CN100512820C - Technique for producing serum gonadotrophin for animal use, and products - Google Patents
Technique for producing serum gonadotrophin for animal use, and products Download PDFInfo
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- CN100512820C CN100512820C CNB2006100167857A CN200610016785A CN100512820C CN 100512820 C CN100512820 C CN 100512820C CN B2006100167857 A CNB2006100167857 A CN B2006100167857A CN 200610016785 A CN200610016785 A CN 200610016785A CN 100512820 C CN100512820 C CN 100512820C
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Abstract
A veterinary dematogonadotropin is prepared from the blood serum of pregnant mare through diluting with distilled water, adding solid ammonium sulfate while stirring, regulating pH value, filtering, dripping metaphosphoric acid to regulate pH value, filtering, ultrafiltrate concentrating, slowly adding pre-cooled alcohol to make 40% of alcohol to deposit, regulating pH value, laying aside for layering, taking supernatant, adding pre-cooled alcohol, regulating pH value, laying aside for layering, removing supernatant, centrifugal separation to obtain deposit, dissolving in physiologic saline, dialyzing, depositing in alcohol, and vacuum drying of the deposit.
Description
Technical field
The invention provides a kind of technique for producing serum gonadotrophin for animal use and products thereof, relate to the improvement of technique for producing serum gonadotrophin for animal use, belong to biological preparation production method technical field for animals.
Background technology
A kind of material with promotion animality function of serum gonadotrophin for animal use for from pregnant mare serum, extracting, this material is found in the pregnant mare serum in nineteen thirty by Cole and Hart the earliest, because of it has the gonad development of promotion function, so called after Pregnant Mare SerumGonadotrophin PMSG Chinese translated name is a pregnant mare serum gonadotrop(h)in (PMSG).Serum gonadotrophin for animal use is the name of Chinese veterinary drug allusion quotation to this material, and its chemical nature is a glycoprotein, is made up of α subunit and β subunit that non-covalent bond links to each other, has 255 aminoacid, and wherein the α subunit contains 96 aminoacid, and the β subunit contains 149 aminoacid.The α subunit sequence of the α subunit of PMSG and horse LH, FSH and TSH is identical, but different with the α subunit sequence of other mammal LH, FSH and TSH.The β subunit of PMSG has exclusive specificity.PMSG is that sugar content is the highest in all mammiferous glycoprotein hormoneses, and its sugar content accounts for 41.7% of whole molecular weight, is mainly galactose, N-acetyl glucosamine and sialic acid.The isoelectric point, IP of PMSG is 2.60-2.65, and molecular weight is 64,030 dalton.The serum gonadotrophin goods are by operations such as the separation of pregnant horse blood, purification, lyophilisation are produced, and China Ministry of Agriculture ministry standard is that goods purity for animals reaches 100IU/mg and gets final product.
At present, the main technique of production serum gonadotrophin is Metaphosphoric acid-ethanol step-by-step precipitation method.The main deficiency of this method is, can obtain higher yields in the medium and small sample extraction of laboratory, but be amplified to 10,000 ml serum/batch the time, yield obviously reduces, usually less than 30%, analyzing reason, may be that one step of Metaphosphoric acid precipitation is bigger because of sample size, the reaction of Metaphosphoric acid and sample is inhomogeneous, and active substance is lost in a large number.
Summary of the invention
Problems such as the present invention discloses a kind of technique for producing serum gonadotrophin for animal use, and the yield that the existing technique for producing serum gonadotrophin for animal use of solution exists is lower.
Technical solution of the present invention may further comprise the steps: get pregnant mare serum (on average tiring more than the 100IU/ml), use the equivalent distilled water diluting, add solid ammonium sulfate while stirring to final concentration 32～36%, transfer PH to 6.0～7.0 with strong aqua ammonia, after fully stirring, filter cloth filters, and clear liquid slowly drips 0.5～1N Metaphosphoric acid while stirring and transfers PH to 3.8～4.6, after fully stirring, filter cloth filters.Clear liquid concentrates (molecular cut off 20KU) through hollow fiber membrane ultrafiltration device, makes volume be concentrated into about 40～50% of original volume, behind this concentrated solution weighing volume, slowly add pre-cooling ethanol, make 40% ethanol precipitation, transfer PH to 6.2～6.8 with strong aqua ammonia, after the static layering, siphon supernatant, weighing volume.Add the pre-cooling medical alcohol to final concentration 80%, transfer PH to 3.6～4.4 with glacial acetic acid, after the static layering, siphonage is inhaled and is abandoned supernatant, the muddy liquid centrifuging and taking precipitation of rest parts.With molten of an amount of normal saline, dialysis is 24 hours in the bag filter of packing into this precipitation, and 80% alcohol precipitation, precipitate are put in the exsiccator negative pressure ventilation and weighed to drying, get a little and carry out biological activity assay, calculated yield and purity.
The present invention compared with prior art has following good effect:
Improved the serum gonadotrophin for animal use yield.Existing Metaphosphoric acid one ethanol step-by-step precipitation method is suitable for the laboratory small lot and extracts, and when being amplified to production scale (more than the 10000ml), then yield generally has only about 30%.The yield of this production technology is about 70%, is higher than present technology more than 2 times.Keep purity on the veterinary drug standard.The product that this technology obtains, purity is 100 ~ 300IU/mg, can satisfy veterinary drug and produce needs.Save ethanol.Product in the middle of this process using Hollow Fiber Ultrafiltration technology concentrates have reduced 60% before making the ethanol amount ratio not use concentration technique, and ethanol is reagent the most valuable in this technology, so has significantly reduced cost.Reduce the use of centrifuge.This process makes full use natural subsidence or filter cloth Filtration separate clear liquid and precipitate, have at utmost reduced the use amount of centrifuge.Centrifuge is an instrument and equipment the most expensive in this technology, therefore adopts this technology cost-saved.
The specific embodiment:
By following examples the present invention is described for example further, and do not limit the present invention in any way, under the prerequisite that does not deviate from technical solution of the present invention, any change or change that those of ordinary skills that the present invention did are realized easily all will fall within the claim scope of the present invention.
Embodiment 1
The 051010 batch of leaching process and result:
Get pregnant mare serum 10000ml adding distil water 10000ml and add solid ammonium sulfate (analytical pure) 600 grams while stirring, behind strong aqua ammonia accent PH to 6.0, filter cloth filters, and adds freshly prepared 1N Metaphosphoric acid in the filtrate, transfers PH to 4.2; Filter cloth filters, collect filtrate, hollow fiber membrane ultrafiltration device (molecular cut off 20KU) ultrafiltration and concentration adds pre-cooling ethanol to final concentration 40% to volume 5000ml, and strong aqua ammonia is transferred PH to 6.2, siphonage sucking-off supernatant, add pre-cooling ethanol to final concentration 80%, glacial acetic acid is transferred PH to 4.0, and siphonage is inhaled and abandoned supernatant, to precipitate with molten of normal saline, dialysis adds pre-cooling ethanol to final concentration 80%, after the static layering, siphonage is inhaled and is abandoned supernatant, precipitation is transferred in the glass dish, placed glass evacuated exsiccator, evacuation drying in the low temperature chamber, weigh, get 10mg and survey biologic activity (rat ovary weightening finish 3+3+3 method).
The result: this mass products property yield is 71.6%, and purity is 268IU/mg.
Embodiment 2
The 051208 batch of leaching process and result:
Get pregnant mare serum 20000ml adding distil water 20000ml, add solid ammonium sulfate (analytical pure) 1200 grams while stirring, note during interpolation big granule is pulverized, slowly add by spoon with little soupspoon, transfer PH to 7.0 with strong aqua ammonia, filter cloth filters, and adds the 1N Metaphosphoric acid, with acidometer monitoring PH to 4.6, filter cloth filters, and hollow fiber membrane ultrafiltration device (molecular cut off 20KU) ultrafiltration and concentration adds pre-cooling ethanol to final concentration 40% to volume 13000ml, strong aqua ammonia is transferred PH to 6.8, siphonage sucking-off supernatant adds pre-cooling ethanol to final concentration 80%, and glacial acetic acid is transferred PH to 4.4, siphonage is inhaled and is abandoned supernatant (reclaiming as waste alcohol), precipitation is dialysed with molten of normal saline, adds pre-cooling ethanol to final concentration 80%, siphonage is inhaled and is abandoned supernatant (reclaiming as waste alcohol), precipitation is transferred in the glass dish, placed glass evacuated exsiccator, evacuation drying in the low temperature chamber, weigh, get 10mg and survey biologic activity (rat ovary weightening finish 3+3+3 method).
The result: this mass products property yield is 69.4%, and purity is 215IU/mg.
Embodiment 3
The 051220 batch of leaching process and result:
Get pregnant mare serum 20000ml adding distil water 20000ml, add solid ammonium sulfate (analytical pure) 1200 grams while stirring, transfer PH to 6.4 with strong aqua ammonia, filter cloth filters, add 1N Metaphosphoric acid (analytical pure), with acidometer monitoring PH to 4.0, filter cloth filters, and collects filtrate, hollow fiber membrane ultrafiltration device (molecular cut off 20KU) ultrafiltration and concentration is to volume 13000ml, add pre-cooling ethanol to final concentration 40%, strong aqua ammonia is transferred PH to 6.2, siphonage sucking-off supernatant, add pre-cooling ethanol, glacial acetic acid is transferred PH to 3.6, and siphonage is inhaled and abandoned supernatant (reclaiming as waste alcohol), molten of normal saline of precipitation, dialysis, the limit adds pre-cooling ethanol to final concentration 80%, and siphonage is inhaled and abandoned supernatant (reclaiming as waste alcohol), and precipitation is transferred in the glass dish, place glass evacuated exsiccator, the evacuation drying is weighed in the low temperature chamber, gets 10mg and surveys biologic activity (rat ovary weightening finish 3+3+3 method).
The result: this mass products property yield is 69.8%, and purity is 220IU/mg.
Claims (1)
1, a kind of technique for producing serum gonadotrophin for animal use, may further comprise the steps: get pregnant mare serum equivalent distilled water diluting, add solid ammonium sulfate while stirring to final concentration 32～36%, transfer PH to 6.0～7.0 with strong aqua ammonia, after fully stirring, filter cloth filters, and clear liquid slowly drips 0.5～1N Metaphosphoric acid while stirring and transfers PH to 3.8～4.6, after fully stirring, filter cloth filters; Clear liquid concentrates through hollow fiber membrane ultrafiltration device, and molecular cut off is 20KU, makes volume be concentrated into 40～50% of original volume, behind this concentrated solution weighing volume, slowly add pre-cooling ethanol, make 40% ethanol precipitation, transfer PH to 6.2～6.8 with strong aqua ammonia, after the static layering, siphon supernatant, weighing volume; Add the pre-cooling medical alcohol to final concentration 80%, transfer PH to 3.6～4.4 with glacial acetic acid, after the static layering, siphonage is inhaled and is abandoned supernatant, the muddy liquid centrifuging and taking precipitation of rest parts; This precipitation with molten of an amount of normal saline, was dialysed 24 hours in the bag filter of packing into, and 80% alcohol precipitation, precipitate are put the extremely dry product that gets of negative pressure ventilation in the exsiccator.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100167857A CN100512820C (en) | 2006-04-21 | 2006-04-21 | Technique for producing serum gonadotrophin for animal use, and products |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100167857A CN100512820C (en) | 2006-04-21 | 2006-04-21 | Technique for producing serum gonadotrophin for animal use, and products |
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| Publication Number | Publication Date |
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| CN1872088A CN1872088A (en) | 2006-12-06 |
| CN100512820C true CN100512820C (en) | 2009-07-15 |
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| CNB2006100167857A Expired - Fee Related CN100512820C (en) | 2006-04-21 | 2006-04-21 | Technique for producing serum gonadotrophin for animal use, and products |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109293735A (en) * | 2018-09-07 | 2019-02-01 | 宁波三生生物科技有限公司 | A kind of pregnant mare serum crude product extract equipment and its extracting method |
| CN111072765A (en) * | 2019-11-22 | 2020-04-28 | 甘肃天祁生物科技有限公司 | Solid-liquid separation method of ethanol/protein suspension in pregnant mare serum gonadotropin crude product extraction process |
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2006
- 2006-04-21 CN CNB2006100167857A patent/CN100512820C/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 孕马血清促性腺激素的提纯研究. 丁若愚，崔青山，陈萸芳，甄英凯，李惟，罗贵民.中国兽医学报，第10卷第2期. 1990 |
| 孕马血清促性腺激素的提纯研究. 丁若愚，崔青山，陈萸芳，甄英凯，李惟，罗贵民.中国兽医学报，第10卷第2期. 1990 * |
| 提纯孕马血清促性腺激素的中试生产研究. 杨兴辉，王秀军，柴希文，尤建东，崔青山，董仁培.中国生化药物杂志，第18卷第1期. 1997 |
| 提纯孕马血清促性腺激素的中试生产研究. 杨兴辉，王秀军，柴希文，尤建东，崔青山，董仁培.中国生化药物杂志，第18卷第1期. 1997 * |
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