CN109651504B - Swainsonine antigen and preparation method thereof - Google Patents
Swainsonine antigen and preparation method thereof Download PDFInfo
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- CN109651504B CN109651504B CN201811566398.XA CN201811566398A CN109651504B CN 109651504 B CN109651504 B CN 109651504B CN 201811566398 A CN201811566398 A CN 201811566398A CN 109651504 B CN109651504 B CN 109651504B
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- FXUAIOOAOAVCGD-FKSUSPILSA-N swainsonine Chemical compound C1CC[C@H](O)[C@H]2[C@H](O)[C@H](O)CN21 FXUAIOOAOAVCGD-FKSUSPILSA-N 0.000 title claims abstract description 40
- 229960005566 swainsonine Drugs 0.000 title claims abstract description 40
- FXUAIOOAOAVCGD-UHFFFAOYSA-N swainsonine Natural products C1CCC(O)C2C(O)C(O)CN21 FXUAIOOAOAVCGD-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 239000000427 antigen Substances 0.000 title claims abstract description 24
- 102000036639 antigens Human genes 0.000 title claims abstract description 24
- 108091007433 antigens Proteins 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- HTSGKJQDMSTCGS-UHFFFAOYSA-N 1,4-bis(4-chlorophenyl)-2-(4-methylphenyl)sulfonylbutane-1,4-dione Chemical compound C1=CC(C)=CC=C1S(=O)(=O)C(C(=O)C=1C=CC(Cl)=CC=1)CC(=O)C1=CC=C(Cl)C=C1 HTSGKJQDMSTCGS-UHFFFAOYSA-N 0.000 claims abstract description 36
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 21
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 21
- 150000001875 compounds Chemical class 0.000 claims abstract description 12
- 230000008878 coupling Effects 0.000 claims abstract description 10
- 238000010168 coupling process Methods 0.000 claims abstract description 10
- 238000005859 coupling reaction Methods 0.000 claims abstract description 10
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 claims abstract description 8
- 230000009471 action Effects 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 8
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000126 substance Substances 0.000 claims abstract description 7
- -1 glutaric acid monoethyl ester acyl chloride Chemical class 0.000 claims abstract description 6
- 239000003513 alkali Substances 0.000 claims abstract description 4
- 230000000382 dechlorinating effect Effects 0.000 claims abstract description 4
- 230000008569 process Effects 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000006482 condensation reaction Methods 0.000 claims 1
- 241001061264 Astragalus Species 0.000 abstract description 15
- 238000003786 synthesis reaction Methods 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 6
- 238000011161 development Methods 0.000 abstract description 4
- 230000003053 immunization Effects 0.000 description 28
- 238000002649 immunization Methods 0.000 description 27
- 241000699670 Mus sp. Species 0.000 description 19
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 12
- JMTMSDXUXJISAY-UHFFFAOYSA-N 2H-benzotriazol-4-ol Chemical compound OC1=CC=CC2=C1N=NN2 JMTMSDXUXJISAY-UHFFFAOYSA-N 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 230000001965 increasing effect Effects 0.000 description 11
- 235000010110 Astragalus glycyphyllos Nutrition 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 230000035484 reaction time Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- NLFBCYMMUAKCPC-KQQUZDAGSA-N ethyl (e)-3-[3-amino-2-cyano-1-[(e)-3-ethoxy-3-oxoprop-1-enyl]sulfanyl-3-oxoprop-1-enyl]sulfanylprop-2-enoate Chemical compound CCOC(=O)\C=C\SC(=C(C#N)C(N)=O)S\C=C\C(=O)OCC NLFBCYMMUAKCPC-KQQUZDAGSA-N 0.000 description 5
- 230000005847 immunogenicity Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 230000002265 prevention Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000005457 ice water Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 235000006533 astragalus Nutrition 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
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- 244000144972 livestock Species 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 210000004233 talus Anatomy 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 1
- MYMNBFURSYZQBR-UHFFFAOYSA-N 5-ethoxy-5-oxopentanoic acid Chemical compound CCOC(=O)CCCC(O)=O MYMNBFURSYZQBR-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241001053158 Oxytropis Species 0.000 description 1
- 208000003141 Plant Poisoning Diseases 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
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- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- DUEPRVBVGDRKAG-UHFFFAOYSA-N carbofuran Chemical compound CNC(=O)OC1=CC=CC2=C1OC(C)(C)C2 DUEPRVBVGDRKAG-UHFFFAOYSA-N 0.000 description 1
- 231100000739 chronic poisoning Toxicity 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
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- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 239000003123 plant toxin Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
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Images
Classifications
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- 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
- 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
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a swainsonine antigen which has the following chemical structural formula:
wherein, BSA is bovine serum albumin. The preparation process comprises the following steps: (1) reacting swainsonine with glutaric acid monoethyl ester acyl chloride to obtain a compound I:
dechlorinating and acidifying the compound I under the action of alkali to obtain a compound II:
Description
Technical Field
The invention relates to a swainsonine antigen and a preparation method thereof.
Background
Locoweed is a kind of toxic plant which is most serious in many areas such as western China, mainly comprises the toxic plants of Astragalus (Astragalus) and acanthopanax (Oxytropis) in leguminous plants, and is distributed in large area in China and other countries in the world. Animals eating locoweed cause chronic poisoning, influence growth and reproduction, even die, cause huge economic loss to the animal husbandry production, and are still not effectively controlled so far. However, the locoweed contains a large amount of nutritional ingredients such as protein, trace elements and the like besides toxic ingredients; meanwhile, the locoweed has developed root system and strong stress resistance, thereby being beneficial to the development of grassland animal husbandry in the aspects of wind prevention and sand fixation, grassland vegetation protection, grassland desertification prevention and the like. In addition, researches also prove that locoweed has an immunoregulation function, can inhibit the growth of tumors, and becomes a hotspot of research of people as a potential new anticancer drug. Therefore, how to reduce the damage of the locoweed to the animal husbandry and reasonably develop and utilize the locoweed draws wide attention.
The immune prevention of the locoweed poisoning of animals opens up a new way for the reasonable development and utilization of the locoweed plants. Swainsonine
Is the main toxic component of locoweed, but as swainsonine is a small molecular hapten and has no immunogenicity, swainsonine molecules must be firstly addedIs modified to be able to couple with a macromolecular carrier to become a complete antigen, so that the antigen has immunogenicity and can be used for animal immunoprophylaxis. Therefore, the chemical synthesis of the swainsonine artificial antigen becomes a key point for researching immunological prevention of locoweed plant poisoning and is also a difficult point.
Disclosure of Invention
The present invention aims to provide a swainsonine antigen and a preparation method thereof according to the current situation of the background technology.
In order to solve the technical problems, the invention provides the following technical scheme:
a swainsonine antigen having the following chemical formula:
Preferably, the coupling ratio of swainsonine to BSA is 28: 1.
The preparation method of the swainsonine antigen comprises the following steps:
(1) reacting swainsonine with glutaric acid monoethyl ester acyl chloride to obtain a compound I:
(2) dechlorinating and acidifying the compound I under the action of alkali to obtain a compound II:
(3) and (2) condensing the compound II with BSA under the action of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole to obtain the swainsonine antigen.
The swainsonine antigen has the advantages of simple synthesis process, easy operation, low cost and high coupling ratio, can generate high-titer antibodies, and opens up a new way for reasonable development and utilization of locoweed plants.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the ultraviolet spectrum of swainsonine-BSA artificial antigen of the present invention identified by ultraviolet scanning.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1 swainsonine antigen preparation method
1.1 materials and methods
1.1.1 principal Material
Swainsonine (SW) is provided by the pharmaceutical research laboratory of Lanzhou institute of livestock and veterinary medicine, national academy of agricultural sciences. Glutaric acid monoethyl ester acid chloride, carbofuran technologies ltd. Bovine Serum Albumin (BSA), rude bio-product. 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (edc. hcl): Shandong-Xia chemical industry Co., Ltd, hydroxy benzotriazole (HOBt): shanghai research Biotech Co., Ltd, American dialysis bag (3500): solarbio, other chemical reagents are analytically pure.
1.1.2 Main Instrument
7-D8505 Freeze dryer, manufactured by SIM, USA. SPECORD 50 UV spectrophotometer, manufactured by Jena Analyzer, Germany. 3K-2 refrigerated centrifuge, manufactured by Sigma.
1.1.3 methods
1.1.3.1SW-BSA synthesis method
(1) Reacting swainsonine with glutaric acid monoethyl ester acyl chloride to obtain a compound I:
(2) dechlorinating and acidifying the compound I under the action of alkali to obtain a compound II:
(3) condensing compound II with BSA under the action of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (edc. hcl) and 1-hydroxybenzotriazole (HOBt) to give compound III:
1.1.3.2 Synthesis of Compound I
Adding swainsonine 90mg (0.52mmol) into a 100mL three-necked bottle, adding dried acetone 20mL, stirring at room temperature, adding glutaric acid monoethyl ester acyl chloride 110.7mg (0.62mmol), heating and stirring at 80 ℃, reacting for 2h, cooling the reaction bottle with ice water, standing in a refrigerator overnight to obtain a solid, filtering the solid, and recrystallizing for 2 times with ethanol 20mL (95%) to obtain the compound I.
1.1.3.3 Synthesis of Compound II
Adding compound I44mg into a 100mL three-necked bottle, adding 0.5mL of 6mol/L NaOH solution, reacting for 3h at room temperature under stirring, adding 1mL of 6mol/L HCl solution, acting for 10min, cooling the reaction bottle with ice water, standing in a refrigerator overnight to obtain a solid, filtering the solid, and recrystallizing for 2 times with 20mL of ethanol (95%) to obtain compound II.
1.1.3.3 Synthesis of Compound III
In a 50mL beaker, 10mg of Compound II was added, and 2mL of Phosphate Buffered Saline (PBS) pH7.4 was added, and after dissolution, 8.3mg of EDC.HCl and 10mg of HOBt were added, and the mixture was allowed to act at room temperature for 10min to obtain solution A.
In a 50mL three-necked flask, 35mg of BSA was added and dissolved in 4mL of Phosphate Buffer Solution (PBS) having a pH of 7.4 to obtain solution B.
In an ice water bath, the solution A is dropwise added into the solution B, and the mixture is stirred for 18 hours at 4 ℃. And after the reaction is finished, taking out the reaction product, putting the reaction product into a dialysis bag, dialyzing the reaction product for 3d by using 500ml PBS with the pH value of 7.4 at the temperature of 4 ℃, changing the solution for 1 time after 6 hours, centrifuging the solution, taking the supernatant, subpackaging the supernatant into 0.5ml centrifuge tubes, preserving the supernatant at the temperature of minus 20 ℃, freezing and drying the product to obtain 4 batches of the reaction product, namely compound III (white powder), and weighing the compound III to calculate the yield. And identifying the compound III by an ultraviolet spectrophotometer.
1.1.4 Synthesis Process optimization
1.1.4.1 influence of raw material ratio
Fixed edc.hcl: compound II (W/W) is 1.5:1, HOBt: the compound II (W/W) is 1:1, and the reaction time is 18 h. Investigation of compound II: the effect of BSA (W/W) on the yield is shown in Table 1.
TABLE 1 influence of raw material formulation on yield
As can be seen from table 1, the product yield increased significantly with increasing amounts of BSA, but when compound II: when BSA (W/W) is increased to 1: 3.5, the yield is not increased obviously and raw materials are wasted. Thus, compound II: BSA (W/W) can be controlled at 1: 2-1: 4, and the optimal ratio is 1: 3.5.
Effect of 1.1.4.21- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCI) on yield
Immobilization compound II: BSA (W/W) 1: 3.5, HOBt: compound II (W/W) was 1:1, reaction time 18h, EDC.HCl: the effect of compound II (W/W) on the yield is shown in Table 2.
Table 2 effect of hcl dosage on yield
As can be seen from Table 2, the yield was somewhat increased with the amount of EDC, but the yield was slightly changed when the amount of EDC.HCl was increased to 1.5: 1. Moreover, the EDC dosage is too much to affect the purification and separation of the product. Thus, edc.hcl: the amount of compound II (W/W) can be controlled in the range of 1.0-2.5: 1, preferably EDC.HCl: compound II (W/W) was 1.5: 1.
1.1.4.3 Effect of reaction time on yield
Fixed edc.hcl: compound II (W/W) is 1.5:1, HOBt: compound II (W/W) is 1:1, compound II: BSA (W/W) 1: 3.5, and the effect of reaction time on yield were examined, and the results are shown in Table 3.
TABLE 3 Effect of reaction time on yield
As can be seen from Table 3, the reaction time has a certain influence on the yield, from 8 to 22h, the product yield increases with increasing reaction time, but not significantly to 18 h. The reaction time can be controlled within 8-22h, and is advantageously set to 18h from the viewpoint of cost and yield.
1.1.4.4 Effect of HOBt on yield
Fixed edc.hcl: compound II (W/W) is 1.5:1, compound II: BS A (W/W) 1: 3.5, reaction time 18h, investigation of HOBt: the effect of compound II on the yield is shown in Table 4.
TABLE 4 Effect of hydroxybenzotriazole (HOBt) on yield
As is clear from Table 4, the yield was slightly increased with the amount of HOBt under the same conditions, but when the amount of HOBt was increased to 1:1, the yield was slightly changed by increasing the amount of HOBt. Therefore, preferred HOBt: the ratio of the compound II (W/W) is 0.5-1.5: 1. Optimal HOBt: the ratio of compound II (W/W) is 1: 1.
1.1.4.5 ultraviolet determination of swainsonine-BSA (III) and calculation of coupling ratio
The ultraviolet scanning identification ultraviolet spectrum result of the swainsonine-BSA artificial antigen is shown in figure 1, the maximum characteristic absorption of hapten II and carrier protein BSA are both 279nm, and under the same concentration, the intensity of the swainsonine-BSA conjugate is obviously enhanced compared with the maximum characteristic absorption peak of BSA, so that a certain spectrum superposition trend is presented. In addition, II is a characteristic peak-to-valley point at 246nm, BSA is a characteristic peak-to-valley point at 253nm, and the characteristic peak-to-valley point of the coupling substance swainsonine-BSA (III) is at 251nm and is between the peak-to-valley points of II and BSA, so that the deviation occurs, a new compound structure is formed in the coupling substance, and the successful coupling of the artificial antigen swainsonine-BSA is proved. The coupling ratio of the artificial antigen swainsonine-BSA is calculated to be about 28: 1.
Example 2 swainsonine-BSA immunogenicity assay in mice
2.1 materials and methods
2.1.1 materials
2.1.1.1 test animal 12 Balb/c male mice, 4-6 weeks old, purchased from the Experimental animals center of Lanzhou university.
2.1.1.2 Main reagents and instruments SW, swainsonine-BSA provided by the institute of veterinary and livestock drug, Lanzhou institute of agricultural science, China; goat anti-mouse IgG-HRP, Huamei bioengineering company; freund's complete adjuvant, Freund's incomplete adjuvant, Sigma Co; tween-20, Amresco Inc.; o-phenylenediamine (OPD), tianjin chemical industries.
2.1.2 immunization of mice with swainsonine-BSA
2.1.2.1 preparation of antigen solution 2mg of swainsonine-BSA was weighed accurately and dissolved in 100ml of physiological saline, 100ml of adjuvant was added and mixed well until white water-in-oil emulsion was formed completely.
2.1.2.2 mouse swainsonine-BSA immunization
After 2.12 Balb/C mice were bred for 1 week, they were randomly divided into 3 groups, i.e., a small dose immunization group (group A), a large dose immunization group (group B) and a control group (group C), 4 mice in each group were numbered 1-4. The experiment was performed 4 times in total: when the mice are immunized for the first time, swainsonine-BSA is completely emulsified by Freund complete adjuvant, the two sides of the neck, the subcutaneous part of the back and the inner side of the hind leg of the mice in the A group and the B group are injected at 5 points, each point is 0.04mL, the immunizing dose is respectively 0.05 mg/group in the A group and 0.10 mg/group in the B group; the 2 nd immunization is carried out on the 30 th day, the adjuvant is Freund incomplete adjuvant, and the A, B groups of immunization approaches and immunization doses are the same as those of the first immunization; the 3 rd immunization is carried out on the 50 th day, the method is the same as the 2 nd immunization, and the A, B groups of immunization doses are 1.5 times of the first immunization; on the 70 th day, the 4 th immunization was carried out by dissolving swainsonine-BSA in physiological saline for injection, and the A, B groups of immunization were performed in the same manner as above, and the immunization dose was 2.5 times that of the first immunization. Specific immunization protocols are shown in table 1. Group C mice were injected subcutaneously with an equal amount of saline simultaneously with 4 immunizations of A, B mice.
2.1.3 mouse serum preparation
At day 10 after the 3 rd and 4 th immunizations, i.e., day 60 and 80 of immunization, mice were decapitated, blood was collected, serum was separated and aliquoted, and stored at-2 ℃ for SW antibody detection.
2.1.4 detection of mouse SW antibodies
The SW antibody titer in mouse serum was detected by indirect hemagglutination assay and enzyme-linked immunosorbent assay (ELISA), respectively.
2.2 results and analysis
2.2.1 IHA detection of mouse SW antibody
The IHA detection result of the mouse SW antibody shows that the SW antibody is generated in the groups A and B after the mice are immunized for 10 days after the 3 rd time, and the antibody titer is 2 respectively3，22，23，23And 24，23，23，23(ii) a 10 days after the 4 th immunization, the antibody titers of group A and group B were 28，27，27，27And 27，26，26，26. It can be seen that the antibody titer of the 4 th immunization was higher than that of the 3 rd, and the antibody titer of group A was higher than that of group B.
2.2.2 Indirect ELISA detection of mouse SW antibodies
The indirect ELISA detection result of the mouse SW antibody shows that the antibody titer of A, B groups of mice is 2 respectively at 10 days after 3 rd immunization of the mice7，26，26，27And 27，27，27，27(ii) a On day 10 after the 4 th immunization, the antibody titers of A, B groups of mice were 210，210，210，210And 210，29，29，29. Compared with the result of the 3 rd immunization, the antibody titer of A, B mice in two groups is obviously increased after the 4 th immunization.
2.3 discussion
In the experiment, swainsonine-BSA is prepared into a vaccine to immunize a Balb/c mouse, and the result shows that after 3 rd immunization, the mouse generates an SW specific antibody; due to the difference among individual mice, the immunogenicity of the swainsonine-BSA to the mice is different, but the immunized mice with high antibody titer are obtained, thereby laying the foundation for the preparation of the SW single-chain antibody. The "spacer" selected in the synthetic antigens of the invention has a significant impact on the immunogenicity of the organism. According to long-term experiments, the separation arm which is too short influences the recognition of hapten by immune system due to the steric hindrance of the carrier, while the separation arm which is too long leads the hapten molecule to be folded due to hydrogen bonds or hydrophobic interaction, and leads the hapten to be close to the carrier protein, thus leading the carrier protein to shield characteristic groups. Therefore, the invention considers the steric hindrance effect between swainsonine and BSA during the coupling design, selects the glutaric acid monoethyl ester acyl chloride as the spacing arm, and is more favorable for enhancing the stimulation of SW-BSA to mice and generating high-titer antibodies.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A swainsonine antigen has a chemical structural formula as follows:
2. The swainsonine antigen as claimed in claim 1 wherein: the coupling ratio of swainsonine to BSA was 28: 1.
3. A process for the preparation of a swainsonine antigen as claimed in claim 1 which comprises:
(1) reacting swainsonine with glutaric acid monoethyl ester acyl chloride to obtain a compound I:
(2) dechlorinating and acidifying the compound I under the action of alkali to obtain a compound II:
(3) and (2) condensing the compound II with BSA under the action of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole to obtain the swainsonine antigen.
4. The production method according to claim 3, characterized in that: the reaction temperature of the step (1) is 80-90 ℃ and the time is 1-4 h.
5. The production method according to claim 3, characterized in that: in the step (3), the mass ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the compound II is 1-2.5:1, the mass ratio of the 1-hydroxybenzotriazole to the compound II is 0.5-1.5:1, and the mass ratio of BSA to the compound II is 2-4: 1.
6. The method of claim 5, wherein: in the step (3), the mass ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the compound II is 1.5:1, the mass ratio of 1-hydroxybenzotriazole to the compound II is 1:1, and the mass ratio of BSA to the compound II is 3.5: 1.
7. The production method according to claim 3, characterized in that: in the step (3), the condensation reaction of the compound II and BSA is carried out at the temperature of 0-4 ℃ for 8-22 h.
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| CN1395967A (en) * | 2002-05-24 | 2003-02-12 | 杨凌大农生物技术有限公司 | Grassland locoism toxin vaccine |
| CN102250238A (en) * | 2011-05-16 | 2011-11-23 | 西北农林科技大学 | Method for synthesizing swainsonine antigen |
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