CN116804054A - Shark single domain antibody targeting helicobacter pylori UreB, and preparation method and application thereof - Google Patents
Shark single domain antibody targeting helicobacter pylori UreB, and preparation method and application thereof Download PDFInfo
- Publication number
- CN116804054A CN116804054A CN202310704139.3A CN202310704139A CN116804054A CN 116804054 A CN116804054 A CN 116804054A CN 202310704139 A CN202310704139 A CN 202310704139A CN 116804054 A CN116804054 A CN 116804054A
- Authority
- CN
- China
- Prior art keywords
- domain antibody
- shark
- shark single
- single domain
- ureb
- 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
- 108010003723 Single-Domain Antibodies Proteins 0.000 title claims abstract description 60
- 241000251730 Chondrichthyes Species 0.000 title claims abstract description 58
- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 17
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 17
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 230000008685 targeting Effects 0.000 title claims abstract description 12
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 17
- 238000004091 panning Methods 0.000 claims abstract description 16
- 238000003259 recombinant expression Methods 0.000 claims abstract description 8
- 239000002299 complementary DNA Substances 0.000 claims abstract description 6
- 239000013604 expression vector Substances 0.000 claims abstract description 6
- 238000002823 phage display Methods 0.000 claims abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 5
- 239000013598 vector Substances 0.000 claims abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 21
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 238000001514 detection method Methods 0.000 claims description 11
- 238000000746 purification Methods 0.000 claims description 8
- 238000000034 method Methods 0.000 claims description 7
- 238000012163 sequencing technique Methods 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 2
- 239000004202 carbamide Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 230000018883 protein targeting Effects 0.000 claims description 2
- 238000003018 immunoassay Methods 0.000 claims 1
- 238000010839 reverse transcription Methods 0.000 claims 1
- 239000000427 antigen Substances 0.000 abstract description 8
- 102000036639 antigens Human genes 0.000 abstract description 8
- 108091007433 antigens Proteins 0.000 abstract description 8
- 239000012634 fragment Substances 0.000 abstract description 7
- 241000123599 Hemiscylliidae Species 0.000 abstract description 2
- 229940126585 therapeutic drug Drugs 0.000 abstract description 2
- 206010019375 Helicobacter infections Diseases 0.000 abstract 1
- 230000001939 inductive effect Effects 0.000 abstract 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 17
- 239000007788 liquid Substances 0.000 description 16
- 239000006228 supernatant Substances 0.000 description 13
- 238000002965 ELISA Methods 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 11
- 241000588724 Escherichia coli Species 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 108010046334 Urease Proteins 0.000 description 5
- 230000027455 binding Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 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 4
- 239000000047 product Substances 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 210000002784 stomach Anatomy 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 240000000220 Panda oleosa Species 0.000 description 3
- 235000016496 Panda oleosa Nutrition 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 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 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 2
- 238000012286 ELISA Assay Methods 0.000 description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 2
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 231100000357 carcinogen Toxicity 0.000 description 2
- 239000003183 carcinogenic agent Substances 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification 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
- 238000011084 recovery Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000003322 Coinfection Diseases 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000008469 Peptic Ulcer Diseases 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 241000187632 Stegostoma fasciatum Species 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 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 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 201000006585 gastric adenocarcinoma Diseases 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 244000000036 gastrointestinal pathogen Species 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229910001453 nickel ion Inorganic materials 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/121—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Helicobacter (Campylobacter) (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- 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
- G01N33/6854—Immunoglobulins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
-
- 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]
-
- 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
- C07K2317/567—Framework region [FR]
-
- 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
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Plant Pathology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
Abstract
The invention discloses a shark single domain antibody targeting helicobacter pylori UreB, a preparation method and application thereof. The shark single domain antibody is 1E3 with the amino acid sequence shown as SEQ ID No.1 and 1G7 with the amino acid sequence shown as SEQ ID No. 2. The invention also provides a preparation method of the shark single-domain antibody, namely, total RNA of immunized striped bamboo shark cells and tissues is extracted and reversely transcribed into cDNA; amplifying the VNAR fragment by taking cDNA as a template, and connecting the VNAR fragment with a vector to construct a phage library; panning positive clones from phage display library that recognize UreB; constructing a recombinant expression vector, and inducing expression of the single-domain antibody to finally obtain the shark single-domain antibody targeting UreB. The UreB-targeted single-domain antibody can be effectively combined with UreB antigen and helicobacter pylori, and lays a foundation for developing therapeutic drugs and diagnostic reagents for helicobacter pylori infection.
Description
Technical Field
The invention belongs to the field of antibody preparation, and in particular relates to a shark single domain antibody targeting helicobacter pylori UreB, and a preparation method and application thereof.
Background
Helicobacter pylori (Helicobacter pylori, h.pyrri) is a gastrointestinal pathogen, which was classified as a class I carcinogen by the world health organization in 1994, and chronic h.pyrri infection was classified as an established carcinogen by the U.S. health and public service department in 2022. H.pyri infection can cause a range of gastrointestinal diseases such as chronic active gastritis, peptic ulcers, gastric adenocarcinoma, and gastric cancer, and about 50% of people worldwide are infected with h.pyri. Current treatment of h.pyri is primarily antibiotic-dependent therapy, but the problem of antibiotic resistance is increasingly becoming a major obstacle to effective treatment of h.pyri. Thus, there is an urgent need to develop new therapies against h.
H.pyrori is capable of producing large amounts of urease, hydrolyzing urea in the stomach to produce NH 3 And CO 2 Neutralizing the acidic environment in the stomach, providing a favorable microenvironment for its survival in the stomach. Meanwhile, ammonia generated by the method is toxic to cells, and can cause obvious tissue damage. Thus, interfering with or blocking the normal function of H.pyrril urease can effectively reduce its colonization and infection of the stomach. Urease consists mainly of a and B subunits, is exposed to the surface of the cell, and the sequence is highly conserved among isolates. UreB is the main structural subunit of urease, exists in the active site of urease, has good immunogenicity, and is a key target for treating and detecting H.pyri infection.
In vivo, researchers have found an antibody (IgNAR) containing only a heavy chain, which has a complete antigen binding activity in its heavy chain variable region, and have cloned and expressed the heavy chain variable region alone by genetic engineering techniques, and the resulting novel antibody is called VNAR, which is also called nanobody because of its small molecular weight. The shark single domain antibody has the characteristics of small molecular weight, stable physicochemical property, easy genetic engineering transformation, capability of recognizing hidden epitope and the like, and has wide application prospect in the aspects of diagnosis and treatment of various diseases and the like.
Disclosure of Invention
The invention aims to provide a shark single domain antibody targeting helicobacter pylori UreB, and a preparation method and application thereof. The invention obtains two single-domain antibodies 1E3 and 1G7 specific to UreB, and the single-domain antibodies have good affinity and targeting.
In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:
the invention provides a shark single domain antibody targeting helicobacter pylori UreB, which has an amino acid sequence shown as SEQ ID No.1 or SEQ ID No. 2.
Further, the shark single domain antibody comprises framework region FR, hypervariable region HV and complementarity determining region CDRs.
Further, the framework regions FR include FR1, FR2, FR3, and FR4; the amino acid sequence of the FR1 is as follows: AQWVEQTPTTTTKEAGESLTINCVLR or
AARVEQTPRTTTKQAGESLTINCVIR; the amino acid sequence of the FR2 is as follows: TYWTFTKKAAAQ or TYWYFTKKGATK; the amino acid sequence of the FR3 is as follows: GRYAETVSFSLRISDLRLEDSGRYYC or GRYAETVSFSLEMSDLRVEDSGTYHC; the amino acid sequence of the FR4 is as follows: EGGGTILTVKP or EGVGTILTVKP.
Further, the hypervariable region HV comprises HV2 and HV4; the amino acid sequence of HV2 is: KEKLSNG or ietrttng; the amino acid sequence of HV4 is: NKASKS.
Further, the complementarity determining regions CDR include CDR1 and CDR3; the amino acid sequence of CDR1 is: DSSCALAS or DSACAMES; the amino acid sequence of CDR3 is: KAYTTGCTKVILNDWNY or EAYSWMPPYCDKNIVYY.
The invention also provides a coding gene of the shark single domain antibody, which has a nucleotide sequence shown as SEQ ID No.3 or SEQ ID No. 4.
The invention also provides a recombinant vector containing the coding gene.
The invention also provides an engineering strain containing the coding gene.
The invention also provides a preparation method of the shark single domain antibody, which comprises the following steps:
(1) Recombinant UreB protein is obtained through recombinant expression and purification of escherichia coli, and immunization is carried out on the striped zebra shark;
(2) Extracting total RNA of peripheral blood lymphocytes or spleen tissues of immunized sharks, reversely transcribing into cDNA, amplifying a sharks nano antibody fragment by PCR, connecting the sharks nano antibody fragment to a phagemid pComb3XSS carrier through T4, and performing electric shock transformation into E.coli TG1 strain to construct a sharks single-domain antibody phage display library;
(3) Amplifying shark single-domain antibody phage display library, enriching and screening UreB protein, identifying positive phage by indirect ELISA, and determining OD 450 And determining that the value is more than 2.1 times of negative value as positive clone, and determining the sequence of the shark single domain antibody targeting UreB through sequencing and sequence alignment.
(4) Cloning the shark single domain antibody sequence in the step (3) into a pET28a recombinant expression vector, transforming into escherichia coli, and obtaining the recombinant shark single domain antibody through induced expression and affinity purification.
Further, the sequence of the PCR amplification primer in the step (2) is shown as SEQ ID No. 5-SEQ ID No. 12.
The invention also provides application of the shark single domain antibody in preparation of a UreB protein targeting agent.
The invention also provides application of the shark single domain antibody in preparing helicobacter pylori immunodetection reagent or detection kit.
Furthermore, the detection reagent or the detection kit comprises a shark single domain antibody 1E3 with an amino acid sequence shown as SEQ ID No.1 or a shark single domain antibody 1G7 with an amino acid sequence shown as SEQ ID No. 2.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the shark single domain antibody 1E3 and 1G7 prepared by the invention has the characteristics of small molecular weight, intracellular expression, high affinity, simple structure and easy expression and purification, can be used for helicobacter pylori detection, and has wide application prospect in the aspects of helicobacter pylori detection reagents and therapeutic drug development.
Drawings
FIG. 1 shows the amplification results of shark single domain antibodies VNAR.
FIG. 2 shows the PCR identification results of the bacterial liquid of the shark single domain antibody phage display library.
FIG. 3 shows the results of ELISA assays of polyclonal phages.
FIG. 4 shows the results of ELISA assay of monoclonal phage.
FIG. 5 shows the purification results of shark single domain antibodies 1E3 and 1G 7; wherein A is the purification result of the single domain antibody 1E 3; b is the purification result of the single domain antibody 1G 7; m: protein Marker,1: whole protein, 2: supernatant, 3: fluid passing through, 4-12: PBS, 20, 50, 75, 100, 200, 300, 400, 500mM imidazole eluent.
FIG. 6 shows the results of indirect ELISA detection of binding of single domain anti-antibodies to antigen UreB.
FIG. 7 shows the results of indirect ELISA detection of binding of single domain antibodies to helicobacter pylori cells.
Detailed Description
For better illustrating the objects, technical solutions and advantages of the present invention, the technical solutions of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.
Example 1: construction of a Single-Domain antibody immune library of UreB
1. Preparation of antigen UreB and immunization of shark
(1) 100ng of the laboratory-deposited pET22b-UreB recombinant expression plasmid was transformed into 100. Mu.L E.coli BL21 (DE 3) competent cells by heat shock and cultured overnight in an incubator at 37℃on LB solid plates containing Kana at a final concentration of 100. Mu.g/mL. On the next day, transfer the culture medium to 100mL LB liquid medium containing Amp according to the volume ratio of 1:100, culture the culture medium to logarithmic phase at 37 ℃, add IPTG with final concentration of 0.1mM for induction, and culture the culture medium at 25 ℃ overnight. The cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in 10mL of lysate (PBS solution containing 20mM imidazole and 1mM PMSF) and sonicated until the solution was clear. The supernatant was centrifuged at 12000rpm at 4℃for 10min and filtered through a 0.45 μm filter. The UreB protein was purified by Ni-NTA affinity chromatography, and the eluate of each component was detected by SDS-PAGE. The eluted target protein was dialyzed against PBS and finally concentrated by ultrafiltration.
(2) First immunization 400 μg of recombinant UreB protein was mixed with an equal volume of Freund's complete adjuvant to immunize striped bamboo shark; the boost was then performed once every two weeks, 5 times in total, after 200 μg of recombinant UreB protein was mixed with an equal volume of Freund's incomplete adjuvant.
2. Single domain antibody library construction
Blood is collected from tail veins of immunized sharks, peripheral blood lymphocytes are extracted, the sharks are killed, spleens of the sharks are taken, RNA of the peripheral blood lymphocytes and spleens of the sharks are extracted, and cDNA is reversely transcribed.
The VNAR fragment was amplified using degenerate primers using cDNA as template. The primer sequences used were:
the amplification system is as follows:
the reaction conditions were as follows:
the gene amplification result was detected by 1% agarose gel electrophoresis, the VNAR fragment was about 500bp (FIG. 1), and the VNAR fragment was recovered by gel. The recovered VNAR fragment and phagemid pComb3XSS were digested with the restriction enzyme sfii, the cleavage system was as follows:
the conditions of the digestion reaction are 50 ℃ overnight, the fragments and the vectors are recovered by a DNA recovery kit after the digestion is completed, then T4 ligation is carried out, the ligation is carried out overnight at 16 ℃, and the ligation products are recovered and concentrated.
Pre-cooling the electric shock cup, melting E.coli TG1 competent cells on ice, adding 5 mu L of the connecting product, gently mixing, placing on ice for 10min, transferring into the electric shock cup, electric shock for 5ms at 1.8kV, immediately adding 950 mu L of Recovery Medium, and immediately culturing at 37 ℃ and 250rpm for 1h. The bacterial liquids are respectively diluted 10 3 、10 4 、10 5 、10 6 100. Mu.L of each of the plates was plated on 2 XYT solid plates containing ampicillin resistance, and the plates were incubated at 37℃overnight in an inverted state. The pool capacity of the constructed shark single domain antibody immune library was calculated based on the number of colonies on the plate.
Randomly picking 10 monoclonals for bacterial liquid PCR identification (figure 2) and sequencing, wherein the effective insertion rate of genes is 100%, comparing the obtained sequences, and the sequence diversity is 80%, and finally obtaining library with the library capacity of 1.28 multiplied by 10 8 CFU。
Example 2: panning of shark Single-Domain antibody immune library
1. Amplification of shark single domain antibody phage original library
Adding 500 μL of library strain into 200mL 2 XYT liquid culture medium (containing 100 μg/mL Amp), and regulating bacterial liquid OD 600 Culturing at about 0.1, 37 deg.C and 200rpm for 1-2 hr. Make OD 600 The cells were collected by adding M13K07 in an amount of 20 times the amount of the cells to infect, standing at 37℃for infection, mixing for 30min, co-infection for 1h, and centrifuging at 5000rpm for 30min at room temperature, and discarding the supernatant. The cells were resuspended in 200mL of fresh 2 XYT liquid medium (50. Mu.g/mL Kana, 100. Mu.g/mL Amp,0.1mM IPTG) and incubated overnight at 30℃and 190 rpm.
The following day, the overnight cultured bacterial liquid was centrifuged at 4900rpm at 4℃for 30min, the supernatant was filtered with a 0.45 μm filter membrane, and a 20% final volume of PEG8000/NaCl solution was added, followed by ice bath for 30min. Then, centrifugation was performed at 6000rpm at 4℃for 20min, the supernatant was discarded, and the mixture was resuspended in 5mL of PBS, and glycerol at a final concentration of 10% was added for storage at-80 ℃.
2. Panning of UreB-targeting shark single domain antibodies
(1) First round panning
(A) Diluting the UreB recombinant protein to 100 mug/mL with PBS, adding 4mL into an immune tube, and coating at 4 ℃ overnight; pouring out the coating liquid, washing the immune tube with PBS for 1 time, filling with PBS containing 5% skimmed milk (5% MPBS), and sealing at 37deg.C for 2 hr; the blocked immune tube is discarded, the blocked liquid is washed for 1 time by PBS, 1mL of phage obtained by preparation and 3mL of 5% MPBS are added, the blocked liquid is blocked by a sealing film, and after rotating and incubating for 1 hour at room temperature, standing and incubating for 1 hour are carried out;
(B) Phage were discarded and the immunization was washed 10 times with PBS (PBST) containing 0.1% Tween-20 and then 2 times with PBS;
(C) Thoroughly draining the residual PBS, adding 1mL of 100mM triethylamine solution, sealing with a sealing film, incubating at room temperature for 10min by rotating, immediately introducing into a centrifuge tube filled with 0.5mL 1M pH7.4 Tris-HCl in advance, washing the immune tube with 250 μl of Tris-HCl, and combining all the washing solutions;
(D) All washes were taken to infect 10mL OD 600 E.coli TG1, OD 5mL between 0.4 and 0.6 600 Infecting immune tubes with E.coli TG1 between 0.4 and 0.6, and incubating in a water bath at 37 ℃ for 30min;
(E) mu.L of the bacterial liquid was 10-fold diluted, and 3 gradients were total diluted, and 100. Mu.L of each gradient was applied to a 2 XYT solid (100. Mu.g/mL Amp,2% glucose) plate, and the remaining initial bacterial liquid was centrifuged at 3000rpm for 10min to collect bacterial cells, resuspended in 0.5mL 2 XYT liquid medium, applied to a solid plate, incubated overnight at 37℃and the number of colonies grown was calculated.
(F) Scraping and collecting colony on solid plate cultured overnight, adding 20 μl of bacterial liquid into 10mL2×TY liquid (100 μg/mL Amp,2% glucose) culture medium, shaking at 37deg.C, and incubating to OD 600 About 0.5, M13K07 with 20 times of the bacterial body amount is added for infection, and the mixture is incubated for 30min in a water bath at 37 ℃.Centrifugation at 4000rpm for 10min, removal of supernatant, resuspension of the cells with 100mL of 2 XYT (100. Mu.g/mL Amp, 50. Mu.g/mL Kana,0.1% glucose) medium, shaking and incubation at 30℃overnight. Phage were collected as described above.
(2) Second round panning
The procedure was the same as the first round of panning, the antigen coating concentration was 10 μg/mL, the blocking solution was 5% MPBST, the plate number was 15, and the rest of the procedure was the same as the first round of panning.
(3) Third round panning
The procedure was the same as the first round of panning, the antigen coating concentration was 2 μg/mL, the plate number was 20, and the rest was the same as the first round of panning.
After three rounds of panning, phages obtained by three rounds of panning were detected by polyclonal phage ELISA, and the results are shown in fig. 3, with specific phages being significantly enriched as the number of panning increases.
(4) Screening positive monoclonal antibodies targeting the UreB by indirect phage ELISA:
(A) 200. Mu.L of fresh 2 XYT/A100 liquid culture medium is added to each well of a 96-well deep-well plate, 190 single colonies are picked from a plate for overnight culture in a third round of panning and cultured in the 96-well plate at a temperature of 37 ℃ with a shaking table of 220rpm for 6 hours, another 96-well deep-well plate is added with fresh 2 XYT (20 times the cell mass M13K07 helper phage, 100. Mu.g/mL Amp,0.1mM IPTG) 100. Mu.L of bacterial liquid in a logarithmic growth phase is added to each well, standing and infection is carried out at 37 ℃ for 30 minutes, and shaking table at a temperature of 30 ℃ with 220rpm is carried out overnight.
(B) Diluting UreB recombinant protein to 1. Mu.g/ml with PBS, and placing 100. Mu.L/Kong Baobei on an ELISA plate at 4deg.C overnight;
(C) The supernatant was discarded, and each well was washed 3 times with 300 μl PBST buffer;
(D) 200. Mu.L/well of 5% MPBS was added and blocked at 37℃for 2h;
(E) PBST cleaning the plate 3 times, wherein the step is the same as the step (C);
(F) Centrifuging a 96-well deep hole plate cultured overnight at 4 ℃ and 3000rpm for 30min, taking supernatant in the 96-well deep hole plate, adding an ELISA plate, taking a culture medium as a blank control, and incubating at 100 mu L/hole and 37 ℃ for 1h;
(G) PBST cleaning the plate 3 times, wherein the step is the same as the step (C);
(H) HRP-labeled Anti-M13 antibody (diluted at 5% MPBS in volume ratio 1:1000) was added at 100. Mu.L/well and incubated at 37℃for 1h;
(I) PBST cleaning the plate 3 times, wherein the step is the same as the step (C);
(J) 100. Mu.L/well TMB substrate was added to the well plate and reacted at room temperature for 5-10min. Add 50. Mu.L/well 1M H 2 SO 4 Terminating the chromogenic reaction, and reading OD by using an ELISA reader 450 Readings at the location.
As shown in FIG. 4, the positive clones with a reading number 2.1 times greater than that of the blank control were obtained, 6 positive monoclonal antibodies were obtained, the 6 positive monoclonal antibodies were sent to the sequencing, and after sequence comparison with the database, 2 shark single domain antibodies with different sequences were obtained, designated 1E3 and 1G7, respectively.
1E3 has an amino acid sequence shown as SEQ ID No.1, and a coded nucleotide sequence shown as SEQ ID No. 3; 1G7 is shown in SEQ ID No.2, the coded nucleotide sequence is shown in SEQ ID No.4, and the sequences of the two are compared with a database, so that the results show that the sequences are all single-domain antibody gene sequences derived from sharks.
Example 3: prokaryotic induction expression and purification of shark single-domain antibody
The 1E3 and 1G7 gene sequences obtained by panning in example 2 were cloned into pET28a expression vector using homologous recombination ClonExpress II One Step Cloning kit, the reaction system is as follows:
the reaction system was left to react at 37℃for 30min, and the ligation product was used for conversion.
The ligation products were directly transformed into E.coli DH 10. Beta. Competent cells and cultured overnight at 37 ℃. The monoclonal is selected, the recombinant expression vector is successfully constructed through sequencing verification, the recombinant expression vector is transformed into E.coli BL21 (DE 3) competence along with plasmid extraction, the expression is induced by IPTG, the monoclonal antibody is purified through ultrasonic disruption and nickel ion affinity chromatography, the purification result is shown in figure 5, and the purified antibody is stored at-80 ℃.
Example 4: affinity detection of single domain antibodies to antigens
Indirect ELISA was used to detect the affinity of the single domain antibodies to the antigen.
mu.g/mL of recombinant UreB protein was added to the ELISA plate at 100. Mu.L per well, and coated overnight at 4 ℃. The supernatant was discarded, and each well was washed 3 times with 300. Mu.L of PBST buffer. 200 μl of 5% MPBS per well was added and blocked at 37deg.C for 2h. After 3 PBST washes, 100 μl of different mass concentrations (150, 30, 6, 1.2, 0.24, 0.048, 0.0096, 0 μg/mL) of shark nanobody per well was added and incubated for 1.5h at 37 ℃. PBST plates were washed 3 times, 100. Mu.L of HRP-anti-HA tag per well (diluted in 5% MPBS at 1:5000 by volume) was added and incubated for 1h at 37 ℃. After PBST washing the plate 3 times, 100. Mu.L of TMB substrate was added to each well, and the reaction was carried out at room temperature for 5-10min. 50 mu L of 1M H are added to each well 2 SO 4 Terminating the chromogenic reaction, and reading OD by using an ELISA reader 450 Readings at the location.
As shown in FIG. 6, 1E3 has a strong binding ability to recombinant UreB protein.
Example 5: indirect ELISA detection of binding of Single-Domain antibodies to helicobacter pylori
After culturing the H.pyri strain under microaerophilic conditions for 2-3 days, the cells were collected and centrifuged at 12000rpm for 5min at room temperature, and the supernatant was discarded. The cells were resuspended in PBS containing 3% (v/v) paraformaldehyde and fixed at 4℃for 24h; centrifugation was performed at 12000rpm at 4℃for 5min, the supernatant was discarded, and the cells were resuspended in PBS and repeated once. According to each hole 10 7 CFU,100 μl coated on an elisa plate, overnight at 4 ℃; the supernatant was discarded, and the rest was the same as in example 4.
As a result, as shown in FIG. 7, the single domain antibody 1E3 was able to bind to helicobacter pylori.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A shark single domain antibody targeting helicobacter pylori UreB, characterized in that the shark single domain antibody has an amino acid sequence as shown in SEQ ID No.1 or as shown in SEQ ID No. 2.
2. The gene encoding a shark single domain antibody of claim 1, wherein the encoding gene has a nucleotide sequence as set forth in SEQ ID No.3 or as set forth in SEQ ID No. 4.
3. A recombinant vector comprising the coding gene of claim 2.
4. An engineered strain comprising the coding gene of claim 2.
5. The method for preparing the shark single domain antibody of claim 1, comprising the steps of:
(1) Recombinant UreB protein is obtained through recombinant expression and purification, and the shark is immunized;
(2) Extracting total RNA of cells or tissues of the immunized shark, carrying out reverse transcription to cDNA, carrying out PCR amplification, connecting a PCR product to a phagemid vector, converting the cells, and constructing a shark single-domain antibody phage display library;
(3) Amplifying the shark single-domain antibody phage display library, panning the shark single-domain antibody phage by utilizing recombinant UreB protein to obtain positive clone, and sequencing and comparing to determine the shark single-domain antibody sequence of the targeting UreB protein;
(4) Cloning the shark single domain antibody sequence in the step (3) into a recombinant expression vector, converting into competent cells, and purifying to obtain the recombinant shark single domain antibody.
6. The method according to claim 5, wherein the sequence of the PCR amplification primer in the step (2) is shown in SEQ ID No.5 to SEQ ID No. 12.
7. The method according to claim 5, wherein the positive clone in step (3) is OD 450 The value was greater than 2.1 times negative for the monoclonal.
8. Use of a shark single domain antibody of claim 1 for the preparation of a urea protein targeting agent.
9. Use of a shark single domain antibody according to claim 1 for the preparation of a helicobacter pylori immunoassay or a detection kit.
10. The use according to claim 9, wherein the detection reagent or kit comprises the shark single domain antibody 1E3 having the amino acid sequence shown in SEQ ID No.1 or the shark single domain antibody 1G7 having the amino acid sequence shown in SEQ ID No. 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310704139.3A CN116804054A (en) | 2023-06-14 | 2023-06-14 | Shark single domain antibody targeting helicobacter pylori UreB, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310704139.3A CN116804054A (en) | 2023-06-14 | 2023-06-14 | Shark single domain antibody targeting helicobacter pylori UreB, and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116804054A true CN116804054A (en) | 2023-09-26 |
Family
ID=88080303
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310704139.3A Pending CN116804054A (en) | 2023-06-14 | 2023-06-14 | Shark single domain antibody targeting helicobacter pylori UreB, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116804054A (en) |
-
2023
- 2023-06-14 CN CN202310704139.3A patent/CN116804054A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111320694B (en) | Nano antibody GN2 composed of variable region of heavy chain antibody and preparation method and application thereof | |
| CN110872351B (en) | Nano antibody GN1 specifically bound with GPC3 protein and preparation method and application thereof | |
| CN113278074B (en) | anti-CEACAM 5 nano antibody | |
| CN113493510A (en) | Bovine-derived single-chain antibody for resisting staphylococcus aureus LukD virulence factor and preparation and application thereof | |
| CN111848790B (en) | Bovine-derived single-chain antibody for resisting staphylococcus aureus and preparation and application thereof | |
| CN108892723B (en) | Single-domain heavy chain antibody for detecting porcine epidemic diarrhea virus, preparation method and application | |
| CN114262377B (en) | Preparation method of anti-human CD70 nano antibody for blocking binding of CD70 and ligand CD27 thereof and coding sequence thereof | |
| CN112625132B (en) | Nano antibody, coding gene, expression vector, host cell and application thereof | |
| CN116396382A (en) | Shark single domain antibody targeting helicobacter pylori HpaA and application thereof | |
| CN112500479B (en) | Preparation of canine II type adenovirus recombinant protein monoclonal antibody | |
| CN110396128B (en) | Preparation of anti-CD 19 nano antibody | |
| CN114106187B (en) | Specific shark single-domain antibody targeting OGT (one glass solution) and preparation method and application thereof | |
| CN116804054A (en) | Shark single domain antibody targeting helicobacter pylori UreB, and preparation method and application thereof | |
| CN114409777A (en) | Specific nano antibody Nb3.27 of colorectal cancer-related bacteroides fragilis toxin protein kinase and application thereof | |
| CN113583119A (en) | Anti-staphylococcus aureus nanobody Nb56, application and kit | |
| CN109762065B (en) | Single-domain heavy chain antibody Nb72 for vibrio fluvialis | |
| CN112250765A (en) | Nano antibody aiming at HER2 and application thereof | |
| CN109762063B (en) | Single-domain heavy chain antibody Nb75 for vibrio fluvialis | |
| CN114106167B (en) | Nanobody for specifically recognizing listeria monocytogenes, recombinant vector, host cell and application of nanobody | |
| CN114369165B (en) | Bovine single-chain antibody of bovine-derived anti-staphylococcus aureus virulence factor GapC, preparation method and application thereof | |
| CN114409792B (en) | anti-EphB 4 nano antibody | |
| CN114891098B (en) | Clostridium perfringens beta toxin nano antibody and application thereof | |
| CN114773465B (en) | Single-domain antibody VHH-3 aiming at S protein of new coronavirus omicron strain, coding sequence and application | |
| CN109762064B (en) | Single-domain heavy chain antibody Nb71 for vibrio fluvialis | |
| CN109824776B (en) | Single-domain heavy chain antibody Nb73 for vibrio fluvialis |
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 |