CN1329413C - Antibody for treating or preventing senile dementia, its expression vector and application in drug preparation - Google Patents
Antibody for treating or preventing senile dementia, its expression vector and application in drug preparation Download PDFInfo
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- CN1329413C CN1329413C CNB200610037930XA CN200610037930A CN1329413C CN 1329413 C CN1329413 C CN 1329413C CN B200610037930X A CNB200610037930X A CN B200610037930XA CN 200610037930 A CN200610037930 A CN 200610037930A CN 1329413 C CN1329413 C CN 1329413C
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Abstract
The present invention discloses an antibody for treating or preventing senile dementia, and an expression carrier and the application thereof for preparing medicines. The amino acid sequence of the antibody is shown as SEQ ID No. 2; a nucleotide sequence coding the antibody is shown as SEQ ID No. 1. The antibody is the specific antibody of beta-amyloid polypeptides, and the antibody and the nucleotide and the expression carrier thereof can be used for preparing medicines for carrying immunotherapy or preventing senile dementia.
Description
Technical field
The invention belongs to the genetically engineered field, relate to the antibody of a kind of treatment or prevention senile dementia, the invention still further relates to expression vector and their application in pharmacy of this antibody.
Background technology
Senile dementia, claim again alzheimer's disease (Alzheimer ' s disease, be a kind of nervus retrogression illness of serious harm human health AD), be one of modal form of senile dementia.AD patient mainly shows as hypomnesis, cognitive disorder and spatial discrimination disappearance, in the terminal stage of a disease even motion occurs and symptoms such as sensory disturbance, epileptic seizures.At present whole world AD number of the infected is up to 1,200 ten thousand, and presents the situation that increases year by year.The method for the treatment of AD at present clinically mainly contains five kinds: the one, and neuroprotective makes it to avoid damage; the 2nd, use the disappearance that anticholinesterase prevents to roll into a ball because of the brain kernel the caused vagusstoff of cell obstacle; the 3rd, the application of non-drug intervention and psychotropic is to improve behavior disorder; the 4th, health care activity, the 5th, to patient's care.Improving patient symptom yet all these methods are, is not etiological treatment.
Typical A D pathology is masked as a large amount of senile plaques and is deposited in the brain.The main component of this senile plaque is beta-amyloid polypeptide 1-(A β), forms by 40～42 amino acid, and be the enzymolysis product of beta-amyloyd precursor protein (APP), wherein with A β
42Have more cytotoxicity.Currently think that the formation of A β and accumulating in the AD pathogenic process plays a crucial role.After A β generates and deposits, but Secondary cases causes generation, oxidation and peroxidatic reaction of lipid, L-glutamic acid source heat toxicity, the inflammatory reaction of neurofibrillary tangles and the cascade reaction that activates apoptotic cell death.All these secondary responses have further amplified the toxic action of A β, so also become the potential target spot of anti-AD nosetiology therapy.But apparent, the real effectively target of treatment AD should be the A β that reduces in the brain.
1999, the people such as Schenk of U.S. Elan company took the lead in reporting, directly injected A β in AD model mice body
1-42, can reduce blood plasma amyloid beta level, suppress A β and be deposited on the existing patch, remove the old patch in the brain.There is report to confirm later on again successively, improves cognitive effect with alleviating maincenter amyloid load behind the solubility A β immune mouse, also having simultaneously.Elan company has developed new generation vaccine AN-1792 rapidly, is applied to AD moulding animal, and effect is remarkable, and the beginning clinical experiment.But in II phase clinical experiment, serious central nervous system aseptic inflammation has appearred in 6% patient, and the test of AD vaccine therapy is forced to stop.Postmortem confirms that the AME master due to the vaccine therapy is by the cell-mediated autoimmune response result of T.
It is generally acknowledged that the therapeutic action behind the A β inoculation body mainly is achieved by the function served as bridge of antibody: exogenous A β can stimulate body to produce specific antibody, the latter combines with A β and forms immune complex, and enter cell by monokaryon-scavenger cell Fc acceptor, make A β obtain degraded.Since it is so, can not inject A β and import A β antibody to reach same result of treatment to body? since two thousand, the transgenic mice of several research groups with anti-amyloid beta antibodies injection overexpression APP successively arranged, the result all finds obviously to alleviate A β load, make the amyloid patch in the brain obtain removing, and no matter be that peripheral intravenous injection or peritoneal injection can both be received positive effect.
Yet mouse source property monoclonal antibody is used in existing report more, is a kind of foreign protein to human body, can cause that people's body immune system produces human antimouse antibody (HAMA), and this has limited its application in human body to a great extent.Because HAMA not only can make the reduction of tiring of antibody, increase the clearance rate of mouse source property monoclonal antibody, influence its result of treatment, more seriously can cause transformation reactions, threaten patient's life security.
In recent years, along with the development of Protocols in Molecular Biology, gene engineering method is that the preparation research of antibody has been opened up a brand-new field, Here it is genetic engineering antibody.Be based upon the revolutionary character progress that appearance that round pcr and phage surface present the phage antibody library technique on (phage display) technical foundation then can claim the genetic engineering antibody field, this technology replaces B cell clone expressing antibodies with bacterial clone, without cytogamy, even, just can not prepare comparatively easily at any antigenic humanized's antibody molecule through immunity.It makes the acquisition of humanized's antibody of high specific, high-affinity become possibility.The antibody molecule of Huo Deing is humanized's a antibody in this way, can be directly used in human body therapy, reduces issuable toxic side effect; Simultaneously less because of its molecular weight again, be easy to penetrate various biological barriers (as hemato encephalic barrier), can arrive diseased region and directly play a role.
Summary of the invention
The antibody that the purpose of this invention is to provide a kind of treatment or prevention senile dementia.
Another object of the present invention provides the expression vector of above-mentioned antibody.
Another object of the present invention provides this antibody, the Nucleotide of this antibody of encoding and the application of their expression vector in pharmacy.
The present invention selects A β as target; use phage antibody library technique, screening obtains the specific phage antibody of anti-A β, and expresses acquisition solubility scFv antibody fragment; and identify its antibody activity, its biological activity of external evaluation and cytoprotection with the method for Western blot.Anti-amyloid beta antibodies and gene fragment and expression vector that screening obtains, the novel drugs that can be used for preparing immunotherapy or prevent senile dementia.
The objective of the invention is to realize by following measures:
The antibody of a kind of treatment or prevention senile dementia, the aminoacid sequence of this antibody is as described in the SEQ ID No.2.
Described antibody is monoclonal antibody.
Described antibody is solubility scFv antibody fragment.
The expression vector of described antibody includes but not limited to plasmid, phagemid, phage, intestinal bacteria.
The encode Nucleotide of above-mentioned described antibody, its sequence is as described in the SEQ ID No.1.
The expression vector of described Nucleotide includes but not limited to plasmid, phagemid, phage, intestinal bacteria.
The application of described antibody in preparation treatment or prevention senile dementia medicine.
The application of described Nucleotide in preparation treatment or prevention senile dementia medicine.
The application of described expression vector in preparation treatment or prevention senile dementia medicine.
Beneficial effect of the present invention:
The present invention is by phage antibody library technique, and screening obtains humanized's single-chain antibody (aminoacid sequence such as SEQ ID NO.2) of a novel directed against amyloid-beta polypeptide (A β).This antibody can suppress and remove the gathering of beta-amyloid polypeptide 1-, the performance cytoprotection.This antibody can be used as a kind of therapeutic antibodies of senile dementia, is used to prepare the medicine for the treatment of senile dementia.The Nucleotide and their expression vector of this antibody of encoding also can be used for preparing the medicine for the treatment of senile dementia.
Description of drawings
Fig. 1 is the ELISA detected result of mono-clonal phage displaying antibody.
Fig. 2 is the anti-A β of the present invention soluble single-chain antibody SDS-PAGE result.
Wherein: M: the molecular weight of albumen standard; 1: cleer and peaceful pericentral siphon chamber extract on the nutrient solution behind the abduction delivering; 2: through the purifying protein composition of His-Trap affinity chromatography.
Fig. 3 is the anti-A β of the present invention soluble single-chain antibody Western blot result.
Fig. 4 is the electromicroscopic photograph of the anti-A β of the present invention soluble single-chain antibody to the influence of A beta peptide aggregation.
Wherein: A, the A β 40 of B:20 μ M hatched for 2 weeks at 37 ℃; C, D:20 μ M E3 single-chain antibody and 20 μ MA β 40 hatched for 2 weeks at 37 ℃ jointly; E, F:20 μ M BSA and 20 μ M A β 40 hatched for 2 weeks at 37 ℃ jointly.Bar(A，C，E)＝500nm，Bar(B，D，F)＝100nm。
Fig. 5 is that the anti-A β of ThT fluorescent quantitative measurement the present invention soluble single-chain antibody influences the A beta peptide aggregation.
Wherein: *: P＜0.05, with the control group fluorescent value comparison of each time point;
*: P＜0.01, with the control group fluorescent value comparison of each time point.
Fig. 6 is the electromicroscopic photograph of the anti-A β of the present invention soluble single-chain antibody depolymerization A beta.
Wherein: A: sophisticated A β 40 assembles fiber; B, C:20 μ M A β 40 fibers continue 37 ℃ separately and hatched 10 days; D:20 μ MBSA and 20 μ M A β, 40 fibers were hatched 10 days at 37 ℃ jointly; E, F:20 μ M E3 single-chain antibody and 20 μ MA β, 40 fibers were hatched 10 days at 37 ℃ jointly.Bar(A，B，D，E)＝500nm，Bar(C，F)＝1μm。
Fig. 7 is the cytotoxic effect that MTT measures A β under the anti-A β of the different concns soluble single-chain antibody condition
Wherein: *: P＜0.05, compare with control group; Δ: P＜0.05 is compared with A β 20 μ M treatment group.
Embodiment
The invention will be further elaborated by the following examples.
Preparation method and identification experiment:
(1), the amplification of people's phage antibody library:
1, with Griffin.1 phage display humanized single-chain antibody library (about 1 * 10
10The clone) be inoculated into 500ml2 * TY-AMP-GLU substratum, 37 ℃, the 220rpm shaking culture is to OD
600Be 0.5 (about 1.5-2h).
2, therefrom get 25ml nutrient solution (about 1 * 10
10Individual bacterium), with the superingection of M13K07 helper phage.Infection proportion is 1: 20 (bacterial count: the helper phage number).In 37 ℃ of water-baths, leave standstill 30min.
3,3300g, 4 ℃ of centrifugal 10min are with the resuspended precipitation of 30ml 2 * TY-AMP-KAN substratum.
4, bacterium liquid is added in 2 * TY-AMP-KAN substratum of the pre-temperature of 470 ml, 30 ℃, the 220rpm shaking culture is spent the night.
5,10800g, 4 ℃ of centrifugal 10min abandon precipitation.
6, collect supernatant, add PEG/NaCl (20% polyoxyethylene glycol-2.5M NaCl, the down with) solution of 1/5 supernatant volume, thoroughly mix back 4 ℃ and leave standstill 1h or longer time with the precipitation phage.
7,3300g, 4 ℃ of centrifugal 30min, throw out are resuspended in 40ml ddH20 and the 8ml PEG/NaCl solution.Thoroughly mix back 4 ℃ and leave standstill 20min or longer time.
8,3300g, 4 ℃ of centrifugal 10min inhale and abandon supernatant.
9, simply centrifugal, absorb remaining PEG/NaCl.
10, throw out is resuspended with 5ml PBS, divides to install in the Eppendorf pipe (being called for short the Ep pipe).
11,11600g, 4 ℃ of centrifugal 10min abandon bacterial debris and impurity, and supernatant is transferred in the new Eppendorf pipe.
12, phage supernatant short-term can be stored in 4 ℃.As the need long storage, then glycerol adding to final concentration is 15%, is stored in-70 ℃.
Phage titre is measured:
1, gets among the PBS that 1 μ l phage supernatant adds 1ml (1: 10
3)
2, get 1 μ l and transform 1ml exponential phase of growth
*(OD
600About 0.4-0.6) E.coli TG1,37 ℃ of water-bath 30min (1: 10
6)
3, get in 2 * TY substratum that 10 μ l add 990 μ l (1: 10
8)
4, get in 2 * TY substratum that 10 μ l add 990 μ l (1: 10
10)
5, get in 2 * TY substratum that 10 μ l add 990 μ l (1: 10
12)
6, from 1: 10
6, 1: 10
8, 1: 10
10, 1: 10
12Respectively get 50 μ l in the bacterium liquid of doubling dilution, be coated with the TYE-AMP-GLU flat board, 37 ℃ of overnight incubation.The bacterial clone number count next day to calculate phage titre.
*: phage/phagemid (phage/phagemid) can the infectious pili positive (F
+) intestinal bacteria, intestinal bacteria must be cultured to (OD exponential phase of growth at 37 ℃
600About 0.4-0.6), at this moment bacterium can produce sex fimbria, thereby possesses higher efficiency of infection.In the process of whole screening and evaluation, all need to prepare the intestinal bacteria of this state.
Preparation step is as follows:
1, intestinal bacteria are drawn the M9 flat board, and as seen 37 ℃ be cultured to bacterium colony.
2, choose mono-clonal from the M9 flat board, be inoculated in 5ml 2 * TY substratum, 37 ℃, the 220rpm shaking culture is spent the night.
3, next day, the bacterium liquid of absorption incubated overnight joins in fresh 2 * TY substratum with 1: 100 ratio, and 37 ℃ of shaking culture are to (OD exponential phase of growth
600About 0.4-0.6), can be used for the conversion of phage.
4, the bacterium liquid short period of time placed on ice before infecting and can strengthen infectious effect, if but surpass 30min, colibacillary sex fimbria will be lost and can not be infected.
(2), prepare secondary phage antibody library:
1, connect (one) 2 step, remaining 475ml nutrient solution continues 37 ℃ of shaking culture 2h.
2,3300g, 4 ℃ of centrifugal 30min are resuspended in bacterial precipitation in 10ml 2 * TY substratum and (contain 15% glycerine), are distributed into 10 Eppendorf pipes, every pipe 1ml.
3, secondary phage antibody library is stored in-70 ℃.Before reusing, identify the positive colony rate and detect phage titre with the method for PCR.
(3), screening A β 1-40 (promptly the A β that is made up of the 1-40 amino acids is called for short A β 40) Humanized antibody specific:
1, with glutaraldehyde method envelope antigen A β 1-40:
(1), handles Nunc immunity pipe 4h with the 100mmol/L sodium phosphate buffer (pH5.0) that contains 0.2% glutaraldehyde (V/V).
(2), manage 2 times with above-mentioned damping fluid washing Nunc.
(3), add 4ml 100mmol/L sodium phosphate buffer (pH 8.0) dissolved A β 1-40 (20 μ g/ml), 37 ℃ of reaction 3h.
(4), use 0.9%NaCl solution washing 2 times.
(5), in the Nunc pipe, fill it up with the PBS (MPBS) that contains 2% skim-milk, 37 ℃ of sealing 2h.
(6), the PBS washing is 3 times.
2, screening specificity humanized monoclonal antibody:
(1), whenever takes turns screening and in the Nunc pipe, add about 10
12T.u. the phage of (colony-forming unit after the transfection) is in 4ml2%MPBS.
(2), the Nunc pipe is placed on the rotary turnplate, continue upset 30min under the room temperature, vertically place again, 90min at least under the room temperature.Abandon unconjugated phage in the supernatant.
(3), the 1st take turns screening with PBS-T (containing 0.1%Tween-20) washing Nunc pipe 10 times, wash 10 times with PBS again.The 2nd takes turns and later on whenever takes turns screening all with PBS-T washing Nunc pipe 20 times, again with PBS washing 20 times.
(4), add the triethylamine of 1ml 100mmol/L, the Nunc pipe is placed continue upset 30min on the rotary turnplate with wash-out specificity bonded phage.The 2nd takes turns and later every the wheel in the screening, adds the pre-wash-out 10min of triethylamine of 1ml 100mmol/L earlier, abandons pre-elutriant, adds the triethylamine wash-out specificity bonded phage of new 1ml 100mmol/L.
(5), in the elution process, in the Ep pipe, prepare the Tris-HCl (pH7.4) of 0.5ml 1.0mol/L in advance, in order in fast and the special phage of wash-out.Phage short-term after the neutralization can be stored in 4 ℃ or be used for transfection E.coli TGl.
(6), get the E.coli TG1 of phage transfection 4.25ml exponential phase of growth of 0.75ml wash-out, 30min is left standstill in 37 ℃ of water-baths.
(7), get the titre that 100 μ l doubling dilutions are measured phage:
Get in 2 * TY substratum that 100 μ l add 900 μ l (1: 10)
Got in 2 * TY substratum that 100 μ l add 900 μ l (1: 10
2)
Got in 2 * TY substratum that 100 μ l add 900 μ l (1: 10
3)
Got in 2 * TY substratum that 100 μ l add 900 μ l (1: 10
4)
From 1: 10,1: 10
2, 1: 10
3, 1: 10
4Respectively get 50 μ l in the bacterium liquid of doubling dilution, be coated with the TYE-AMP-GLU flat board, 37 ℃ of overnight incubation.The bacterial clone number count next day to calculate phage titre.
(8), the bacterium 3300g that infects of residue, 4 ℃ of centrifugal 10min are with the resuspended bacterial precipitation of 500 μ l, 2 * TY substratum.
(9), 500 μ l bacterium liquid all are coated with the dull and stereotyped several piece of TYE-AMP-GLU, 30 ℃ of overnight incubation or to bacterium colony as seen.
(10), add 5-6ml 2 * TY (containing 15% glycerine) on the flat board, gently scrape all bacterium colonies, mixing with the glass sorter.
(11), therefrom get 100 μ l and add 100ml 2 * TY-AMP-GLU, detect its OD
600≤ 0.1.Residue bacterium liquid can be stored in-70 ℃.
(12) 37 ℃, the 220rpm shaking culture is to OD
600Be 0.5 (about 2h).
(13), therefrom get 10ml, with the superingection of M13K07 helper phage.Infection proportion is 1: 20 (bacterial count: the helper phage number).37 ℃ of water-bath 30min.
(14), 3300g, 4 ℃ of centrifugal 10min, precipitation is resuspended with 50ml 2 * TY-AMP-KAN substratum.30 ℃, the 220rpm shaking culture is spent the night.
(15), 10800g, 4 ℃ of centrifugal bacterium liquid 10min abandon precipitation.
(16), collect supernatant, add the PEG/NaCl of 1/5 volume, ice thoroughly mixes back 4 ℃ and leaves standstill more than the 1h.
(17) 10800g, 4 ℃ of centrifugal 30min, precipitation is resuspended in 40ml ddH
20 and 8ml PEG/NaCl solution in.Thoroughly mix back 4 ℃ and leave standstill 20min or longer time.
(18), 10800g, 4 ℃ of centrifugal 10min inhale and to abandon supernatant, and are simply centrifugal, absorb remaining PEG/NaCl.
(19), precipitation is resuspended with 2ml PBS, divide to install in 2 Ep pipes.The centrifugal 10min of 11600g abandons bacterial debris and impurity, and supernatant is transferred in the new Ep pipe.Wherein 1ml can be stored in 4 ℃.1ml can be used for the next round screening in addition.
(20), repeat above screening step, carry out five altogether and take turns " absorption-wash-out-amplification " enrichment and screen.
(4), the ELISA of mono-clonal phage displaying antibody identifies:
1, the preparation of mono-clonal phage antibody:
(1), with the 5th E.coli TG1 that takes turns phage transfection exponential phase of growth of wash-out in the screening, be coated with the TYE-AMP-GLU flat board, 37 ℃ of overnight incubation.
(2), get 96 porocyte culture plates, every hole adds 100 μ l, 2 * TY-AMP-GLU.58 colony inoculations of random choose are to 96 orifice plates from the flat board of incubated overnight, and 37 ℃, the 250rpm shaking culture is spent the night.
(3), get another 96 porocyte culture plate, every hole adds 200 μ l, 2 * TY-AMP-GLU, 2 blocks of plates of transferase 45 μ l to the respectively from the 1st each hole of plate.37 ℃, 250rpm shaking culture 1h.Plate 1 can glycerol adding be to final concentration 15% in every hole, and-70 ℃ frozen.
(4), again in every hole, add 25 μ l, 2 * TY-AMP-GLU, wherein contain 1 * 10
9The M13K07 helper phage of pfu carries out superingection.
(5), 37 ℃ leave standstill 30min, continue 37 ℃, 250rpm shaking culture 1h.
(6), the centrifugal 10min of 1800g, absorb supernatant.
(7), the precipitation in every hole uses 200 μ l, 2 * TY-AMP-KAN resuspended respectively, 30 ℃, the 250rpm shaking culture is spent the night.
(8), the centrifugal 10min of 1800g, get supernatant and be used for ELISA and detect.
ELISA detects:
(1), handles ELISA Sptting plate 4h with the 100mmol/L sodium phosphate buffer (pH5.0) that contains 0.2% glutaraldehyde (V/V).
(2), wash the ELISA Sptting plate 2 times with above-mentioned damping fluid.
(3), add in every hole 100 μ l, 20 μ g/ml with 100mmol/L sodium phosphate buffer (pH8.0) dissolved A β 1-40, add the same buffer dissolved in the every hole of control group, isocyatic BSA, 37 ℃ of reaction 3h.
(4), use 0.9%NaCl solution washing 2 times.
(5), fill it up with in every hole and contain 2%MPBS, 37 ℃ of sealing 2h.
(6), the PBS washing is 3 times.
(7), add 20 μ l mono-clonal phage supernatants respectively in every hole, reaction is 3 hours under the room temperature.
(8), clean 3 times with the PBS-T that contains 0.05%Tween-20.
(9), add the anti-M13 antibody 100 μ l of the horseradish peroxidase-labeled of 1: 4000 times of dilution in every hole, reaction is 3 hours under the room temperature.
(10) PBS-T cleans 3 times.
(11), add the tmb substrate liquid of 100 μ l in every hole, react 10min under the room temperature.
(12), every hole, the clear back of colour developing adds the sulfuric acid termination reaction of 50 μ l 1mol/L.
(13) light absorption value (A of the every hole 450nm of mensuration
450), negative control group replaces the phage antibody clone with the M13K07 helper phage.
(5), the phage antibody dna sequence dna is identified
1, the extraction of pHEN2 phagemid and purifying: with reference to the purification kit specification sheets operation in a small amount of the precious biotech firm in Dalian plasmid DNA.
2,1% agarose gel electrophoresis is identified the plasmid extraction effect.
3, PCR reaction.
Primer:
Upstream: LMB3:5 '-CAG GAA ABA GCT ATG AC-3 ' (SEQ ID No.3)
Downstream: Fd seq1:5 '-GAA TTT TCT GTA TGA ACC-3 ' (SEQ ID No.4)
Reaction system:
10 * PCR reaction buffer, 2 μ l
25mmol/L?MgCl
2 1.5μl
2mmol/L?dNTPs 1.5μl
Upstream primer (10 μ M) 0.5 μ l
Downstream primer (10 μ M) 0.5 μ l
Taq enzyme 0.2 μ l
ddH2O 13μl
Reaction conditions:
95℃ 5min
72℃ 7min
4,1% agarose gel electrophoresis.
5, the positive colony that 750bp left and right sides band occurs, phagemid are served extra large Ying Jun Bioisystech Co., Ltd and are measured sequence.
(6), abduction delivering and the purifying of anti-A β solubility ScFv
1, mono-clonal phage Transformed E .coli HB2151
(1), according to above qualification result, select positive colony, the mono-clonal phage is increased the PEG/NaCl gathering with aforesaid method.
(2), with being coated with the TYE-AMP-GLU flat board, 37 ℃ of overnight incubation behind the epacmastic E.coli HB2151 of accumulative mono-clonal phagemid transformation index.
2, the anti-A β of IPTG abduction delivering solubility scFv
(1), mono-clonal bacterium (be step 1 preparation mono-clonal phage Transformed E .coli HB2151) is inoculated in 5ml2 * TY-AMP-GLU substratum, 37 ℃, the 220rpm shaking culture is spent the night.
(2), therefrom draw 200 μ l and be inoculated in 20ml 2 * TY-AMP-GLU substratum, 37 ℃, the 220rpm shaking culture is to OD
600Be 0.5 (about 2h).
(3), therefrom draw 10ml and be inoculated in 1000ml 2 * TY-AMP-GLU substratum, 37 ℃, the 220rpm shaking culture is to OD
600Be 0.9 (about 3h).
(4), when reaching the OD value that needs, 4 ℃, 4500rpm, centrifugal 15min abandons nutrient solution, bacterial precipitation is resuspended with isopyknic fresh 2 * TY-AMP nutrient solution.And add IPTG to final concentration 0.1mmol/L, carry out abduction delivering.
(5), 20 ℃, 220rpm continues shaking culture 16-24h.
(6), 4500rpm, 4 ℃ of centrifugal 30min collect to go up cleer and peaceful bacterial precipitation respectively.
3, the nutrient solution component protein concentrates
(1), the beaker that will fill 1000ml nutrient solution supernatant places on ice.Limit rule mild stirring, the limit adds 561g ammonium sulfate slowly, and this step should be finished in 5～10min;
(2), continue to stir 30min on ice.
(3), 4500rpm, 4 ℃ of centrifugal 30min.
(4), abandon supernatant, precipitation is suspended among the PBS of 1-2 times of throw out volume, residual insoluble substance may be a metaprotein, remove by centrifugal, nutrient solution supernatant component.
4, bacterium pericentral siphon component is extracted
(1), heavy molten bacterial precipitation (being produced by step 2 (6)) is in the sucrose of 50ml 30mM Tris-HCl pH8.0 20%.Add 100 μ l 0.5M EDTApH8.0 (final concentration is 1mM).Add magnetic stirring bar, slowly stir 10min under the room temperature.
(2), 4 ℃ of centrifugal 10min collecting cells of 10000g, remove supernatant liquor.
(3), the MgSO of the freezing 5mM of 50ml again
4Thoroughly heavy molten precipitation slowly stirs suspension 10min on ice.This moment, periplasm protein was released in the damping fluid.
(4), 4 ℃ of centrifugal 10min of 10000g, collect supernatant liquor (pericentral siphon part).
5, the anti-A β of His-Trap affinity chromatography column purification scFv soluble antibody (convenient for explaining, called after E3 scFv or E3 single-chain antibody)
Operate with reference to His-Trap affinity column specification sheets.
(1), will extract nutrient solution supernatant component after concentrating and cell pericentral siphon component with binding buffer liquid dialysed overnight.With 0.45 μ m filter membrane degerming, prevent to stop up affinity column.
(2), the 5ml deionized water adds affinity column.
(3), 0.5ml 0.1mol/L single nickel salt injects post.
(4), 5ml deionization washing affinity column.
(5), 10ml binding buffer liquid balance chromatography column.
(6), the filtered sample in the step (1) adds affinity column.Flow velocity is 1ml/min.
(7), 10ml binding buffer liquid adds affinity column, flush away non-specific binding albumen.
(8), contain each 1ml of elutriant of 100mM, 200mM, 300mM, 400mM, 500mM imidazoles, add affinity column successively, flush away specificity bonded albumen, 500 μ l/ pipe Fractional Collections elutriant.
(9), 10ml binding buffer liquid adds affinity column.
(10) 5ml contains the binding buffer liquid adding chromatography column of 0.05M EDTA, removes nickel ion.
(11), 10ml deionization washing affinity column.
(12) 5ml 20% ethanol adds affinity column, the sealing chromatography column, and 4 degree are preserved.
6, the anti-A β of SDS-PAGE preliminary evaluation soluble antibody.
(1) gel preparation:
| 12% separation gel: | 5% concentrates glue: | ||
| ddH 2O | 1.6ml | ?1.5ml | |
| 30% acrylamide | 2.0ml | ?300μl | |
| 1.5M?Tris(pH8.8) | 1.3ml | 1M?Tris(pH6.8) | ?750μl |
| 10％SDS | 50μl | ?30μl | |
| 10% ammonium persulphate (APS) | 50μl | ?50μl | |
| Tetramethyl Ethylene Diamine (TEMED) | 5μl | ?5μl | |
(2), access method 5 (8) is respectively managed elutriant 10 μ l+2 * sample-loading buffers 10 μ l mixings, boiling water bath 5min.
(3), go up sample, 75V constant voltage electrophoresis enters separation gel to the tetrabromophenol sulfonphthalein indicator, 110V constant voltage electrophoresis is to the tetrabromophenol sulfonphthalein completely dissolve.
(4), coomassie brilliant blue R250 dyeing 30min.
(5), destainer decolours transparently to background color, observes purification effect.
7, dialysis desalting.
(7), the Western blot of solubility ScFv antibody identifies
1, Tricine SDS-PAGE electrophoresis
(1), gel formula:
| Separation gel (15%) | Concentrate glue | ||
| ?ddH 2O | ?1.1ml | 1.5ml | |
| 30% acrylamide | ?2.5ml | 300μl | |
| ?1.5M?Tris(pH?8.8) | ?1.3ml | 1M?Tris(pH6.8) | 750μl |
| ?10％SDS | ?50μl | 30μl | |
| 10% ammonium persulphate | ?50μl | 50μl | |
| Tetramethyl Ethylene Diamine | ?5μl | 5μl | |
(2), get 10 μ l sample liquid and 10 μ l, 2 * sample loading buffer mixing, boil sex change, the centrifugal 1min of 1000rpm.The amount that adds sample protein (A β 1-42 or BSA) in every well is 20 μ g.
(3), take out comb, plate is placed electrophoresis chamber, fill it up with the Tricine electrophoretic buffer, go up sample successively.100V constant voltage electrophoresis 90min.
2, change film
(1), after electrophoresis finishes, cut concentrated glue, glue is dipped in balance 10-20min in the albumen transfering buffering liquid, prepares the pvdf membrane of a suitable size simultaneously.
(2) correctly laying electricity changes box, adds an amount of albumen transfering buffering liquid, protein band is shifted electrotransfer to pvdf membrane, 0.3A, 55min with wet commentaries on classics method.
(3) after electricity changes end, film is taken out from electricity commentaries on classics box, label orientation is simply washed with PBS.
3, sealing
Film is dipped in the confining liquid (MPBS) incubated at room 2 hours, the nonspecific proteins binding site on the blocking-up pvdf membrane.
4, antibody incubation
(1), add the antibody supernatant behind the purifying of dilution in 1: 5,4 ℃ are shaken down and spend the night.
(2), PBS-T washes film 5min * 3 time.
(3), add anti-c-myc antibody (dilution in 1: 200) and hatch 2h for 37 ℃.
(4), PBS-T washes film 5min * 3 time.Remove a unnecessary anti-reaction solution and the antibody of non-specific adsorption.
(5), add the goat anti-mouse igg (dilution in 1: 200) of horseradish peroxidase-labeled, hatch 2h for 37 ℃.
(6), PBS-T washes film 5min * 3 time.
5, ECL colour developing: face with preceding the ECL liquid A that develops the color is mixed with B, even dropping is on the film surface, the lucifuge sheet that exposes to the sun, processing in developing solution, the stop bath, observations.
(8), transmission electron microscope (TEM) observation
1, A β uses the preparation of liquid
(1), the A beta polypeptides is dissolved in 1% the sodium hydroxide solution, be made into the A β storage liquid of concentration greater than 5mg/ml.
(2), BCA method protein quantification
The preparation of working fluid: get A liquid and B liquid in the BCA protein quantification detection kit (PIERCE company), by 1: 50 (V/V) preparation working fluid.
Add the A β storage liquid and 0,0.5,1.0,1.5 of 10 μ l dilution, the standard protein solution of the several different concns of 2.0mg/ml in the enzyme plate respectively, every hole adds working fluid 200 μ l, mixing, 37 ℃ of incubation 30min, the CliniBio-128 microplate reader reads wavelength 562nm place light absorption value, the drawing standard curve calculates A β storage liquid concentration.
(3), face with preceding usefulness and comprise 0.02%NaN
350mmol/LPBS dilution, pH 7.5.A β 0.5mg/ml was hatched 3 days for 37 ℃ continuously, can make it form fibril aggregation.
2, TEM observation A beta
(1), experiment grouping:
Control group: A β 20 μ M;
ScFv treatment group: A β 20 μ M, E3 scFv 20 μ M;
BSA control group: A β 20 μ M, BSA 20 μ M.
(2), 37 ℃ leave standstill and hatched for 2 weeks.
(3), hatch end after, get 10 μ l protein solns and drip on the carbon film of copper mesh seasoning.
(4), the acetic acid uranium of 2% (W/V) dyeing.
(5), the JEM-1010 transmission electron microscope observation, the operating voltage of Electronic Speculum is 100kV, ratio of enlargement is 15,000-50,000.
(9), thioflavine T (ThT) fluorescent quantitation detects
1, experiment grouping:
Control group: A β 20 μ M;
E3 (1: 1) group: A β 20 μ M, E3 scFv 20 μ M;
E3 (1: 10) group: A β 20 μ M, E3 scFv 2 μ M.
2, hatched for 0,1,2 weeks for 37 ℃.
3, get sample after 25 μ l are hatched, (be dissolved in 50mM PBS, pH6.5) mixing with the diluent of 75 μ l, 10 μ M ThT.(establish 5 multiple holes for every group, and set up blank, promptly do not contain the diluent of 10 μ M ThT of sample).
4, hatch 10min for 37 ℃, utilize Spectra MAXGEMINI EM microplate fluorescent quantitation instrument to measure the fluorescence reading.(excitation wavelength: 450nm, emission wavelength: 482nm)
(10), E3 scFv (E3 soluble antibody) is to the provide protection of SK-N-SH cell A β toxic damages
1, the cultivation of neuroblastoma (SK-N-SH Neuroblastoma cell): the SK-N-SH cell inoculation places saturated humidity, 5%CO in the high sugared nutrient solution of the DMEM that contains 10% calf serum, 100IU/ml gentamicin
2, cultivate in 37 ℃ the incubator.
2, passage and kind plate: discard nutrient solution, with D-Hank ' s liquid washed cell 2 times, add 0.25% trypsinase-0.02%EDTA mixed solution and cover cell surface, room temperature effect 2～3min, when seeing cellular contraction, edge clear with the inverted microscope observation, add the high sugared nutrient solution of the DMEM that contains 10% calf serum and end the pancreatin activity, the cell on the piping and druming bottle wall is made single cell suspension, and adjusting cell density is 1 * 10
5/ ml.100 μ l/ hole kinds are in 96 orifice plates.Place saturated humidity, 5%CO
2, cultivate in 37 ℃ the incubator.
3, the cell mitochondrial succinic dehydrogenase activity is measured: behind cell inoculation 24h, change to contain the nutrient solution of 15-30 μ M A β, continue to cultivate 72h, add 5mg/ml MTT solution 10 μ l then, 37 ℃ are continued to cultivate 4h, supernatant is abandoned in suction, add DMSO 150 μ l/ holes, light shaking 10 minutes, dissolve purple crystallization, on the enzyme-linked immunoassay instrument, read wavelength 490nm place absorbancy, reflect mitochondrial respiratory function with this.
4, the E3 soluble antibody is to the provide protection of SK-N-SH cell A β toxic damages:
(1), experiment grouping and processing:
The normal nutrient solution of control group: DMEM;
A β group: the DMEM nutrient solution that contains 20 μ M A β;
E3 treatment group: the DMEM nutrient solution that contains 2-20 μ M E3 scFv and 20 μ M A β.
(2), behind the cell inoculation 24h, change with different conditioned mediums, continue to cultivate 72h according to above-mentioned grouping.
(3), change the high sugared nutrient solution of fresh DMEM, add 5mg/ml MTT solution 10 μ l, 37 ℃ are continued to cultivate 4h, supernatant is abandoned in suction, add DMSO 150 μ l/ holes, light shaking 10 minutes, dissolve purple crystallization, on the enzyme-linked immunoassay instrument, read wavelength 490nm place absorbancy, reflect mitochondrial respiratory function with this.
The result:
(1), screening A β 40 specific human endogenous antibody results:
Through the five affine screenings of taking turns, 58 clones of random choose, amplification cultivation is also collected phage antibody, carries out ELISA and detects.In the detection, replace phage antibody as negative control, eliminate the non-specific binding of phage and A beta polypeptides with the M13K07 helper phage.The ELISA detected result is seen Fig. 1.
(2), the gene of phage antibody and protein sequence:
E3 mono-clonal phage antibody obtains its concrete antibody gene fragment through the gene sequencer order-checking, does not wherein contain terminator codon TAG, TAA, TGA.Antibody heavy chain variable region and chain variable region gene are learnt in translation in view of the above, (National Center for Biotechnology Information, the hypervariable region and the rack area aminoacid sequence of this antibody learnt in immunoglobulin (Ig) BLAST database comparison www.ncbi.nlm.nih.gov) to utilize NCBI.As shown in table 1.
(3), Western blot identifies solubility scFv antibody:
1, the anti-A β of His-Trap affinity chromatography column purification soluble single-chain antibody
IPTG induces 2L E.coli HB2151 to express soluble antibody, gets cleer and peaceful pericentral siphon chamber extracting solution on the spissated nutrient solution, the His-Trap affinity chromatography, and the 12%SDS-PAGE electrophoresis, the purifying protein band of the visible about 31kD of molecular weight, as shown in Figure 2.
2, Western blot identifies anti-A β soluble single-chain antibody
Western blot detected result shows that anti-A β soluble antibody can combine with synthetic A beta polypeptides, with the BSA no cross reaction.As shown in Figure 3.
The nucleotide sequence of table 1 E3 single-chain antibody (E3 scFv) CDRs and FRs (SEQ ID NO.1) and aminoacid sequence (SEQ ID NO.2).
CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTCTGGT
Q V Q L Q E S G P G L V K P S E T L S L T C A V S G
H-CDR1
TACTCCATCAGC
AGTGGTTACTACTGGGGCTGGATCCGGCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGG
AGTATC
Y S I S S G Y Y W G W I R Q P P G K G L E W I G S I
H-CDR2
TATCATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAG
Y H S G S T Y Y N P S L K S R V T I S V D T S K N Q
H-CDR3
TTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCAAGA
GATCTTGGTAGTTCTGTG
F S L K L S S V T A A D T A V Y Y C A R D L G S S V
Linker
AGTTGGGGCCAAGGTACCCTGGTCACCGTCTCGAGT
GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTAGTGCA
S W G Q G T L V T V S S G G G G S G G G G S G G S A
CTTGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCAGTTGT
CGGATG
L D I Q M T Q S P S S L S A S V G D R V T I S C R M
L-CDR1
AGTCAGGGCATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTAT
GCTGCA
S Q G I S N Y L A W Y Q Q K P G K V P K L L I Y A A
L-CDR2
TCCACTTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGC
S T L Q S G V P S R F S G S G S G T D F T L T I S S
L-CDR3
CTGCAGCCTGAAGATGTTGCAACTTATTACTGT
CAAAAGTATAACAGTGCCCTCGTTACGTTCGGCCAAGGGACCAAG
L Q P E D V A T Y Y C Q K Y N S A L V T F G Q G T K
CTGGAAATCAAACGT
L E I K R
(4) anti-A β soluble single-chain antibody is to the influence of A β aggregation in vitro
1, anti-A β soluble single-chain antibody suppresses the gathering of A β
TEM observes discovery, and the A β 40 of 20 μ M hatched for 2 weeks at 37 ℃, can assemble to form sophisticated fiber.As Fig. 4 A, shown in the B, sophisticated fiber can form netted.The E3 single-chain antibody of volumetric molar concentrations such as adding hatched for 2 weeks at 37 ℃ jointly with A β 40, can obviously suppress A β 40 and assemble the formation fiber, and A β 40 mainly presents amorphous structure, as Fig. 4 C, D.And in the control group of volumetric molar concentration BSA such as adding, A β 40 still assembles the formation mature fibers, as Fig. 4 E, and F.
ThT is a kind of fluorescence dye, and it can combine with mutual β-laminated structure specificity in the Amyloid, can be used as a quantitative analysis index of A beta peptide aggregation.The A β 40 of 20 μ M continues to hatch through 7-14 days 37 ℃, and ThT fluorescence value of reading of sample obviously increases, as shown in Figure 5.But after the anti-A β soluble single-chain antibody of different volumetric molar concentrations was hatched jointly, the test group ThT fluorescence value of reading was starkly lower than the control group fluorescent value with time point.As shown in Figure 5.
2, the short A β mature fibers disaggregation of anti-A β soluble single-chain antibody
20 μ M A β 40 assemble fibers with etc. after the anti-A β soluble single-chain antibody of volumetric molar concentration hatches 1 day jointly, the A beta obviously reduces.As shown in Figure 6, with respect to A β control group and BSA treatment group, the A beta structure of E3 single-chain antibody treatment group takes place obviously to change, and albumen mainly presents amorphous structure in the visual field, does not see that tangible A β 40 assembles fiber.
(5) anti-A β soluble single-chain antibody suppresses the cytotoxic effect of A β
MTT result shows that 20 μ M accumulative as can suppress the activity of the succinodehydrogenase of SK-N-SH cell, and pair cell has tangible toxic action.And add jointly in the cell culture fluid with anti-A β soluble single-chain antibody, a suppresses obviously to reduce to the activity of succinodehydrogenase.As shown in Figure 7, the toxicity restraining effect of anti-A β soluble single-chain antibody is the significant concn dependency.
Embodiment 2: preparation of drug combination
Antibody of the present invention (SEQ ID No.2) or its expression vector of treatment significant quantity (are included but not limited to plasmid, phagemid, phage, intestinal bacteria, down together), perhaps the encode Nucleotide (SEQ ID No.1) of this antibody or its expression vector mixes the back separately or mutually and forms pharmaceutical composition with the auxiliary material that pharmaceutically allows, this pharmaceutical composition can use separately, also can use with other medicament mixed that does not influence this pharmaceutical composition function.The preparation of these pharmaceutical compositions can be any formulation that pharmaceutically allows, and includes but not limited to tablet, pill, capsule, injection, oral liquid or liposome.The preparation method of these preparations is technology known to a person of ordinary skill in the art.These pharmaceutical compositions can be used for the treatment of or prevent senile dementia.
＜110〉Nanjing Medical University
＜120〉antibody and the expression vector and the application in pharmacy of a kind of treatment or prevention senile dementia
<160>4
<210>1
<211>720
<212>DNA
＜213〉artificial sequence
<400>
cag?gtg?cag?ctg?cag?gag?tcg?ggc?cca?gga?ctg?gtg?aag?cct?tcg?gag 48
Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu
1 5 10 15
acc?ctg?tcc?ctc?acc?tgc?gct?gtc?tct?ggt?tac?tcc?atc?agc?agt?ggt 96
Thr?Leu?Ser?Leu?Thr?Cys?Ala?Val?Ser?Gly?Tyr?Ser?Ile?Ser?Ser?Gly
20 25 30
tac?tac?tgg?ggc?tgg?atc?cgg?cag?ccc?cca?ggg?aag?ggg?ctg?gag?tgg?144
Tyr?Tyr?Trp?Gly?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp
35 40 45
att?ggg?agt?atc?tat?cat?agt?ggg?agc?acc?tac?tac?aac?ccg?tcc?ctc?192
Ile?Gly?Ser?Ile?Tyr?His?Ser?Gly?Ser?Thr?Tyr?Tyr?Asn?Pro?Ser?Leu
50 55 60
aag?agt?cga?gtc?acc?ata?tca?gta?gac?acg?tcc?aag?aac?cag?ttc?tcc?240
Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser
65 70 75 80
ctg?aag?ctg?agc?tct?gtg?acc?gcc?gca?gac?acg?gcc?gtg?tat?tac?tgt?288
Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
gca?aga?gat?ctt?ggt?agt?tct?gtg?agt?tgg?ggc?caa?ggt?acc?ctg?gtc?336
Ala?Arg?Asp?Leu?Gly?Ser?Ser?Val?Ser?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
acc?gtc?tcg?agt?ggt?gga?ggc?ggt?tca?ggc?gga?ggt?ggc?tct?ggc?ggt?384
Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly
115 120 125
agt?gca?ctt?gac?atc?cag?atg?acc?cag?tct?cca?tcc?tcc?ctg?tct?gca?432
Ser?Ala?Leu?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala
130 135 140
tct?gta?gga?gac?aga?gtc?acc?atc?agt?tgt?cgg?atg?agt?cag?ggc?att?480
Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Ser?Cys?Arg?Met?Ser?Gln?Gly?Ile
145 150 155 160
agc?aat?tat?tta?gcc?tgg?tat?cag?cag?aaa?cca?ggg?aaa?gtt?cct?aag?528
Ser?Asn?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Val?Pro?Lys
165 170 175
ctc?ctg?atc?tat?gct?gca?tcc?act?ttg?caa?tca?ggg?gtc?cca?tct?cgg?576
Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg
180 185 190
ttc?agt?ggc?agt?gga?tct?ggg?aca?gat?ttc?act?ctc?acc?atc?agc?agc?624
Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser
195 200 205
ctg?cag?cct?gaa?gat?gtt?gca?act?tat?tac?tgt?caa?aag?tat?aac?agt?672
Leu?Gln?Pro?Glu?Asp?Val?Ala?Thr?Tyr?Tyr?Cys?Gln?Lys?Tyr?Asn?Ser
210 215 220
gcc?ctc?gtt?acg?ttc?ggc?caa?ggg?acc?aag?ctg?gaa?atc?aaa?cgt 720
Ala?Leu?Val?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg
225 230 235
<210>2
<211>239
<212>PRT
＜213〉artificial sequence
<400>
Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu
1 5 10 15
Thr?Leu?Ser?Leu?Thr?Cys?Ala?Val?Ser?Gly?Tyr?Ser?Ile?Ser?Ser?Gly
20 25 30
Tyr?Tyr?Trp?Gly?Trp?Ile?Arg?Gln?Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp
35 40 45
Ile?Gly?Ser?Ile?Tyr?His?Ser?Gly?Ser?Thr?Tyr?Tyr?Asn?Pro?Ser?Leu
50 55 60
Lys?Ser?Arg?Val?Thr?Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser
65 70 75 80
Leu?Lys?Leu?Ser?Ser?Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Arg?Asp?Leu?Gly?Ser?Ser?Val?Ser?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly
115 120 125
Ser?Ala?Leu?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala
130 135 140
Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Ser?Cys?Arg?Met?Ser?Gln?Gly?Ile
145 150 155 160
Ser?Asn?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Val?Pro?Lys
165 170 175
Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg
180 185 190
Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser
195 200 205
Leu?Gln?Pro?Glu?Asp?Val?Ala?Thr?Tyr?Tyr?Cys?Gln?Lys?Tyr?Asn?Ser
210 215 220
Ala?Leu?Val?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg
225 230 235
<210>3
<211>17
<212>DNA
＜213〉artificial sequence
<400>
caggaaabag?ctatgac 17
<210>4
<211>18
<212>DNA
＜213〉artificial sequence
<400>
gaattttctg?tatgaacc?18
Claims (10)
1, the antibody of a kind of treatment or prevention senile dementia, the aminoacid sequence of this antibody is as described in the SEQ ID No.2.
2, antibody according to claim 1 is characterized in that described antibody is solubility scFv antibody fragment.
3, the expression vector of the described antibody of claim 1.
4, expression vector according to claim 3 is characterized in that described expression vector is plasmid, phagemid, phage or intestinal bacteria.
5, the Nucleotide of coding claim 1 described antibody, its sequence is as described in the SEQ ID No.1.
6, the expression vector of the described Nucleotide of claim 5.
7, expression vector according to claim 6 is characterized in that described expression vector is plasmid, phagemid, phage or intestinal bacteria.
8, the application of the described antibody of claim 1 in preparation treatment or prevention senile dementia medicine.
9, the application of the described Nucleotide of claim 5 in preparation treatment or prevention senile dementia medicine.
10, claim 4 or the 6 described expression vectors application in preparation treatment or prevention senile dementia medicine.
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| US8295792B2 (en) | 2006-06-14 | 2012-10-23 | Research In Motion Limited | Control of switcher regulated power amplifier modules |
| US8606199B2 (en) | 2006-06-14 | 2013-12-10 | Blackberry Limited | Control of switcher regulated power amplifier modules |
| US8761305B2 (en) | 2006-06-14 | 2014-06-24 | Blackberry Limited | Input drive control for switcher regulated power amplifier modules |
| US8620238B2 (en) | 2010-07-23 | 2013-12-31 | Blackberry Limited | Method of power amplifier switching power control using post power amplifier power detection |
| US9276545B2 (en) | 2010-07-23 | 2016-03-01 | Blackberry Limited | Method of power amplifier switching power control using post power amplifier power detection |
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| CN1803842A (en) | 2006-07-19 |
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