CN116375857A - Single-chain antibody for detecting Tau protein, and preparation method and application thereof - Google Patents
Single-chain antibody for detecting Tau protein, and preparation method and application thereof Download PDFInfo
- Publication number
- CN116375857A CN116375857A CN202310329690.4A CN202310329690A CN116375857A CN 116375857 A CN116375857 A CN 116375857A CN 202310329690 A CN202310329690 A CN 202310329690A CN 116375857 A CN116375857 A CN 116375857A
- Authority
- CN
- China
- Prior art keywords
- variable region
- chain variable
- tau protein
- chain antibody
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010026424 tau Proteins Proteins 0.000 title claims abstract description 89
- 102000013498 tau Proteins Human genes 0.000 title claims abstract description 89
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 239000012634 fragment Substances 0.000 claims abstract description 18
- 108010075254 C-Peptide Proteins 0.000 claims abstract description 12
- 108010047041 Complementarity Determining Regions Proteins 0.000 claims abstract description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 65
- 108090000623 proteins and genes Proteins 0.000 claims description 39
- 238000000034 method Methods 0.000 claims description 24
- 210000004408 hybridoma Anatomy 0.000 claims description 20
- 239000013612 plasmid Substances 0.000 claims description 17
- 102000004169 proteins and genes Human genes 0.000 claims description 16
- 239000002299 complementary DNA Substances 0.000 claims description 14
- 108020004414 DNA Proteins 0.000 claims description 13
- 241000894006 Bacteria Species 0.000 claims description 12
- 230000014509 gene expression Effects 0.000 claims description 12
- 238000003259 recombinant expression Methods 0.000 claims description 11
- 239000013613 expression plasmid Substances 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 102000053602 DNA Human genes 0.000 claims description 9
- 230000003248 secreting effect Effects 0.000 claims description 9
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 238000010353 genetic engineering Methods 0.000 claims description 7
- 238000003786 synthesis reaction Methods 0.000 claims description 7
- 230000002194 synthesizing effect Effects 0.000 claims description 7
- 238000010276 construction Methods 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 230000003698 anagen phase Effects 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 230000006698 induction Effects 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000007857 nested PCR Methods 0.000 claims description 3
- 230000009465 prokaryotic expression Effects 0.000 claims description 3
- 238000003757 reverse transcription PCR Methods 0.000 claims description 3
- 238000002731 protein assay Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 6
- 238000001514 detection method Methods 0.000 abstract description 24
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 229920001184 polypeptide Polymers 0.000 description 55
- 150000001413 amino acids Chemical group 0.000 description 53
- 102000004196 processed proteins & peptides Human genes 0.000 description 40
- 239000000243 solution Substances 0.000 description 26
- 210000004897 n-terminal region Anatomy 0.000 description 19
- 239000007788 liquid Substances 0.000 description 18
- 230000027455 binding Effects 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 17
- 238000002965 ELISA Methods 0.000 description 16
- 239000000499 gel Substances 0.000 description 15
- 238000012546 transfer Methods 0.000 description 15
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 14
- 229940098773 bovine serum albumin Drugs 0.000 description 14
- 239000012528 membrane Substances 0.000 description 13
- 235000018102 proteins Nutrition 0.000 description 13
- 238000012286 ELISA Assay Methods 0.000 description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 10
- 230000003053 immunization Effects 0.000 description 10
- 238000002649 immunization Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 9
- 210000004899 c-terminal region Anatomy 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 208000024827 Alzheimer disease Diseases 0.000 description 6
- 239000000427 antigen Substances 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000011161 development Methods 0.000 description 6
- 230000018109 developmental process Effects 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 239000011248 coating agent Substances 0.000 description 5
- 238000000576 coating method Methods 0.000 description 5
- 230000002163 immunogen Effects 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 238000010168 coupling process Methods 0.000 description 4
- 238000010839 reverse transcription Methods 0.000 description 4
- 235000020183 skimmed milk Nutrition 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 102000029749 Microtubule Human genes 0.000 description 3
- 108091022875 Microtubule Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000007910 cell fusion Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000005847 immunogenicity Effects 0.000 description 3
- 210000004688 microtubule Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 208000037259 Amyloid Plaque Diseases 0.000 description 2
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 2
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000010805 cDNA synthesis kit Methods 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013861 fat-free Nutrition 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000006951 hyperphosphorylation Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 208000015122 neurodegenerative disease Diseases 0.000 description 2
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 235000010265 sodium sulphite Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 230000007470 synaptic degeneration Effects 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 229940125373 Gamma-Secretase Inhibitor Drugs 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 206010044221 Toxic encephalopathy Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000011126 aluminium potassium sulphate Nutrition 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000013373 clone screening Methods 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000003540 gamma secretase inhibitor Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 230000016273 neuron death Effects 0.000 description 1
- 230000007135 neurotoxicity Effects 0.000 description 1
- 231100000228 neurotoxicity Toxicity 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229940050271 potassium alum Drugs 0.000 description 1
- GRLPQNLYRHEGIJ-UHFFFAOYSA-J potassium aluminium sulfate Chemical compound [Al+3].[K+].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRLPQNLYRHEGIJ-UHFFFAOYSA-J 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000018883 protein targeting Effects 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- AKHNMLFCWUSKQB-UHFFFAOYSA-L sodium thiosulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=S AKHNMLFCWUSKQB-UHFFFAOYSA-L 0.000 description 1
- 235000019345 sodium thiosulphate Nutrition 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010863 targeted diagnosis Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Neurosurgery (AREA)
- Cell Biology (AREA)
- Neurology (AREA)
- Plant Pathology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
The application relates to the technical field of immunology, and particularly discloses a single-chain antibody for detecting Tau protein, a preparation method and application thereof, wherein the single-chain antibody comprises a heavy chain variable region and a light chain variable region, and a flexible connecting peptide for connecting the heavy chain variable region and the light chain variable region; the complementarity determining regions of the heavy chain variable region have the fragments shown in SEQ ID Nos. 1 to 3, and the complementarity determining regions of the light chain variable region have the fragments shown in SEQ ID Nos. 4 to 6. The single-chain antibody has higher sensitivity and specificity, and the detection limit is as low as 50pg/mL, so that the single-chain antibody can be applied to detection of an actual sample.
Description
Technical Field
The application belongs to the technical field of genetically engineered antibodies, and particularly relates to a single-chain antibody for detecting Tau protein, and a preparation method and application thereof.
Background
Alzheimer's Disease (AD) is a progressive degenerative disease of the central nervous system and is the most common cause of dementia, so that senile dementia is also called onset, disease progression is chronic, and the period from pathological changes to obvious symptoms can last for decades, and finally cognitive dysfunction is caused. Two major typical pathological changes in alzheimer's disease include: intracellular amyloid plaque (amyoidplatae) formation and extracellular neurofibrillary tangles (NeurofibrillaryTangles, NFTs), with subsequent synaptic loss and neuronal death. Amyloid plaques are mainly composed of beta Amyloid (Amyloid beta, aβ), and NFTs are caused to be abnormal Tau protein as a main component. Most of the medicines (Abeta vaccine and gamma secretase inhibitor) aiming at Abeta have been announced to fail in clinical researches, and more researches point to that Tau protein hyperphosphorylation is a direct cause of neurotoxicity generated by Alzheimer disease, and the binding force between Tau protein hyperphosphorylation and tubulin is only 1/10 of that of normal Tau protein, so that the biological function of promoting microtubule assembly and the effect of maintaining microtubule stability are lost, the structure of a neuron microtubule is widely damaged, normal axon transport is damaged, synapse loss is finally caused, and the function of the neuron is damaged, and cerebral neurodegenerative disease occurs. Thus, tau protein is the most promising therapeutic target for alzheimer's disease.
Along with the development of genetic engineering technology, therapeutic antibody medicines gradually occupy the global market, and the development of anti-Tau protein monoclonal antibodies and genetic engineering antibodies is one of the directions of Tau protein targeting diagnosis and treatment, so that a certain technology and material foundation can be laid for diagnosis and treatment of Alzheimer patients.
Immunoglobulins (Ig) or antibodies are important components of the immune system, and they play a dual role in terms of structure. They are capable of site-specifically binding to an antigen via an antigen binding fragment on the one hand and of activating other cells of the immune system via the Fc-segment on the other hand to mediate immune responses. They can be classified into polyclonal antibodies (multiple antibodies recognize multiple epitopes of an antigen) and monoclonal antibodies (mAbs). The latter is a highly specific antibody that recognizes only a specific epitope of an antigen and is widely used in immunohistochemistry, flow cytometry, immunoblotting and related techniques. In addition, monoclonal antibodies have also become an important component of cancer therapy in the last 20 years. Their medical use can be extended to chronic inflammatory diseases, transplantation and infections (e.g. HIV monoclonal antibodies have a significant effect in the treatment of HIV disease in humans).
Monoclonal antibodies (MAbs) have the advantages of strong specificity and high affinity, but the existing production technology has the defects of high production cost, difficult industrial scale up and quality control and the like, and the small molecular antibodies in genetic engineering are ideal substitution products of the monoclonal antibodies. The recombinant antibody is a third generation antibody after the polyclonal antibody and the monoclonal antibody, the gene structure of the antibody is modified by utilizing a recombinant DNA technology, and then the recombinant antibody gene is transferred into a prokaryotic or eukaryotic expression system for expression, so that the recombinant antibody is formed. Compared with the preparation of polyclonal antibodies and monoclonal antibodies, the recombinant antibody technology has the following characteristics and advantages: (1) no immunization of the animal is required; (2) the antibody engineering bacteria are more stable than hybridoma cells, are easy to store, and are suitable for large-scale industrial production; (3) specific antibodies with high affinity can be obtained. The single chain antibody (ScFv) has the advantages of small molecular weight, strong tissue penetrating power, short in vivo circulation half-life, low immunogenicity, easy genetic engineering improvement and the like, and plays an important role in clinical diagnosis, treatment, prevention and the like of diseases. At present, no report on recombinant single-chain antibodies against Tau protein exists.
Disclosure of Invention
The invention aims to provide a recombinant single-chain antibody aiming at a Tau protein N-terminal region, and a preparation method and application of the recombinant single-chain antibody. The single-chain antibody can detect Tau protein with high specificity, high sensitivity and high accuracy.
The applicant couples the amino acid sequence (SEQ ID No. 14) of the N end region of the Tau protein with BSA (bovine serum albumin) to form an immune complex, utilizes the BSA-polypeptide (immunogen) to immunize animals to prepare hybridoma cells, screens a hybridoma cell strain capable of secreting monoclonal antibodies for recognizing the N end region of the Tau protein by an indirect ELISA method, analyzes the affinity with the polypeptide (SEQ ID No. 14) of the N end region of the Tau protein to obtain a hybridoma cell strain with stronger binding force with the polypeptide (SEQ ID No. 14) of the N end region of the Tau protein, determines and analyzes the antibody sequence of the hybridoma cell strain, designs recombinant plasmids, expresses the recombinant plasmids by escherichia coli Zhou Zhiqiang, and finally obtains the single-chain antibody for detecting the Tau protein.
As a first embodiment of the present application, there is provided a single chain antibody for detecting Tau protein, comprising a polypeptide comprising a heavy chain variable region V H And light chain variable region V L And for linking the heavy chain variable region V H And the light chain variable region V L Is a flexible linking peptide of (a);
the heavy chain variable region V H Has CDR1 in the complementarity determining region of (a) H 、CDR2 H And CDR3 H Is a fragment of said CDR1 H 、CDR2 H And CDR3 H The amino acid sequences of (2) are respectively shown as SEQ ID No. 1-3;
the light chain variable region V L Has CDR1 in the complementarity determining region of (a) L 、CDR2 L And CDR3 L Is a fragment of said CDR1 L 、CDR2 L And CDR3 L The amino acid sequences of (2) are respectively shown as SEQ ID No. 4-6.
In this embodiment, the heavy chain variable region V H The amino acid sequence of (2) is preferably as shown in SEQ ID No. 7.
In this embodiment, the light chain variable region V L The amino acid sequence of (2) is preferably as shown in SEQ ID No. 8.
In this embodiment, the flexible connecting peptide is an amino acid sequence (G 4 S) n The peptide chain is shown, wherein n is an integer.
Preferably, the flexible connecting peptide is an amino acid sequence (G 4 S) 3 The peptide chain shown.
In this embodiment, the heavy chain variable region V H Is linked to the light chain variable region V by a flexible linking peptide at the N-terminus L Is connected with the C end of the L-shaped metal tube; or said light chain variable region V L Is linked to the heavy chain variable region V by a flexible linking peptide H Is connected to the C terminal of (C).
In this embodiment, the single chain antibody follows the light chain variable region V L Flexible connecting peptide-heavy chain variable region V H The amino acid sequence is shown as SEQ ID No. 9.
As a second embodiment of the present application, a DNA molecule is provided, which encodes the single chain antibody described above.
As a third embodiment of the present application, there is provided a recombinant plasmid comprising the CDR1 as described above H 、CDR2 H And CDR3 H And the CDR1 L 、CDR2 L And CDR3 L Is a fragment of (a).
Preferably, the recombinant plasmid comprises the heavy chain variable region V having the amino acid sequence shown in SEQ ID No.7 H And a light chain variable region V having an amino acid sequence shown in SEQ ID No.8 L 。
Preferably, the recombinant plasmid comprises the single-chain antibody with the amino acid sequence shown as SEQ ID No. 9.
As a fourth embodiment of the present application, there is provided a kit for detecting Tau protein, comprising the single-chain antibody described above; or comprising said DNA molecule; or comprises the recombinant plasmid.
As a fifth embodiment of the present application, a method for preparing the single-chain antibody is provided, so that the single-chain antibody for detecting Tau protein can be prepared better, and the prepared single-chain antibody can be used for targeted diagnosis and treatment of Tau protein better.
The preparation method comprises the following steps:
1) Synthesis of cDNA templates
Extracting total RNA from hybridoma cell strains secreting anti-Tau protein monoclonal antibodies in logarithmic growth phase, and synthesizing cDNA by reverse transcription PCR;
2) PCR amplification of light chain variable region V L Gene and heavy chain variable region V H Gene
Designing the heavy chain variable region V using the obtained cDNA as a template H And the light chain variable region V L Is subjected to PCR to obtain the heavy chain variable region V H And the light chain variable region V L Fragments;
3) Synthesis of Single chain antibodies
Ligation of the heavy chain variable region V by Flexible connecting peptide Using overlap extension PCR H And the light chain variable region V L Fragments, synthesizing to obtain the single-chain antibody gene;
4) Construction of recombinant engineering bacteria
Recombining the single-chain antibody nucleotide sequence into a prokaryotic expression vector, constructing a recombinant expression plasmid for expressing the single-chain antibody, and introducing the recombinant expression plasmid into genetic engineering bacteria to construct recombinant engineering bacteria;
5) Expression of Single chain antibodies
And (3) performing induction culture on the obtained recombinant engineering bacteria to obtain the single-chain antibody and purifying.
Further, since the single chain antibody follows the light chain variable region V L Flexible connecting peptide-heavy chain variable region V H In the sequence of (2) in the light chain variable region V in the single chain antibody L 3' end of (2) and the heavy chain variable region V H Part of the flexible connecting peptide sequence is added to the 5' -end of the sequence.
At the same time, the light chain variable region V for facilitating subsequent cloning expression of the single chain antibody L Primer V of (2) L -F introduction of cleavage site and protecting base, said heavy chain variable region V H Primer V of (2) H Introduction into RCleavage site and protecting base.
Further, the light chain variable region V is designed L The primer of (a) is a nucleotide sequence shown as SEQ ID No. 10-11, and the heavy chain variable region V H The primer of (2) is a nucleotide sequence shown as SEQ ID No. 12-13.
Further, the PCR reaction system was 25. Mu.L: 2 XPCR mix 12.5. Mu.L, template cDNA 2. Mu.L, 25. Mu.M upstream and downstream primers 1. Mu.L each, ddH 2 O 8.5μL。
Further, the PCR amplification procedure: pre-denaturation at 95℃for 3min; denaturation at 94℃for 40s, annealing at 64℃for 40s, extension at 72℃for 1min,30 cycles; finally, the extension is carried out for 10min at 72 ℃.
As a sixth embodiment of the present application, there is provided the use of said single chain antibody or said DNA molecule or said recombinant plasmid in the preparation of a Tau protein assay product.
The beneficial effects of this application are:
the heavy chain variable region V of the monoclonal antibody is obtained by carrying out gene analysis on the obtained monoclonal antibody H And heavy chain variable region V L Further, the heavy chain variable region V is obtained by recombinant antibody technology H And heavy chain variable region V L According to heavy chain variable region V by flexible connecting peptide H Flexible connecting peptide-light chain variable region V L And (3) constitute a single chain antibody. The single chain antibody is convenient to obtain, has small molecular weight, can form functional antibody molecules without glycosylation, and can be expressed in prokaryotic cells. Compared with the traditional monoclonal antibody production technology, the method has low cost and short period.
Meanwhile, the single-chain antibody can specifically target the N-terminal region of the Tau protein, can detect 6 isomers of the Tau protein at the same time, and has stronger applicability. The detection limit of the single-chain antibody is as low as 50pg/mL, and the OD of ELISA detection of the Tau protein N-terminal region polypeptide is proved by experiments 450 The value can reach 3.718, and the OD detected by ELISA of the complete Tau protein 450 The value can reach 3.649, and the polypeptide is not combined with the polypeptide at the C-terminal region of the Tau protein, so that the polypeptide can be applied to detection of actual samples.
Drawings
FIG. 1 is an electrophoresis diagram of a Tau-ScFv single-chain antibody in example 2 of the present application;
FIG. 2 is an electrophoretogram of the immunological detection of Tau protein in example 3 of the present application.
Detailed Description
The following description of the present application will be made clearly and fully with reference to specific embodiments of the present application, it being apparent that the described embodiments are only some, but not all, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
Because the Tau protein has 6 isomers, the anti-Tau protein antibody prepared by taking the complete Tau protein as an immunogen is difficult to determine by combining an antigenic determinant of the complete Tau protein; if the anti-Tau protein antibody is prepared aiming at a certain Tau protein isomer, the obtained anti-Tau protein antibody has poor adaptability and has a certain influence on the accuracy of a detection result, and meanwhile, if the accurate detection of a sample is to be realized, the anti-Tau protein antibodies aiming at different isomers are combined for use, so that the detection cost is high, and the popularization and the use are not facilitated. The immune complex (BSA-polypeptide) containing the Tau protein N-region polypeptide is obtained by taking the Tau protein N-region polypeptide as an immunogen and performing composite coupling with bovine serum albumin, wherein the amino acid of the Tau protein N-region polypeptide is specifically shown as (SEQ ID No. 14). Injecting the immune complex (BSA-polypeptide) into mouse, cell fusion, screening out hybridoma cell strain capable of secreting monoclonal antibody for recognizing Tau protein N end region, analyzing the gene sequence of the monoclonal antibody to obtain heavy chain variable region V of monoclonal antibody H The amino acid sequence of (a) is shown as SEQ ID No.7, and the light chain variable region V L The amino acid sequence of (C) is shown as SEQ ID No. 8.
In some specific embodiments, the present application constructs a single chain antibody based on the light and heavy chain sequences of the resulting monoclonal antibody, the single chain antibodyChain antibodies comprise heavy chain variable region V H And light chain variable region V L And for linking the heavy chain variable region V H And the light chain variable region V L Is a flexible Linker of the peptide Linker.
In the heavy chain variable region V H Comprising complementarity determining regions CDR1 with amino acid sequences shown in SEQ ID No. 1-3 respectively H 、CDR2 H And CDR3 H Or the complementarity determining region CDR1 H 、CDR2 H And CDR3 H One or more amino acid sequences of amino acid sequences having an identity of more than 80% to the amino acid sequences shown in SEQ ID Nos. 1 to 3, respectively, specifically, one or more amino acid sequences of amino acid sequences having an identity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%, respectively. In the light chain variable region V L Comprises complementarity determining regions CDR1 with amino acid sequences shown in SEQ ID No. 4-6 respectively L 、CDR2 L And CDR3 L Or the complementarity determining region CDR1 L 、CDR2 L And CDR3 L One or more amino acid sequences of amino acid sequences having an identity of more than 80% to the amino acid sequences shown in SEQ ID Nos. 4 to 6, respectively, specifically, one or more amino acid sequences of amino acid sequences having an identity of 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.
Further, the heavy chain variable region V H The amino acid sequence shown in SEQ ID No.7, or one or more amino acid sequences having more than 80% identity with the amino acid sequence shown in SEQ ID No.7, specifically, one or more amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity. Further, the light chain variable region V L One or more of the amino acid sequences shown in SEQ ID No.8 or the amino acid sequences with more than 80% of identity with the amino acid sequence shown in SEQ ID No.8A plurality of amino acid sequences, such as in particular one or more of amino acid sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% identity.
Preferably, the heavy chain variable region V H The amino acid sequence of (2) is preferably as shown in SEQ ID No. 7. The light chain variable region V L The amino acid sequence of (2) is preferably as shown in SEQ ID No. 8.
The flexible connecting peptide is an amino acid sequence (G) 4 S) n The peptide chain is shown, wherein n is an integer. Specifically, the flexible connecting peptide is an amino acid sequence (G 4 S) 2 The peptide chain shown, or the flexible connecting peptide is an amino acid sequence (G 4 S) 3 The peptide chain shown. Preferably, the flexible connecting peptide is an amino acid sequence (G 4 S) 3 The peptide chain shown.
In the amino acid structure of the single chain antibody, the heavy chain variable region V H Is linked to the light chain variable region V by a flexible linking peptide at the N-terminus L The C-terminal of the Linker peptide Linker is specifically linked to the light chain variable region V L The C-terminus of the Linker peptide Linker is linked to the heavy chain variable region V H Is linked to the N-terminus of the single chain antibody, the sequence structure of the single chain antibody is that of the light chain variable region V L Flexible Linker-heavy chain variable region V H Is formed by sequentially connecting the above components. Or said light chain variable region V L Is linked to the heavy chain variable region V by a flexible linking peptide H The C-terminus of the Linker peptide Linker is specifically linked to the light chain variable region V L The N-terminus of the Linker peptide Linker is linked to the heavy chain variable region V H Is linked to the C-terminal of the single chain antibody, the sequence structure of the single chain antibody is that of the heavy chain variable region V H Flexible Linker-light chain variable region V L Is formed by sequentially connecting the above components.
Preferably, the sequence structure of the single chain antibody follows the light chain variable region V L Flexible connecting peptide-heavy chain variable region V H The amino acid sequence is shown as SEQ ID No. 9.
In some specific embodiments, the method of preparing the single chain antibody comprises the steps of:
1) Synthesis of cDNA templates
Extracting total RNA from hybridoma cell strains in logarithmic growth phase secreting anti-Tau protein N-region polypeptide monoclonal antibodies, and synthesizing cDNA by reverse transcription PCR; wherein total RNA of the hybridoma cell line was extracted using Trizol (TRIzol reagent available from Invitrogen corporation), and cDNA was synthesized using the total RNA as a template according to the procedure described in the reverse transcription kit (reverse transcription cDNA Synthesis kit available from TaKaRa corporation) using random primers.
Wherein, the hybridoma cell strain secreting the anti-Tau protein N-region polypeptide monoclonal antibody is obtained by the following method:
(1) immunogen preparation
The Tau protein N-terminal region polypeptide with the amino acid sequence shown as SEQ ID No.14 is coupled with BSA (bovine serum albumin). A Tau protein N-terminal region polypeptide shown in SEQ ID No.14 is synthesized, a cysteine is added at the C end for connection with BSA during synthesis, and then the synthesized polypeptide is coupled with BSA by an SPDP coupling method to obtain BSA-polypeptide, namely immunogen, the specific operation is completed by the company of Hefat national peptide biotechnology.
Immunogenicity of the above BSA-polypeptides was verified by ELISA. ELISA assay plates were coated with 100. Mu.L of BSA-polypeptide at a concentration of 5. Mu.g/mL, 100. Mu.L of commercial monoclonal antibody (Purified anti-Tau, brand: bioLegend, cat. 806501) at a concentration of 1. Mu.g/mL was added, 100. Mu.L of HRP-goat anti-mouse IgG secondary antibody (Thermo filter) diluted 1:5000 was added per well, and the OD at 450nm was measured. BSA was used as a control. The results show that the separate carrier protein BSA hardly binds to commercial monoclonal antibodies, while the BSA-polypeptide has good binding capacity to commercial monoclonal antibodies, indicating that the BSA-polypeptide is successfully prepared and has good immunogenicity for use in subsequent immunization procedures.
(2) Immunization of animals
Female Balb/c mice (Jiangsu Jiugang Biotechnology Co., ltd.) of 4-6 weeks old were immunized with BSA-polypeptide, 20. Mu.L was collected from the orbit before immunization, and serum was obtained after centrifugation, and subsequently used as a negative control.
At the time of primary immunization, each mouse was injected with 50. Mu.g of BSA-polypeptide, diluted to a volume of 200. Mu.L with physiological saline, mixed with an equal volume of Freund's complete adjuvant, and fully emulsified and injected subcutaneously in the abdomen. The mice were boosted subcutaneously 1 time every 14 days at a dose of 25. Mu.g of BSA-polypeptide, diluted to a volume of 200. Mu.L with physiological saline, mixed with an equal volume of Freund's incomplete adjuvant, and injected after sufficient emulsification. The eyebox was bled 7 days after the 3 rd booster immunization, serum antibody titers were detected by ELISA, impact immunization was performed on mice meeting the requirements, and 50. Mu.g of BSA-polypeptide was intraperitoneally injected into each of the mice at a dose of 100. Mu.L diluted with physiological saline at the time of injection, to obtain immunized mice. After immunization, 20 μl of the immunized mice were collected from the orbits, and serum was obtained after centrifugation, and subsequently used as a positive control. Cell fusion was performed after 3 days.
(3) Cell fusion
Freshly picked spleens of immunized mice were placed on a cell sieve, crushed and filtered, mixed with SP2/0 cells (myeloma cells) in a quantitative ratio of 3:1, centrifuged at 1000rpm at room temperature for 5min, and the supernatant discarded. The tube containing the centrifuged cells was placed in a warm water bath at 37℃and 1mL of polyethylene glycol solution (Sigma, P7181-5X 5 mL) was slowly added while stirring the cells. In a water bath, the mixture was allowed to stand for 1min, 10mL of serum-free DMEM medium (Hyclone, SH30243.01B) was slowly added, and the supernatant was discarded after centrifugation at 1000rpm for 5min. Serum-containing DMEM medium was added and cells were inoculated into 96-well cell culture plates at a cell density of 1X 10 by 150. Mu.L per well 5 Placing at 37deg.C and 5% CO in each hole 2 Is cultured in an incubator of (a). After 24h, 50. Mu.L of HAT (Gibco, 21060017) containing medium was added. Thereafter, the liquid is changed every 3 to 5 days.
(4) Cell screening
When the cell density reached about 50%, cell supernatants were collected and hybridoma cell lines capable of secreting monoclonal antibodies recognizing the N-terminal region of Tau protein were screened by indirect ELISA, as follows:
the Tau protein N-terminal region polypeptide shown in SEQ ID No.14 is coupled with KLH by an SPDP coupling method,the specific procedure for obtaining KLH-polypeptide was carried out by the company Hefei national peptide biotechnology Co. ELISA assay plates were coated with 100. Mu.L of KLH-polypeptide at a concentration of 0.5. Mu.g/mL and allowed to stand at 37℃for 1h. The cells were washed 4 times with PBST, 100. Mu.L of the culture supernatant of the hybridoma cells was added to KLH-polypeptide-coated ELISA detection wells, and at the same time, immunized mouse serum (diluted 1:10000) after animal immunization was used as a positive control, mouse serum before animal immunization was used as a negative control, and incubated at 37℃for 1 hour. After washing 4 times with PBST, 100. Mu.L of horseradish peroxidase HRP-labeled affinity purified goat anti-mouse IgG (H+L) F (ab') 2 fragment (Jackson, 115-036-003, diluted 1:5000 with 1% BSA) was added to each well and incubated at 37℃for 1H. After washing 4 times with PBST, TMB (Solarbio, PR 1210-2X 250 mL) was added for color development, and OD at 450nm was measured. OD (optical density) 450 A positive clone was determined to have a value of greater than 0.5.
(5) Establishment of hybridoma cell lines
After 3 subclones and indirect ELISA screening, 8 hybridoma cell lines secreting antibodies specifically recognizing the N-terminal region of the Tau protein were obtained, and the clone numbers were designated N-1, N-2, N-3, N-4, N-5, N-6, N-7 and N-8 in sequence. The affinity between the antibody secreted by the 8 hybridoma cell lines and the Tau protein N-terminal region polypeptide shown in SEQ ID No.14 is detected by an indirect ELISA method, and the binding capacity between the hybridoma cell line N-6 and the Tau protein N-terminal region polypeptide shown in SEQ ID No.14 is strongest, so that the antibody secreted by the hybridoma cell line N-6 is selected as a monoclonal antibody for recognizing the Tau protein N-terminal region and used for subsequent experiments.
The hybridoma cell line N-6 was subjected to antibody sequence determination and analysis by Nanjing Tedder Biotechnology Co. Determining and analyzing the sequence, wherein the amino acid sequence of a heavy chain variable region of the monoclonal antibody for identifying the N-terminal region of the Tau protein is shown as SEQ ID No.7, and the nucleotide sequence is shown as SEQ ID No.15; the amino acid sequence of the light chain variable region is shown as SEQ ID No.8, and the nucleotide sequence is shown as SEQ ID No.16.
Through analysis, the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3 respectively; the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, respectively.
2) PCR amplification of light chain variable region V L Gene and heavy chain variable region V H Gene
Designing the heavy chain variable region V using the obtained cDNA as a template H And the light chain variable region V L Is subjected to PCR to obtain the heavy chain variable region V H And the light chain variable region V L Fragments.
For amplifying the heavy chain variable region V H The primer pair of (2) is V H -F and V H -R for amplifying said light chain variable region V L The primer pair of (2) is V L -F and V L R, wherein V L -F、V H R contains NdeI and HindIII cleavage sites, respectively; v (V) H -F、V L R contains a complementary Linker sequence. Primers were synthesized by synfat national peptide biotechnology limited:
V L -F：5'-GGAATTCCATATGGAATTCCGACGTGGTGCTGACCC AGAC-3'(SEQ ID No.10)
V L -R：5'-CCACCGCCACCGAGCCCTCCACCCAGTTGGCGTAG TTGGTGGTG-3'(SEQ ID No.11)
V H -F：5'-ACCTAGGCCGCCACCGCCAAGGCCTGACCGGCAA CTTCTTC-3'(SEQ ID No.12)
V H -R：5'-CCCAAGCTTGGGTGTTCTTGGCGTTGTCCCT-3'(SEQ ID No.13)。
3) Expression of Single chain antibodies
Ligation of the heavy chain variable region V by Flexible connecting peptide Using overlap extension PCR H And the light chain variable region V L And synthesizing fragments to obtain the single-chain antibody gene, connecting the nucleotide sequence of the single-chain antibody to pET22b (+) to construct and obtain recombinant expression plasmid for expressing the single-chain antibody, wherein the gene structure is VL-Linker-VH-His.
The recombinant expression plasmid comprises the CDR1 H 、CDR2 H And CDR3 H And the CDR1 L 、CDR2 L And CDR3 L Is a fragment of (a). Preferably, the recombinant expression plasmid comprises the heavy chain variable region shown in SEQ ID No.7, so thatAnd the light chain variable region shown in SEQ ID No. 8.
4) Expression of Single chain antibodies
And (3) introducing the recombinant expression plasmid into genetic engineering bacteria to construct recombinant engineering bacteria, and performing induction culture to obtain the single-chain antibody and purifying.
In some specific embodiments, a kit is provided, which comprises the single-chain antibody, and can be used for detecting and identifying 6 Tau protein isomers because the single-chain antibody can specifically identify the Tau protein N-terminal region, so that the monoclonal antibody for identifying the Tau protein N-terminal region can effectively detect all isomers of the Tau protein, has stronger applicability and ensures the accuracy of detection results. Or the kit comprises a coding sequence encoding the heavy chain variable region V H And the light chain variable region V L Is a DNA molecule of (a). Or the kit comprises DNA molecules encoding CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region. Or comprises the recombinant expression plasmid.
In principle, the single chain antibody or the heavy chain variable region V is encoded H And the light chain variable region V L The recombinant plasmid or the DNA molecule encoding CDR1, CDR2 and CDR3 of the heavy chain variable region and CDR1, CDR2 and CDR3 of the light chain variable region or the recombinant expression plasmid can be used to prepare any product suitable for detecting Tau protein.
EXAMPLE 1 construction of Tau-ScFv prokaryotic expression System
1) Construction of Tau-ScFv Single-chain antibody Gene
The obtained hybridoma cell line N-6 secreting the anti-Tau protein monoclonal antibody was used to extract total RNA by Trizol method (TRIZOL Reagent was purchased from TaKaRa), and cDNA was synthesized by using the extracted total RNA as a template and using random primers according to the procedure described in the reverse transcription kit (reverse transcription cDNA synthesis kit was purchased from TaKaRa). Amplifying the heavy chain variable region V by PCR method using the obtained cDNA as template H Light chain variable region V L And connecting the peptide gene fragments, purifying by a gel recovery kit (TaKaRaAgarose Gel DNA Purification Kit) to obtain the 3-segment gene fragments. The saidThe PCR reaction system was 25. Mu.L: 2 XPCR mix 12.5. Mu.L, template cDNA 2. Mu.L, 25. Mu.M upstream and downstream primers 1. Mu.L each, ddH 2 O8.5. Mu.L. PCR amplification procedure: pre-denaturation at 95℃for 3min; denaturation at 94℃for 40s, annealing at 64℃for 40s, extension at 72℃for 1min,30 cycles; finally, the extension is carried out for 10min at 72 ℃. Heavy chain variable region V containing Linker sequence was prepared by recombinant chain extension reaction (SOE-PCR) H And light chain variable region V L Gene ligation is a Tau-scFv gene (light chain variable region V L Flexible connecting peptide-heavy chain variable region V H ). The PCR reaction products were analyzed by 1.5% agarose gel electrophoresis.
Overlapping extension PCR products were taken and primer V was used L -F and V H R was amplified from the Tau-ScFv gene, the target gene was recovered by a PCR product recovery kit, and double-digested with the vector pET22b (+) was performed, and a T4 DNAligenase ligation system was prepared at a molar ratio of vector to target gene of 1:10 (0.03 pmol:0.3 pmol), followed by reaction at 16℃overnight.
E.coli DH5 alpha competent cells are transformed by the pET-Tau-ScFv recombinant plasmid, and after AMP resistance screening, the plasmid is extracted and sent to a company for sequencing, which shows that the pET-Tau-ScFv recombinant plasmid is successfully constructed. Taking 200 mu l of escherichia coli DH5 alpha competent cell suspension from a refrigerator at the temperature of-70 ℃, thawing the suspension at room temperature, immediately placing the suspension on ice after thawing, adding a connection product into competent cells, uniformly mixing, and placing the suspension on ice for 30min; heat-shocking in water bath at 42 deg.C for 90 seconds or water bath at 37 deg.C for 5 minutes, rapidly cooling on ice for 3-5 minutes, and adding 1ml LB liquid medium (without Amp) into the tube; shaking culture at 37 ℃ for 1 hour after uniform mixing, so that the bacteria are recovered to a normal growth state, and the antibiotic resistance gene (Ampr) encoded by the plasmid is expressed; centrifuging at 8000rpm for 1min, discarding most supernatant, keeping about 50-100 μl, shaking up bacterial liquid, coating 100 μl on a screening plate containing Amp, standing for half an hour in front, inverting the culture dish after bacterial liquid is completely absorbed by culture medium, and culturing at 37deg.C for 16-24 hr. Two controls were simultaneously made, control 1, replacing the DNA solution with sterile double distilled water of the same volume, the other operations being the same as above. This group should normally have no colonies present on LB plates containing antibiotics. Control group 2, which replaced the DNA solution with the same volume of sterile double distilled water, but which was plated with 5. Mu.l of bacterial solution on LB plates without antibiotics, normally resulted in a large number of colonies. Selecting monoclonal colony with good growth condition on the plate, and screening the strain by using a positive clone screening method. Positive clones were transferred to 5mL of ampicillin-resistant liquid medium, incubated at 37℃overnight at 180rpm, plasmids were extracted and identified using a plasmid miniprep kit.
EXAMPLE 2 purification of Tau-ScFv Single chain antibody expression
The strain sequenced correctly in example 1 above was picked up, cultured overnight in liquid LB medium containing AMP, plasmids were extracted and transformed into E.coli BL21 (DE 3), the bacterial solution was spread on ampicillin-resistant agar plates and cultured overnight at 37 ℃. The following day, single colonies were picked up in 5mL LB-A liquid medium at 37℃at 220rpm for 12-16h, positive clones were identified by colony PCR, and inducible expression of recombinant proteins was prepared. Picking the positive clone strain obtained in the previous step, shake culturing in fresh liquid LB culture medium containing AMP at 37 ℃ and 200rpm until the bacterial log phase is reached, namely OD600 = 0.8, adding IPTG to the final concentration of 1mM, shake culturing at 22 ℃ and 200rpm for 12-16h, centrifuging the bacterial liquid at 4 ℃ and 5000rpm for 12min, discarding the supernatant, re-suspending the bacterial body by 0.1M pre-cooled phosphate buffer (phosphate buffer saline, PBS) and washing; the cells were suspended in PBS buffer, 1mM protease inhibitor PMSF was added, the bacterial solution was placed on ice and sonicated using a sonicator until the cells were completely disrupted. 100 mu L of whole bacterial liquid is added into 5 XSDS loading buffer solution, and is kept stand at 100 ℃ for 5min for SDS-PAGE electrophoresis detection, a pipettor is carefully added into a gel aperture, 30mA constant current is carried out in an electrophoresis tank until bromophenol blue is discharged out of the bottom of the gel, and the gel is dyed and decolored and then the expression of target protein is analyzed in a gel electrophoresis system. The recombinant protein is purified by Ni-NTA (GE Healthcare, chicago) affinity chromatography, purified Tau-ScFv recombinant protein is renatured by gradual dialysis, finally, the recombinant protein is dialyzed by ultrapure water and freeze-dried, and the purity and the protein concentration are detected by SDS-PAGE after the concentration is adjusted.
Results: SDS-PAGE shows that obvious protein bands appear in the strain induced by IPTG, and meanwhile, the purified and renatured scFv recombinant protein bands are single and have higher concentration, which indicates that the Tau-ScFv single-chain antibody has successful recombinant expression and can be used for the subsequent experiments (figure 1).
EXAMPLE 3 identification of Tau-ScFv Single chain antibody by immunoblotting
1) Sample processing
The method comprises the steps of synthesizing complete Tau protein, the amino acid sequence of which is shown as SEQ ID No.17, entrusting the synthesis of complete Tau protein by the Hefei national peptide biotechnology Co-Ltd, adding concentrated Tau protein suspension into sample buffer solution containing sodium dodecyl sulfate in equal proportion, and boiling for 3-5 min. Adding the treated sample into a sample loading hole, adding 10 mu l of the sample into each hole, and carrying out electrophoresis under a constant current condition; the low current (30-40 mA) is used at the beginning, after the sample is concentrated into a line in the concentrated gel part, the current (50-70 mA) is increased until the bromophenol blue indicator reaches the bottom edge, the electrophoresis can be stopped, and the gel is taken out.
2) Transfer film
Preparation: transfer buffer (tris5.8g glycine 2.9g SDS 0.37g methanol 200ml v=1l), clips for transfer membrane, two sponges, one dropper, 2 filter papers, one nitrocellulose membrane (NC membrane). Disposable PE gloves are needed to be worn when the filter paper and the membrane are sheared, so that the membrane is prevented from being polluted by proteins on hands.
Before transferring, NC membrane should be soaked in methanol solution for 5-10 seconds until it is soaked, and balanced in balancing solution (methanol is used for fixing macromolecular protein, so that small molecular substances are easy to transfer out). The clamp for transferring the membrane, two spongy pads, a dropper, 2 pieces of filter paper and an NC membrane are soaked in a transfer buffer solution, then SDS-PAGE gel is taken out, concentrated gel is scraped off lightly, and a corner of the gel is used as a mark to distinguish the loading sequence. The gel was immersed in a transfer buffer (25 mmol/L Tris-Base,192mmol/L glycine, 20% methanol, pH 8.3) for about 5min to equilibrate the ionic strength. The clamp is opened to place the black electrode (cathode) at the bottom, a sponge gasket is placed, and the glass rod is rolled back and forth for several times to roll away the bubbles inside. Placing 1 piece of filter paper soaked by transfer buffer solution on a sponge gasket, aligning, and then using a glass rod as a roller to extrude all bubbles, and if necessary, dripping transfer film liquid for wetting. The gel immersed in the transfer membrane solution was taken out and laid flat on a filter paper, and all bubbles were removed. NC film was placed on polyacrylamide gel and rolled back and forth for several times to exclude all bubbles, taking care to mark the front of the film (a corner of the film could be cut or marked on a corner of the film with a sign pen). A piece of filter paper soaked with transfer buffer was placed over the membrane, again to ensure that no air bubbles remained. Finally, another Zhang Haimian pad is covered, an anode plate (white) is covered, and the gel is clamped, so that certain pressure is ensured to the gel. The clips were placed in the transfer tank with the black of the clips facing the black of the tank and the white of the clips facing the red of the tank, and transferred at a constant pressure of 110V for 60min. The transfer tank is placed into ice water for transfer. And stirring the electric transfer liquid by using a magnetic stirrer in the film transfer process.
2) Immunological detection
The membrane was taken, shaken in a 1 XPBST solution for five minutes with the membrane facing up, washed once, and transferred to a plate containing a blocking solution (bottle of PBST buffer containing 10% nonfat dry milk; nonfat dry milk 5g, 100ml of PBST) and blocked by shaking on a decolorizing shaker at room temperature for 1 hour. The membrane was removed and washed in 1 XPBST solution for 5 minutes, shaken, washed twice, placed in 1 XPBST buffer (containing 5% skimmed milk), and simultaneously incubated with Tau-ScFv single chain antibody and HRP-His tag antibody in buffer for 60 minutes, washed at least three times with 1 XPBST for 5 minutes/time. In a darkroom, 1X developing solution (50 ml) and fixing solution (50 ml) were respectively put into a plastic box; taking out X-ray film under red light, cutting with paper cutter to proper size (25 px larger than the length and width of film); opening an X-ray film clamp, putting the film into the X-ray film clamp, uniformly smearing luminous liquid on the film, then putting the X-ray film on the film, closing the X-ray film clamp, exposing for 3min, opening the X-ray film clamp, taking out the X-ray film, quickly immersing into developing liquid for developing, and immediately stopping developing after obvious strips appear. Immediately after development, the X-ray film is immersed in the fixing solution for 5 to 10 minutes until the film is transparent, and the residual fixing solution is washed out with tap water and dried at room temperature. Wherein, developer solution (5X): 375ml of tap water, 1.55g of miracle, 22.5g of sodium sulfite, 33.75g of sodium carbonate and 20.95g of potassium bromide, and supplementing water to 500 ml. Fixing liquid: 700ml of tap water, 240g of sodium thiosulfate, 15g of sodium sulfite, 12.6ml of glacial acetic acid, 7.5g of boric acid and 15g of potassium alum, adding water to a volume of 1000ml and preserving at room temperature. Luminescent liquid: 2ml of luminol reagent and 1.3ml of hydrogen peroxide.
As shown in FIG. 2, the Tau-ScFv single-chain antibody can realize specific recognition of Tau protein without impurity bands.
Example 4Tau-ScFv Single chain antibody affinity
And detecting the antigen binding activity of the Tau-ScFv single-chain antibody by adopting a competitive ELISA method, namely detecting the affinity of the Tau-ScFv single-chain antibody.
(1) Binding objects
The binding subjects are Tau protein N-terminal region polypeptides, tau protein C-terminal region polypeptides and intact Tau proteins.
Wherein the amino acid sequence of the Tau protein N-terminal region polypeptide is shown as SEQ ID No.14, and the amino acid sequence of the Tau protein C-terminal region polypeptide is shown as SEQ ID No. 18. The two polypeptides are respectively coupled with KLH through an SPDP coupling method to obtain the KLH-N end region polypeptide and the KLH-C end region polypeptide, and the specific operation is completed by the company of the biological technology of the peptide of the Hefei nationality.
Wherein the amino acid sequence of the complete Tau protein is shown as SEQ ID No. 17. Cloning the nucleotide sequence of the complete Tau protein into an expression vector pCDNA 3.4, constructing a recombinant vector, performing transient expression in 293T cells to verify successful expression of the protein, and purifying to obtain the complete Tau protein.
(2) The specific detection method comprises the following steps:
ELISA assay plates were coated with 100. Mu.L of the KLH-N end region polypeptide, KLH-C end region polypeptide and intact Tau protein at a concentration of 0.5. Mu.g/mL, respectively, and allowed to stand at 37℃for 1h. The ELISA assay plate was blocked with 5% BSA after washing 4 times with PBST, allowed to stand at 37℃for 1h, and washed 4 times with PBST to prepare a KLH-N end region polypeptide-coated ELISA assay plate, a KLH-C end region polypeptide-coated ELISA assay plate, and a complete Tau protein-coated ELISA assay plate.
The Tau-ScFv single-chain antibody and the commercial monoclonal antibody (Purified anti-Tau, brand: bioLegend, cat. No. 806501) provided in the present application were diluted with 1% BSA respectively to a final concentration of 0.5. Mu.g/mL, 100. Mu.L of the diluted antibodies were added to KLH-N end region polypeptide-coated ELISA assay plates, KLH-C end region polypeptide-coated ELISA assay plates, and detection wells of the whole Tau protein-coated ELISA assay plates, respectively, and 1% BSA was used as a negative control, and left standing at 37℃for 1 hour.
After washing 4 times with PBST, 100. Mu.L of HRP-His tag antibody (diluted 1:5000 with 1% BSA) was added to each well, incubated at 37℃for 1h, washed 4 times with PBST, developed with TMB, and OD at 450nm was determined.
TABLE 1 binding Capacity of Tau-ScFv Single chain antibodies
As can be seen from Table 1, the Tau-ScFv single-chain antibody in the present application has good affinity with the polypeptide of the N-terminal region of the Tau protein, and the OD after binding 450 The numerical value can reach 3.718, which is higher than 3.453 of commercial monoclonal antibodies, and shows that the Tau-ScFv single-chain antibody has better binding capability with the Tau protein N-terminal region polypeptide. Meanwhile, the Tau-ScFv single-chain antibody and OD of Tau protein C-terminal region polypeptide of the application 450 Only 0.013, which is lower than the commercial monoclonal antibody, indicates that the Tau-ScFv single-chain antibody of the application does not bind to the polypeptide in the C-terminal region of the Tau protein. In addition, the Tau-ScFv single-chain antibody has good affinity with the complete Tau protein, but the binding capacity is lower than that of the polypeptide in the N-terminal region of the Tau protein, presumably because the complete Tau protein forms a corresponding higher structure, so that the binding between the Tau-ScFv single-chain antibody and a corresponding antigenic determinant is influenced to a certain extent.
EXAMPLE 5Tau-ScFv Single chain antibody sensitivity
(1) Binding objects
The binding object is KLH-N end region polypeptide, and the amino acid sequence of the Tau protein N end region polypeptide is shown as SEQ ID No. 14.
(2) Detection method
The KLH-N-domain polypeptide was diluted to final concentrations of 500ng/mL, 50ng/mL, 5ng/mL, 500pg/mL, 50pg/mL and 5pg/mL, ELISA assay plates were coated with 100. Mu.L of the different concentrations of KLH-N-domain polypeptide dilutions described above, and ELISA assays were performed for specific procedures as described in example 4.
(3) Detection result
The sensitivity test results of the Tau-ScFv single-chain antibody and commercial monoclonal antibody (Purified anti-Tau, brand: bioLegend, cat. No. 806501) of the present application are shown in Table 2.
TABLE 2 sensitivity of Tau-ScFv Single chain antibodies
As can be seen from Table 2, the Tau-ScFv single-chain antibody in the application has good sensitivity compared with commercial monoclonal antibodies, the sensitivity is improved by one order of magnitude, and the Tau protein with the concentration as low as 50pg/mL in the sample can be effectively detected.
Example 6 ELISA method-based kit for detecting Tau protein
The embodiment provides a kit for detecting Tau protein, which comprises the following components: tau-ScFv single-chain antibody (purified Tau-ScFv single-chain antibody of example 2), ELISA plate, blocking solution (5% skim milk powder), HRP-His tag antibody (optimal dilution 1:6400), color development solution (tetramethylbenzidine), stop solution (2 mol/L H) 2 SO 4 ) Coating solution (20 mmol/LpH8.5, tris-HCl) and washing solution (PBST solution).
The using method of the kit is as follows:
(1) purifying the monoclonal antibody coating: diluting the Tau-ScFv single-chain antibody to 1:800 by using a coating solution, wherein the temperature is 4 ℃ overnight, 100 mu L of the coating solution is per well, and the temperature is 4 ℃ refrigerator overnight;
(2) closing: 200 mu L of 5% skim milk powder is added to each well, and incubated for 1h at 37 ℃;
(3) adding an antigen: adding 100 mu L of sample into each hole, and incubating for 1-2 h at 37 ℃;
(4) adding enzyme-labeled secondary antibodies: HRP-goat anti-mouse IgG secondary antibody was diluted to 1:6400 with 5% skim milk powder, incubated at 37 ℃ for 1.5h, 100 μl per well;
(5) developing a substrate: adding 100 mu L of color development liquid into each hole, and developing for 10min;
(6) terminating the reaction: 50. Mu.L/well of concentrated sulfuric acid (2 mol/L) was added to each well, and OD was measured 450 nm reaction value.
Washing the plate 3-5 times with washing liquid between the steps (1) - (4) for 10min each time.
The kit of this example detects whether 10 clinical samples express Tau protein, while using commercial monoclonal antibodies as controls.
Samples were lysed, ELISA plates were coated with the lysate, and ELISA was performed using a kit and a commercial monoclonal antibody (Purified anti-Tau, brand: bioLegend, cat. No. 806501), respectively, and the results are shown in Table 3.
TABLE 3 detection results of Tau protein expression in clinical samples
As can be seen from table 3, for the above 10 clinical samples, the consistency of the detection results of the Tau protein ELISA detection kit of the present application and the commercial monoclonal antibodies is 100%, which indicates that the Tau protein ELISA detection kit has good accuracy and specificity, and has equivalent property to the commercial products in use, and can be applied to practical clinical detection.
In conclusion, the Tau-ScFv single-chain antibody constructed by the method has the specificity and accuracy similar to those of commercial antibodies, but has better sensitivity and binding capacity with Tau protein, the detection limit is as low as 50pg/mL, and the method can be applied to detection of actual samples and has important application value in relevant theoretical research and clinical detection.
Claims (10)
1. A single chain antibody for detecting Tau protein, characterized in that said single chain antibody comprises a heavy chain variable region comprising V H And light chain variable region V L And for linking the heavy chain variable region V H And the light chain variable region V L Is a flexible linking peptide of (a);
the heavy chain variable region V H Has CDRs in the complementarity determining region of (C)1 H 、CDR2 H And CDR3 H Is a fragment of said CDR1 H 、CDR2 H And CDR3 H The amino acid sequences of (2) are respectively shown as SEQ ID No. 1-3;
the light chain variable region V L Has CDR1 in the complementarity determining region of (a) L 、CDR2 L And CDR3 L Is a fragment of said CDR1 L 、CDR2 L And CDR3 L The amino acid sequences of (2) are respectively shown as SEQ ID No. 4-6.
2. A single chain antibody for detecting Tau protein of claim 1, wherein said heavy chain variable region V H The amino acid sequence of (a) is preferably as shown in SEQ ID No. 7; the light chain variable region V L The amino acid sequence of (2) is preferably as shown in SEQ ID No. 8.
3. A single chain antibody for detecting Tau protein of claim 1, wherein said flexible linker peptide is an amino acid sequence (G 4 S) 3 The peptide chain shown.
4. A single chain antibody for detecting Tau protein of claim 1, wherein said single chain antibody follows light chain variable region V L Flexible connecting peptide-heavy chain variable region V H The amino acid sequence is shown as SEQ ID No. 9.
5. A DNA molecule encoding the single chain antibody of any one of claims 1 to 4.
6. A recombinant plasmid comprising the single-chain antibody of any one of claims 1 to 4 or the DNA molecule of claim 5.
7. A kit for detecting Tau protein, characterized in that it comprises the single-chain antibody of any one of claims 1 to 4 or the DNA molecule of claim 5.
8. The method for producing a single-chain antibody for detecting Tau protein of claim 1, comprising:
1) Synthesis of cDNA templates
Extracting total RNA from hybridoma cell strains secreting anti-Tau protein monoclonal antibodies in logarithmic growth phase, and synthesizing cDNA by reverse transcription PCR;
2) PCR amplification of light chain variable region V L Gene and heavy chain variable region V H Gene
Designing the heavy chain variable region V using the obtained cDNA as a template H And the light chain variable region V L Is subjected to PCR to obtain the heavy chain variable region V H And the light chain variable region V L Fragments;
3) Synthesis of Single chain antibodies
Ligation of the heavy chain variable region V by Flexible connecting peptide Using overlap extension PCR H And the light chain variable region V L Fragments, synthesizing to obtain the single-chain antibody gene;
4) Construction of recombinant engineering bacteria
Recombining the single-chain antibody nucleotide sequence into a prokaryotic expression vector, constructing a recombinant expression plasmid for expressing the single-chain antibody, and introducing the recombinant expression plasmid into genetic engineering bacteria to construct recombinant engineering bacteria;
5) Expression of Single chain antibodies
And (3) performing induction culture on the obtained recombinant engineering bacteria to obtain the single-chain antibody and purifying.
9. The method of claim 8, wherein the light chain variable region V L The primer of (a) is a nucleotide sequence shown as SEQ ID No. 10-11, and the heavy chain variable region V H The primer of (2) is a nucleotide sequence shown as SEQ ID No. 12-13.
10. Use of a single chain antibody according to any one of claims 1 to 4 or a DNA molecule according to claim 5 or a recombinant plasmid according to claim 6 for the preparation of a Tau protein assay product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310329690.4A CN116375857A (en) | 2023-03-30 | 2023-03-30 | Single-chain antibody for detecting Tau protein, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310329690.4A CN116375857A (en) | 2023-03-30 | 2023-03-30 | Single-chain antibody for detecting Tau protein, and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116375857A true CN116375857A (en) | 2023-07-04 |
Family
ID=86980073
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310329690.4A Pending CN116375857A (en) | 2023-03-30 | 2023-03-30 | Single-chain antibody for detecting Tau protein, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116375857A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2013106270A (en) * | 2010-07-14 | 2014-08-20 | Мерк Шарп Энд Домэ Корп. | MONOCLONAL ANTIBODY AGAINST ADDL AND ITS APPLICATION |
| WO2015200806A2 (en) * | 2014-06-27 | 2015-12-30 | C2N Diagnostics Llc | Humanized anti-tau antibodies |
| CN115724967A (en) * | 2022-09-21 | 2023-03-03 | 天津鸿宇泰生物科技有限公司 | Monoclonal antibody for identifying Tau protein N-terminal region and application thereof |
-
2023
- 2023-03-30 CN CN202310329690.4A patent/CN116375857A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2013106270A (en) * | 2010-07-14 | 2014-08-20 | Мерк Шарп Энд Домэ Корп. | MONOCLONAL ANTIBODY AGAINST ADDL AND ITS APPLICATION |
| WO2015200806A2 (en) * | 2014-06-27 | 2015-12-30 | C2N Diagnostics Llc | Humanized anti-tau antibodies |
| JP2020054356A (en) * | 2014-06-27 | 2020-04-09 | シー・２・エヌ・ダイアグノスティクス・エル・エル・シー | Humanized anti-tau antibody |
| CN115724967A (en) * | 2022-09-21 | 2023-03-03 | 天津鸿宇泰生物科技有限公司 | Monoclonal antibody for identifying Tau protein N-terminal region and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| ERIN E. CONGDON: "Tau-targeting therapies for Alzheimer disease", 《NATURE REVIEWS》, vol. 14, 31 July 2018 (2018-07-31), pages 399 - 415, XP093186392, DOI: 10.1038/s41582-018-0013-z * |
| 匡宇竹: "阿尔茨海默症治疗型单链抗体的构建、表达优化与体内外活性研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》, 15 February 2023 (2023-02-15), pages 071 - 134 * |
| 王世真: "《分子核医学》", vol. 2, 31 March 2004, 中国协和医科大学出版社, pages: 255 - 257 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2299889C2 (en) | Conformational anomalous forms of tau proteins and specific antibodies to them | |
| JPH03500005A (en) | Method for producing recombinant DNA protein | |
| CN108997499B (en) | Anti-human PD-L1 antibody and application thereof | |
| CN106866820B (en) | Monoclonal antibody for capturing tumor cells and resisting human keratin 18 and application thereof | |
| CN114605543B (en) | Monoclonal antibody of HLA-G isomer molecule HLA-G5 and HLA-G6 and application thereof | |
| CN111153992A (en) | Monoclonal antibody of mycobacterium tuberculosis LAM and application thereof | |
| CN111705066B (en) | Genetically modified TIGIT protein, monoclonal antibody and application thereof | |
| JP4651495B2 (en) | Monoclonal antibody specifically reacting with ISG15 protein, hybridoma producing the same, and method for detecting cancer and virus infection | |
| JP4714265B2 (en) | Neuroglobin enzyme immunoassay kit and use thereof | |
| CN113480646B (en) | Preparation and application of monoclonal antibody specifically binding H1.4 frameshift mutant protein | |
| CN116375857A (en) | Single-chain antibody for detecting Tau protein, and preparation method and application thereof | |
| CN106674350B (en) | Preparation and application of liver cancer marker single-chain antibody | |
| JP3920556B2 (en) | Abbreviated midkine (tMK) protein-specific monoclonal antibody and use thereof | |
| JP2002234900A (en) | Anti-tenacin c monoclonal antibody and hybridoma producing the same | |
| CN111763255A (en) | Genetically modified VEGFA protein, monoclonal antibody thereof and application | |
| CN106589128B (en) | Preparation and application of liver cancer marker monoclonal antibody | |
| CN110054675B (en) | Immunogenic polypeptide, anti-TTC 36 antibody CP4-3 and application | |
| CN116813766B (en) | High-permeability anti-calmodulin fusion nano-antibody and preparation method and application thereof | |
| CN110540591A (en) | anti-Glycoprotein A33 (glycoprotin A33) monoclonal antibody and immunodetection application thereof | |
| CN110054674B (en) | Immunogenic polypeptide, anti-TTC 36 antibody AP2-19 and application | |
| CN117402251B (en) | Antibody for resisting small G protein RBJ and application thereof | |
| CN110054676B (en) | Immunogenic polypeptide, anti-TTC 36 antibody AP3-5 and application | |
| CN117624353B (en) | Antibodies that bind smooth muscle myosin heavy chain and uses thereof | |
| CN110760483B (en) | Preparation and application of anti-TNF-alpha monoclonal antibody with cattle and sheep cross reaction | |
| CN111349170B (en) | Monoclonal antibody of immune related GTPase family M (IRGM) and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |