CN105647848A - Separation and preparation method of deer serum - Google Patents
Separation and preparation method of deer serum Download PDFInfo
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- CN105647848A CN105647848A CN201610103829.3A CN201610103829A CN105647848A CN 105647848 A CN105647848 A CN 105647848A CN 201610103829 A CN201610103829 A CN 201610103829A CN 105647848 A CN105647848 A CN 105647848A
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Abstract
The invention discloses a separation and preparation method of deer serum. According to the method, the deer whole blood obtained after sterile blood sampling from a cleaned and disinfected to-be-sampled deer is left to stand and subjected to centrifugal separation, fibrous protein is removed, then the whole blood is inactivated, the concentration of IgG and the content of glucose are reduced, obtained serum is irradiated with a certain dosage of gamma-rays, viruses and mycoplasmas are removed, the serum is irradiated with ultraviolet rays for sterilization while being filtered by an asbestos-free Chua's filter disc, deer serum filtrate is obtained, finally, the deer serum filtrate is filtered with a filtering membrane, and a finished product, the deer serum, is obtained. The preparation method is simple, convenient, quick, stable in process and suitable for large-scale production; the obtained deer serum does not contain fibrous protein, the concentration of IgG and the content of glucose are low, pollution of the viruses, germs and mycoplasmas can be completely eliminated, and the prepared deer serum can be used for preparation of a culture medium containing serum cells.
Description
Technical field
The present invention relates to the production technology that a kind of Sanguis cervi is clear, particularly relate to the method for separating and preparing that a kind of Sanguis cervi is clear.
Background technology
Current cell culture process and method are widely used to the fields such as medicine, medical treatment, biological engineering, genetic engineering, production of vaccine, and wherein cell culture fluid becomes, in the production and research of biological technology products, the biomaterial that can not be substituted. The cell culture fluid circulated on Present Domestic market, is mainly the cell culture fluid containing calf serum or hyclone and the cell culture fluid of some serum-frees. The Ox blood serum overwhelming majority wherein used is from states such as Australia, New Zealand, and domestic derived therefrom is concentrated mainly on bottom, market, and raw material comes from mostly butchers Sanguis Bovis seu Bubali.
After there is bovine spongiform encephalopathy event in the world in recent years, cell culture fluid industry is defined structural impact and impact root, if especially detecting bovine spongiform encephalopathy poison in cell culture fluid can produce extremely serious consequence. At medicine, medical treatment, biomaterial on Vehicles Collected from Market, the field such as genetic engineering, production of vaccine, the various cell culture mediums based on the serum of cattle source used are in the face of crazy Sanguis Bovis seu Bubali is it may happen that popular risk is objective, it is therefore desirable to safer cell culture fluid substitutes cattle source serum cell culture fluid. But owing to the blood serum medium cultivated for precious cell is mostly from external import, price comparison is expensive, causes bigger cost burden to use unit. Therefore, under above-mentioned background, a kind of direct substitution calf serum how is provided, the animal serum of hyclone becomes a big important topic of current cell culture fluid technical field.
Summary of the invention
In order to overcome produced problem in above-mentioned prior art, the invention provides the method for separating and preparing that a kind of Sanguis cervi is clear, this preparation method is simple and efficient, process stabilizing and be suitable to produce in enormous quantities, and gained Sanguis cervi can be used in clearly the preparation containing serum cell culture fluid.
The present invention is to solve that its technical problem be the technical scheme is that
The method for separating and preparing that a kind of Sanguis cervi is clear, comprises the steps:
(1) blood sampling: disinfectant solution being adopted to carry out after deer body whole body cleaning-sterilizing processes until blood sampling deer, sterile blood sampling method takes deer whole blood to centrifuge bottle, centrifuge bottle is placed at 4��5 DEG C constant temperature standing 5��6h;
(2) separate: will through the deer whole blood after (1) processes is at 4��5 DEG C when, adopt the centrifugal 40��50min of rotating speed of 2000��3000rpm, from centrifuge bottle, serum is extracted under vacuum conditions after centrifugal end, it is placed at-25��-30 DEG C by the serum of this extraction gained to save backup, obtains the serum of freezing;
(3) fibrin is removed: be placed in 4��5 DEG C of environment by the freezing serum processing gained through (2) and melt, filtered by multilamellar sterile gauze by gained serum after thawing, collect filtrate;
(4) inactivation: gained filtrate will be processed through (3) and be placed at 56��60 DEG C water bath with thermostatic control 30��40min, and rock filtrate in water-bath process at any time;
(5) IgG concentration and glucose content are reduced: adopt chromatography that IgG concentration decreases below 5 �� g/ml the filtrate after (4) process; Then the dialyzer that molecular weight of albumen is 12000��14000 of can dialysing is adopted to dialyse to glucose content less than 0.5mg/ml in the NaCl aqueous solution of 0.15��0.18mol/L;
(6) virus and mycoplasma and aseptic filtration are removed: the gamma-rays adopting dosage to be 30��45KGy the serum after (5) process irradiates; Then being loaded by above-mentioned serum bleeds in slurry steriliser continuously, and irradiate this slurry steriliser of bleeding continuously equipped with serum with ultraviolet with 45��50 ��, irradiate and serum is obtained filtrate by EK level without asbestos Cai Shi filter disc simultaneously, then again this filtrate is filtered by 0.22��0.25 ��m of filter membrane, filter through 100��150nm filter membrane after, obtain Sanguis cervi clear.
Its further technical scheme is:
Step (1) is treated blood sampling deer be that health is healthy, the mental status good, without the one in the adult deer of epidemic situation history, fawn and tire deer.
Step (1) adopts 2wt.% iodine tincture and 75vol.% ethanol treat blood sampling deer successively as disinfectant solution and carry out deer body whole body cleaning-sterilizing.
Step (3) adopt eight layers of sterile gauze be filtered melting gained serum.
Filtrate is placed in water bath with thermostatic control 30min at 56 DEG C by step (4).
The flow velocity starching steriliser of bleeding continuously described in step (6) is 250-270mL/min.
The invention also discloses the Sanguis cervi that a kind of above-mentioned method for separating and preparing prepares gained clear.
The invention also discloses and prepared the Sanguis cervi of gained clearly containing the application in serum cell culture medium by above-mentioned preparation method.
The method have the benefit that: preparation method of the present invention is simple and efficient, process stabilizing and be suitable to produce in enormous quantities, and gained Sanguis cervi is clearly without fibrin, IgG concentration and glucose content are low, can the pollution of thoroughly preventing virus, pathogenic bacteria and mycoplasma, the Sanguis cervi preparing gained can be used in clearly the preparation containing serum cell culture fluid.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in further detail. Following specific embodiment is selected the newborn Cervus nippon Temminck do not taken food, and check deer body archives: include maternal age, body weight, health status, mental status, the nearly 1 year vaccine injection situation with or without epidemic situation, nearly 1 year, and foregoing is registered into the blood archives treating blood sampling deer.
Specific embodiment one
(1) blood sampling: by above-mentioned choose treat that deer body is carried out whole body cleaning-sterilizing process by the blood sampling deer iodine tincture that adopts concentration to be 2wt.% successively and ethanol that concentration is 75vol.%, after process terminates, use aseptic blood taking needle to carry out collecting whole blood from the vein of deer body after cleaning-sterilizing, and the whole blood collected left in centrifuge bottle and at 4 DEG C, stand 6h;
(2) separate: will through the deer whole blood after (1) processes is at 4 DEG C when, adopt the centrifugal 50min of rotating speed of 2000rpm, from centrifuge bottle, serum is extracted under vacuum conditions after centrifugal end, save backup at the serum of this extraction gained is placed in-25 DEG C, obtain the serum of freezing;
(3) fibrin is removed: being placed in the refrigerator of 4 DEG C by the freezing serum processing gained through (2) and melt, after melting, gained serum is filtered by eight layers of sterile gauze and removes fibrin, collects filtrate;
(4) inactivation: will be placed at 56 DEG C through (3) process gained filtrate and strictly control water bath with thermostatic control 40min under water temperature condition, rock filtrate in water-bath process at any time and keep its filtrate uniform, it is to avoid filtrate produces precipitation;
(5) IgG concentration and glucose content are reduced: adopt current conventional use of chromatography that IgG concentration decreases below 5 �� g/ml the filtrate after (4) process; Then the dialyzer that molecular weight of albumen is 12000 of can dialysing is adopted to dialyse to glucose content less than 0.5mg/ml in the NaCl aqueous solution of 0.15mol/L;
(6) virus and mycoplasma and aseptic filtration are removed: the gamma-rays adopting dosage to be 30KGy the serum after (5) process irradiates; Then above-mentioned serum loaded in slurry steriliser of bleeding continuously and adopt ultraviolet radiation with 45�� angle degree, the flow velocity starching steriliser of wherein bleeding continuously is 250mL/min, irradiate and serum is obtained filtrate by EK level without asbestos Cai Shi filter disc simultaneously, after this filtrate is filtered by 0.22 ��m of filter membrane, filter then through 100nm filter membrane, obtain deer blood serum sample 1. The above-mentioned Sanguis cervi preparing gained is saved in clearly freezen protective in the refrigerator of-20 DEG C.
Specific embodiment two
(1) blood sampling: by above-mentioned choose treat that deer body is carried out whole body cleaning-sterilizing process by the blood sampling deer iodine tincture that adopts concentration to be 2wt.% successively and ethanol that concentration is 75vol.%, after process terminates, use aseptic blood taking needle to carry out collecting whole blood from the vein of deer body after cleaning-sterilizing, and the whole blood collected left in centrifuge bottle and at 5 DEG C, stand 5h;
(2) separate: will through the deer whole blood after (1) processes is at 5 DEG C when, adopt the centrifugal 40min of rotating speed of 3000rpm, from centrifuge bottle, serum is extracted under vacuum conditions after centrifugal end, save backup at the serum of this extraction gained is placed in-30 DEG C, obtain the serum of freezing;
(3) fibrin is removed: being placed in the refrigerator of 5 DEG C by the freezing serum processing gained through (2) and melt, after melting, gained serum is filtered by eight layers of sterile gauze and removes fibrin, collects filtrate;
(4) inactivation: will be placed at 60 DEG C through (3) process gained filtrate and strictly control water bath with thermostatic control 30min under water temperature condition, rock filtrate in water-bath process at any time and keep its filtrate uniform, it is to avoid filtrate produces precipitation;
(5) IgG concentration and glucose content are reduced: adopt current conventional use of chromatography that IgG concentration decreases below 5 �� g/ml the filtrate after (4) process; Then the dialyzer that molecular weight of albumen is 14000 of can dialysing is adopted to be in that in the NaCl aqueous solution of 0.18mol/L to dialyse to glucose content less than 0.5mg/ml;
(6) virus and mycoplasma and aseptic filtration are removed: the gamma-rays adopting dosage to be 45KGy the serum after (5) process irradiates; Then above-mentioned serum loaded in slurry steriliser of bleeding continuously and adopt ultraviolet radiation with 50 �� of angles, the flow velocity starching steriliser of wherein bleeding continuously is 270mL/min, irradiate and serum is obtained filtrate by EK level without asbestos Cai Shi filter disc simultaneously, after this filtrate is filtered by 0.25 ��m of filter membrane, filter then through 150nm filter membrane, obtain deer blood serum sample 2.The above-mentioned Sanguis cervi preparing gained is saved in clearly freezen protective in the refrigerator of-20 DEG C.
Specific embodiment three
(1) blood sampling: by above-mentioned choose treat that deer body is carried out whole body cleaning-sterilizing process by the blood sampling deer iodine tincture that adopts concentration to be 2wt.% successively and ethanol that concentration is 75vol.%, after process terminates, use aseptic blood taking needle to carry out collecting whole blood from the vein of deer body after cleaning-sterilizing, and the whole blood collected left in centrifuge bottle and at 4 DEG C, stand 5h;
(2) separate: will through the deer whole blood after (1) processes is at 4 DEG C when, adopt the centrifugal 45min of rotating speed of 2500rpm, from centrifuge bottle, serum is extracted under vacuum conditions after centrifugal end, save backup at the serum of this extraction gained is placed in-25 DEG C, obtain the serum of freezing;
(3) fibrin is removed: being placed in the refrigerator of 4 DEG C by the freezing serum processing gained through (2) and melt, after melting, gained serum is filtered by eight layers of sterile gauze and removes fibrin, collects filtrate;
(4) inactivation: will be placed at 56 DEG C through (3) process gained filtrate and strictly control water bath with thermostatic control 35min under water temperature condition, rock filtrate in water-bath process at any time and keep its filtrate uniform, it is to avoid filtrate produces precipitation;
(5) IgG concentration and glucose content are reduced: adopt current conventional use of chromatography that IgG concentration decreases below 5 �� g/ml the filtrate after (4) process; Then the dialyzer that molecular weight of albumen is 13000 of can dialysing is adopted to be in that in the NaCl aqueous solution of 0.17mol/L to dialyse to glucose content less than 0.5mg/ml;
(6) virus and mycoplasma and aseptic filtration are removed: the gamma-rays adopting dosage to be 40KGy the serum after (5) process irradiates; Then above-mentioned serum loaded in slurry steriliser of bleeding continuously and adopt ultraviolet radiation with 48 �� of angles, the flow velocity starching steriliser of wherein bleeding continuously is 260mL/min, irradiate and serum is obtained filtrate by EK level without asbestos Cai Shi filter disc simultaneously, after this filtrate is filtered by 0.22 ��m of filter membrane, filter then through 120nm filter membrane, obtain deer blood serum sample 3. The above-mentioned Sanguis cervi preparing gained is saved in clearly freezen protective in the refrigerator of-20 DEG C.
Specific embodiment four
(1) blood sampling: by above-mentioned choose treat that deer body is carried out whole body cleaning-sterilizing process by the blood sampling deer iodine tincture that adopts concentration to be 2wt.% successively and ethanol that concentration is 75vol.%, after process terminates, use aseptic blood taking needle to carry out collecting whole blood from the vein of deer body after cleaning-sterilizing, and the whole blood collected left in centrifuge bottle and at 4 DEG C, stand 6h;
(2) separate: will through the deer whole blood after (1) processes is at 4 DEG C when, adopt the centrifugal 45min of rotating speed of 2000rpm, from centrifuge bottle, serum is extracted under vacuum conditions after centrifugal end, save backup at the serum of this extraction gained is placed in-28 DEG C, obtain the serum of freezing;
(3) fibrin is removed: being placed in the refrigerator of 5 DEG C by the freezing serum processing gained through (2) and melt, after melting, gained serum is filtered by eight layers of sterile gauze and removes fibrin, collects filtrate;
(4) inactivation: will be placed at 56 DEG C through (3) process gained filtrate and strictly control water bath with thermostatic control 30min under water temperature condition, rock filtrate in water-bath process at any time and keep its filtrate uniform, it is to avoid filtrate produces precipitation;
(5) IgG concentration and glucose content are reduced: adopt current conventional use of chromatography that IgG concentration decreases below 5 �� g/ml the filtrate after (4) process;Then the dialyzer that molecular weight of albumen is 13000 of can dialysing is adopted to be in that in the NaCl aqueous solution of 0.16mol/L to dialyse to glucose content less than 0.5mg/ml;
(6) virus and mycoplasma and aseptic filtration are removed: the gamma-rays adopting dosage to be 40KGy the serum after (5) process irradiates; Then above-mentioned serum loaded in slurry steriliser of bleeding continuously and adopt ultraviolet radiation with 45�� angle degree, the flow velocity starching steriliser of wherein bleeding continuously is 270mL/min, irradiate and serum is obtained filtrate by EK level without asbestos Cai Shi filter disc simultaneously, after this filtrate is filtered by 0.22 ��m of filter membrane, filter then through 150nm filter membrane, obtain deer blood serum sample 4. The above-mentioned Sanguis cervi preparing gained is saved in clearly freezen protective in the refrigerator of-20 DEG C.
Above-mentioned deer blood serum sample 1 to the sample 4 collected is done osmotic pressure detection, pH value detection, protein content detection, antibacterial and fungal detection, mycoplasma and Viral diagnosis, the detection method adopted adopts the common detection methods that those skilled in the art use to detect, and testing result is referring to described in table 1.
Table 1
| Sample 1 | Sample 2 | Sample 3 | Sample 4 | |
| Osmotic pressure (mOsmol/kg) | 280 | 310 | 305 | 286 |
| PH value | 7.26 | 7.43 | 7.29 | 7.55 |
| Protein content (g/L) | 38 | 41 | 40 | 45 |
| Antibacterial | Do not detect | Do not detect | Do not detect | Do not detect |
| Coliphage | Negative | Negative | Negative | Negative |
| Mycoplasma | Negative | Negative | Negative | Negative |
| Virus | Negative | Negative | Negative | Negative |
The Sanguis cervi preparing gained in above-mentioned specific embodiment 1 to specific embodiment 4 can apply to clearly in the preparation containing serum cell culture medium.
Claims (8)
1. the method for separating and preparing that a Sanguis cervi is clear, it is characterised in that: comprise the steps:
(1) blood sampling: disinfectant solution being adopted to carry out after deer body whole body cleaning-sterilizing processes until blood sampling deer, sterile blood sampling method takes deer whole blood to centrifuge bottle, centrifuge bottle is placed at 4��5 DEG C constant temperature standing 5��6h;
(2) separate: will through the deer whole blood after (1) processes is at 4��5 DEG C when, adopt the centrifugal 40��50min of rotating speed of 2000��3000rpm, from centrifuge bottle, serum is extracted under vacuum conditions after centrifugal end, it is placed at-25��-30 DEG C by the serum of this extraction gained to save backup, obtains the serum of freezing;
(3) fibrin is removed: be placed in 4��5 DEG C of environment by the freezing serum processing gained through (2) and melt, filtered by multilamellar sterile gauze by gained serum after thawing, collect filtrate;
(4) inactivation: gained filtrate will be processed through (3) and be placed at 56��60 DEG C water bath with thermostatic control 30��40min, and rock filtrate in water-bath process at any time;
(5) IgG concentration and glucose content are reduced: adopt chromatography that IgG concentration decreases below 5 �� g/ml the filtrate after (4) process; Then the dialyzer that molecular weight of albumen is 12000��14000 of can dialysing is adopted to dialyse to glucose content less than 0.5mg/ml in the NaCl aqueous solution of 0.15��0.18mol/L;
(6) virus and mycoplasma and aseptic filtration are removed: the gamma-rays adopting dosage to be 30��45KGy the serum after (5) process irradiates; Then being loaded by above-mentioned serum bleeds in slurry steriliser continuously, and irradiate this slurry steriliser of bleeding continuously equipped with serum with ultraviolet with 45��50 ��, irradiate and serum is obtained filtrate by EK level without asbestos Cai Shi filter disc simultaneously, then again this filtrate is filtered by 0.22��0.25 ��m of filter membrane, filter through 100��150nm filter membrane after, obtain Sanguis cervi clear.
2. the method for separating and preparing that Sanguis cervi according to claim 1 is clear, it is characterised in that: step (1) is treated blood sampling deer be that health is healthy, the mental status good, without the one in the adult deer of epidemic situation history, fawn and tire deer.
3. the method for separating and preparing that Sanguis cervi according to claim 1 is clear, it is characterised in that: step (1) adopts 2wt.% iodine tincture and 75vol.% ethanol treat blood sampling deer successively as disinfectant solution and carry out deer body whole body cleaning-sterilizing.
4. the method for separating and preparing that Sanguis cervi according to claim 1 is clear, it is characterised in that: step (3) adopt eight layers of sterile gauze be filtered melting gained serum.
5. the method for separating and preparing that Sanguis cervi according to claim 1 is clear, it is characterised in that: filtrate is placed in water bath with thermostatic control 30min at 56 DEG C by step (4).
6. the method for separating and preparing that Sanguis cervi according to claim 1 is clear, it is characterised in that: the flow velocity of slurry steriliser of bleeding continuously described in step (6) is 250��270mL/min.
7. the Sanguis cervi that a method for separating and preparing according to any claim in claim 1 to 6 prepares gained is clear.
8. the application that Sanguis cervi according to claim 7 is clear, it is characterised in that: described Sanguis cervi is clearly containing the application in serum cell culture medium.
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| CN201610103829.3A CN105647848A (en) | 2016-02-25 | 2016-02-25 | Separation and preparation method of deer serum |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104650221A (en) * | 2015-03-20 | 2015-05-27 | 西南大学 | Preparation method of high-capacity serum antibody |
| CN106404483A (en) * | 2016-08-31 | 2017-02-15 | 上海科华生物工程股份有限公司 | Preparation method of blood serum |
| CN110777107A (en) * | 2019-11-05 | 2020-02-11 | 内蒙古维克生生物技术股份有限公司 | Method for producing horse serum without lipoprotein |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104650221A (en) * | 2015-03-20 | 2015-05-27 | 西南大学 | Preparation method of high-capacity serum antibody |
| CN106404483A (en) * | 2016-08-31 | 2017-02-15 | 上海科华生物工程股份有限公司 | Preparation method of blood serum |
| CN110777107A (en) * | 2019-11-05 | 2020-02-11 | 内蒙古维克生生物技术股份有限公司 | Method for producing horse serum without lipoprotein |
| CN110777107B (en) * | 2019-11-05 | 2023-10-13 | 内蒙古维克生生物技术股份有限公司 | Production method for removing lipoprotein from horse serum |
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