LU101600B1 - Single-chain antibody TRSAB1 against TORSV and preparation method thereof - Google Patents
Single-chain antibody TRSAB1 against TORSV and preparation method thereof Download PDFInfo
- Publication number
- LU101600B1 LU101600B1 LU101600A LU101600A LU101600B1 LU 101600 B1 LU101600 B1 LU 101600B1 LU 101600 A LU101600 A LU 101600A LU 101600 A LU101600 A LU 101600A LU 101600 B1 LU101600 B1 LU 101600B1
- Authority
- LU
- Luxembourg
- Prior art keywords
- trsab1
- chain antibody
- torsv
- variable region
- expression
- Prior art date
Links
- 238000002360 preparation method Methods 0.000 title description 5
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- 125000000539 amino acid group Chemical group 0.000 claims abstract description 4
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 36
- 230000014509 gene expression Effects 0.000 claims description 26
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 18
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 14
- 229910001453 nickel ion Inorganic materials 0.000 claims description 14
- 241000588724 Escherichia coli Species 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 6
- 239000004327 boric acid Substances 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 6
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 6
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 6
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 6
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 6
- 230000001939 inductive effect Effects 0.000 claims description 6
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229940099596 manganese sulfate Drugs 0.000 claims description 6
- 239000011702 manganese sulphate Substances 0.000 claims description 6
- 235000007079 manganese sulphate Nutrition 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000011684 sodium molybdate Substances 0.000 claims description 6
- 235000015393 sodium molybdate Nutrition 0.000 claims description 6
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 239000012137 tryptone Substances 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229960001763 zinc sulfate Drugs 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 238000003780 insertion Methods 0.000 claims description 3
- 230000037431 insertion Effects 0.000 claims description 3
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- 241000620209 Escherichia coli DH5[alpha] Species 0.000 claims description 2
- 238000010276 construction Methods 0.000 claims description 2
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- 238000011068 loading method Methods 0.000 claims description 2
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- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 abstract description 8
- 101710125418 Major capsid protein Proteins 0.000 abstract description 8
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- 101710083689 Probable capsid protein Proteins 0.000 abstract description 8
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- 229910000403 monosodium phosphate Inorganic materials 0.000 description 9
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 9
- 239000007853 buffer solution Substances 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 241000700605 Viruses Species 0.000 description 6
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- FPPNZSSZRUTDAP-UWFZAAFLSA-N carbenicillin Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)C(C(O)=O)C1=CC=CC=C1 FPPNZSSZRUTDAP-UWFZAAFLSA-N 0.000 description 1
- 229960003669 carbenicillin Drugs 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1009—Picornaviridae, e.g. hepatitis A virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Dentistry (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Communicable Diseases (AREA)
- Peptides Or Proteins (AREA)
Claims (7)
1. Un anticorps à chaîne unique TRsAb1 contre ToRSV, ayant une séquence d'acides aminés comme indiqué dans SEQ ID NO.1.
2. Anticorps à chaîne unique TRsAb1 selon la revendication 1, consistant en une région d'étiquette histidine 15aa, une région variable de chaîne lourde ToRSV de 180aa, un peptide de liaison 15aa et une région variable de chaîne légère ToRSV de 117aa réticulée, connectées en séquence, avec une longueur de 327 résidus d'acides aminés, où les séquences de la région variable de chaîne lourde de 180aa et de la région variable de chaîne légère de 117aa sont dérivées de la séquence d'un anticorps monoclonal produit par la souche cellulaire d'hybridome CGMCC18321.
3. Anticorps à chaîne unique TRsAb1 selon la revendication 1, dans lequel la séquence du gène codant de l'anticorps à chaîne unique TRsAb1 est telle que représentée dans SEQ ID NO.2.
4. Anticorps à chaîne unique TRsAb1 selon la revendication 3, dans lequel la séquence du gène codant de l'anticorps à chaîne unique TRsAb1 a une longueur de 981 pb, dans laquelle la séquence du gène codant de la région d'étiquette histidine a une longueur de 45 pb, le gène codant la séquence de la région variable de la chaîne lourde ToRSV de 180aa a une longueur de 540 pb, la séquence du gène codant du peptide de liaison est de 45 pb et la séquence du gène codant de la région variable de la chaîne légère du ToRSV 117aa a une longueur de 351 pb.
5. Anticorps à chaîne unique TRsAb1 selon la revendication 3, dans lequel l'amont de la séquence du gène codant de l'anticorps à chaîne unique TRsAb1 comprend en outre des éléments d'expression: promoteurs Lac, opérons Lac et sites de liaison aux ribosomes (RBS).
6. Anticorps à chaîne unique TRsAb1 selon la revendication 4, dans lequel un vecteur d'expression d'anticorps à chaîne unique TRsAb1 est PUC57, son site d'insertion est un site de coupure de l'enzyme de restriction EcoRV, et sa souche hôte d'expression est une souche E. coli DH5alpha.
7. Anticorps à chaîne unique TRsAb1 selon la revendication 1, dans lequel un procédé de production de l'anticorps à chaîne unique TRsAb1 comprend les étapes suivantes: a. construction d'un vecteur d'expression d'anticorps à chaîne unique TRsAb1: insertion d'une séquence de gène représentée dans SEQ ID NO. 2 dans un site ECORV sur un vecteur PUC57 pour obtenir un vecteur d'expression TRsAb1 d'anticorps à chaîne unique; b. introduire le vecteur d'expression d'anticorps à chaîne unique TRsAb1 dans E. coli DH5a pour obtenir une souche d'expression d'anticorps à chaîne unique TRSAB1; c. expression auto-induite de l'anticorps à chaîne unique TRsAb1: inoculation de la souche d'expression d'anticorps à chaîne unique TRSAB1 dans un milieu d'expression auto-inducteur, et fermentation à une température de 16 à 40°C pendant 12 à 24 heures pour obtenir un bouillon de fermentation du produit, dans lequel un litre du milieu d'expression d'auto-induction comprend 1,0 à 5,0 g d'extrait de levure, 5,0 à 10,0 g de tryptone, 5,0 à 15,0 g de poudre de maïs, 0,1 à 1,5 g de saccharose, 4,0 à 10,0 g de phosphate dipotassique, 3,5 à 9,0 g de dihydrogénophosphate de potassium, 2,0 à 6,0 g d'hydrogénophosphate de diammonium, 0,2 à 1,6 g de sulfate de magnésium, 2,0 à 5,0 mg de chlorure de calcium, 0,5 à 3,5 mg de chlorure de cobalt, 0,5 à 1,5 mg de chlorure de cuivre, 1,2-5,0 mg de sulfate de manganèse, 3,2-8,0mg de molybdate de sodium, 0,1-0,5 mg d'acide borique, 1,0-8,0 mg de LU101600 chlorure ferrique, 0,5-3,0 mg de sulfate de zinc, 1,0-10,0 g de du glycérol et à 300 mg d'isopropylthio-B-D-galactoside (IPTG); etd. séparation et purification de I'anticorps à chaîne unique TRsAb1: centrifugation du bouillon de fermentation obtenu à l'étape (c), collecte des cellules bactériennes, remise en suspension dans une solution tampon phosphate, mise en échographie, puis re-centrifugation, collecte du surnageant, charger le surnageant sur une colonne d'ions nickel, éluer avec de l'imidazole à gradient de concentration, collecter l'éluat d'anticorps TRsAb1 à chaîne unique, et effectuer la dialyse et la lyophilisation pour obtenir l'anticorps TRsAb1 à chaîne unique.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU101600A LU101600B1 (en) | 2020-01-09 | 2020-01-09 | Single-chain antibody TRSAB1 against TORSV and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU101600A LU101600B1 (en) | 2020-01-09 | 2020-01-09 | Single-chain antibody TRSAB1 against TORSV and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| LU101600B1 true LU101600B1 (en) | 2020-05-18 |
Family
ID=70732373
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| LU101600A LU101600B1 (en) | 2020-01-09 | 2020-01-09 | Single-chain antibody TRSAB1 against TORSV and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| LU (1) | LU101600B1 (fr) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL2027066B1 (en) * | 2020-12-08 | 2021-09-02 | Univ Jiliang China | Single-chain antibody trab1 against trsv and preparation method thereof |
-
2020
- 2020-01-09 LU LU101600A patent/LU101600B1/en active IP Right Grant
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL2027066B1 (en) * | 2020-12-08 | 2021-09-02 | Univ Jiliang China | Single-chain antibody trab1 against trsv and preparation method thereof |
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