CN1329413C - Antibody for treating or preventing senile dementia, its expression vector and application in drug preparation - Google Patents

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

An antibody for treating or preventing senile dementia, its expression vector and its application in pharmacy

technical field

The invention belongs to the field of genetic engineering and relates to an antibody for treating or preventing senile dementia. The invention also relates to the expression vector of the antibody and their application in pharmacy.

Background technique

Senile dementia, also known as Alzheimer's disease (Alzheimer's disease, AD), is a neurodegenerative disease that seriously endangers human health and is one of the most common forms of senile dementia. AD patients are mainly manifested as memory loss, cognitive impairment and loss of spatial discrimination ability, and even motor and sensory dysfunction, seizures and other symptoms in the late stage of the disease. At present, the incidence of AD in the world is as high as 12 million, and it shows a trend of increasing year by year. At present, there are mainly five clinical methods for the treatment of AD: one is to protect the nerves from damage, the other is to use cholinesterase inhibitors to prevent the loss of acetylcholine caused by the brain nucleus mass cell disorder, and the third is non-drug intervention. and the application of psychotropic drugs to improve behavioral disorders, the fourth is health care activities, and the fifth is care for patients. However, all these methods are to improve the patient's symptoms, not to treat the cause.

The typical pathological sign of AD is the deposition of a large number of senile plaques in the brain. The main component of this senile plaque is β-amyloid polypeptide (Aβ), which is composed of 40-42 amino acids and is the enzymatic hydrolysis product of β-amyloid precursor protein (APP), among which Aβ ₄₂ is more cytotoxic. It is currently believed that the formation and accumulation of Aβ play a key role in the pathogenesis of AD. After the generation and deposition of Aβ, it can secondary cause the formation of neurofibrillary tangles, oxidation and lipid peroxidation, glutamate-derived excitotoxicity, inflammation and activation of apoptotic cell death cascade. All these secondary responses further amplify the toxic effects of Aβ and thus also become potential targets for anti-AD etiological therapies. However, it is obvious that the truly effective target for the treatment of AD should be to reduce the Aβ in the brain.

In 1999, Schenk et al. from Elan Corporation of the United States first reported that direct injection of Aβ _1-42 into AD model mice could reduce the level of plasma amyloid β, inhibit the deposition of Aβ on existing plaques, and remove the β-amyloid in the brain. age plaque. Later reports confirmed that immunization of mice with soluble Aβ could not only reduce the central amyloid burden, but also improve cognition. Elan quickly developed a new type of vaccine AN-1792, which was applied to AD model animals, and the effect was remarkable, and clinical trials began. However, in the phase II clinical trial, 6% of patients developed severe aseptic inflammation of the central nervous system, and the AD vaccine treatment trial was forced to terminate. Autopsy confirmed that aseptic meningoencephalitis caused by vaccine therapy was mainly the result of T cell-mediated autoimmune reaction.

It is generally believed that the therapeutic effect of Aβ after inoculation into the body is mainly achieved through the bridging effect of antibodies: exogenous Aβ can stimulate the body to produce specific antibodies, which combine with Aβ to form antigen-antibody complexes, and pass through monocyte-macrophage Fc The receptor enters the cell and Aβ is degraded. That being the case, is it possible to inject Aβ antibody into the body without injecting Aβ to achieve the same therapeutic effect? Since 2000, several research groups have injected transgenic mice overexpressing APP with anti-Aβ antibodies, and found that the Aβ load can be significantly reduced, and the amyloid plaques in the brain can be cleared. Or intraperitoneal injection can receive obvious effect.

However, the existing reports mostly use mouse-derived monoclonal antibody, which is a foreign protein for the human body and can cause the human immune system to produce human anti-mouse antibody (HAMA), which largely limits its use in the human body. Applications. Because HAMA can not only reduce the titer of the antibody, increase the clearance rate of the mouse-derived monoclonal antibody, and affect its therapeutic effect, but more seriously, it can cause an allergic reaction and threaten the life safety of the patient.

In recent years, with the development of molecular biology technology, genetic engineering methods have opened up a new field for the preparation of antibodies, which is genetic engineering antibodies. The emergence of phage antibody library technology based on PCR technology and phage display technology can be called a revolutionary progress in the field of genetically engineered antibodies. This technology uses bacterial clones instead of B cell clones to express antibodies without cell fusion. , and even without immunization, human antibody molecules against any antigen can be prepared more conveniently. It makes it possible to obtain highly specific and high affinity humanized antibodies. The antibody molecules obtained by this method are human-derived antibodies, which can be directly used in human treatment to reduce possible toxic and side effects; at the same time, because of their small molecular weight, they are easy to penetrate various biological barriers (such as the blood-brain barrier), It can reach the lesion site and play a role directly.

Contents of the invention

The object of the present invention is to provide an antibody for treating or preventing senile dementia.

Another object of the present invention is to provide the expression vector of the above antibody.

Another object of the present invention is to provide the application of the antibody, the nucleotides encoding the antibody and their expression vectors in pharmacy.

The present invention selects Aβ as the target, applies phage antibody library technology, obtains specific phage antibody against Aβ by screening, expresses and obtains soluble scFv antibody fragments, and uses Western blot method to identify its antibody activity, and its biological activity and cell protection effect in vitro . The anti-Aβ antibody obtained by screening, its gene fragment and expression vector can be used to prepare new drugs for immunotherapy or prevention of senile dementia.

The object of the present invention is achieved through the following measures:

An antibody for treating or preventing senile dementia, the amino acid sequence of the antibody is as described in SEQ ID No.2.

The antibody is a monoclonal antibody.

The antibody is a soluble scFv antibody fragment.

Said antibody expression vectors include but not limited to plasmids, phagemids, phages, and Escherichia coli.

The nucleotide encoding the above-mentioned antibody has a sequence as described in SEQ ID No.1.

The nucleotide expression vectors include, but are not limited to, plasmids, phagemids, phages, and Escherichia coli.

The application of the antibody in the preparation of medicines for treating or preventing senile dementia.

Application of said nucleotide in preparation of medicine for treating or preventing senile dementia.

The application of the expression vector in the preparation of medicines for treating or preventing senile dementia.

Beneficial effects of the present invention:

The present invention obtains a novel anti-beta-amyloid polypeptide (Aβ) humanized single-chain antibody (amino acid sequence such as SEQ ID NO.2) by screening the phage antibody library technology. The antibody can inhibit and release the aggregation of β-amyloid polypeptide and play a protective role in cells. The antibody can be used as a therapeutic antibody for senile dementia, and can be used for preparing medicine for treating senile dementia. The nucleotides encoding the antibody and their expression vectors can also be used to prepare medicaments for treating senile dementia.

Description of drawings

Figure 1 is the ELISA detection results of monoclonal phage display antibodies.

Fig. 2 is the SDS-PAGE result of the anti-Aβ soluble single chain antibody of the present invention.

Among them: M: protein molecular weight standard; 1: culture supernatant and periplasmic cavity extract after induction of expression; 2: purified protein components through His-Trap affinity chromatography.

Fig. 3 is the Western blot result of the anti-Aβ soluble single-chain antibody of the present invention.

Fig. 4 is an electron micrograph of the effect of the anti-Aβ soluble single-chain antibody of the present invention on Aβ aggregation.

Among them: A, B: 20 μM Aβ40 incubated at 37°C for 2 weeks; C, D: 20 μM E3 single-chain antibody and 20 μM Aβ40 were incubated at 37°C for 2 weeks; E, F: 20 μM BSA and 20 μM Aβ40 were incubated at 37°C for 2 weeks week. Bar (A, C, E) = 500 nm, Bar (B, D, F) = 100 nm.

Fig. 5 is ThT fluorescence quantitative measurement of the effect of the anti-Aβ soluble single-chain antibody of the present invention on Aβ aggregation.

Among them: *: P<0.05, compared with the fluorescence value of the control group at each time point; ^** : P<0.01, compared with the fluorescence value of the control group at each time point.

Fig. 6 is an electron micrograph of the depolymerization of Aβ fibers by the anti-Aβ soluble single-chain antibody of the present invention.

Among them: A: mature Aβ40 aggregated fibers; B, C: 20 μM Aβ40 fibers were incubated alone at 37°C for 10 days; D: 20 μM Aβ40 fibers were incubated with 20 μM Aβ40 fibers at 37°C for 10 days; E, F: 20 μM E3 single-chain antibody and 20 μM Aβ40 fibers were co-incubated at 37°C for 10 days. Bar (A, B, D, E) = 500 nm, Bar (C, F) = 1 μm.

Figure 7 is the cytotoxic effect of Aβ under the condition of different concentrations of anti-Aβ soluble single-chain antibody measured by MTT

Where: *: P<0.05, compared with the control group; Δ: P<0.05, compared with the Aβ 20 μM treatment group.

Detailed ways

The present invention is described further below by embodiment.

Example 1

Preparation method and identification experiment:

(1) Amplification of the human phage antibody library:

1. Inoculate the Griffin.1 phage display human single-chain antibody library (about 1×10 ¹⁰ clones) into 500ml 2×TY-AMP-GLU medium, culture at 37°C with shaking at 220rpm until the OD ₆₀₀ is 0.5 (about 1.5- 2h).

2. Take 25ml of culture solution (about 1×10 ¹⁰ bacteria) and superinfect with M13K07 helper phage. The infection ratio was 1:20 (number of bacteria: number of helper phages). In a water bath at 37°C, let stand for 30 minutes.

3. Centrifuge at 3300g for 10min at 4°C, and resuspend the pellet with 30ml 2×TY-AMP-KAN medium.

4. Add the bacterial liquid to 470 ml of pre-warmed 2×TY-AMP-KAN medium, culture at 30°C and shake at 220rpm overnight.

5. Centrifuge at 10800g at 4°C for 10min, and discard the precipitate.

6. Collect the supernatant, add 1/5 supernatant volume of PEG/NaCl (20% polyethylene glycol-2.5M NaCl, the same below) solution, mix thoroughly and let stand at 4°C for 1 hour or longer to precipitate phage.

7. Centrifuge at 3300g for 30min at 4°C, and resuspend the precipitate in 40ml ddH20 and 8ml PEG/NaCl solution. After thorough mixing, let stand at 4°C for 20 minutes or longer.

8. Centrifuge at 3300g for 10 minutes at 4°C, and discard the supernatant.

9. Briefly centrifuge to remove the remaining PEG/NaCl.

10. The precipitate was resuspended with 5ml PBS and distributed into Eppendorf tubes (referred to as Ep tubes).

11. Centrifuge at 11600g for 10min at 4°C, discard bacterial debris and impurities, and transfer the supernatant to a new Eppendorf tube.

12. The phage supernatant can be stored at 4°C for a short period of time. For long-term storage, add glycerol to a final concentration of 15%, and store at -70°C.

Phage titer determination:

1. Take 1 μl of phage supernatant and add it to 1ml of PBS (1:10 ³ )

2. Take 1 μl of E.coli TG1 transformed into 1ml of exponential growth phase ^* (OD ₆₀₀ about 0.4-0.6), and bathe in 37°C water for 30 minutes (1:10 ⁶ )

3. Add 10 μl to 990 μl 2×TY medium (1:10 ⁸ )

4. Add 10 μl to 990 μl 2×TY medium (1:10 ¹⁰ )

5. Add 10 μl to 990 μl 2×TY medium (1:10 ¹² )

6. Take 50 μl each of 1:10 ⁶ , 1:10 ⁸ , 1:10 ¹⁰ , and 1:10 ¹² -fold dilutions, spread on TYE-AMP-GLU plates, and incubate overnight at 37°C. The number of bacterial clones was counted the next day to calculate the phage titer.

^* : Phage/phagemid (phage/phagemid) can infect pili positive (F ⁺ ) Escherichia coli, Escherichia coli must be cultured at 37°C to the exponential growth phase (OD ₆₀₀ about 0.4-0.6), at this time the bacteria will Produce sexual pili, which have a high infection efficiency. Escherichia coli in this state needs to be prepared during the entire screening and identification process.

The preparation steps are as follows:

1. Streak E. coli on an M9 plate and culture at 37°C until colonies are visible.

2. Pick a single clone from the M9 plate, inoculate it in 5ml 2×TY medium, and cultivate overnight at 37°C with shaking at 220rpm.

3. The next day, absorb the overnight cultured bacterial solution, add it to fresh 2×TY medium at a ratio of 1:100, and cultivate it with shaking at 37°C until the exponential growth phase (OD ₆₀₀ is about 0.4-0.6), which can be used for phage transformation .

4. Putting the bacterial solution on ice for a short time before infection can enhance the infection effect, but if it exceeds 30 minutes, the sex pili of E. coli will be lost and cannot be infected.

(2) Preparation of secondary phage antibody library:

1. Following step (1) 2, the remaining 475ml of culture medium continued to be shaken at 37°C for 2 hours.

2. Centrifuge at 3300g for 30min at 4°C, resuspend the bacterial pellet in 10ml of 2×TY medium (containing 15% glycerol), and divide into 10 Eppendorf tubes, each with 1ml.

3. Store the secondary phage antibody library at -70°C. Before using again, use PCR method to identify positive clone rate and detect phage titer.

(3) Screening of Aβ1-40 (Aβ composed of amino acids 1-40, referred to as Aβ40) specific humanized antibody:

1. Coating antigen Aβ1-40 with glutaraldehyde method:

(1) Nunc immunotubes were treated with 100 mmol/L sodium phosphate buffer (pH 5.0) containing 0.2% glutaraldehyde (V/V) for 4 hours.

(2) Wash the Nunc tube twice with the above buffer solution.

(3) Add 4ml of Aβ1-40 (20μg/ml) dissolved in 100mmol/L sodium phosphate buffer (pH 8.0), and react at 37°C for 3h.

(4) Wash twice with 0.9% NaCl solution.

(5) Fill the Nunc tube with PBS (MPBS) containing 2% skimmed milk powder, and block at 37° C. for 2 hours.

(6), wash with PBS 3 times.

2. Screening of specific human monoclonal antibodies:

(1) For each round of screening, add about 10 ¹² tu (colony forming units after transfection) of phage to 4 ml of 2% MPBS in a Nunc tube.

(2) Place the Nunc tube on the rotating turntable, turn it over for 30 minutes at room temperature, and then place it vertically for at least 90 minutes at room temperature. Discard unbound phage in the supernatant.

(3) In the first round of screening, the Nunc tube was washed 10 times with PBS-T (containing 0.1% Tween-20), and then washed 10 times with PBS. In the second and subsequent rounds of screening, Nunc tubes were washed 20 times with PBS-T, and then washed 20 times with PBS.

(4), add 1ml of 100mmol/L triethylamine, place the Nunc tube on the rotating turntable and continuously overturn for 30min to elute the specifically bound phage. In the second and subsequent rounds of screening, first add 1ml of 100mmol/L triethylamine to pre-elute for 10min, discard the pre-elution solution, and add a new 1ml of 100mmol/L triethylamine to elute the specifically bound phage.

(5) During the elution process, prepare 0.5 ml of 1.0 mol/L Tris-HCl (pH 7.4) in the Ep tube in advance to quickly neutralize the eluted specific phage. The neutralized phages can be stored at 4°C for a short period of time or used to transfect E.coli TG1.

(6) Take 0.75ml of the eluted phage to transfect 4.25ml of E.coli TG1 in the exponential growth phase, and let stand in a water bath at 37°C for 30min.

(7), take 100 μl doubling dilution and determine the titer of phage:

Add 100 μl to 900 μl 2×TY medium (1:10)

Add 100 μl to 900 μl 2×TY medium (1:10 ² )

Add 100 μl to 900 μl 2×TY medium (1:10 ³ )

Add 100 μl to 900 μl 2×TY medium (1:10 ⁴ )

Take 50 μl from each of the 4-fold dilutions of 1:10, 1:10 ² , 1:10 ³ , and 1:10 ^4-fold dilutions, spread them on TYE-AMP-GLU plates, and incubate overnight at 37°C. The number of bacterial clones was counted the next day to calculate the phage titer.

(8) 3300 g of the remaining infected bacteria were centrifuged at 4°C for 10 min, and the bacterial pellet was resuspended in 500 μl of 2×TY medium.

(9) Apply 500 μl of bacterial solution to several pieces of TYE-AMP-GLU plates and incubate at 30° C. overnight or until colonies are visible.

(10), add 5-6ml 2×TY (containing 15% glycerol) on the plate, gently scrape off all colonies with a glass sorter, and mix well.

(11) Take 100 μl of it and add it to 100 ml 2×TY-AMP-GLU, and check its OD ₆₀₀ ≤0.1. The remaining bacterial liquid can be stored at -70°C.

(12) Culture at 37° C. with shaking at 220 rpm until the OD ₆₀₀ is 0.5 (about 2 hours).

(13), take 10ml from it, and superinfect with M13K07 helper phage. The infection ratio was 1:20 (number of bacteria: number of helper phages). 37°C water bath for 30min.

(14) Centrifuge at 3300g for 10min at 4°C, and resuspend the pellet with 50ml of 2×TY-AMP-KAN medium. Culture overnight at 30°C with shaking at 220rpm.

(15), 10800g, centrifuge the bacterial solution at 4°C for 10min, and discard the precipitate.

(16) Collect the supernatant, add 1/5 volume of PEG/NaCl, mix thoroughly on ice, and let stand at 4°C for more than 1 hour.

(17) Centrifuge at 10800 g at 4°C for 30 min, and resuspend the pellet in 40 ml ddH ₂ 0 and 8 ml PEG/NaCl solution. After thorough mixing, let stand at 4°C for 20 minutes or longer.

(18) Centrifuge at 10800g for 10 min at 4°C, discard the supernatant, centrifuge briefly, and remove the remaining PEG/NaCl.

(19), the precipitate was resuspended with 2ml PBS, and divided into 2 Ep tubes. Centrifuge at 11600g for 10min, discard bacterial debris and impurities, and transfer the supernatant to a new Ep tube. 1ml of which can be stored at 4°C. Another 1ml can be used for the next round of screening.

(20) The above screening steps were repeated, and a total of five rounds of "adsorption-elution-amplification" enrichment screening were performed.

(4) ELISA identification of monoclonal phage display antibodies:

1. Preparation of monoclonal phage antibody:

(1) Transfect E.coli TG1 in the exponential growth phase with the phage eluted in the fifth round of screening, spread on TYE-AMP-GLU plate, and culture overnight at 37°C.

(2) Take a 96-well cell culture plate and add 100 μl 2×TY-AMP-GLU to each well. 58 colonies were randomly selected from the plate cultured overnight and inoculated into a 96-well plate, and cultivated overnight at 37° C. with shaking at 250 rpm.

(3) Take another 96-well cell culture plate, add 200 μl 2×TY-AMP-GLU to each well, and transfer 5 μl from each well of the first plate to the second plate. 37°C, 250rpm shaking culture for 1h. Plate 1 can be added to each well with glycerol to a final concentration of 15%, and frozen at -70°C.

(4) Add 25 μl of 2×TY-AMP-GLU to each well, which contains 1×10 ⁹ pfu of M13K07 helper phage for superinfection.

(5) Stand still at 37°C for 30 minutes, and continue to culture at 37°C with shaking at 250 rpm for 1 hour.

(6) Centrifuge at 1800 g for 10 min, and suck off the supernatant.

(7) The precipitates in each well were resuspended with 200 μl 2×TY-AMP-KAN, and cultured overnight at 30°C with shaking at 250 rpm.

(8) Centrifuge at 1800 g for 10 min, and take the supernatant for ELISA detection.

ELISA detection:

(1) Treat the ELISA reaction plate with 100 mmol/L sodium phosphate buffer solution (pH5.0) containing 0.2% glutaraldehyde (V/V) for 4 hours.

(2) Wash the ELISA reaction plate twice with the above buffer solution.

(3) Add 100 μl 20 μg/ml of Aβ1-40 dissolved in 100 mmol/L sodium phosphate buffer (pH 8.0) to each well, add the same concentration of BSA dissolved in the same buffer to each well of the control group, 37 ℃ reaction 3h.

(4) Wash twice with 0.9% NaCl solution.

(5) Fill each well with 2% MPBS and block at 37°C for 2 hours.

(6), wash with PBS 3 times.

(7) Add 20 μl monoclonal phage supernatant to each well and react at room temperature for 3 hours.

(8) Wash 3 times with PBS-T containing 0.05% Tween-20.

(9) 100 μl of horseradish peroxidase-labeled anti-M13 antibody diluted 1:4000 times was added to each well, and reacted at room temperature for 3 hours.

(10) PBS-T washed 3 times.

(11) Add 100 μl of TMB substrate solution to each well, and react at room temperature for 10 minutes.

(12) After the color development is clear, add 50 μl of 1mol/L sulfuric acid to each well to terminate the reaction.

(13) Measure the absorbance value (A ₄₅₀ ) at 450 nm in each well, and use M13K07 helper phage to replace the phage antibody clone in the negative control group.

(5) DNA sequence identification of phage antibody

1. Extraction and purification of pHEN2 phagemids: operate according to the instructions of the plasmid DNA mini purification kit of Dalian Bao Biology Co., Ltd.

2. 1% agarose gel electrophoresis to identify the plasmid extraction effect.

3. PCR reaction.

Primers:

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 2μl

25mmol/L MgCl ₂ 1.5μl

2mmol/L dNTPs 1.5μl

Upstream primer (10μM) 0.5μl

Downstream primer (10μM) 0.5μl

Template DNA 1 μl

Taq enzyme 0.2μl

ddH2O 13μl

             

Total volume 20μl

Reaction conditions:

95°C 5min

72℃ 7min

4. 1% agarose gel electrophoresis.

5. For positive clones with a band of about 750bp, send the phagemid to Shanghai Yingjun Biotechnology Co., Ltd. for sequence determination.

(6) Induced expression and purification of anti-Aβ soluble ScFv

1. Transformation of E.coli HB2151 with monoclonal phage

(1), according to the above identification results, select positive clones, use the aforementioned method to amplify the monoclonal phage, and gather PEG/NaCl.

(2) E.coli HB2151 in the exponential growth phase was transformed with the aggregated monoclonal phagemids, then coated with TYE-AMP-GLU plates, and cultured overnight at 37°C.

2. IPTG induced expression of anti-Aβ soluble scFv

(1) Monoclonal bacteria (that is, the monoclonal phage transformed with E. coli HB2151 prepared in step 1) were inoculated in 5 ml of 2×TY-AMP-GLU medium, cultured overnight at 37° C. with shaking at 220 rpm.

(2) Take 200 μl from it and inoculate it into 20 ml 2×TY-AMP-GLU medium, culture at 37° C. with shaking at 220 rpm until the OD ₆₀₀ is 0.5 (about 2 hours).

(3) Take 10 ml of it and inoculate it into 1000 ml 2×TY-AMP-GLU medium, culture at 37° C. with shaking at 220 rpm until the OD ₆₀₀ is 0.9 (about 3 hours).

(4) When the required OD value is reached, centrifuge at 4°C at 4500 rpm for 15 minutes, discard the culture medium, and resuspend the bacterial pellet with an equal volume of fresh 2×TY-AMP culture medium. And add IPTG to the final concentration of 0.1mmol/L to induce expression.

(5) Continue shaking culture at 20°C and 220rpm for 16-24h.

(6) Centrifuge at 4500 rpm for 30 min at 4° C., and collect supernatant and bacterial precipitates respectively.

3. Protein concentration of culture medium components

(1) Place the beaker containing 1000ml culture supernatant on ice. Stir regularly and gently, while slowly adding 561g of ammonium sulfate, this step should be completed within 5 to 10 minutes;

(2) Continue stirring on ice for 30 min.

(3) Centrifuge at 4500 rpm for 30 min at 4°C.

(4) Discard the supernatant, and suspend the precipitate in PBS with 1-2 times the volume of the precipitate. The remaining insoluble matter may be denatured protein, which is removed by centrifugation to obtain the supernatant fraction of the culture medium.

4. Bacterial periplasmic components extraction

(1), redissolve the bacterial precipitate (produced by step 2 (6)) in 50ml 30mM Tris-HCl pH8.0 20% sucrose. Add 100 μl 0.5M EDTA pH 8.0 (final concentration is 1 mM). Add a magnetic stirring bar and stir slowly at room temperature for 10 min.

(2) Collect the cells by centrifugation at 10,000 g at 4°C for 10 min, and remove the supernatant.

(3) Then 50ml of frozen 5mM MgSO ₄ completely redissolve the precipitate, and slowly stir the suspension on ice for 10min. At this point the periplasmic proteins are released into the buffer.

(4) Centrifuge at 10,000 g at 4°C for 10 min, and collect the supernatant (periplasmic part).

5. Purification of anti-Aβ scFv soluble antibody by His-Trap affinity chromatography column (named as E3 scFv or E3 single-chain antibody for convenience of expression)

Operate according to the instructions of the His-Trap affinity chromatography column.

(1) The supernatant fraction and the periplasmic fraction of the extracted and concentrated culture solution were dialyzed overnight with a binding buffer. Sterilize with a 0.45 μm filter membrane to prevent clogging of the affinity chromatography column.

(2) Add 5ml of deionized water to the affinity chromatography column.

(3), 0.5ml 0.1mol/L nickel sulfate is injected into the column.

(4) Wash the affinity chromatography column with 5ml of deionized water.

(5) Equilibrate the chromatographic column with 10 ml of binding buffer.

(6), the filtered sample in step (1) is added to an affinity chromatography column. The flow rate was 1 ml/min.

(7) Add 10ml of binding buffer to the affinity chromatography column to wash away non-specific binding proteins.

(8) Add 1ml each of the eluents containing 100mM, 200mM, 300mM, 400mM, and 500mM imidazole to the affinity chromatography column in order to wash away the specifically bound protein, and collect the eluent in sections at 500 μl/tube.

(9) Add 10ml of binding buffer to the affinity chromatography column.

(10) Add 5ml of binding buffer containing 0.05M EDTA to the chromatographic column to remove nickel ions.

(11) Wash the affinity chromatography column with 10 ml of deionized water.

(12) Add 5ml of 20% ethanol to the affinity chromatography column, seal the chromatography column, and store at 4°C.

6. Preliminary identification of anti-Aβ soluble antibody by SDS-PAGE.

(1) Gel preparation:

12% separating gel: 5% stacking gel: ddH ₂ O 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 persulfate (APS) 50μl 50μl   Tetramethylethylenediamine (TEMED) 5μl 5μl

(2) Take 10 μl of the eluent from each tube of method 5 (8) and 10 μl of 2× loading buffer, mix well, and bathe in boiling water for 5 minutes.

(3) Sample loading, 75V constant voltage electrophoresis until the bromophenol blue indicator enters the separation gel, 110V constant voltage electrophoresis until the bromophenol blue completely disappears.

(4) Coomassie Brilliant Blue R250 staining for 30 minutes.

(5) Decolorize the decolorizing solution until the background color is transparent, and observe the purification effect.

7. Dialysis and desalination.

(7), Western blot identification of soluble ScFv antibody

1. Tricine SDS-PAGE electrophoresis

(1), Gel formula:

Separating Gel (15%) stacking gel ddH ₂ O 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 persulfate 50μl 50μl Tetramethylethylenediamine 5μl 5μl

(2) Take 10 μl sample solution and 10 μl 2× loading buffer, mix well, boil to denature, and centrifuge at 1000 rpm for 1 min. The amount of sample protein (Aβ1-42 or BSA) added to each sample well was 20 μg.

(3) Pull out the comb, place the plate in the electrophoresis tank, fill up with Tricine electrophoresis buffer, and load samples in sequence. 100V constant voltage electrophoresis for 90min.

2. Transfer film

(1) After electrophoresis, cut off the stacking gel, soak the gel in protein transfer buffer for 10-20min, and prepare a PVDF membrane of appropriate size.

(2) Correctly install the electrotransfer box, add an appropriate amount of protein transfer buffer, and transfer the protein bands to the PVDF membrane by wet transfer method, 0.3A, 55min.

(3) After electroporation, take the membrane out of the electroporation box, mark the direction, and wash briefly with PBS.

3. Closed

Immerse the membrane in blocking solution (MPBS) and incubate at room temperature for 2 hours to block the non-specific protein binding sites on the PVDF membrane.

4. Antibody Incubation

(1) Add the purified antibody supernatant diluted 1:5 and shake overnight at 4°C.

(2) Wash the membrane with PBS-T for 5 min×3 times.

(3) Add anti-c-myc antibody (1:200 dilution) and incubate at 37°C for 2h.

(4) Wash the membrane with PBS-T for 5 min×3 times. Remove excess primary antibody reaction solution and non-specifically adsorbed antibodies.

(5) Add horseradish peroxidase-labeled goat anti-mouse IgG (diluted at 1:200), and incubate at 37° C. for 2 h.

(6) Wash the membrane with PBS-T for 5 min×3 times.

5. ECL color development: before use, mix ECL color development solution A and B, drop evenly on the surface of the film, avoid light exposure, develop photos in developer and fixer solution, and observe the results.

(8), transmission electron microscope (TEM) observation

1. Preparation of Aβ application solution

(1) Dissolving the Aβ polypeptide in 1% sodium hydroxide solution to prepare an Aβ storage solution with a concentration greater than 5 mg/ml.

(2), BCA protein quantification

Preparation of the working solution: take the A and B solutions in the BCA protein quantitative detection kit (PIERCE company), and prepare the working solution at a ratio of 1:50 (V/V).

Add 10 μl of diluted Aβ stock solution and standard protein solutions with different concentrations of 0, 0.5, 1.0, 1.5, and 2.0 mg/ml into the microtiter plate, add 200 μl of working solution to each well, mix well, and incubate at 37°C for 30 minutes , CliniBio-128 microplate reader reads the absorbance at a wavelength of 562nm, draws a standard curve, and calculates the concentration of the Aβ stock solution.

(3) Before use, dilute with 50 mmol/LPBS containing 0.02% NaN ₃ , pH 7.5. Aβ0.5mg/ml, continuously incubated at 37°C for 3 days, can make it form fibrous aggregates.

2. TEM observation of Aβ fibers

(1), Experimental grouping:

Control group: Aβ 20μM;

scFv treatment group: Aβ 20μM, E3 scFv 20μM;

BSA control group: Aβ 20 μM, BSA 20 μM.

(2) Incubate at 37°C for 2 weeks.

(3) After the incubation, take 10 μl of the protein solution and drop it on the carbon film of the copper grid, and let it dry naturally.

(4), 2% (W/V) uranyl acetate staining.

(5) JEM-1010 transmission electron microscope observation, the operating voltage of the electron microscope is 100kV, and the magnification ratio is 15,000-50,000.

(9) Fluorescence quantitative detection of Thioflavin T (ThT)

1. Experimental group:

Control group: Aβ 20μM;

E3 (1:1) group: Aβ 20 μM, E3 scFv 20 μM;

E3 (1:10) group: Aβ 20 μM, E3 scFv 2 μM.

2. Incubate at 37°C for 0, 1, and 2 weeks.

3. Take 25μl of the incubated sample and mix it with 75μl of 10μM ThT diluent (dissolved in 50mM PBS, pH 6.5). (Each group is set up 5 duplicate wells, and sets up blank control, promptly does not contain the dilution of 10 μ M ThT of sample).

4. Incubate at 37°C for 10 minutes, and use the Spectra MAXGEMINI EM Microplate Fluorescence Quantitative Instrument to measure the fluorescence reading. (excitation wavelength: 450nm, emission wavelength: 482nm)

(10), the protective effect of E3 scFv (E3 soluble antibody) on SK-N-SH cell Aβ toxicity damage

1. Culture of neuroblastoma (SK-N-SH Neuroblastoma cell): SK-N-SH cells were inoculated in DMEM high-glucose medium containing 10% calf serum and 100IU/ml gentamycin, placed in saturated Humidity, 5% CO ₂ , 37°C incubator.

2. Cell subculture and seed plate: Discard the culture medium, wash the cells twice with D-Hank's solution, add 0.25% trypsin-0.02% EDTA mixture to cover the cell surface, let it act for 2-3 minutes at room temperature, and observe the cells with an inverted microscope When the contraction and the edges are clear, add DMEM high-glucose medium containing 10% calf serum to stop the trypsin activity, blow the cells on the bottle wall to make a single-cell suspension, and adjust the cell density to 1×10 ⁵ /ml. 100 μl/well was seeded in a 96-well plate. Culture in an incubator with saturated humidity, 5% CO ₂ , and 37°C.

3. Determination of cell mitochondrial succinate dehydrogenase activity: 24 hours after cell inoculation, replace with culture medium containing 15-30 μM Aβ, continue to cultivate for 72 hours, then add 10 μl of 5 mg/ml MTT solution, continue to cultivate at 37°C for 4 hours, aspirate and discard Add 150 μl/well of DMSO to the supernatant, shake gently for 10 minutes to dissolve the purple crystals, and read the absorbance at a wavelength of 490 nm on an enzyme-linked immunoassay instrument to reflect the respiratory function of mitochondria.

4. The protective effect of E3 soluble antibody on Aβ toxicity damage of SK-N-SH cells:

(1), experimental grouping and processing:

Control group: DMEM normal culture medium;

Aβ group: DMEM culture solution containing 20 μM Aβ;

E3 treatment group: DMEM medium containing 2-20 μM E3 scFv and 20 μM Aβ.

(2) After 24 hours of cell inoculation, the cells were replaced with different conditioned medium according to the above grouping, and culture was continued for 72 hours.

(3) Replace with fresh DMEM high-glucose culture medium, add 10 μl of 5 mg/ml MTT solution, continue to incubate at 37°C for 4 hours, discard the supernatant, add 150 μl/well of DMSO, shake gently for 10 minutes, dissolve the purple crystals, and dissolve the purple crystals in the enzyme The absorbance at the wavelength of 490nm was read on the immunoassay to reflect the respiratory function of mitochondria.

result:

(1) Results of screening Aβ40-specific human-derived antibodies:

After five rounds of affinity screening, 58 clones were randomly selected, amplified and cultured, and phage antibodies were collected for ELISA detection. In the detection, the M13K07 helper phage was used instead of the phage antibody as a negative control to eliminate the non-specific binding between the phage and the Aβ polypeptide. The results of the ELISA test are shown in Figure 1.

(2) Gene and protein sequence of phage antibody:

The E3 monoclonal phage antibody is sequenced by a gene sequencer to obtain its specific antibody gene fragment, which does not contain stop codons TAG, TAA, and TGA. According to this translation, the heavy chain variable region and light chain variable region genes of the antibody are known, and the antibody is known by using the immunoglobulin BLAST database of NCBI (National Center for Biotechnology Information, www.ncbi.nlm.nih.gov) Amino acid sequences of the hypervariable region and scaffold region. As shown in Table 1.

(3), Western blot identification of soluble scFv antibodies:

1. Purification of anti-Aβ soluble single chain antibody by His-Trap affinity chromatography column

IPTG induces 2L E.coli HB2151 to express soluble antibody, take the concentrated culture supernatant and periplasmic cavity extract, His-Trap affinity chromatography, 12% SDS-PAGE electrophoresis, and a purified protein band with a molecular weight of about 31kD can be seen, such as Figure 2 shows.

2. Identification of anti-Aβ soluble single chain antibody by Western blot

The results of Western blot showed that the anti-Aβ soluble antibody could bind to the synthetic Aβ polypeptide and had no cross-reaction with BSA. As shown in Figure 3.

Table 1 Nucleotide sequence (SEQ ID NO.1) and amino acid sequence (SEQ ID NO.2) of CDRs and FRs of E3 single-chain antibody (E3 scFv).

                       

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 AGTGGTTACTACTGGGGC TGGATCCGGCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGG 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

TATCATAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGT CGAGTCACCATATCAGTAGACACGTCCAAAGAACCAG

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

TTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCAAGAGATCTTGGTAGTTCTGTG

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

AGT TGGGGCCAAGGTACCCTGGTCACCGTCTCGAGT 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

AGTCAGGGCATTAGCAATTATTTAGCC TGGTATCAGCAGAAACCAGGGAAAGTTCCTAAGCTCCTGATCTAT GCTGCA

S Q G I S N Y L A W Y Q 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

CTGCAGCCTGAAGATGTTGCAACTTATTACTGTCAAAAGTATAACAGTGCCCTCGTTACG TTCGGCCAAGGGACCAAG

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) Effect of anti-Aβ soluble single chain antibody on Aβ aggregation in vitro

1. Anti-Aβ soluble single chain antibody inhibits the aggregation of Aβ

TEM observation found that 20μM Aβ40 could aggregate to form mature fibers when incubated at 37℃ for 2 weeks. As shown in Fig. 4A,B, mature fibers can form a network. Adding an equimolar concentration of E3 single-chain antibody and incubating with Aβ40 at 37°C for 2 weeks can significantly inhibit the aggregation of Aβ40 to form fibers. Aβ40 mainly presents an amorphous structure, as shown in Figure 4C, D. In the control group added with equimolar concentration of BSA, Aβ40 still aggregated to form mature fibers, as shown in Figure 4E,F.

ThT is a fluorescent dye that can specifically bind to the interactive β-sheet structure in amyloid, and can be used as a quantitative analysis indicator of Aβ aggregation. After continuous incubation at 37° C. for 7-14 days with 20 μM Aβ40, the ThT fluorescence reading value of the sample increased significantly, as shown in FIG. 5 . However, after co-incubation with different molar concentrations of anti-Aβ soluble single-chain antibody, the ThT fluorescence reading value of the test group was significantly lower than that of the control group at the same time point. As shown in Figure 5.

2. Anti-Aβ soluble single-chain antibody promotes disaggregation of Aβ mature fibrils

After co-incubating 20 μM Aβ40 aggregated fibers with an equimolar concentration of anti-Aβ soluble single-chain antibody for 1 day, the Aβ fibers were significantly reduced. As shown in Figure 6, compared with the Aβ control group and the BSA treatment group, the Aβ structure of the E3 single-chain antibody treatment group changed significantly, and the protein in the field of view mainly presented an amorphous structure, and no obvious Aβ40 aggregation fibers were seen.

(5) Anti-Aβ soluble single-chain antibody inhibits the cytotoxicity of Aβ

MTT results showed that 20 μM aggregated Aβ protein could inhibit the activity of succinate dehydrogenase in SK-N-SH cells, and had obvious toxic effects on cells. However, when the anti-Aβ soluble single-chain antibody was added to the cell culture medium, the inhibition of the activity of the Aβ protein on succinate dehydrogenase was significantly reduced. As shown in Figure 7, the toxicity inhibitory effect of the anti-Aβ soluble single chain antibody was obviously concentration-dependent.

Embodiment 2: the preparation of pharmaceutical composition

A therapeutically effective amount of the antibody of the present invention (SEQ ID No.2) or its expression vector (including but not limited to plasmid, phagemid, phage, Escherichia coli, the same below), or the nucleotide encoding the antibody (SEQ ID No.1) or its expression vectors are used alone or mixed with pharmaceutically acceptable adjuvants to form a pharmaceutical composition. The pharmaceutical composition can be used alone or mixed with other drugs that do not affect the function of the pharmaceutical composition. The preparation of these pharmaceutical compositions can be in any pharmaceutically acceptable dosage form, including but not limited to tablets, pills, capsules, injections, oral liquids or liposomes. Methods for the preparation of these formulations are well known to those of ordinary skill in the art. These pharmaceutical compositions can be used for treating or preventing senile dementia.

<110> Nanjing Medical University

<120>An antibody for treating or preventing senile dementia, its expression vector and its application in pharmacy

<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. An antibody for treating or preventing senile dementia, the amino acid sequence of the antibody is as described in SEQ ID No.2. 2. The antibody according to claim 1, characterized in that the antibody is a soluble scFv antibody fragment. 3. The expression vector of the antibody of claim 1. 4. The expression vector according to claim 3, characterized in that the expression vector is a plasmid, phagemid, phage or Escherichia coli. 5. The nucleotide encoding the antibody of claim 1, whose sequence is as described in SEQ ID No.1. 6. The expression vector of the nucleotide as claimed in claim 5. 7. The expression vector according to claim 6, characterized in that the expression vector is a plasmid, phagemid, phage or Escherichia coli. 8. The use of the antibody of claim 1 in the preparation of medicaments for treating or preventing senile dementia. 9. The use of the nucleotide according to claim 5 in the preparation of medicaments for treating or preventing senile dementia. 10. The use of the expression vector according to claim 4 or 6 in the preparation of medicaments for treating or preventing senile dementia.

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