CN110437330B - Preparation method of natamycin complete antigen - Google Patents
Preparation method of natamycin complete antigen Download PDFInfo
- Publication number
- CN110437330B CN110437330B CN201910715945.4A CN201910715945A CN110437330B CN 110437330 B CN110437330 B CN 110437330B CN 201910715945 A CN201910715945 A CN 201910715945A CN 110437330 B CN110437330 B CN 110437330B
- Authority
- CN
- China
- Prior art keywords
- solution
- natamycin
- hours
- fermentation
- complete antigen
- 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.)
- Expired - Fee Related
Links
- 229960003255 natamycin Drugs 0.000 title claims abstract description 45
- NCXMLFZGDNKEPB-FFPOYIOWSA-N natamycin Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C[C@@H](C)OC(=O)/C=C/[C@H]2O[C@@H]2C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 NCXMLFZGDNKEPB-FFPOYIOWSA-N 0.000 title claims abstract description 44
- 235000010298 natamycin Nutrition 0.000 title claims abstract description 44
- 239000004311 natamycin Substances 0.000 title claims abstract description 44
- 239000000427 antigen Substances 0.000 title claims abstract description 30
- 102000036639 antigens Human genes 0.000 title claims abstract description 30
- 108091007433 antigens Proteins 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 239000000243 solution Substances 0.000 claims abstract description 53
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 23
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 23
- 238000000034 method Methods 0.000 claims abstract description 18
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 12
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 12
- 210000000440 neutrophil Anatomy 0.000 claims abstract description 12
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical compound ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 claims abstract description 11
- DCPMPXBYPZGNDC-UHFFFAOYSA-N hydron;methanediimine;chloride Chemical compound Cl.N=C=N DCPMPXBYPZGNDC-UHFFFAOYSA-N 0.000 claims abstract description 11
- WNLRTRBMVRJNCN-UHFFFAOYSA-N adipic acid Chemical compound OC(=O)CCCCC(O)=O WNLRTRBMVRJNCN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 102000013382 Gelatinases Human genes 0.000 claims abstract description 9
- 108010026132 Gelatinases Proteins 0.000 claims abstract description 9
- 235000011037 adipic acid Nutrition 0.000 claims abstract description 5
- 239000001361 adipic acid Substances 0.000 claims abstract description 5
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000007864 aqueous solution Substances 0.000 claims abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 62
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- 238000000855 fermentation Methods 0.000 claims description 28
- 230000004151 fermentation Effects 0.000 claims description 28
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 27
- 102000013519 Lipocalin-2 Human genes 0.000 claims description 26
- 108010051335 Lipocalin-2 Proteins 0.000 claims description 26
- 101150051118 PTM1 gene Proteins 0.000 claims description 15
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 10
- 238000001514 detection method Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 6
- 239000001301 oxygen Substances 0.000 claims description 6
- 229910052760 oxygen Inorganic materials 0.000 claims description 6
- 229920000936 Agarose Polymers 0.000 claims description 5
- 229910052759 nickel Inorganic materials 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 238000003786 synthesis reaction Methods 0.000 claims description 4
- 241000894006 Bacteria Species 0.000 claims description 3
- 108010010803 Gelatin Proteins 0.000 claims description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 239000007433 bsm medium Substances 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 3
- 239000003480 eluent Substances 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 230000009145 protein modification Effects 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000012216 screening Methods 0.000 claims description 3
- 102000004357 Transferases Human genes 0.000 claims description 2
- 108090000992 Transferases Proteins 0.000 claims description 2
- 239000000706 filtrate Substances 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims 2
- 229910021529 ammonia Inorganic materials 0.000 claims 1
- 229920000159 gelatin Polymers 0.000 claims 1
- 239000008273 gelatin Substances 0.000 claims 1
- 235000019322 gelatine Nutrition 0.000 claims 1
- 235000011852 gelatine desserts Nutrition 0.000 claims 1
- 235000011187 glycerol Nutrition 0.000 description 15
- 235000013305 food Nutrition 0.000 description 10
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 8
- 230000003053 immunization Effects 0.000 description 6
- 238000002649 immunization Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 241000233866 Fungi Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 125000003700 epoxy group Chemical group 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 238000003317 immunochromatography Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 241001506991 Komagataella phaffii GS115 Species 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- NCXMLFZGDNKEPB-UHFFFAOYSA-N Pimaricin Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCC(C)OC(=O)C=CC2OC2CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 NCXMLFZGDNKEPB-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101100241858 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) OAC1 gene Proteins 0.000 description 1
- 241000970906 Streptomyces natalensis Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- -1 amino, carboxyl Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000843 anti-fungal effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000002587 enol group Chemical group 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002636 mycotoxin Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000009965 odorless effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 150000004965 peroxy acids Chemical class 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 238000005554 pickling Methods 0.000 description 1
- 101150092906 pmt1 gene Proteins 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000009597 pregnancy test Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 235000013580 sausages Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 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
- 230000009967 tasteless effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
- 238000002562 urinalysis Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Mycology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a preparation method of a natamycin complete antigen, which is characterized by comprising the following steps: (1) Dissolving adipic acid in MES solution, sequentially adding carbodiimide hydrochloride and N-hydroxy thiosuccinimide, oscillating at room temperature, adding MES solution of neutrophil gelatinase or bovine serum albumin, and oscillating at room temperature; adding iodoacetamide for reaction, and dialyzing overnight to obtain modified carrier protein; (2) The carbodiimide hydrochloride and the N-hydroxyl thiosuccinimide added into the modified carrier protein are taken, placed on ice after being lightly oscillated, added with 50wt% of DMSO aqueous solution containing the natamycin, oscillated for 2 hours at room temperature and dialyzed in PB solution overnight to obtain the natamycin complete antigen, and the method has the advantage that the natamycin complete antigen is not easy to agglomerate after being placed for a long time.
Description
Technical Field
The invention relates to the field of complete antigen preparation, in particular to a preparation method of a natamycin complete antigen.
Background
In the processing of food, people usually add preservatives to the food to prolong the preservation time of the food and prevent the food from being polluted by fungi. Natamycin (also called Natamycin or pimaricin) with molecular formula C 33 H 47 NO 13 The molecular weight is 665.73, and the preservative is odorless, tasteless, low in dosage and high in safety and edibility. Natamycin is slightly soluble in water and relatively insoluble in most organic solvents. Usually slightly soluble in methanol solution. Solubility is 30-100mg/L at room temperature, and solubility is improved at pH lower than 3 or higher than 9, but stability is reduced. Natamycin (Natamycin), a natural antifungal compound produced by controlled fermentation of streptomyces natalensis (a white to milky crystalline powder with no odor or taste), generally exists in a enol form. It can inhibit growth of yeast and mold, inhibit generation of mycotoxin, and reduce food spoilage probability. Therefore, the processes of fermentation of the yoghourt, cheese fermentation, ham sausage pickling and the like can not be influenced. Natamycin is currently the only international componentThe natural food preservative is approved to be added, has high efficiency, safety, no toxicity, no side effect and no influence on the taste, color and flavor of food.
Although natamycin has been approved by the country for additive use in food production, the addition of natamycin as an antibiotic is also strictly controlled and tracked over a long period of time. Because of this, a large amount of natamycin is added to the food, and a large amount of samples need to be monitored, so that a simple and rapid detection method is crucial to the detection of natamycin. At present, methods for detecting natamycin mainly comprise an ultraviolet spectrophotometry method, a thin layer chromatography method, a high performance liquid chromatography method, an ultra high performance liquid chromatography method and an ultra high performance liquid chromatography-tandem mass spectrometry method, wherein the high performance liquid chromatography method is most widely applied.
The traditional detection and monitoring means is mainly the combination of chromatographic mass spectrometry, and has better analysis sensitivity and accuracy. However, the liquid phase separation of the glucoside substances is difficult, one set of equipment can often reach three to four million, and less than one hundred samples are detected every day. The extremely high monitoring cost and the lower detection speed waste a large amount of manpower, material resources and financial resources, and simultaneously greatly limit the monitoring efficiency and the monitoring popularization of related departments.
The immunoassay method is a method for identifying and detecting target molecules in a sample by utilizing the specificity of an antibody and measuring by a subsequent sensing technology, has the characteristics of low cost, high speed, high throughput and the like, and is widely applied to aspects such as drug urinalysis, pregnancy test, in-vitro diagnosis and the like. The development of immune products for food safety detection is a great heat in the field of food safety at present, and the mainstream immunoassay method mainly comprises the following steps: enzyme-linked immunosorbent assay (ELISA), colloidal Gold Immunochromatography (GICT) and fluorescent Immunochromatography (IFC).
Natamycin is a small molecule which cannot directly produce an antibody, so that natamycin needs to be coupled to carrier protein to be made into a complete antigen so as to stimulate the production of the antibody; meanwhile, the antigen coupled on the carrier protein is also favorable for being adsorbed on an enzyme label plate or an NC membrane, thereby being convenient for subsequent product development. So far, a colloidal gold test strip of natamycin has been developed, but the synthesis of a complete natamycin antibody is not reported. However, natamycin has a complex structure and has multiple functional groups such as amino, carboxyl and epoxy groups, and particularly, the epoxy group in natamycin is easy to open a ring or react with the amino group of natamycin in a peracid or alkali environment, so that resistance inactivation is caused. In the previous research, the coupled complete antigen is often easy to precipitate in the storage process, so that the antigenicity and the subsequent preparation of the kit are influenced.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of a natamycin complete antigen which is not easy to agglomerate after long-term storage.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of natamycin complete antigen comprises the following steps:
(1) Carrier protein modification
Dissolving 20mg of adipic acid in 5mL of MES solution with pH value of 6.5, adding 50mg of carbodiimide hydrochloride (EDC HCl) and 50mg of N-hydroxythiosuccinimide in sequence, slowly shaking at room temperature for 20 minutes, adding 5mL of MES solution containing 20mg of Neutrophil Gelatinase (NGAL) or Bovine Serum Albumin (BSA), and slowly shaking at room temperature for 2 hours; then 2mg of iodoacetamide is added to react for two hours, and the mixture is dialyzed in 20mM MES solution overnight and is dialyzed twice, thus obtaining the modified carrier protein;
(2) Complete antigen synthesis
Taking 10mg of modified carrier protein Bovine Serum Albumin (BSA), adding 0.04mg of carbodiimide hydrochloride and 0.05mg of N-hydroxy thiosuccinimide (NHS); or taking 10mg of modified carrier protein Neutrophil Gelatinase (NGAL), adding 0.08 mg of carbodiimide hydrochloride (EDC HCl) and 0.1mg of N-hydroxy thiosuccinimide (NHS); after gently shaking for 20min, the mixture was placed on ice, and a 50wt% DMSO aqueous solution containing 5mg of natamycin was added thereto, and the mixture was shaken at room temperature for 2 hours and dialyzed overnight in a 20mM PB solution with pH of 7.4, to obtain a natamycin complete antigen.
The preparation method of the neutrophil gelatinase comprises the following steps:
(1) Construction of NGAL expression vectors
(ii) cleaving a fragment of NGAL protein: <xnotran> QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG, N 6X his tag CACCATCACCACCATCAC, EcoR I BamH I , pHIL-S1 , Sal I , GS115 , MD ; </xnotran>
(2) Screening of high expression clones
Picking 20 clones from the plate of step (1), and adding to 5mL of BMGY medium respectively; after 24 hours, methanol was added every 24 hours to a final concentration of 1 wt%; after 72 hours, centrifuging 2mL bacterial liquid at 10000r/min for 1min, taking the supernatant, using an NGAL detection kit, selecting high-expression clone, scribing and preserving bacteria;
(3) NGAL protein fermentation and purification
Selecting high-expression clones from the plate activated in the step (2), inoculating the clones in 5mL YNB medium containing 1vt% of glycerol, culturing at 28 ℃ for 24 hours, transferring to 500mL fresh YNB medium containing 1vt% of glycerol, continuously culturing for 24 hours, transferring to 5L BSM medium containing 20mL PTM1 solution and 4vt% of glycerol, starting tank fermentation, adjusting pH to 5.0 with ammonia water, adding 100mL 50% glycerol containing 1.2vt of PTM1 solution when DO rebounds and rising, and reducing dissolved oxygen; adding 100mL of 50% glycerol containing 1.2vt percent PTM1 solution when DO rebounds, replenishing four times in total, adjusting the pH value of the fermentation solution to 6.0 after the last replenishing, adding 20mL of methanol when the last glycerol is consumed, beginning to reduce dissolved oxygen after 3 hours, adding 50mL of methanol containing 1.2vt percent PTM1 solution when DO rebounds every time, until the fermentation is finished, sampling every two hours to detect the fermentation concentration after the methanol is induced for 4 hours, and after the fermentation is carried out for 72 hours, the concentration of NGAL does not rise any more and can be discharged; taking the fermentation liquor at 10000rpm, centrifuging at 4 ℃ for 20min, keeping the supernatant, adding imidazole with the final concentration of 10mM, standing overnight at 4 ℃, filtering with a 0.45um membrane, taking the filtrate, and purifying with an NTA nickel agarose column to obtain the neutrophil gelatin transferase protein.
The equilibrium solution of the NTA nickel agarose column is PBS solution with pH =6.0 and 20mM imidazole, and the eluent is PBS solution with pH =6.0 and 100mM imidazole.
Compared with the prior art, the invention has the advantages that: the invention discloses a preparation method of natamycin complete antigen for the first time, because excessive amino or exposed sulfhydryl on carrier protein reacts slowly with natamycin epoxy group, excessive cross-linking and finally forming deposition, thus, excessive adipic acid is added to change the amino on the surface of carrier protein into carboxyl under the catalysis of EDC; and then adding excessive iodoacetamide to seal the sulfydryl on the upper surface of the carrier protein, and finally dialyzing to remove small molecules to obtain the modified carrier protein. And (3) mixing the modified carrier protein, EDC hydrochloride and NHS according to the mole number of 1:5:10 MES solution with pH value of 6.5 is added in sequence to react for 20min, and finally excessive natamycin is added to react for 2 h and dialyzed overnight to obtain transparent complete antigen which is not easy to coagulate after long-term standing. The carrier protein used was neutrophil gelatinase transferase (NGAL), which has been shown to be highly immunogenic in new zealand white. Experiments prove that even if the surface amino group is blocked, the NGAL can also cause great immune activity in vivo and is more suitable to be used as modified carrier protein.
Drawings
FIG. 1 is a plot of NGAL concentration in the supernatant of a fermentation broth versus fermentation time;
FIG. 2 is an SDS-PAGE analysis of the purification of NGAL antigen;
FIG. 3 is a natamycin competitive inhibition ELISA assay.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Example 1
1. Construction of NGAL expression vectors
Truncating the fragment of NGAL protein: <xnotran> QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG, N 6X his tag CACCATCACCACCATCAC. </xnotran> Finally, the fragment is cloned to a pHIL-S1 vector through EcoR I and BamH I sites. Then linearization is carried out through restriction enzyme Sal I, electric transduction is carried out to the Pichia pastoris GS115 strain, and an MD culture medium plate is paved;
2. screening of high expression clones
Picking 20 clones from the plate of step (1) and adding to 5mL of BMGY medium, respectively; after 24 hours, methanol was added every 24 hours to a final concentration of 1 wt%; after 72 hours, centrifuging 2mL bacterial liquid at 10000r/min for 1min, taking the supernatant, using a nine-strong-organism NGAL detection kit (latex enhanced immunoturbidimetry), picking high-expression clones, scribing and preserving bacteria.
3. NGAL protein fermentation and purification
From the plate activated in step 2, colonies were picked and inoculated into 5mL YNB medium (containing 1vt% glycerol); after 24 hours of incubation at 28 degrees, the cells were transferred to 500mL of fresh YNB medium (containing 1vt% glycerol) and incubated for another 24 hours, then 5L of BSM medium (containing 20mL of PTM1 solution and 4vt% glycerol) was transferred and tank fermentation was started. Adjusting pH =5.0 with ammonia water, adding 100mL of 50% glycerol (each liter contains 12mL of PTM1 solution) when DO rises, and reducing dissolved oxygen; when DO rebounds, adding 100mL of 50% glycerol (each liter contains 12mL of PTM1 solution), supplementing for four times totally, and after the last supplementing, adjusting the pH value of the fermentation solution to be 6.0. After the last glycerol was consumed, 20mL of methanol was added. After about 3 hours, the dissolved oxygen began to drop. After each subsequent DO rebound, 50mL of methanol (12 mL of PTM1 solution per liter of methanol) was added until fermentation was complete. After 4 hours of methanol induction, sampling every two hours to detect the fermentation concentration, generally after 72 hours of fermentation, the concentration of NGAL does not rise any more, and then the fermentation tank can be taken out, at the moment, the concentration is about 360mg/L, as shown in figure 1, the concentration of NGAL in the supernatant of the fermentation liquid is a curve changing along with time. The fermentation broth was centrifuged at 10000rpm for 20min at 4 ℃ and the supernatant was retained, imidazole was added to a final concentration of 10mM, allowed to stand overnight at 4 ℃, filtered through a 0.45um membrane and then purified by an NTA nickel agarose column. The equilibrium solution was a PBS solution containing 20mM imidazole (pH = 6.0), and the eluent was a PBS solution containing 100mM imidazole (pH = 6.0), and after purification, NGAL was analyzed by SDS-PAGE, and the results are shown in fig. 2.
The preparation method of the BSM culture medium (containing PTM1 solution and 4wt% of glycerol) comprises the following steps: sterilizing BSM inorganic culture medium under high pressure, and adding 4.0ml of filter-sterilized PTM1 (microelement) per liter to obtain BSM culture medium (containing PTM1 solution and 4wt% of glycerin). BSM inorganic medium: 85% of H 3 PO 4 26.7ml/L，CaSO 4 •2H 2 O 0.93g/L，
K 2 SO 4 18.2g/L，MgSO 4 •2H 2 14.9g/L of O, 4.13g/L of KOH, 40g/L of glycerol and 4.0ml/L of PMT;
PMT1 (1L) solution formulation: cuSO 4 •5H 2 O 6.0g/L ，KI 0.088g/L，MnSO 4 •H 2 O 3.0g/L，Na 2 MoO 4 •2H 2 O 0.2g/L，H 3 BO 3 0.02g/L，CoCl 2 •6H 2 O 0.5g/L，ZnCl 2 20.0g/L，FeSO 4 •7H 2 O65.0 g/L, biotin 0.2g/L, concentrated H 2 SO 4 5.0mL/L。
Example 2
1. Carrier protein modification
Dissolving 20mg of adipic acid in 5mL of MES solution with pH value of 6.5, adding 50mg of carbodiimide hydrochloride (EDC HCl) and 50mg of N-hydroxythiosuccinimide in sequence, slowly shaking at room temperature for 20 minutes, adding 5mL of MES solution containing 20mg of Neutrophil Gelatinase (NGAL) or Bovine Serum Albumin (BSA), and slowly shaking at room temperature for 2 hours; then 2mg of iodoacetamide is added to react for two hours, and the mixture is dialyzed in 20mM MES solution overnight and is dialyzed twice, thus obtaining the modified carrier protein;
2. complete antigen synthesis
Taking 10mg of modified carrier protein Bovine Serum Albumin (BSA), adding 0.04mg of carbodiimide hydrochloride and 0.05mg of N-hydroxy thiosuccinimide (NHS); or taking 10mg of the modified carrier protein Neutrophil Gelatinase (NGAL), adding 0.08 mg of carbodiimide hydrochloride (EDC HCl) and 0.1mg of N-hydroxy thiosuccinimide (NHS); after gently shaking for 20min, placing on ice, adding 50wt% DMSO aqueous solution containing 5mg of natamycin, shaking for 2 hours at room temperature, and dialyzing in 20mM PB solution with pH of 7.4 overnight to obtain natamycin complete antigen.
Example 3
1. New Zealand big ear white immunization
The protein concentration of the natamycin-NGAL is calculated by the total protein amount, namely the total protein concentration is multiplied by the adding volume to obtain the adding amount of the natamycin-NGAL. When the new zealand white fungus of 6-8 weeks old is immunized for the first time, the natamycin-NGAL immunogen prepared in example 2 is diluted to 1mg/mL (diluted by 0.01mol/L PBS), mixed with Freund's complete adjuvant in equal volume and fully emulsified, and 5 new zealand white fungus are inoculated to the neck and back at subcutaneous multiple points, wherein the inoculation antigen dose is 2.5 mg/mouse with 1mL per mouse; after 14 days, 2 nd immunization is carried out, the volume of the immune agent is emulsified with the same volume of the immunogen by Freund incomplete adjuvant, the dose of the immune agent is the same as that of the first immunization, and the number of times of boosting immunization is 4.
2. Identification of the immune potency
After one week of the last booster (total 5 immunizations), the carotid artery of the rabbit was sampled and the plasma was about 80mL, centrifuged, and the serum (about 40 mL) was collected and measured for titer by the classical chessboard method and sensitivity by the indirect competitive ELISA method.
1) The method for measuring the antiserum titer comprises the following steps:
(1) Coating: the coated antigens (natamycin-BSA ) were diluted to 0.2. Mu.g/mL with 0.05mol/L carbonate buffer (pH 9.6), 100. Mu.L/well were added to two enzyme-labeled plates, and incubated for 2 hours at 37 ℃. Decanting off the liquid in the wells, washing the plate with PBST buffer (pH 7.4) for 3 times, and spin-drying the washing solution;
(2) And (3) sealing: adding 150 μ L of blocking solution (containing 2wt% skimmed milk) into each well, blocking at 37 deg.C for 1 hr, spin-drying the liquid in the well, washing the plate with PBST buffer solution (pH 7.4) for 3 times, and patting dry the washing solution;
(3) And (3) antiserum dilution: mu.L of 0.02M PBS (pH 7.4) was added to each well, followed by 50. Mu.L of diluted natamycin antiserum, starting from 1: 100. 1: 300. 1: 900. 1: 2700. 1: 8100. 1: 24300. 1: 72900. 1:216700, etc. 8 gradients;
(4) Adding antiserum: adding rabbit serum which is diluted according to the step (3) and immunized by the natamycin-NGAL into the hole coated with the natamycin-BSA antigen; otherwise, adding the natamycin-BSA immune serum diluted according to the step (3); the amount of sample was 50. Mu.L per well, incubated at 37 ℃ for 30min, washed 3 times with PBST buffer (pH 7.4), and patted dry. Incubate 30min, wash 3 times with PBST buffer (pH 7.4), and pat dry. Meanwhile, setting the serum of a rabbit without immunization as a negative control;
(5) Adding an enzyme-labeled secondary antibody: adding 100 μ L HRP-goat anti-rabbit IgG (diluted 5000 times with PBS) into each well, incubating for 30min at 37 ℃, washing for 3 times with PBST buffer solution (pH 7.4), and patting dry;
(6) Color development: a horseradish peroxidase substrate 3,3', 5' -tetramethylbenzidine solution and 30% hydrogen peroxide by mass are mixed according to a volume ratio of 1. Then 50. Mu.L of stop solution (2 mol/L H) was added to each well 2 SO 4 )；
(7) And (3) reading determination: the absorbance (OD) was read with a microplate reader at a wavelength of 450 nm. The result shows that the antibody titer in the natamycin-NGAL immune serum is about 1:5000. therefore, the natamycin-NGAL complete antigen has higher advantages than the natamycin-BSA complete antigen in the production of natamycin antibodies.
3. The method for measuring the sensitivity of the antiserum comprises the following steps:
(1) The coating and sealing processes are the same as those in the measurement of the antiserum titer;
(2) Adding standard substances and antibodies: each well was added 50 μ L natamycin standard solution and 50 μ L of a solution prepared according to 1:2700 diluted serum dilutions were incubated at 37 ℃ for 30min, then washed 3 times with PBST solution and patted dry. The solvent of the standard solution is PBS buffer solution, the concentrations of the standard solutions are respectively 0, 0.03, 0.1, 0.3, 0.9, 2.7, 8.1 and 24.3ng/mL, and three concentrations are in parallel;
(3) Adding an enzyme-labeled secondary antibody: adding 100 mu L of enzyme-labeled secondary antibody diluent into each hole, incubating for 30min at 37 ℃, then washing for 3 times by using PBST solution, and patting dry;
(4) Color development: adding 100 mu L of color development liquid into each hole, and incubating for 15min at 37 ℃;
(5) And (4) terminating: adding 50 mu L of 2mol/L concentrated sulfuric acid into each hole;
(6) Reading: by OD 450 Measuring OD value of each hole by nm wavelength;
plotted as-log 10 (competitor) value against OD 450 As a result of the ordinate, as shown in FIG. 3, the Kd value was approximately 1ng/mL, and the curve showed a high affinity. The above ELISA results were combined to show that natamycin was successfully labelled to NGAL and BSA. Wherein, natamycin-NGAL is suitable to be used as immunogen to induce and generate antibody; and natamycin-BSA is suitable as a detection antigen for the evaluation of the immune effect.
PBS buffer formulation: 0.2g KH 2 PO 4 ，2.90g Na 2 HPO 4 4g of NaCl, adjust the pH to 6.0 and make the volume constant to 1L.
The formula of the sealing liquid comprises: adding 1g of skimmed milk powder into 50mL of the PBS solution, and stirring uniformly.
PBST solution formula: adding 0.5mL of Tween 20 into 1L of the PBS, and stirring.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also appreciate that they may make various changes, modifications, additions and substitutions within the spirit and scope of the invention.
Claims (3)
1. A preparation method of a natamycin complete antigen is characterized by comprising the following steps:
(1) Carrier protein modification
Dissolving 20mg of adipic acid in 5mL of MES solution with the pH value of 6.5, then sequentially adding 50mg of carbodiimide hydrochloride and 50mg of N-hydroxythiosuccinimide, slowly oscillating at room temperature for 20 minutes, then adding 5mL of MES solution containing 20mg of neutrophil gelatin transferase or bovine serum albumin, and slowly oscillating at room temperature for 2 hours; then 2mg of iodoacetamide is added to react for two hours, and the mixture is dialyzed in 20mM MES solution overnight and is dialyzed twice, thus obtaining the modified carrier protein;
(2) Complete antigen synthesis
Taking 10mg of modified carrier protein bovine serum albumin, and adding 0.04mg of carbodiimide hydrochloride and 0.05mg of N-hydroxy thiosuccinimide; or taking 10mg of modified carrier protein neutrophil gelatinase, and adding 0.08 mg of carbodiimide hydrochloride and 0.1mg of N-hydroxy thiosuccinimide; after gently shaking for 20min, placing on ice, adding 50wt% DMSO aqueous solution containing 5mg of natamycin, shaking for 2 hours at room temperature, and dialyzing in 20mM PB solution with pH of 7.4 overnight to obtain natamycin complete antigen.
2. The method for preparing natamycin complete antigen according to claim 1, characterized in that the method for preparing neutrophil gelatinase is as follows:
(1) Construction of NGAL expression vectors
(ii) cleaving a fragment of NGAL protein: <xnotran> QDSTSDLIPAPPLSKVPLQQNFQDNQFQGKWYVVGLAGNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCDYWIRTFVPGCQPGEFTLGNIKSYPGLTSYLVRVVSTNYNQHAMVFFKKVSQNREYFKITLYGRTKELTSELKENFIRFSKSLGLPENHIVFPVPIDQCIDG, N 6X his tag CACCATCACCACCATCAC, EcoR I BamH I , pHIL-S1 , Sal I , GS115 , MD ; </xnotran>
(2) Screening of high expression clones
Picking 20 clones from the plate of step (1), and adding to 5mL of BMGY medium respectively; after 24 hours, methanol was added every 24 hours to a final concentration of 1 wt%; after 72 hours, centrifuging 2mL bacterial liquid at 10000r/min for 1min, taking the supernatant, using an NGAL detection kit, selecting high-expression clone, scribing and preserving bacteria;
(3) NGAL protein fermentation and purification
Selecting clones from the plate activated in the step (2), inoculating the clones into 5mL of YNB medium containing 1vt% of glycerol, after 24-hour incubation at 28 ℃ transferring the clones into 500mL of fresh YNB medium containing 1vt% of glycerol, continuing the incubation for 24 hours, transferring the clones into 5L of BSM medium containing 20mL of PTM1 solution and 4vt% of glycerol, starting the on-tank fermentation, adjusting pH =5.0 with ammonia, and when DO rebounds up, adding 100mL of 50% glycerol containing 1.2vt% of PTM1 solution, and reducing dissolved oxygen; adding 100mL of 50% glycerol containing 1.2vt percent PTM1 solution when DO rebounds, replenishing four times in total, adjusting the pH value of the fermentation solution to 6.0 after the last replenishing, adding 20mL of methanol when the last glycerol is consumed, beginning to reduce dissolved oxygen after 3 hours, adding 50mL of methanol containing 1.2vt percent PTM1 solution when DO rebounds every time, until the fermentation is finished, sampling every two hours to detect the fermentation concentration after the methanol is induced for 4 hours, and after the fermentation is carried out for 72 hours, the concentration of NGAL does not rise any more and can be discharged; and taking the fermentation liquor at 10000rpm, centrifuging for 20min at 4 ℃, keeping the supernatant, adding imidazole with the final concentration of 10mM, standing overnight at 4 ℃, filtering by using a 0.45um membrane, taking the filtrate, and purifying by using an NTA nickel agarose column to obtain the neutrophil gelatin transferase protein.
3. The method for preparing natamycin complete antigen according to claim 2, characterized in that: the equilibrium solution of the NTA nickel agarose column is PBS solution with pH =6.0 and 20mM imidazole, and the eluent is PBS solution with pH =6.0 and 100mM imidazole.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910715945.4A CN110437330B (en) | 2019-08-05 | 2019-08-05 | Preparation method of natamycin complete antigen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910715945.4A CN110437330B (en) | 2019-08-05 | 2019-08-05 | Preparation method of natamycin complete antigen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110437330A CN110437330A (en) | 2019-11-12 |
| CN110437330B true CN110437330B (en) | 2022-11-18 |
Family
ID=68433171
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910715945.4A Expired - Fee Related CN110437330B (en) | 2019-08-05 | 2019-08-05 | Preparation method of natamycin complete antigen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110437330B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06321998A (en) * | 1993-05-10 | 1994-11-22 | Kikkoman Corp | Anti-fumonisin monoclonal antibody, hybridoma, hapten antigen and their production |
| CN104479007A (en) * | 2014-11-18 | 2015-04-01 | 浙江大学 | Carbodiimide synthesis method for citrinin-protein carrier artificial coupled antigen |
| CN104560886A (en) * | 2014-12-25 | 2015-04-29 | 江南大学 | Anti-natamycin monoclonal antibody hybridoma cell strain and application thereof |
| CN105017411A (en) * | 2015-07-06 | 2015-11-04 | 浙江大学 | Synthetic method for artificially coupling antigen through aflatoxin B1-carrier proteins |
| CN105037526A (en) * | 2015-08-07 | 2015-11-11 | 西北农林科技大学 | Gentamicin antigen synthetic method and gentamicin-IgY antibody preparation method |
-
2019
- 2019-08-05 CN CN201910715945.4A patent/CN110437330B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH06321998A (en) * | 1993-05-10 | 1994-11-22 | Kikkoman Corp | Anti-fumonisin monoclonal antibody, hybridoma, hapten antigen and their production |
| CN104479007A (en) * | 2014-11-18 | 2015-04-01 | 浙江大学 | Carbodiimide synthesis method for citrinin-protein carrier artificial coupled antigen |
| CN104560886A (en) * | 2014-12-25 | 2015-04-29 | 江南大学 | Anti-natamycin monoclonal antibody hybridoma cell strain and application thereof |
| CN105017411A (en) * | 2015-07-06 | 2015-11-04 | 浙江大学 | Synthetic method for artificially coupling antigen through aflatoxin B1-carrier proteins |
| CN105037526A (en) * | 2015-08-07 | 2015-11-11 | 西北农林科技大学 | Gentamicin antigen synthetic method and gentamicin-IgY antibody preparation method |
Non-Patent Citations (2)
| Title |
|---|
| 土霉素完全抗原的制备与鉴定;徐桂连等;《食品工业科技》;20110401(第04期);全文 * |
| 盐酸金霉素人工抗原的合成及鼠源多抗血清的制备;李靓等;《河南农业科学》;20120715(第07期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110437330A (en) | 2019-11-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US8507214B2 (en) | Elisa kit for detecting lincomycin | |
| CN101413955B (en) | ELISA test box for detecting zearalenone and preparing and detecting method thereof | |
| CN102967709B (en) | Detect enzyme linked immunological kit and the application thereof of zearalenone medicine | |
| US10428139B2 (en) | GM hybridoma cell, monoclonal antibody, kit and preparation method and use thereof | |
| CN111304174A (en) | Triazolone monoclonal antibody hybridoma cell strain B11S and application thereof | |
| CN113717949A (en) | Hybridoma cell strain capable of secreting ketoconazole monoclonal antibody and application thereof | |
| CN110437330B (en) | Preparation method of natamycin complete antigen | |
| CN110724192B (en) | Hybridoma cell strain secreting serpentine monoclonal antibody and application thereof | |
| WO2018103630A1 (en) | Hybridoma cell strain c1 for secreting anti-paromomycin monoclonal antibody and use thereof | |
| CN108794507B (en) | Rifaximin hapten, artificial antigen, preparation method and application thereof | |
| CN111334479A (en) | Chlorhydroxypyridine monoclonal antibody hybridoma cell strain TYL and application thereof | |
| CN111273015A (en) | Enzyme linked immunosorbent assay kit for detecting Gymnodinium breve toxin and preparation and application thereof | |
| CN116675696A (en) | Saxitoxin monoclonal antibody, and preparation method and application thereof | |
| CN106544324B (en) | Monoclonal antibody of mycoplasma hyopneumoniae and application thereof | |
| CN112904008B (en) | ELISA kit for detecting impurities such as protein A in biological product and application thereof | |
| CN115057926A (en) | Monoclonal antibody capable of recognizing Phomopsis oblonga and hybridoma cell strain PoF7 thereof | |
| CN101571549B (en) | Vero cell HCP test kit and application thereof | |
| CN110117286B (en) | Heterocyclic amine 8-MeIQx hapten and antibody as well as preparation method and application thereof | |
| CN111443202B (en) | ELISA kit for detecting anticoagulant rodenticide, preparation and application thereof | |
| CN113637642A (en) | Hybridoma cell strain capable of secreting monoclonal antibody of dicofol and application of hybridoma cell strain | |
| CN112011516A (en) | Sirolimus monoclonal antibody hybridoma cell strain and application thereof | |
| Petruzzelli et al. | Preparation and characterization of a monoclonal antibody specific for the β‐agonist clenbuterol | |
| CN111377888A (en) | Rhododendrin mollis toxin III hapten as well as preparation method and application thereof | |
| CN110618269A (en) | Preparation and application of emamectin benzoate monoclonal antibody | |
| CN105301253B (en) | It is enriched with immunomagnetic beads of the toxin of T 2 and preparation method and application |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20221018 Address after: No. 8, Huikang Road, Haishu District, Ningbo, Zhejiang 315012 Applicant after: Ningbo Customs Technology Center Address before: 315800 no.219 South Changjiang Road, Beilun District, Ningbo City, Zhejiang Province Applicant before: Beilun customs of the people's Republic of China |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20221118 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |