CN110054682A - A kind of preparation method and application of Steroid antigen - Google Patents
A kind of preparation method and application of Steroid antigen Download PDFInfo
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- CN110054682A CN110054682A CN201910288479.6A CN201910288479A CN110054682A CN 110054682 A CN110054682 A CN 110054682A CN 201910288479 A CN201910288479 A CN 201910288479A CN 110054682 A CN110054682 A CN 110054682A
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- 239000000427 antigen Substances 0.000 title claims abstract description 60
- 102000036639 antigens Human genes 0.000 title claims abstract description 60
- 108091007433 antigens Proteins 0.000 title claims abstract description 60
- 150000003431 steroids Chemical class 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- MUMGGOZAMZWBJJ-DYKIIFRCSA-N Testostosterone Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 MUMGGOZAMZWBJJ-DYKIIFRCSA-N 0.000 claims abstract description 69
- 229960003604 testosterone Drugs 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 23
- 150000002500 ions Chemical class 0.000 claims abstract description 20
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims abstract description 18
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims abstract description 14
- CJQNBXFUHQZFOE-VYAQIDIUSA-N testosterone hemisuccinate Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@H](CC4)OC(=O)CCC(O)=O)[C@@H]4[C@@H]3CCC2=C1 CJQNBXFUHQZFOE-VYAQIDIUSA-N 0.000 claims abstract description 11
- 238000011160 research Methods 0.000 claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 238000004090 dissolution Methods 0.000 claims abstract description 9
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000012153 distilled water Substances 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- FALRKNHUBBKYCC-UHFFFAOYSA-N 2-(chloromethyl)pyridine-3-carbonitrile Chemical compound ClCC1=NC=CC=C1C#N FALRKNHUBBKYCC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229940014800 succinic anhydride Drugs 0.000 claims abstract description 6
- 239000000376 reactant Substances 0.000 claims abstract description 3
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- -1 testosterone amber Acid esters Chemical class 0.000 claims description 16
- 238000009395 breeding Methods 0.000 claims description 9
- 230000001488 breeding effect Effects 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000004990 Smectic liquid crystal Substances 0.000 claims description 3
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 239000002274 desiccant Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims 1
- 150000002148 esters Chemical class 0.000 claims 1
- 235000019687 Lamb Nutrition 0.000 abstract description 21
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- 238000005516 engineering process Methods 0.000 abstract description 4
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- 239000002245 particle Substances 0.000 description 6
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- 238000010884 ion-beam technique Methods 0.000 description 5
- 230000003637 steroidlike Effects 0.000 description 5
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000001568 sexual effect Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
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- 238000011161 development Methods 0.000 description 3
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- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
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- 241001465754 Metazoa Species 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
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- 229940098773 bovine serum albumin Drugs 0.000 description 1
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- 239000000745 gonadal hormone Substances 0.000 description 1
- 230000001456 gonadotroph Effects 0.000 description 1
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 210000002394 ovarian follicle Anatomy 0.000 description 1
- 238000011170 pharmaceutical development Methods 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
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- 239000012086 standard solution Substances 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- 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
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Genetics & Genomics (AREA)
- Reproductive Health (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Epidemiology (AREA)
- Endocrinology (AREA)
- Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Gynecology & Obstetrics (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pregnancy & Childbirth (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Steroid Compounds (AREA)
Abstract
The invention discloses a kind of preparation methods of Steroid antigen, and described method includes following steps: (1) testosterone being dissolved in anhydrous pyridine, succinic anhydride is added after dissolution, reaction obtains testosterone succinate；(2) after the testosterone succinate that step (1) obtains being dissolved in n,N-Dimethylformamide, the phosphate buffer of BSA is added, reacts；(3) reactant of step (2) is placed on the turntable of heavy ion research device biological irradiation, carries out Ion Irradiation on Multi-walled Carbon；(4) by step (3) irradiation after product distilled water and PBS dialyse, be then lyophilized to get.This method passes through Ion Irradiation on Multi-walled Carbon testosterone and BSA, it is successfully realized coupling, prepare Steroid antigen, gained Steroid antigen by inoculation can manual control ewe produce the technology of twin lamb(s), which has the characteristics that at low cost, effect is good, simple and easy to do, without side-effects.
Description
Technical field
The invention belongs to field of biotechnology for animals, and in particular to a kind of preparation method and application of Steroid antigen.
Background technique
The development and exploitation of polymer material are that scholars endeavour the heat subject carried out for many years.Ion irradiation can be led
It causes material microstructure and macro property to change, thus be used to be transformed in the research work of polymer material.Early stage is main
The particle (such as gamma-rays, electronics) of low ionising radiation is used to make irradiation bomb.The study found that irradiation generated in the polymer
Effect mainly has the fracture and crosslinking of molecular link, the release of gas molecule and new formation of chemical bond etc., the fracture of molecular link and
Crosslinking changes molecular weight and its distribution of material, to influence the macro property of material.In general, the crosslinking of strand makes point
Son amount increases, and causes the soluble of biomaterial to reduce, trap reduces.These phenomenons that low ionising radiation causes have obtained very
Good research, and have suitable extensive commercial application.Heavy ion is in substance mainly so that electron ionization and the excitation of target atom
Mode off-energy and low ionising radiation particle unlike, heavy ion have very high electronstoppingpower (linear energy
Branch value LET).This allows for heavy ion irradiation and electric charge transfer and energy is caused to deposit, thus when ion passes through medium, along it
As soon as path will leave a continuous columnar ionization region, therefore generate and be crosslinked and can form stable complex, and not
It will cause the loss of activity of biology, this effect is that the complex studied in biological medicine passes through a kind of means.
Antigen is made in steroid hormone with chemically synthesized method, inoculation ewe is generated by immune response
Antibody can specifically neutralize original corresponding hormone in ewe body, so that its original bioactivity is lost or is reduced, cause to give birth to
It grows the intrinsic balance multilated of endocrine system and a series of adjustment occurs.Negative feedback of the gonadal hormone to hypothalamo pituitary
Decrease promotes hypothalamus to secrete more gonadotropin releasing hormones, and hypophysis secretes more gonadotropic hormone, and then accelerates sexual gland
Development, reaches raising number of eggs ovulated, improves the effect of breeding potential.
There are some R&D institutions to carry out the research of Steroid antigen both at home and abroad, prepare the antigen of multiple types, all using change
Learn synthetic antigen, mainly send one's regards at present it is entitled, BSA in conjunction with the molecule of steroidal than being mostly 1:20 or so because Percentage bound it is low and
Dissolvent residual seriously affects immune effect and sheep body health.
Summary of the invention
The first purpose of this invention is to provide a kind of preparation method of Steroid antigen to overcome drawbacks described above.
A second object of the present invention is to provide the purposes according to Steroid antigen obtained by the above method.
The purpose of the present invention is achieved by the following technical scheme:
A kind of preparation method of Steroid antigen, described method includes following steps:
(1) testosterone is dissolved in anhydrous pyridine, succinic anhydride is added after dissolution, reaction obtains testosterone succinate；
(2) after the testosterone succinate that step (1) obtains being dissolved in n,N-Dimethylformamide, the phosphate of BSA is added
Buffer, reaction；
(3) reactant of step (2) is placed on the turntable of heavy ion research device biological irradiation, carries out carbon ion spoke
According to；
(4) by step (3) irradiation after product distilled water and PBS dialyse, be then lyophilized to get.
Further, in step (1), the testosterone succinate specific the preparation method comprises the following steps:
It weighs testosterone 52mg and is dissolved in 3mL anhydrous pyridine, 90mg succinic anhydride is added after completely dissolution and keeps away under room temperature
Light generation is reacted for 24 hours, and then with pyridine is dried with nitrogen, faint yellow grease-like compound is made；By smectic compound with 5wt%'s
100mL NaHCO3Solution dissolution, washed with ether, then with hydrochloric acid be adjusted to pH be 7.2 after, be layered after centrifugation, abandon supernatant, receive
Collect lower liquid 86mL, 5g anhydrous sodium sulfate is added to be dried overnight, filtration drying agent obtains faint yellow after solvent volatilization completely
Solid-like object 67mg, as testosterone succinate.
Further, in step (2), the weight ratio of the testosterone succinate and BSA is 6:11, reaction under stiring into
Row 6h.
Further, in step (3), irradiation dose is respectively 5-80Gy, dosage rate 20Gy/min.
Further, irradiation dose 40Gy.
Further, in step (4), distilled water dialysis time is 3d, and PBS dialysis time is 3d.
Further, the Steroid antigen has following structural formula:
It is used to improve the breeding potential of sheep according to the resulting Steroid antigen of any of the above-described method.
The invention has the following advantages:
The preparation method of Steroid antigen provided by the invention is successfully realized idol by Ion Irradiation on Multi-walled Carbon testosterone and BSA
Connection, prepares Steroid antigen, gained Steroid antigen by inoculation can manual control ewe produce the technology of twin lamb(s), the biology system
Agent has the characteristics that at low cost, effect is good, simple and easy to do, without side-effects.
Detailed description of the invention
Fig. 1 is the ultraviolet spectra of testosterone, BSA and testo-BSA artificial antigen.
Fig. 2 is the infrared spectroscopy of testosterone, BSA and testo-BSA artificial antigen.
Specific embodiment
Hereinafter, preferred embodiments of the present invention will be described, it should be understood that preferred embodiment described herein is only used
In the description and interpretation present invention, it is not intended to limit the present invention.
Embodiment 1
Ion Irradiation on Multi-walled Carbon prepares the preparation method of Steroid antigen
By breeding immunology and reproductive endocrinology method, manual control ewe produces the technology of twin lamb(s), is emerging at present
With at low cost, biological self reproducing technology that effect is good, simple and easy to do, without side-effects.A Steroid antigen is constructed, steroidal is made
Antigen can be improved ewe ovulation rate and lambing percentage.The present invention is successfully realized idol by Ion Irradiation on Multi-walled Carbon testosterone and BSA
Connection, prepares Steroid antigen.
1. experimental material:
Testosterone (content 98%): it is purchased from Shanghai Guan Dao bioengineering Co., Ltd；
Bovine serum albumin(BSA) (BSA): (content 96%) Yancheng Sai Bao Biotechnology Co., Ltd；
Remaining reagent is analytical reagents.
2. irradiance method
National Laboratory, the CAS Institute of Modern Physics Lanzhou heavy ion research device (HIRFL) biological irradiation is whole
It is carried out on end.The 12C6+ ion beam that primary power is 20MeV/u can piece by the nickel window of beam current tube, ionisation chamber, air and drop
Irradiation sample afterwards monitors injection rate with air ionization chamber, and sample replacement and data acquisition are controlled by computer, and all processes exist
It is carried out under room temperature and Atmospheric Condition, irradiation dose is respectively 5,10,20,40,80Gy, dosage rate 20Gy/min.
3. preparation method
Testosterone 52mg is accurately weighed with electronic balance and is dissolved in 3mL anhydrous pyridine, and 90mg succinic anhydride is added after completely dissolution,
Under room temperature, it is protected from light oscillating reactions for 24 hours.Then pyridine is dried up with nitrogen evaporator, faint yellow grease-like compound 95mL is made.
The 100mL NaHCO for being 5% by smectic object mass fraction3Solution dissolution, the washing of the ether of 10mL × 3, then use
It after 5wt%HCl is adjusted to pH7.2, is layered after centrifugation, abandons supernatant, collected lower liquid 86mL, add 5g anhydrous sodium sulfate dried
Night, filtration drying agent obtain faint yellow solid shape object 67mg, as testosterone on a rotary evaporator by after solvent volatilization completely
Succinate.
5mL is added dropwise to after 36mg testosterone succinate is dissolved with 3mL N,N-dimethylformamide (DMF) to contain
In the phosphate buffer (PBS, configuration method are shown in biochemistry textbook) of 66mgBSA, rotor is stirred to react 6h.It has reacted
Products obtained therefrom is fitted into 10mL plastic bottle by Cheng Hou, is placed on the turntable of heavy ion research device biological irradiation, according to different doses
Amount irradiated, by after irradiation product be packed into bag filter, first with distilled water dialyse 3d, then with PBS dialyse 3d.After, freeze
The dry testosterone immunogene (testo-BSA) for saving preparation.
The preparation route of Steroid antigen is as follows:
4. UV scanning identification prepares testosterone and BSA standard items with the PBS buffer solution containing 8% methanol.Then take 0.5mg's
Testo-BSA immunogene is dissolved in PBS, with the concentration and characteristic absorption peak of determined by ultraviolet spectrophotometry BSA, and adjusts testosterone-
The protein concentration of BSA immunogene is consistent with BSA standard solution, and ultraviolet full scan, root are carried out in 220-400nm wave-length coverage
According to the Variation Features of absorption peak judge coupling whether succeed, and calculate protein in conjunction with the molecule of testosterone than.
As shown in Figure 1, testo-BSA haptens has typical absorption at 285nm and 345nm, and BSA is in 275nm and 280nm
There is maximum absorption band at place, and the maximum absorption band of testo-BSA immunogene is then located at 274nm and 278nm, this illustrates testosterone-
BSA artificial antigen conjugate maximum absorption band moves to left, and has different characteristic ultraviolet absorptions from its precursor substance, tentatively shows idol
It is unified into function.According to the formula of textbook can calculate BSA in conjunction with the molecule of testosterone than.
5. the testo-BSA immunogene after the thorough dialysis of infrared spectroscopy identification；BSA；Testosterone haptens makees infrared spectroscopy inspection
It surveys.Sample 2mg is taken, dry KBr is added and is placed in agate mortar, is ground under infrared light irradiation；It mixes and is made thickness 1mm's
Transparent KBr sample wafer, upper machine testing.
As shown in Figure 2, BSA and testo-BSA immunogene have in the region of 2500-3200cm-1 and 1660-1500cm-1
There is similar absorption peak, this is the characteristic peak of amino acid in BSA, illustrates to contain BSA in the immunogene of synthesis.Testosterone standard items exist
There is a strong absworption peak at 3300cm-1, this is the characteristic peak of hydroxyl, and artificial antigen testo-BSA shows half without this absorption peak
The success of antigen derivatization.Meanwhile testo-BSA and testosterone standard items have similar absorption peak in 2800cm-1, and BSA is here
Without absorption peak, prove to contain testosterone in testo-BSA.Determine therefrom that testo-BSA artificial antigen is coupled successfully.
6. the determination of irradiation dose
Irradiation carries out in heavy ion research device (HIRFL) biological irradiation terminal.Primary power is the 12C6 of 20MeV/u
+ ion beam irradiation sample after the nickel window of beam current tube, ionisation chamber, air and drop energy piece, is monitored with air ionization chamber and is injected
Amount, sample replacement and data acquisition are controlled by computer, and all processes carry out under room temperature and Atmospheric Condition, irradiate agent
Amount is respectively 5,10,20,40,80Gy, dosage rate 20Gy/min.Irradiation results are shown in Table 1.
The percentage composition and yield that testo-BSA artificial antigen is coupled after table 1 irradiates
| Irradiation dose/Gy | Protein in conjunction with the molecule of testosterone than | Yield % |
| 5 | 1:16.5 | 62.11 |
| 10 | 1:19.5 | 64.77 |
| 20 | 1:26.5 | 65.25 |
| 40 | 1:30.5 | 68.44 |
| 80 | 1:28.5 | 65.16 |
7. conclusion and prospect
The present invention prepares the research of the preparation method of Steroid antigen using Ion Irradiation on Multi-walled Carbon, the results showed that 40Gy irradiation group
The yield and the combination equal highest of number of the Steroid antigen of preparation, BSA is in conjunction with the molecule of testosterone than being mostly 1:30.5, clinical data table
Bright twinning rate also highest obtains comparatively ideal as a result, a kind of method for creating preparation for preparing Steroid antigen, is bio-pharmaceutical
Development provide one kind and open one's minds.The interaction of 12C6+ ion beam and biological target is a sufficiently complex process,
It mainly include ionization, excitation, scattering process, absorption etc., main process is that charged particle is injected into biology
In target, charged particle in target material electronics and atom occur a series of collision, this process with ion energy
It gradually decreases and changes.When the energy of incident ion is higher, inelastic collision mainly occurs with drug molecule or atom, makes biology
Ionization or excitation occur for molecule, and biomolecule is caused to ionize and crosslink.The characteristic inspires the Ke Yixuan when preparing cross-linking agent
Use this method.Certainly it should be also made good use of in further work when particle energy reduces nuclear elastic collision to a certain extent
It hits and causes target atom displacement or the fracture of molecular link and recombination occurs and combines, screen the active component etc. of new biology, emphasis closes
Note nuclear stopping power when particle energy loses totally steeply rises, and energy loss reaches the peak value i.e. peak Bragg, in this area
The maximum probability of macromolecular is changed or is combined into target molecule.In short, heavy ion beam is energy to the effect of biomolecule
Amount transmitting, quality deposition, charging neutrality and the comprehensive functions such as exchange, the sequence and structure for eventually leading to molecule between biology change
Become.Steroid antigen is prepared by 12C6+ ion beam irradiation, combines its intermolecular chemical bond well, internal in sheep can also
With extraordinary absorption, induction of ovulation effect is given full play to.
Application of the testosterone Steroid antigen in sheep produces
The present invention is directed to improve the breeding rate of mutton sheep, the economic benefit of Fine wool Sheep is further played.Inventor with it is sweet
Respectful Yuzhong herding centre for spreading cooperation selected two Yang Chang in the Yuzhong Beishan Mountain in 2016, had carried out the comparison of testosterone Steroid antigen
Experiment, scientific evaluation effect of the testosterone Steroid antigen to sheep reproductive capacity, provides foundation to be widely applied.
One, test material and method
1. experimental cultivar
The Hybrid sheep of Small-fat-tail sheep ewe and purebred Poll Dorset ram.
2. testosterone Steroid antigen
Testosterone Steroid antigen selects the product in above-described embodiment, and the antigen of 2mg is matched in every 100mL distilled water, is usually set
It is saved backup in 4 DEG C of refrigerators.
3. testing sheep quantity and grouping
The Yuzhong Beishan Mountain has selected two sheep pilot to select healthy adult ewe totally 416.By age, parity and weight
Respectively it is divided into two groups Deng random, one group is processing group, injects testosterone Steroid antigen, another group is not injected testosterone Steroid antigen, is right
According to group.Test, the sheep compareed are only same a group, and it is identical that manage bar part is superintended and directed in raising.
4. testosterone Steroid antigen is injected
Processing group ewe is in first 7 weeks and 4 weeks every each injections testosterone Steroid antigen 2ml bred (neck is subcutaneous).
5. breeding
Artificial insemination is routinely carried out using fresh semen.
6. feeding management
The processing of 6.1 two pilots or control group ewe, respectively same a group, i.e., its herd, supplementary feeding, breeding and lambing etc.
Condition is identical, all to have ear tag and number for examination ewe, and accurately performs the work such as record registration.
6.2 weighing.All same day for injecting testosterone Steroid antigen in first time for examination ewe weigh primary on an empty stomach by only.
6.3 feed supplement.For trying all pregnancy ewes, started to mend in antenatal 6 weeks and feed the fine fodder (mixing such as corn, dregs of fat sum number skin
Material).No matter compare or processing group ewe, it is every 200 grams of feed supplement daily, until lambing terminates.
Two, experimental results
1. lambing situation
Two pilots for trying ewe successively normal lambing, terminate substantially to lambing.The result shows that testosterone steroidal is to raising
The breeding potential of meat ewe has positive effect.Such as ewe totally 200 of the processing of testosterone steroidal, 194 tire of lambing of being impregnated, common property lamb 274
Only, breeding potential 137.0%, processing group double lamb rate accounts for 41.24% (80/194).See Table 2 for details.
2 testosterone Steroid antigen comparative experiments lambing result of table (only, tire, %)
As can be seen from Table 2, the lambing percentage of processing group ewe is apparently higher than control group.That is testosterone Steroid antigen processing group ewe
It produces double lamb rate and improves 35.84% than control group 1；2 are improved 27.24%, and two o'clock averagely improves 31.74%.Prove testosterone steroid
Body antigen produces the reproductive capacity of meat type hybrid ewe to improving, and has fairly obvious effect.
2. lamb situation
2.1 from the survival rate of lamb: handling lamb obtained, the basic phase of survival rate and control group through Steroid antigen
Together, all survivals.
The litter weight at birth of 2.2 lambs: lamb litter weight at birth, between processing and control group, either single, double lamb, or male and female
The birth weight of sheep is without significant difference, the lamb birth weight of two pilots, and single lamb is greater than twin lamb(s), and public lamb is greater than ewe lamb, all meets
Universal law.Thus it proves, testosterone Steroid antigen has no adverse effects to the embryonic development of lamb.With regard to the nascent of two pilot lambs
Compare again, no matter the lamb of processing group or control group, 2 are slightly larger than 1 site, this may be related with the ewe age of two o'clock, 2
For the ewe of site up to 6 years old, the ewe of Nutrition and Metabolism power 1 site lighter not as good as the age causes development of fetus by certain
It influences.
Three, conclusions and discussion
1. the reproductive capacity that Steroid antigen can effectively improve meat type Hybrids sheep.As under the same conditions, production is substantially increased
Twinning rate.This method is simple and easy to do, and effect stability is reliable, without side-effects.
2. the polyembryony mechanism of Steroid antigen.Sheep is usually mostly single tire.Under field conditions (factors), promoting sexual gland hormone stimulation folliculus is sent out
It educates and ovulates, adjust estrogen secretion.When internal estrogen level reduces, then promoting sexual gland hormone content can be promoted to improve, made
2-3 ovarian follicle is reached maturity simultaneously, and ewe is made to produce twin lamb(s).And Steroid antigen is a kind of artificial synthesized to stay alcohol parahormone.To mother
Sheep injecting immune makes to form reproductive hormone antibody in body, thus reduces free levels of reproductive hormones, and stimulation promotees sexual gland and swashs
Element secretion accelerates the folliculus in sheep ovary mature, causes to ovulate simultaneously, so greatly improving fertilization and biparous possibility.By
After with twin lamb hormone immunity inoculation, make the levels of reproductive hormones naturally dissociated reduction, and then rush property is stimulated to promote hormone secretion, this
It is external conditions, inner region is the normal development of folliculus in sheep ovary.When flock of sheep malnutrition, the effect of said preparation is little, this
It may be why with same treatment method, two pilots produce the different bar reason for it of twin lamb(s) effect.It can be seen that
So that testosterone steroidal is played good result, ewe must have been supported first, interior gene basis has been accomplished fluently, can be only achieved expected purpose.
It is merely a preferred embodiment of the present invention, is not intended to restrict the invention, although referring to aforementioned implementation described in upper
Invention is explained in detail for example, for those skilled in the art, still can be to foregoing embodiments
Documented technical solution is modified or equivalent replacement of some of the technical features.It is all in spirit of the invention
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of preparation method of Steroid antigen, which is characterized in that described method includes following steps:
(1) testosterone is dissolved in anhydrous pyridine, succinic anhydride is added after dissolution, reaction obtains testosterone succinate；
(2) after the testosterone succinate that step (1) obtains being dissolved in n,N-Dimethylformamide, the phosphate-buffered of BSA is added
Liquid, reaction；
(3) reactant of step (2) is placed on the turntable of heavy ion research device biological irradiation, carries out Ion Irradiation on Multi-walled Carbon；
(4) by step (3) irradiation after product distilled water and PBS dialyse, be then lyophilized to get.
2. the preparation method of Steroid antigen according to claim 1, which is characterized in that in step (1), the testosterone amber
Acid esters specific the preparation method comprises the following steps:
It weighs testosterone 52mg and is dissolved in 3mL anhydrous pyridine, 90mg succinic anhydride is added after completely dissolution and is protected from light vibration under room temperature
It swings reaction for 24 hours, then with pyridine is dried with nitrogen, faint yellow grease-like compound is made；By smectic compound with 5wt%'s
100mL NaHCO3Solution dissolution, washed with ether, then with hydrochloric acid be adjusted to pH be 7.2 after, be layered after centrifugation, abandon supernatant, receive
Collect lower liquid 86mL, 5g anhydrous sodium sulfate is added to be dried overnight, filtration drying agent obtains faint yellow after solvent volatilization completely
Solid-like object 67mg, as testosterone succinate.
3. the preparation method of Steroid antigen according to claim 1, which is characterized in that in step (2), the testosterone amber
The weight ratio of acid esters and BSA are 6:11, and reaction carries out 6h under stiring.
4. the preparation method of Steroid antigen according to claim 1, which is characterized in that in step (3), irradiation dose difference
For 5-80Gy, dosage rate 20Gy/min.
5. the preparation method of Steroid antigen according to claim 1, which is characterized in that irradiation dose 40Gy.
6. the preparation method of Steroid antigen according to claim 1, which is characterized in that in step (4), when distilled water is dialysed
Between be 3d, PBS dialysis time be 3d.
7. the preparation method of Steroid antigen according to claim 1, which is characterized in that the Steroid antigen has following
Structural formula:
8. being used to improve the breeding potential of sheep according to the resulting Steroid antigen of claim 1-7 either method.
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|---|---|---|---|---|
| US4749567A (en) * | 1985-10-29 | 1988-06-07 | The Director General Of The Ministry Of Agriculture And Fisheries | Method and product for increasing fertility in sheep using milk protein conjugates |
| CN103251934A (en) * | 2013-05-30 | 2013-08-21 | 中国农业科学院兰州畜牧与兽药研究所 | Production process of sheep and goat steroid hormone antigen twins vaccine |
| CN104840424A (en) * | 2015-05-05 | 2015-08-19 | 中国农业科学院兰州畜牧与兽药研究所 | Hypericin albumin nanoparticle and escherichia coli serum antibody compound and preparation method and application thereof |
| CN106645760A (en) * | 2016-12-28 | 2017-05-10 | 河南科技学院 | Metandienone antigen, application thereof and test paper card |
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2019
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4749567A (en) * | 1985-10-29 | 1988-06-07 | The Director General Of The Ministry Of Agriculture And Fisheries | Method and product for increasing fertility in sheep using milk protein conjugates |
| CN103251934A (en) * | 2013-05-30 | 2013-08-21 | 中国农业科学院兰州畜牧与兽药研究所 | Production process of sheep and goat steroid hormone antigen twins vaccine |
| CN104840424A (en) * | 2015-05-05 | 2015-08-19 | 中国农业科学院兰州畜牧与兽药研究所 | Hypericin albumin nanoparticle and escherichia coli serum antibody compound and preparation method and application thereof |
| CN106645760A (en) * | 2016-12-28 | 2017-05-10 | 河南科技学院 | Metandienone antigen, application thereof and test paper card |
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