CN101921331A - Method for preparing thymosin - Google Patents
Method for preparing thymosin Download PDFInfo
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- CN101921331A CN101921331A CN 201010255435 CN201010255435A CN101921331A CN 101921331 A CN101921331 A CN 101921331A CN 201010255435 CN201010255435 CN 201010255435 CN 201010255435 A CN201010255435 A CN 201010255435A CN 101921331 A CN101921331 A CN 101921331A
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- zadaxin
- homogenate
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- thymosin
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Abstract
The invention relates to a method for preparing thymosin. The method comprises the following steps of: washing thymosin clean with a buffer solution with the pH of 3.5-4.0, then adding the buffer solution with the pH of 3.5-4.0 and homogenizing at low temperature to prepare a thymosin homogenate solution; carrying out thermotropy centrifugation on the obtained homogenate solution to remove protein impurities; then regulating the pH value of a supernatant to 4.5-5 with hydrochloric acid; centrifuging once again, and regulating the pH of the supernatant to be neutral; filtering the obtained neutral supernatant by using different filter membranes to remove impurities and bacteria; and freeze-drying to obtain a finished thymosin product. The method has the advantages of improving the activity and the yield of the thymosin and reducing the production cost.
Description
Technical field
The present invention relates to a kind of preparation method of Zadaxin, belong to the biologics preparing technical field.
Background technology
Zadaxin is one group of polypeptide that the thymic tissue excretory has physiologically active, has the effect of adjusting and enhancing human body cell immunologic function.Zadaxin is to adopt biochemical method to extract from animal thymus tissue.Publication number is that the Chinese patent of CN1721440A discloses a kind of method for preparing Zadaxin, the steps include: that (1) obtains homogenate with the high speed shear crusher machine with fresh thymic tissue down at 20 ℃～23 ℃; (2) regulate the pH value between 4.5～4.8 with dilute hydrochloric acid; (3) homogenate was placed 3～4 hours under 20 ℃～23 ℃ conditions; (4) in 60 ℃～65 ℃ water-baths, heat 30min, naturally cool to room temperature; (5) filter; (6) filtrate is that 10000 tubular fibre column purification, degerming obtain the Zadaxin product with molecular weight cut-off.
Above-mentioned preparation method does not add the activity that damping fluid comes the arrestin enzyme when high-speed homogenization, and proteolytic enzyme can decompose or destroy Zadaxin, and then reduces the yield and the activity of Zadaxin; Homogenized temperature is 20 ℃～23 ℃ in addition, also is difficult for keeping the activity of Zadaxin.
Summary of the invention
In order to overcome the weak point of above-mentioned technology, the object of the invention is to provide a kind of method for preparing Zadaxin.
In order to realize the object of the invention, technical program of the present invention lies in providing a kind of method for preparing Zadaxin, step is as follows:
1) gets fresh thymus gland, with the damping fluid washes clean of pH3～3.5;
2) add pH3.5～4.0 damping fluids, under 8 ℃～10 ℃ condition,, obtain homogenate with the homogenate of high speed homogenizer;
3) above-mentioned homogenate is placed on earlier under 0 ℃～4 ℃ conditions, and then is placed under-18 ℃～-25 ℃ conditions;
4) will freeze the back homogenate and in 70～80 ℃ water-bath, heat 10～15min;
5) rapidly above-mentioned homogenate is placed on cold condition down to freezing fully, takes out to put in the room temperature then and melt multigelation;
6) with the homogenate behind the above-mentioned multigelation, centrifugal, get supernatant liquor, regulate pH value 4.5～5 with dilute hydrochloric acid, recentrifuge is got supernatant, regulates the pH value to neutral;
7) above-mentioned neutral supernatant liquor is filtered through pulp board earlier, the filtrate that obtains is used the Hollow Fiber Ultrafiltration post ultrafiltration of molecular weight cut-off 100 000, and the filtrate that obtains is used molecular weight cut-off 10 000 ultra-filtration membrane ultrafiltration again, and the filtrate that obtains is mycoderm filtration after filtration at last;
8) filtrate of getting after the degerming is surveyed protein content, and freeze-drying obtains the Zadaxin finished product.
Being 36h～48h 0 ℃～4 ℃ following storage periods in the step 3), is 36h～48h-18 ℃～-25 ℃ following storage periods.
Low temperature described in the step 5) is-15 ℃～-20 ℃.
Centrifuging temperature described in the step 6) is 4 ℃.
The aperture of the bacteriological filtration film described in the step 7) is 0. 22 μ m.
Beneficial effect of the present invention: 1) adopt the damping fluid of pH3～3.5 to wash, suppressed the activity of enzyme effectively.
2) homogenate is placed 36h-48h then under-18 ℃～-25 ℃ conditions after placing 36h～48h under 0 ℃～4 ℃ conditions, causes the activity of Zadaxin to descend to prevent Zadaxin from entering low temperature suddenly.
3) adopt low-temperature homogenate and low-temperature centrifugation, help the activity of inhibitory enzyme on the one hand, keep the activity of Zadaxin on the other hand.
4) adopt low pH value to carry out homogenate, help the complete fragmentation of thymocyte.
5) with hydrochloric acid the pH value of homogenate being transferred to is 4.5～5, can make the foreign protein precipitation, and inhibitory enzyme improves the activity of Zadaxin to the destruction of Zadaxin.
6) adopt ultra-filtration membrane further to remove impurities and bacteria, the qualification rate of product improves, and the content of Zadaxin is improved.
Embodiment
Get the fresh calf thymus of 1kg, remove fat and reticular tissue, with the damping fluid washes clean of pH3～3.5, the damping fluid that adds pH3.5～4 of 1000 mL, under 8 ℃～10 ℃, use refiner with 10000 rpm/min speed homogenate, 10 min, up to becoming milky white liquid; The homogenate that obtains is placed 36h～48h earlier under 0 ℃～4 ℃ conditions, place 36h～48h then under-18 ℃～-25 ℃ conditions, and the homogenate that will freeze is put into 70～80 ℃ of water-baths, thermostatically heating 10 min～15min; Take out homogenate rapidly and be placed on-15 ℃～-20 ℃ conditions down to freezing, take out then to put room temperature thawing, multigelation 3 times fully; Under 4 ℃ of conditions, centrifugal 20 min of 7000rpm/min get supernatant liquor, and with dilute hydrochloric acid adjust pH to 4.5～5, under 4 ℃ of conditions, centrifugal 20 min of 7000rpm/min get supernatant liquor once more, regulate the pH value to neutral with 4 mol/ L NaOH; Getting neutral supernatant liquor filters through pulp board, filtrate is used the Hollow Fiber Ultrafiltration post ultrafiltration of molecular weight cut-off 100 000, the filtrate that obtains is passed through molecular weight cut-off 10 000 ultra-filtration membrane ultrafiltration again, the filtrate that obtains is 0. 22 μ m bacteriological filtration membrane filtrations through the aperture, the filtrate of getting after the degerming is surveyed protein content, freeze-drying obtains the Zadaxin finished product.Measuring Zadaxin content is 16.21mg/ml, and Thymosin alpha 1 content is 3.2%.External rosette experiment, diluted sample is to 0.002mg/ml, and activity increases by 37.5%.
Claims (5)
1. the preparation method of a Zadaxin, it is characterized in that: described preparation method may further comprise the steps:
1) gets fresh thymus gland, with the damping fluid washes clean of pH3～3.5;
2) add pH3.5～4.0 damping fluids, under 8 ℃～10 ℃ condition, use homogenizer homogenate, obtain homogenate;
3) above-mentioned homogenate is placed on earlier under 0 ℃～4 ℃ conditions, and then is placed under-18 ℃～-25 ℃ conditions;
4) will freeze the back homogenate and in 70～80 ℃ water-bath, heat 10 min～15min;
5) rapidly above-mentioned homogenate is placed on cold condition down to freezing fully, takes out to put in the room temperature then and melt multigelation;
6) with the homogenate behind the above-mentioned multigelation, centrifugal, get supernatant liquor, regulate pH value 4.5～5 with dilute hydrochloric acid, recentrifuge is got supernatant, regulates the pH value to neutral;
7) above-mentioned neutral supernatant liquor is filtered through pulp board earlier, the filtrate that obtains is used the Hollow Fiber Ultrafiltration post ultrafiltration of molecular weight cut-off 100 000, and the filtrate that obtains is passed through molecular weight cut-off 10 000 ultra-filtration membrane ultrafiltration again, and the filtrate that obtains is mycoderm filtration after filtration at last;
8) filtrate of getting after the degerming is surveyed protein content, and freeze-drying obtains the Zadaxin finished product.
2. the preparation method of Zadaxin according to claim 1 is characterized in that: being 36h～48h 0 ℃～4 ℃ following storage periods in the step 3), is 36h～48h-18 ℃～-25 ℃ following storage periods.
3. the preparation method of Zadaxin according to claim 1, it is characterized in that: the low temperature described in the step 5) is-15 ℃～-20 ℃.
4. the preparation method of Zadaxin according to claim 1, it is characterized in that: the centrifuging temperature described in the step 6) is 4 ℃.
5. the preparation method of Zadaxin according to claim 1, it is characterized in that: the aperture of the bacteriological filtration film described in the step 7) is 0. 22 μ m.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010255435 CN101921331A (en) | 2010-08-17 | 2010-08-17 | Method for preparing thymosin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010255435 CN101921331A (en) | 2010-08-17 | 2010-08-17 | Method for preparing thymosin |
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| CN101921331A true CN101921331A (en) | 2010-12-22 |
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| CN 201010255435 Pending CN101921331A (en) | 2010-08-17 | 2010-08-17 | Method for preparing thymosin |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103172725A (en) * | 2013-04-02 | 2013-06-26 | 黑龙江迪龙制药有限公司 | Preparation method of thymosin rich in thymosin alpha 1 |
| CN105541996A (en) * | 2015-12-31 | 2016-05-04 | 天津泰创生物科技有限公司 | Method for extracting thymosin, medium molecular thymus protein and DNA from thymus tissue |
| CN105777891A (en) * | 2014-12-16 | 2016-07-20 | 康普药业股份有限公司 | Method for improving extraction efficiency of thymosin |
| CN112143768A (en) * | 2020-09-29 | 2020-12-29 | 北京赛升药业股份有限公司 | Method for jointly preparing DNA and thymosin by using calf thymus |
| CN112450316A (en) * | 2020-12-02 | 2021-03-09 | 通化承诚药业有限公司 | Preparation method of calf thymus peptide powder |
| CN114805541A (en) * | 2022-05-27 | 2022-07-29 | 福建农业职业技术学院 | Preparation method of pig thymosin |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1390853A (en) * | 2002-05-16 | 2003-01-15 | 西安迪赛生物药业有限责任公司 | Process for preparing thymic peptide |
| CN1712064A (en) * | 2004-06-23 | 2005-12-28 | 北京赛生药业有限公司 | Production of high-concentration thymic peptide solution and large-specification thymic peptide preparation |
| CN1721440A (en) * | 2004-07-12 | 2006-01-18 | 张宜俊 | Process for preparing thymosin |
-
2010
- 2010-08-17 CN CN 201010255435 patent/CN101921331A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1390853A (en) * | 2002-05-16 | 2003-01-15 | 西安迪赛生物药业有限责任公司 | Process for preparing thymic peptide |
| CN1712064A (en) * | 2004-06-23 | 2005-12-28 | 北京赛生药业有限公司 | Production of high-concentration thymic peptide solution and large-specification thymic peptide preparation |
| CN1721440A (en) * | 2004-07-12 | 2006-01-18 | 张宜俊 | Process for preparing thymosin |
Non-Patent Citations (2)
| Title |
|---|
| 《中国生化药物杂志》 尹德瑛等 不同动物来源胸腺肽的制备及生化性质的研究 , 第02期 * |
| 《黑龙江医药》 20061231 高思英 胸腺肽的提取与性质研究 443-445 1-5 第19卷, 第6期 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103172725A (en) * | 2013-04-02 | 2013-06-26 | 黑龙江迪龙制药有限公司 | Preparation method of thymosin rich in thymosin alpha 1 |
| CN105777891A (en) * | 2014-12-16 | 2016-07-20 | 康普药业股份有限公司 | Method for improving extraction efficiency of thymosin |
| CN105541996A (en) * | 2015-12-31 | 2016-05-04 | 天津泰创生物科技有限公司 | Method for extracting thymosin, medium molecular thymus protein and DNA from thymus tissue |
| CN112143768A (en) * | 2020-09-29 | 2020-12-29 | 北京赛升药业股份有限公司 | Method for jointly preparing DNA and thymosin by using calf thymus |
| CN112450316A (en) * | 2020-12-02 | 2021-03-09 | 通化承诚药业有限公司 | Preparation method of calf thymus peptide powder |
| CN114805541A (en) * | 2022-05-27 | 2022-07-29 | 福建农业职业技术学院 | Preparation method of pig thymosin |
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Application publication date: 20101222 |
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