CN108484761B - Single-chain antibody for specifically recognizing and inducing depolymerization of oligomer and fibril of Abeta 42, single-chain antibody gene and application thereof - Google Patents
Single-chain antibody for specifically recognizing and inducing depolymerization of oligomer and fibril of Abeta 42, single-chain antibody gene and application thereof Download PDFInfo
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Abstract
A single-chain antibody and a single-chain antibody gene for specifically recognizing and combining Abeta 42 oligomer and fibril belong to the technical field of genetic engineering antibodies. In particular to a single-chain antibody HT7 of a human anti-Abeta 42 oligomer comprising a heavy chain variable region and a light chain variable region, and the amino acid sequence of the single-chain antibody is shown as SEQ ID NO. 3. Meanwhile, a gene of a coding single-chain antibody HT7 with a nucleotide sequence shown in SEQ ID NO.4 and a gene engineering expression vector of the single-chain antibody HT7 constructed by the gene and vectors such as pET-28a, pET-41b, pMA5, pPZW103 and the like are provided. The single-chain antibody HT7 can specifically recognize and combine A beta 42 oligomers and fibrils, reduce the levels of the A beta 42 oligomers and fibrils, effectively inhibit the neurocytotoxicity of the A beta 42 oligomers and fibrils, and can be widely applied to preparation of drugs for resisting neurotoxic A beta 42 or Alzheimer's disease.
Description
Technical Field
The invention belongs to the technical field of genetic engineering antibodies, and particularly relates to a single-chain antibody for specifically recognizing and inducing depolymerization of oligomers and fibrils of beta-amyloid (Abeta 42), a gene encoding the single-chain antibody, and application of the single-chain antibody in preparation of anti-neurotoxic Abeta 42 drugs or anti-Alzheimer's disease drugs.
Background
Alzheimer's Disease (AD) is a neurodegenerative disease whose main and original pathological feature is progressive aggregation and deposition of a β 42. There are several views on the pathogenesis of AD, of which the a β 42 toxicity theory is generally accepted. It has been demonstrated that a β 42 oligomers and fibrils formed by aggregation of a β 42 monomers are neurotoxic and are the main causative agent of AD. However, most of the antibodies against toxic a β 42 reported internationally are directed against the primary sequence of a β 42, and these antibodies bind to a β 42 monomers, oligomers, fibrils or fibers, and cannot specifically (or exclusively) bind to a β 42 oligomers or fibrils and inhibit their toxicity, thus being prone to cause major side effects in the treatment of AD. In addition, the reported antibody specifically targeting a β 42 oligomers or fibrils can specifically recognize and bind to a specific a β 42 aggregate, but cannot simultaneously and effectively induce depolymerization of a β 42 oligomers and fibrils, and cannot effectively suppress or eliminate toxicity of a β 42 oligomers and fibrils for a long period of time.
In the reports of passive immunotherapy for AD, humanized monoclonal antibodies are mostly adopted, but the clinical application of the antibodies is limited because the antibodies are large in molecules, difficult to pass through the blood brain barrier or poor in specificity and cause side effects. To overcome these limitations, screening of single-chain antibodies that specifically recognize and bind neurotoxic Α β 42 oligomers and fibrils, starting from the construction of small-molecule human single-chain antibodies, is a hot spot in the current diagnosis or treatment of AD.
The single chain antibody (scFv) is a small-molecule genetic engineering antibody, which is a small-molecule recombinant antibody formed by connecting a natural antibody heavy chain variable region (VH) and a natural antibody light chain variable region (VL) through a section of Linker by using a genetic engineering method on a DNA level. Compared with the complete antibody molecule, the antibody has the following characteristics: contains complete antibody variable regions and has complete antigen binding sites; the Fc segment of antibody molecules is not contained, so the immunogenicity is weak, and the antibody is not easy to generate immune reaction when being used for a human body; the molecular weight is small, the penetrating power is strong, the blood brain barrier can be easily penetrated, and the kit is suitable for diagnosis or treatment of AD; the half-life period of the circulation in vivo is short, and the circulation in vivo is easy to remove from the blood circulation; the functional antibody molecule can be formed without glycosylation modification, so that the method is favorable for genetic engineering operation and mass production of prokaryotic expression systems. Therefore, the single-chain antibody is the most reported and promising genetic engineering antibody against AD at present.
Although single-chain antibodies against A beta 42 oligomers have been reported internationally (Sebellela A et al, 2017, J neurochem. doi: 10.1111/jnc.14118; Yoshihara T et al, 2008, J Biochem,143, 475-. So far, no report of a single-chain antibody which specifically binds to a β 42 oligomers and fibrils and can effectively induce depolymerization of the a β 42 oligomers and fibrils is seen in China.
Disclosure of Invention
The purpose of the present invention is to provide a single-chain antibody that specifically recognizes and binds to A beta 42 oligomers and fibrils and can efficiently induce depolymerization of the A beta 42 oligomers and fibrils, and a gene encoding the single-chain antibody.
The invention not only solves the technical problem that the prior anti-Abeta 42 antibody can be combined with all Abeta 42 forms including Abeta 42 monomer without specificity, but also solves the technical problem that the prior single-chain antibody of anti-Abeta 42 oligomer has poor specificity to Abeta 42 fibril and does not induce Abeta 42 oligomer and fibril depolymerization. The invention successfully screens out the single-chain antibody which is specifically combined with the Abeta 42 oligomer and fibril but not combined with the Abeta 42 monomer and fibril by utilizing the genetic engineering antibody technology and can induce the depolymerization of the Abeta 42 oligomer and fibril. The characteristic of the single-chain antibody of the invention for specifically binding to the a β 42 oligomer and fibril is not related to the primary structure of a β 42, but is a conformation-dependent single-chain antibody based on the conformation specific to the a β 42 oligomer and fibril.
The A beta 42 oligomer and fibril of the present invention is formed by the aggregation of 2 to dozens of A beta 42 monomers, the molecules are mainly combined by hydrogen bonds, hydrophobic bonds and Van der Waals force, and the molecular weight is from 9kDa to 100 kDa.
The invention provides a single-chain antibody HT7 of humanized anti-Abeta 42 oligomer and fibril comprising a heavy chain variable region (VH) and a light chain variable region (VL), the amino acid sequence of the single-chain antibody is shown in SEQ ID NO.3, wherein the VH has the amino acid sequence shown in SEQ ID NO.1, the VL has the amino acid sequence shown in SEQ ID NO.2, and the molecular weight of HT7 is about 29 kDa.
The invention also provides a gene of the single-chain antibody HT7 for encoding the human anti-Abeta 42 oligomer and fibril, and the nucleotide sequence is shown in SEQ ID NO. 4.
Furthermore, the single-chain antibody HT7 can effectively inhibit the aggregation of A beta 42 monomers, can depolymerize A beta 42 oligomers and fibrils, can obviously reduce the toxicity of the A beta 42 oligomers and fibrils to nerve cells, and has wide application in preparing anti-Alzheimer's disease drugs.
Furthermore, the gene of the single-chain antibody HT7 for coding the humanized anti-Abeta 42 oligomer and fibril can be recombined with pET-28a, pET-41b, pMA5 or pPZW103 to construct a gene engineering expression vector of the single-chain antibody HT7, and the HT7 gene and the HT7 gene engineering expression vector have wide application in preparing anti-Alzheimer disease drugs.
The single-chain antibody HT7(scFv HT7) disclosed by the invention can specifically recognize and combine A beta 42 oligomers and fibrils, and has wide application prospects as a clinical diagnosis preparation for detecting the A beta 42 oligomers and fibrils and an immune preparation for treating AD.
Drawings
FIG. 1: sequencing spectrogram of pET28a-HT7 recombinant expression vector;
FIG. 2: SDS-PAGE profile of purified single chain antibody HT7, wherein M: a protein Marker; 1: a control group; 2: expressed HT 7; 3: purified HT 7;
FIG. 3: a Dot-blot analysis spectrogram recognized by the single-chain antibody HT7 and different forms of Abeta 42;
FIG. 4: a Western blot analysis spectrogram for recognizing the single-chain antibody HT7 and Abeta 42 aggregates with different molecular weights;
FIG. 5: a graph of the affinity of HT7 to A beta 42 oligomers measured by ELISA;
FIG. 6: analyzing the spectrogram of inhibiting A beta 42 aggregation and inducing A beta 42 aggregate depolymerization by using a single-chain antibody HT7 through a ThT-F method; wherein, M: monomer, O: oligomer, P: fibril, F: fibers; p < 0.01, p < 0.05.
FIG. 7: a graph for determining the concentration-dependent inhibition of A beta 42 cytotoxicity of a single-chain antibody HT7 by the MTT method.
Detailed Description
Example 1 preparation of Abeta 42 oligomers, fibrils and Abeta 42 fibers
Abeta 42 monomer (purchased from Sigma, USA) was dissolved in ice-pre-chilled Hexafluoroisopropanol (HFIP) to a concentration of 1mg/mL, sonicated in an ice-water bath for 10min, dried under vacuum, and frozen at-20 ℃. When the Abeta 42 protein is used, the obtained Abeta 42 monomer is dissolved by dimethyl sulfoxide (DMSO) to the concentration of 1mg/mL, Abeta 42 is diluted into a phosphate buffer solution (pH7.4 and 50mM) with the concentration of 10 mu M, the final concentration of Abeta 42 is 10 mu M, the solution is respectively incubated at 37 ℃ for 3 to 12 hours to form Abeta 42 oligomers, the solution is incubated for 12 to 24 hours to form Abeta 42 fibrils, and the solution is incubated for 36 to 37 hours to form the aggregation state of Abeta 42 mature fibers. All a β 42 aggregation states were confirmed by electron microscopy. Due to the irreversibility of A beta 42 forming aggregates, various aggregates of A beta 42 are prepared as-is.
EXAMPLE 2 screening of Positive clones
(1) Peripheral blood lymphocyte RNA extraction and reverse transcription synthesis cDNA
Separating and extracting peripheral blood lymphocytes: peripheral blood samples of 10mL from Alzheimer's patients were collected using an ethylenediaminetetraacetic acid dipotassium salt (EDTA-2K) anticoagulant tube (Amersham pharmacia Biotech) and diluted with a balanced salt buffer (D' Hanks) free of calcium and magnesium ions (from Biyuntian Biotech research institute) at a volume ratio of 1:1 volume dilution to dilute blood and lymphocyte isolates 2: 1 (volume ratio) Polysucrose-diatrizoate dextran lymphocyte isolate (from Sigma, USA) was added. Centrifuge at 2000rpm/min for 20min at room temperature. After centrifugation, the mononuclear cell layer was aspirated. The cells were washed with D' Hanks and centrifuged at 1000rpm/min for 10min to obtain peripheral blood lymphocytes.
Peripheral blood lymphocyte RNA extraction: at 1X 1071mL of phenol-guanidinium isothiocyanate total RNA extraction reagent was added to each peripheral blood lymphocyte
Reagent, from Invitrogen corporation), left at 4 ℃ for 5 min. 200 μ L of chloroform was added and the solution was turned upside down until a white slurry appeared. Place on ice for 5 min. Centrifuging at 12000rpm/min for 15min at 4 ℃. The upper aqueous phase was transferred to another centrifuge tube, an equal volume of isopropanol was added, and incubation was performed on ice for 10 min. Centrifuging at 12000rpm/min at 4 deg.C for 10 min. The supernatant was discarded and the pellet (containing RNA) was washed with 1mL of 75% (volume fraction) ethanol. Centrifuging at 12000rpm/min for 5min at 4 deg.C to obtain RNA precipitate. After air drying, dissolving with appropriate amount of Tris-EDTA buffer (TE) or RNase-free deionized water for use.
Reverse transcription to synthesize cDNA: the method for synthesizing cDNA using the above-extracted RNA under the action of reverse transcriptase was as follows, taking 4. mu.L (1000. mu.g/. mu.L) of the above-prepared RNA solution and adding to a 0.1mL centrifuge tube, adding 1. mu.L of oligothymidine Primer (Oligo dT Primer) and 1. mu.L of deoxyribonucleoside triphosphate mixture (dNTP), finally supplementing to 10. mu.L with RNase-free deionized water, incubating at 65 ℃ for 5min, and rapidly cooling in an ice bath. To the tube, reverse transcription Buffer (PrimeScript II Buffer) 4. mu. L, RNA enzyme Inhibitor (RNase Inhibitor) 0.5. mu.L and reverse transcriptase (PrimeScript II RTase) 1. mu.L were added, and 20. mu.L of the mixture was supplemented with RNase-free deionized water and mixed slowly. The reverse transcription was carried out at 42 ℃ for 45min and 95 ℃ for 5min, and then cooled on ice.
(2) Amplification of DNA fragments encoding VH and VL
Primers for amplifying DNA fragments encoding VH and VL were designed and synthesized based on the conserved sequences of VH and VL, and the sequences were as follows:
5′CCTGAGCCACCTCCGCCAGAACCGCCTCCACCTGAAGAGACGGT VHA1
5′TGGCGGAGGTGGCTCAGGCGGTGGAGGATCGGATATCCAGATGA VLS1
the underlined 5 'CATATG sequence in VHS1 is the recognition site for endonuclease Nde I, and the underlined 3' GCGGCCGC sequence in VLA1 is the recognition site for endonuclease Not I.
The amplification process is as follows:
diluting the cDNA prepared in the step (1) by 50 times with RNase-free deionized water, and adding 1 μ L of the diluted cDNA into a PCR system for amplifying DNA fragments encoding VH and VL respectively to obtain a DNA fragment amplification system encoding VH, wherein the PCR system comprises: VHS1 (25. mu. mol/L) 0.4. mu.L, VHA1 (25. mu. mol/L) 0.4. mu.L; amplification System for DNA fragments encoding VL: VLS1 (25. mu. mol/L) 0.4. mu.L, VLA1 (25. mu. mol/L) 0.4. mu.L; the two systems are respectively added with 0.1 mu L of DNA polymerase (r-Taq), 0.8 mu L of dNTPs, 1 mu L of 10 XBuffer and 6.3 mu L of deionized water without RNase for respectively carrying out the amplification of target fragments: at 94 deg.C for 5min, at 94 deg.C for 30s, at 55 deg.C for 30s, at 72 deg.C for 1min for 30 cycles, and at 72 deg.C for 10 min. Finally, DNA fragments encoding VH and VL were recovered with gel recovery kits (purchased from Shanghai Bioengineering Co., Ltd.), respectively.
(3) Splicing and amplification of DNA fragments encoding scFv
The splicing system is as follows: 2 μ L of DNA fragment encoding VH [ from step (2) ], 1 μ L of DNA fragment encoding VL [ from step (2) ], 0.8 μ L of dNTPs, 1 μ L of 10 XBuffer, 0.1 μ L of r-Taq DNA polymerase, 5.1 μ L of RNase-free deionized water, effecting splicing of DNA fragments encoding scFv under the following conditions: at 94 deg.C for 5min, at 94 deg.C for 50s, at 55 deg.C for 50s, at 72 deg.C for 1min, for 30 cycles, at 72 deg.C for 10min, and at 4 deg.C.
Amplification of DNA fragments encoding scFv: the spliced DNA fragment encoding scFv was diluted 50-fold, and 5. mu.L of the DNA fragment was added to the following amplification system: VHS 12.5. mu.L, VLA 12.5. mu.L, r-Taq DNA polymerase 0.5. mu.L, dNTPs 4. mu.L, 10 XBuffer 5. mu.L, RNase-free deionized water 30.5. mu.L, and amplification conditions are as follows: at 94 deg.C for 5min, at 94 deg.C for 30s, at 55 deg.C for 30s, at 72 deg.C for 1min, and for 30 cycles, at 72 deg.C for 10 min. 1 μ L of the amplification product was subjected to 1% agarose gel electrophoresis, and the rest was subjected to gel electrophoresis and recovered with a gel recovery kit (purchased from Shanghai Bioengineering Co., Ltd.).
(4) Screening for Positive clones
The Nde I and Not I cleavage sites designed in the primers were matched with Nde I and Not I of the prokaryotic expression vector pET28a (a general E.coli expression vector containing a strong T7 promoter, a C-terminal histidine tag, a kanamycin resistance gene, and the like, available from Novagen, Invitrogen, and the like) used in the present invention, and were suitable for efficient expression in E.coli. The humanized scFv gene fragment was cloned into expression vector pET28a between NdeI and NotI cleavage sites using NdeI and NotI cleavage sites, and ligated to obtain a recombinant expression vector.
The construction steps of the expression vector are as follows:
double enzyme digestion reaction: 1.0. mu.g of pET28a vector and the scFv gene fragment obtained above were mixed with an appropriate amount of deionized water to make the total volume 18. mu.L, 2-3 units of restriction enzyme Nde I and 2-3 units of Not I were added to each of them, and 2. mu.L of the corresponding 10 XH buffer was added to each of them, and after mixing, the mixture was kept in a 37 ℃ water bath for 2-3 hours, and then the objective DNA (purchased from Shanghai Bioengineering Co., Ltd.) was recovered by gel electrophoresis and then by using a gel recovery kit.
Ligation of the DNA fragment encoding scFv to the pET28a vector: taking 0.5 mu g of pET28a vector DNA recovered in the above step, adding 2-10 times of molar weight of scFv coding gene fragment obtained in the above step and 2 mu L of 10 XT 4DNA ligase buffer solution, adding deionized water to be set to 20 mu L, finally adding 1 unit of T4DNA ligase, uniformly mixing and instantly centrifuging to enable liquid drops to gather at the bottom of a tube, and placing the tube in a water bath at 16 ℃ overnight to obtain the connected recombinant expression vector pET28 a-scFv.
Transformation of E.coli BL21(DE3) competent cells with the recombinant expression vector pET28a-scFv [ E.coli BL21(DE3) competent cells preparation method refer to "molecular cloning laboratory Manual" (second edition, scientific Press) page 49 ], ice bath 30 minutes later, 42 ℃ water bath heat preservation 1 minutes, immediately taken out and placed in ice bath cooling 2 minutes. After 200. mu.L of LB liquid medium preheated at 37 ℃ was added and cultured with shaking at 37 ℃ and 150rpm for 60 minutes, 100. mu.L of the culture was taken out and spread on LB agar plates containing kanamycin (Kan), and after culturing at 37 ℃ for 12 hours, transformed colonies appeared.
Single clones were picked up on a 96-well bacterial culture plate (purchased from Corning, USA) and 200. mu.L of LB liquid medium containing 100. mu.g/mL kanamycin (Kan) was added to each well. The cells were cultured overnight at 37 ℃ with shaking at 200 rpm. Sucking 2 μ L of the bacterial liquid from each well, adding into another new 96-well bacterial culture plate, adding 200 μ L LB culture medium containing 100 μ g/mL Kan into each well, and shaking at 37 deg.C and 200rpm to OD6000.8 to 1.0. A volume of glycerol was added to the first plate to a final concentration of 15% and stored at-80 ℃. Isopropyl- β -D-thiogalactoside (IPTG) (from Sigma, USA) was added to each well of the second 96-well plate to a final concentration of 0.05mM, and induced at 20 ℃ for 12h with shaking at 160rpm, and then centrifuged at 4000rpm for 10min in a 96-well plate centrifuge, and the medium was discarded to collect the cells. Adding thallus lysate into a 96-well plate, performing ice-bath lysis for 1 hour, centrifuging at 4000rpm for 10 minutes, and collecting total protein after lysis.
A96-well plate (from Corning, USA) was coated with 10. mu.g/mL of A.beta.42 oligomer (from example 1) at 100. mu.L/well for 16-18h at 4 ℃. Discard the antigen solution, add 100. mu.L of 1% (volume ratio) Bovine Serum Albumin (BSA) to each well, and block at 37 ℃ for 1 h. Phosphate Buffered Saline (PBS) [ containing 0.1% (by volume) Tween-20]The plate was washed three times, 100. mu.L of the total protein collected above was added to each well, and incubated at 37 ℃ for 2 h. Positive control wells plus commercial Abeta antibody B4 (available from Santa cruz, USA), negative control wells plus BAnd (SA). PBS [ containing 0.1% (by volume) Tween-20]The plate was washed six times. mu.L of 1:2000 (vol.) diluted anti-His mab (available from Santa cruz, USA) was added and incubated at 37 ℃ for 1 h. PBS [ containing 0.1% (by volume) Tween-20]The plate was washed six times. mu.L of a Horse Radish Peroxidase (HRP) -labeled goat anti-rabbit IgG antibody (purchased from Dr. China) diluted 1:4000 (by volume) was added and incubated at 37 ℃ for 1 h. PBS [ containing 0.1% (by volume) Tween-20]The plate was washed six times. Adding 50 μ L/well of 3,3',5,5' -Tetramethylbenzidine (TMB) (available from Amresco, USA), and reacting at room temperature in dark for 15min, adding 30 μ L2 mol/L H per well2SO4The reaction was stopped and the OD value (wavelength 450nm) was measured with a microplate reader. And (4) judging a result: the OD value is more than 3 times of that of the negative control, and the OD value of the blank control is less than 0.2. And through result identification, a monoclonal strain HT7 with strong binding force with the antigen is screened out for subsequent experiments.
EXAMPLE 3 DNA sequencing of the Single-chain antibody HT7 Gene
The plasmid pET28a-HT7 was extracted and submitted to the Shanghai Producer sequencing department for sequence determination, the result is shown in FIG. 1 (pET28a-HT7 vector sequencing map, wherein the gene coding HT7 is nucleotide No. 120-869). The measured HT7 gene sequence and the immunoglobulin gene sequence in GeneBank are compared and analyzed, and the results prove that: the obtained HT7 clone has a DNA sequence coding for scFv, an initiation codon of ATG and a termination codon of TGA, the nucleotide sequence is shown in SEQ ID NO.4, the DNA sequence comprises a DNA sequence coding for a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody, and the deduced corresponding amino acid sequences (SEQ ID NO.1 and SEQ ID NO.2) have typical antibody variable region structures.
EXAMPLE 4 expression and preparation of the Single chain antibody HT7
E.coli BL21(DE3) containing pET28a-HT7 plasmid was cultured overnight at 37 ℃ with shaking. Inoculating at a ratio of 1:100, and shake-culturing OD at 37 deg.C600When the strain is equal to 0.8-1.0, IPTG is added to induce the expression of HT7, the strain is induced by shaking at the temperature of 20 ℃ and the rpm of 160 for 12 hours, and then the strain is centrifuged at the temperature of 4 ℃ and the rpm of 5000 for 5min to collect the strain. The cells were resuspended in PBS (pH7.4), sonicated and centrifuged to collect the supernatant. Applying Ni to the collected supernatant2+NTA column purification of the human single-chain antibody HT 7: will be at the topThe clear liquid slowly flows through Ni2 +NTA column, then column-washed with 8 column volume buffer (20mM phosphate buffer, 500mM sodium chloride, 20mM imidazole), and finally eluted with elution buffer (20mM phosphate buffer, 500mM sodium chloride, 250mM imidazole) human single-chain antibody HT7 of the present invention. FIG. 2 shows the SDS-PAGE identification of the prepared human single-chain antibody HT7, and the purified human single-chain antibody HT7 has a molecular weight of about 29.0 kDa.
Example 5 Dot-blot detection of the binding specificity of the Single-chain antibody HT7 to Abeta 42 monomers and aggregates
A beta 42 monomer, oligomer, fibril and fiber are put on a nitrocellulose membrane, the membrane is sealed by 5 percent (volume ratio) of skimmed milk powder, and the mixture is placed at room temperature for 1 h. After 1h incubation with the addition of the single chain antibody HT7, the membranes were washed 3 times with PBS for 10min each. Further adding a His-tag antibody [1:1000 (vol.), incubation at 37 ℃ for 1h, washing the membranes with PBS (containing 0.1% (vol.). sup.Tween-20) 3 times for 10min each. The membrane was added to HRP-labeled rabbitIgG [1:5000 (volume ratio) ] and incubated at 37 ℃ for 1 h. PBS [ containing 0.1% (volume ratio) Tween-20] washing the membrane 3 times, each time for 10 min. The membrane was washed with PBS 1 time for 10 min. The membrane was developed with the substrate luminescence and color development kit (ECL) (available from the bio-technical institute on cloudy days), and as shown in fig. 3, it showed distinct spots only at a β 42 oligomers and fibrils, and almost no spots in a β 42 monomers and fiber morphology.
Example 6 Western blot detection of the recognition of Ass 42 aggregates by the Single-chain antibody HT7
The Abeta 42 mixture (monomer, oligomer, fibril and fiber volume ratio is 1: 1: 1) is separated by non-denaturing gel electrophoresis, transferred to polyvinylidene fluoride (PVDF) membrane, and blocked with 5% (volume ratio) skimmed milk for 1 h. The membranes were washed 3 times for 10min each with PBS [ containing 0.1% (vol/vol) Tween-20 ]. The membrane was placed in a hybridization bag, to which a single-chain antibody HT7 was added, and to a control group, an anti-Abeta 42 antibody (B4) (available from Santa Cruz Co., Ltd.) [1:1000 (volume ratio) ], and after 2h of hybridization at room temperature, the membrane was washed 3 times with PBS [ containing 0.1% (volume ratio) Tween-20], each for 10 min. The membranes were placed in hybridization bags, and anti-histidine-Tag antibody (anti His-Tag) (available from Santa Cruz Co.) [1:1000 (volume ratio) ] antibody was added to the experimental groups, and after hybridization at room temperature for 1h, the membranes were washed 3 times with PBS [ containing 0.1% (volume ratio) Tween-20], 10min each time. Corresponding HRP-labeled IgG (1: 5000 (volume ratio)) was added to each fraction, and hybridization was carried out at room temperature for 1 hour. PBS [ containing 0.1% (volume ratio) Tween-20] washing the membrane 3 times, each time for 10 min. ECL was added for color development, and the results are shown in FIG. 4: HT7 can specifically recognize the region with the molecular weight of 18-55 KDa, namely A beta 42 oligomer and fibril region, and has no obvious combination to A beta 42 monomer and A beta 42 mature fiber form.
Example 7 determination of the affinity of the Single-chain antibody HT7
The affinity of the single-chain antibody HT7 was determined by indirect ELISA and was determined as the equilibrium dissociation constant (K) of the antigen-antibody complexD) And (4) showing. The ELISA plate was coated with Abeta 42 oligomer or fibril at a concentration of 1. mu.g/mL overnight at 4 ℃, blocked with 1% BSA at 37 ℃ for 1h, and the single-chain antibodies HT7 and 10 were incubated in the other plate-10～10-4mixing Abeta 42 oligomer or fibril at mol/L concentration, adding blocking solution with equal volume, incubating at room temperature for 1 hr, adding the mixed solution into antigen coated well, incubating at 37 deg.C for 2 hr, and adding PBS (containing 0.1% (volume ratio) Tween-20]After washing, 100. mu.L of 1:2000 (vol.) diluted anti His-Tag antibody was added to each well, and incubated at 37 ℃ for 1h in PBS [ containing 0.1% (vol.) Tween-20]Washing, adding TMB to develop color, and measuring Optical Density (OD) at 450nm450) The results of a β 42 oligomers are shown in fig. 5, and the results of a β 42 fibrils are similar to (less than) a β 42 oligomers. KDTo achieve maximum OD450The A.beta.42 oligomer or fibril concentration at 50% of the value is known from experimental data, KD oligomersAnd KD fibrilAre all 3.1 × 10-6M。
Example 8 inhibition of A.beta.42 aggregation by the Single chain antibody HT7
Monomers, oligomers, fibrils and fibers of A beta 42 were mixed with a single-chain antibody HT7 at an equimolar ratio, incubated at 37 ℃, and the aggregation degree of A beta 42 was measured by thioflavin T fluorescence staining (ThT-F) at 0h, 3h, 12h, 24h and 48h, respectively, and the results are shown in FIG. 6. FIG. 6 shows that: 1, the single-chain antibody HT7 can remarkably aggregate A beta 42 monomers and oligomers; 2, the HT7 single-chain antibody can remarkably induce depolymerization of Abeta 42 oligomer and fibril; 3: the HT7 single-chain antibody has certain inhibiting effect on the aggregation of A beta 42 fibers and certain effect of inducing the depolymerization of the A beta 42 fibers. From the above results it can be derived: the single-chain antibody HT7 not only can inhibit the aggregation of A beta 42, but also can effectively induce the depolymerization of the formed A beta 42 oligomer and fibril.
Example 9 Single chain antibodies inhibit the cytotoxic effects of Abeta 42 oligomers and fibrils on cells
SH-SY5Y cells were seeded into multi-well cell culture plates at 5000 cells per well, 100. mu.L per well volume, and after the cells were cultured at 37 ℃ for 24h, the following 17 experiments were performed:
after the above 17 groups were incubated at 37 ℃ for 20h, 20 μ L of 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyltetrazolium bromide (MTT) (purchased from sigma, usa) solution (5mg/mL) was added to each well, incubation was continued for 4h at 37 ℃, the culture was terminated, and the survival rate of the cells was detected by the MTT method, and the results are shown in fig. 7, in which the single-chain antibody HT7 inhibited the toxic effect of a β 42 monomers, oligomers, fibrils and fibers on the cells in a concentration-dependent manner, and the survival rate of a β 42 oligomers and fibrils was significantly reduced, while the survival rate of the corresponding cells was significantly increased after the single-chain antibody HT 7. In contrast, the single chain antibody HT7 has limited inhibitory effect on the toxicity of a β 42 monomers and fibers. These results also demonstrate that the single-chain antibody HT7 has a strong affinity for a β 42 oligomers and fibrils, and is able to effectively bind to a β 42 oligomers and fibrils and inhibit their toxic effects on cells.
Example 10 in vitro modeling of blood-brain Barrier and detection of Single chain antibodies penetrating the blood-brain Barrier
The in vitro blood brain barrier model is established by a method of co-culturing human umbilical vein endothelial cells HUVEC and glioma cells C6.
The co-culture method is as follows: HUVEC culture conditions of human umbilical vein endothelial cells: to Ham's F12K medium was added a final concentration of 2mM L-glutamine, 1.5g/L sodium bicarbonate, 0.03mg/mL endothelial growth factor support (ECGS), and 10% fetal bovine serum.
Culturing conditions of glioma cells C6: DMEM-F12 medium was supplemented with fetal bovine serum to a final concentration of 10%.
Firstly, inoculating 45,000 cells/hole C6 on the bottom surface of a PET (polyethylene terephthalate) membrane, after the cells are attached to the PET membrane, setting a transwell chamber right, adding a proper amount of culture medium into a receiving pool, inoculating 30,000 cells/hole on the front surface of the PET membrane after 2-3 days, inoculating HUVEC cells at 37 ℃ and 5% CO2And co-culturing for 5-7 days under the saturated humidity condition.
And (3) identification:
a. observation under an optical microscope
b. Leakage test: the difference between the liquid level inside and outside the transwell chamber was 0.5cm or more, and the change in the difference between the liquid levels was observed after 4 hours of culture.
c. Transendothelial resistance value (TEER) was measured: HUVEC and C6 cells were co-cultured and two-sided resistance values were measured using MilliCell-ERS Voltohmmeter. Fresh medium was changed every day while measuring TEER values until it was confirmed microscopically that the cells had completely fused and TEER values had stabilized.
Detection of single chain antibodies crossing the blood brain barrier: when HUVEC/C6 cells are co-cultured for 5-7 days, the TEER value is up to 630ohm/cm2And after stabilization, carrying out a single-chain antibody cross blood brain barrier transport detection experiment. Mu.g of the single-chain antibody HT7 was added to the chamber. At 5min, 15min, 30min, 60min, 90min, and 120min, 60. mu.L of sample was aspirated outside the chamber, added to the microplate, and coated overnight at 4 ℃. ELISA method to detect single chain antibody HT7 penetration blood brain barrier. As a result: the efficiency of the single-chain antibody HT7 in penetrating the blood-brain barrier increases with time within 60 minutes, and the penetration rate approaches the peak value at 60min, and then almost assumes a stable state. The results demonstrate that the single chain antibody HT7 is able to penetrate the blood brain barrier efficiently.
<110> Jilin university
<120> single-chain antibody, single-chain antibody gene and use thereof for specifically recognizing and inducing depolymerization of oligomer and fibril of Abeta 42
<160> 4
<210> 1
<211> 126
<212> PRT
<213> VH
<400> 1
Met Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
1 5 10 15
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Val Thr Ser
20 25 30
Tyr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
35 40 45
Met Gly Gly Ile Asn Thr Asn Thr Gly Asn Pro Ala Tyr Ala Pro Gly
50 55 60
Phe Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala
65 70 75 80
His Leu Gln Ile Gly Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe
85 90 95
Cys Ala Arg Ser Ser Ser Gly Tyr Gln Gly Ser Ser Tyr Tyr Phe Asp
100 105 110
Met Asp Val Trp Asp Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 2
<211> 108
<212> PRT
<213> VL
<400> 2
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Val Ser Gly Thr His Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr His Tyr Cys Gln Gln Ser Ser Ser Thr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 3
<211> 249
<212> PRT
<213> HT7
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Met Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
1 5 10 15
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Val Thr Ser
20 25 30
Tyr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
35 40 45
Met Gly Gly Ile Asn Thr Asn Thr Gly Asn Pro Ala Tyr Ala Pro Gly
50 55 60
Phe Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala
65 70 75 80
His Leu Gln Ile Gly Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe
85 90 95
Cys Ala Arg Ser Ser Ser Gly Tyr Gln Gly Ser Ser Tyr Tyr Phe Asp
100 105 110
Met Asp Val Trp Asp Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln
130 135 140
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
145 150 155 160
Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr Leu Asn Trp
165 170 175
Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile Tyr Ala Ala
180 185 190
Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Val Ser
195 200 205
Gly Thr His Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
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Ala Thr His Tyr Cys Gln Gln Ser Ser Ser Thr Pro Leu Thr Phe Gly
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Gly Gly Thr Lys Val Glu Ile Lys Arg
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atg cag gtg cag ctg gtg cag tct gga gct gag gtg aag aag cct ggg
Met Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
1 5 10 15
gcc tca gtg aag gtc tcc tgc aag gct tct ggt tac acc gtt acc agt
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Val Thr Ser
20 25 30
tat ggt atc agc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg
Tyr Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
35 40 45
atg gga ggg atc aac acc aat act ggg aac cca gcg tat gcc ccg ggc
Met Gly Gly Ile Asn Thr Asn Thr Gly Asn Pro Ala Tyr Ala Pro Gly
50 55 60
ttc aca gga cga ttt gtc ttc tcc ttg gac acc tct gtc agc acg gca
Phe Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala
65 70 75 80
cac ctg cag atc ggc agc cta aag gct gag gac acc gcc gtt tat ttc
His Leu Gln Ile Gly Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe
85 90 95
tgt gcg agg agt tcg agt ggt tat cag ggt tcc tcc tac tac ttc gat
Cys Ala Arg Ser Ser Ser Gly Tyr Gln Gly Ser Ser Tyr Tyr Phe Asp
100 105 110
atg gac gtc tgg gac caa ggg aca acg gtc acc gtc tct tca ggt gga
Met Asp Val Trp Asp Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly
115 120 125
ggc ggt tct ggc gga ggt ggc tca ggt ggt gga gga tcg gat atc cag
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln
130 135 140
atg act cag tct cca tcc tcc ctg tct gcg tct gta gga gac aga gtc
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val
145 150 155 160
acc atc act tgc cgg gca agt cgg acc att agc agt tat tta aat tgg
Thr Ile Thr Cys Arg Ala Ser Arg Thr Ile Ser Ser Tyr Leu Asn Trp
165 170 175
tat cag cag aaa cca ggg aaa ccc cct aaa ctc ctg atc tat gct gca
Tyr Gln Gln Lys Pro Gly Lys Pro Pro Lys Leu Leu Ile Tyr Ala Ala
180 185 190
tcc att tta caa agt ggg gtc cca tca agg ttc agt ggc agt gta tct
Ser Ile Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Val Ser
195 200 205
ggg aca cat ttc act ctc acc atc agc agt ctg caa cct gaa gat ttt
Gly Thr His Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe
210 215 220
gca acc cac tac tgt caa cag agt tcc agt acc ccg ctt act ttc ggc
Ala Thr His Tyr Cys Gln Gln Ser Ser Ser Thr Pro Leu Thr Phe Gly
225 230 235 240
gga ggg acc aaa gtg gag atc aaa cgt
Gly Gly Thr Lys Val Glu Ile Lys Arg
245
Claims (10)
1. A single chain antibody HT7, characterized by: the amino acid sequence is shown in SEQ ID NO. 3.
2. The use of the single chain antibody HT7 of claim 1 for the manufacture of a medicament for inhibiting A β 42 monomer aggregation.
3. Use of the single chain antibody HT7 of claim 1 for the preparation of a medicament for inhibiting aggregation and disaggregating A β 42 oligomers and fibrils.
4. The use of the single chain antibody HT7 of claim 1 for the manufacture of a medicament for reducing the neurotoxicity of A β 42 oligomers and fibrils.
5. Use of the single chain antibody HT7 of claim 1 for the manufacture of a medicament against neurotoxic A β 42.
6. Use of the single chain antibody HT7 according to claim 1 for the manufacture of a medicament against Alzheimer's disease.
7. A gene encoding the single chain antibody HT7 of claim 1, characterized in that: the nucleotide sequence is shown in SEQ ID NO. 4.
8. A genetically engineered expression vector characterized by: is an expression vector of the single-chain antibody HT7 constructed by the gene encoding the single-chain antibody HT7 of claim 7 and pET-28a, pET-41b, pMA5 or pPZW103 vector.
9. Use of the gene of claim 7 for the preparation of an anti-neurotoxic Α β 42 drug or an anti-alzheimer's disease drug.
10. The use of the genetically engineered expression vector of claim 8 in the preparation of an anti-neurotoxic Α β 42 drug or an anti-alzheimer's disease drug.
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