of Phosphorylated tau be respectively 0.363 ±
0.011,0.114 ± 0.023, i.e. the accumulation optical density of the total Protein tau of model group and Phosphorylated tau is significantly higher than blank
Group shows model construction success.
Embodiment 3: effect of the anti-tau antibody to Phosphorylated tau in Alzheimer disease mouse
Based on previous embodiment as a result, selection monoclonal antibody E2, B8 and A2 reduces Alzheimer disease to study it
The effect of Phosphorylated tau in mouse.
If embodiment 2 constructs Alzheimer disease mouse model and naive mice model, 40 A Erbihaimo are chosen
Disease model mouse is randomly divided into 4 groups, every group 10, gives physiological saline, list by tail vein injections respectively as experimental group
Clonal antibody E2, B8 and A2 (500 μ g/ mouse)；It chooses 10 normal mouses and 10 blank group model mices passes through tail respectively
Physiological saline (500 μ g/ mouse), the free feeding of experiment mice and drinking-water are given in portion's intravenous injection.Experimental group, normal group and blank
Group is administered every other day respectively after being administered for the first time, is spaced 5 days and is administered after administration 5 times, then be administered 6 times, the 50th day after experiment starts
Mouse is put to death, brain is taken and separates hippocampal tissue.
Tau protein abnormal phosphorylation in Hippocampus of Mice is detected by western blot, i.e. extraction hippocampal tissue is total
Determining the protein quantity is carried out after albumen, quantitative albumen is subjected to PAGE gel electrophoresis, transferring film is exempted from after the completion of electrophoresis
Epidemic disease reaction, chemiluminescence, developing and fixing, molecular weight and net optical density using gel image analysis software analysis target stripe
It is worth and determines the content of Phosphorylated tau.
The results are shown in Table 2:
Compared with normal group,∞P < 0.01；Compared with model group,※P < 0.01
That is, the results show that unexpectedly, monoclonal antibody E2 can be played very relative to monoclonal antibody B2 and A2
The good effect for reducing Hyperphosphorylationof Protein tau in Alzheimer disease mouse, can be used for preventing and/or treating A Erci
The relevant neurodegenerative diseases of Protein taus Hyperphosphorylationof such as sea silent disease, traumatic brain injury.
Embodiment 4: anti-tau antibody influence ethological for Alzheimer disease mouse model
If embodiment 2 constructs Alzheimer disease mouse model, chooses 20 model mices and be divided into 2 groups, one group is given list
Clonal antibody E2 (500 μ g/ mouse), one group is given physiological saline (500 μ g/ mouse), carries out morris water maze laboratory.
Water maze laboratory are as follows: water maze is diameter 100cm, and the round pool of high 50cm, depth of water 30cm, water temperature is maintained at 26
± 1 DEG C, the place of entry of all directions four is indicated on pool wall, pond is divided into four quadrants, be referred to as first, second, third and fourth as
Limit, placing a diameter in first quartile center is 9cm, and the circular non-opaque platform of high 29cm, deck roof is lower than water surface 1cm, fan
The video camera being connected with computer is mounted with above palace, synchronous recording mouse movement track, labyrinth External reference object is protected during training
It holds constant.
Model mice and normal mouse are administered referring to embodiment 3, the 5th day beginning morris water after the completion of last time is administered
Maze experiment.Allow mouse free swimming 2min for first day the 1st time, first day the 2nd time beginning formal training, then training 2 daily
Secondary, totally 4 days, one place of entry of selection into the water towards pool wall by mouse observed and recorded mouse and finds and climb up when training
Route map, required time and the swimming rate of platform need to be led to platform if mouse fails to find platform in 120s,
It is allowed to stop on platform 20 seconds, data acquisition and processing (DAP) is completed by the labyrinth Morris image east monitoring processing system.
Experimental result is shown: the mouse quantity of monoclonal antibody E2 experimental group discovery platform is as test number of days gradually increases
Add, and the mouse quantity of physiological saline experimental group discovery platform is not shown with test number of days and increased trend, as a result
See Fig. 4, the above results show that the ability of learning and memory of monoclonal antibody E2 experimental mice has improvement compared with physiological saline experimental group,
Monoclonal antibody E2 can be used in preventing and/or treating the Protein taus Hyperphosphorylationofs such as Alzheimer disease, traumatic brain injury
Relevant neurodegenerative disease.
Embodiment 5: anti-tau antibody influence ethological for Alzheimer disease mouse model
Transfer to the raw work in Shanghai that monoclonal antibody is sequenced hybridoma E2, sequencing result shows monoclonal antibody E2's
Light-chain amino acid sequence is as shown in SEQ ID NO:1, and heavy chain amino acid sequence is as shown in SEQ ID NO:2.
Optionally, humanization processing can be carried out to monoclonal antibody E2 by antibody humanization's technology of this field routine,
It is excessive to allow it to the Protein taus such as the Alzheimer disease, the traumatic brain injury that are particularly suited for preventing and/or treating the mankind
The relevant neurodegenerative disease of phosphorylation.
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
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Claims (8)
1. a kind of anti-Protein tau antibody or its antigen-binding portion thereof, it is characterised in that: the antibody can specifically bind phosphoric acid
Change Protein tau, and the light-chain amino acid sequence of the antibody is as shown in SEQ ID NO:1, heavy chain amino acid sequence such as SEQ
Shown in ID NO:2.
2. a kind of coding nucleotide, it is characterised in that: the encoding nucleoside acid encoding antibody described in claim 1 or its antigen
Bound fraction.
3. a kind of recombinant expression carrier, it is characterised in that: the carrier includes coding nucleotide as claimed in claim 2.
4. a kind of hybridoma, it is characterised in that: the hybridoma is carried comprising recombinant expression as claimed in claim 3
Body.
5. anti-Protein tau antibody is in the drug of the relevant neurodegenerative disease of preparation treatment Hyperphosphorylationof Protein tau
Purposes, it is characterised in that: the light-chain amino acid sequence of the anti-Protein tau antibody is as shown in SEQ ID NO:1, heavy chain amino
Sequence is as shown in SEQ ID NO:2.
6. purposes according to claim 5, it is characterised in that: the neurodegenerative disease is Alzheimer disease, wound
Wound property cerebral injury.
7. purposes according to claim 5, it is characterised in that: the drug further includes pharmaceutically acceptable carrier.
8. purposes according to claim 5, it is characterised in that: the carrier is preferably excipient, stabilizer.
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| Publication Number | Publication Date |
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| CN110028583A true CN110028583A (en) | 2019-07-19 |
| CN110028583B CN110028583B (en) | 2020-09-11 |
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| CN110679549A (en) * | 2019-11-05 | 2020-01-14 | 南通大学 | Construction method of Alzheimer disease mouse model |
| CN113005096A (en) * | 2021-01-19 | 2021-06-22 | 华中科技大学 | Hybridoma cell strain secreting anti-serine phosphorylation tau protein monoclonal antibody |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110679549A (en) * | 2019-11-05 | 2020-01-14 | 南通大学 | Construction method of Alzheimer disease mouse model |
| CN110679549B (en) * | 2019-11-05 | 2021-08-20 | 南通大学 | Construction method of Alzheimer disease mouse model |
| CN113005096A (en) * | 2021-01-19 | 2021-06-22 | 华中科技大学 | Hybridoma cell strain secreting anti-serine phosphorylation tau protein monoclonal antibody |
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