CN102977184B - Purification method of protein group of 30 kD in silkworm pupa - Google Patents
Purification method of protein group of 30 kD in silkworm pupa Download PDFInfo
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- CN102977184B CN102977184B CN201210452817.3A CN201210452817A CN102977184B CN 102977184 B CN102977184 B CN 102977184B CN 201210452817 A CN201210452817 A CN 201210452817A CN 102977184 B CN102977184 B CN 102977184B
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- 241000255789 Bombyx mori Species 0.000 title claims abstract description 29
- 238000000746 purification Methods 0.000 title claims abstract description 26
- 241000382353 Pupa Species 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 title abstract description 19
- 102000004169 proteins and genes Human genes 0.000 title abstract description 19
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 51
- 239000012528 membrane Substances 0.000 claims abstract description 33
- 239000006228 supernatant Substances 0.000 claims abstract description 32
- 239000007788 liquid Substances 0.000 claims abstract description 23
- 239000012530 fluid Substances 0.000 claims description 20
- 238000012870 ammonium sulfate precipitation Methods 0.000 claims description 14
- 239000002244 precipitate Substances 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 abstract description 10
- 239000000126 substance Substances 0.000 abstract description 4
- BIGPRXCJEDHCLP-UHFFFAOYSA-N ammonium bisulfate Chemical compound [NH4+].OS([O-])(=O)=O BIGPRXCJEDHCLP-UHFFFAOYSA-N 0.000 abstract 1
- 238000005119 centrifugation Methods 0.000 abstract 1
- 239000013049 sediment Substances 0.000 abstract 1
- 239000002953 phosphate buffered saline Substances 0.000 description 25
- 238000003760 magnetic stirring Methods 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 8
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 8
- 235000011130 ammonium sulphate Nutrition 0.000 description 8
- 239000008176 lyophilized powder Substances 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000007787 solid Substances 0.000 description 6
- 230000003068 static effect Effects 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 235000021245 dietary protein Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 102000015781 Dietary Proteins Human genes 0.000 description 1
- 108010010256 Dietary Proteins Proteins 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 1
- -1 compound amino acid Chemical class 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 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 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 108010025964 lipophorin Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000003161 proteinsynthetic effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000009938 salting Methods 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- 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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
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- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
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Abstract
The invention discloses a purification method of a protein group of 30 kD in silkworm pupa, and belongs to the technical field of purification of protein. The method comprises the purification steps: carrying out cell disruption on silkworm pupa, taking supernatant to conduct ammonia sulfate fractional precipitation after centrifugation of cell disruption liquid, suspending the sediment again and utilizing an ultrafiltration membrane to filter substances having a molecular weight of 80kD to 100kD, utilizing the ultrafiltration membrane to filter flow liquid having a molecular weight of 30kD to 50kD, collecting retaining liquid, and finally obtaining the protein group of 30 kD. The purification method of the protein group of 30 kD in the silkworm pupa reduces damage and loss on interest protein, and the simplest steps can be utilized to obtain large amount of purified interest protein. The purification method of the protein group of 30 kD in the silkworm pupa is simple in operation and low in cost.
Description
Technical field
The present invention relates to purified technology of protein field, be specifically related to the purification process of 30kD albumen group in a kind of silkworm pupa.
Background technology
Pupa albumen is a kind of protein resource of high-quality, contains 18 seed amino acids, and wherein 8 kinds of essential amino acid content exceed 40% of total amino acid content, and its albumen is very easily hydrolyzed digested, is a kind of more satisfactory dietary protein origin.Research shows, silkworm chrysalis protein and amino acid have special physiologically active, and its polypeptide has the serum cholesterol of reduction and antioxygenation.It is reported, silkworm compound amino acid has obvious promotion male rat childhood body weight gain and blood transportation oxygen and nutritive substance ability, promotes body protein synthetic, accelerate metabolism, improve the multiple physiologically actives such as body immunity and suprarenal gland function, can be biochemical pharmacy and food protein cheap raw material is provided.
The 30kD albumen group who has 70% left and right in silkworm pupa, this albumen group has lipophorin function, participates in suppressing the programmed cell death of silkworm, and can suppress human cell's programmed death.
At present all comparatively loaded down with trivial details for 30kD albumen group's purification process, and purification efficiency neither be very high.Chinese patent application (publication number 102424701) discloses a kind of purification process of human growth hormone-like protein in silkworm pupa, need to pass through the essential step such as homogenate, centrifugal, ammonium sulfate precipitation, protein purification instrument purifying, affinitive layer purification, ultrafiltration and concentration.
Summary of the invention
Loaded down with trivial details in order to solve in prior art 30kD albumen group purification step complexity, cost is higher, the defect that purification efficiency is low, the invention provides the purification process of 30kD albumen group in a kind of new silkworm pupa, Main Basis molecular weight of albumen size, by simple ammonium sulfate precipitation, centrifugal and ultra-filtration membrane bag hyperfiltration process, the 30kD albumen group of acquisition.
30kD albumen group's purification process in silkworm pupa provided by the invention, purification step is: get silkworm pupa and carry out smudge cells, after cytoclasis liquid is centrifugal, get supernatant and carry out ammonium sulfate precipitation, precipitate resuspended after through the ultrafiltration of 80kD ~ 100kD ultra-filtration membrane, effluent liquid passes through the ultrafiltration of 30kD ~ 50kD ultra-filtration membrane again, collect trapped fluid, finally obtain 30kD albumen group.
The silkworm chrysalis product that silkworm pupa can use fresh silkworm chrysalis, process through super-dry or lyophilize etc.
Preferably, purifying carries out under the temperature condition of 0 ~ 4 DEG C.30kD albumen group, for having bioactive albumen group, preferably can remain under cold condition and carry out avoiding being degraded in purification process, guarantees that final protein of purifying still has biological activity and keeps higher purity.
Preferably, described smudge cells uses the method for homogenate, the PBS of use and silkworm pupa mixing homogenate.PBS is phosphate buffered saline buffer, fills a prescription as follows: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4, pH7.4 ~ 7.6.The PBS that preferably uses precooling, precooling refers to before experiment first lowers the temperature, for example, be placed on 4 DEG C of Temperature drop in refrigerators.
PBS volume is preferably ten times of silkworm chrysalis volume, can fully dissolve the albumen in silkworm chrysalis.The more use ultrasonic fragmentation of method of cell disruption, stamp mill crush method, multigelation method and chemical method etc., with regard to 30kD albumen group of the present invention, use homogenate method can meet the requirement of broken abundant and unlikely albumen inactivation, also can be farthest cost-saving, the method just can obtain by simple experimental installation and operation the optimizing materials that contains target protein in a large number, and low price is conducive to the foundation of large scale purification process.
Preferably, described centrifugal speed is more than 8000rpm.More preferably 8000 ~ 12000rpm.30kD albumen group is the albumen group of molecular weight, if rotating speed is too low, cannot isolate this albumen group.
Preferably, before the step of described ammonium sulfate precipitation, also comprise the step of 9 layers of filtered through gauze of the supernatant after centrifugal cytoclasis liquid.This step is mainly used in removing the composition such as insoluble substance and grease in supernatant liquor, is convenient to carrying out smoothly of subsequent purification, and this step also can be added on demand in follow-up step.
Preferably, described ammonium sulfate precipitation saturation ratio is 25% (NH
4)
2sO
4salt is molten, (the NH that is 65% by saturation ratio
4)
2sO
4saltout.In the time that segmentation is saltoutd, add salt concn and generally represent with saturation ratio, the saturation ratio of saturated solution is decided to be 100%.Molten and the salting point of the salt of ammonium sulfate is optimum 30kD albumen group's concentration, has improved to greatest extent 30kD albumen group's purifying rate.And the pH value to ammoniumsulphate soln is without any restrictions, is convenient to solution preparation, has simplified purification step.
Preferably, precipitate resuspended after through the ultrafiltration of 100kD ultra-filtration membrane, effluent liquid passes through the ultrafiltration of 30kD ultra-filtration membrane again.Find through test of many times, use 100kD and 30kD ultra-filtration membrane, can improve 30kD albumen group's purifying rate.
Preferably, described collection trapped fluid is to use the PBS of 3 ~ 10 times of trapped fluid volumes repeatedly to rinse ultrafiltration system, collects elutant.Can obtain like this target protein of maximum.PBS volume the best is 10 times of trapped fluid volume.
The present invention has following beneficial effect:
Purification process of the present invention has reduced damage and the loss to target protein, can utilize the simplest step to obtain a large amount of purer target proteins, not only simply but also save a large amount of manpower and materials.The present invention can provide the facility on purification process for the further research of 30kD albumen group function, and offers reference for the separation and purification of other albuminoids.
Brief description of the drawings
Fig. 1 is the 30kD albumen group purifying figure of embodiment 1, M: molecular weight of albumen standard; 1:80kD ultra-filtration membrane bag effluent liquid; 2:50kD ultra-filtration membrane bag trapped fluid.
Fig. 2 is the 30 kD albumen group purifying figure of embodiment 2, M: molecular weight of albumen standard; 1: silkworm chrysalis lyophilized powder; 2: the centrifugal rear supernatant solution of silkworm chrysalis lyophilized powder; Albumen under 3:45% saturation ratio ammonium sulfate precipitation; 4 and 5:65% saturation ratio ammonium sulfate precipitation under albumen; Albumen under 6:80% saturation ratio ammonium sulfate precipitation.
Fig. 3 is the 30kD albumen group purifying figure of embodiment 3, M: molecular weight of albumen standard; 1: supernatant before the ultrafiltration of ultra-filtration membrane bag; 2:100kD ultra-filtration membrane bag trapped fluid; 3:100kD ultra-filtration membrane bag effluent liquid; 4:30kD ultra-filtration membrane bag trapped fluid; 5:30kD ultra-filtration membrane bag effluent liquid.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described, can be implemented, but illustrated embodiment is not as a limitation of the invention so that those skilled in the art can better understand the present invention also.Except as otherwise noted, all Science and Technology terms used herein have the identical meanings of conventionally understanding with the technical field of the invention personnel.
PBS formula: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4, pH7.4 ~ 7.6.
1. homogenate and centrifugal
Get 100g silkworm chrysalis, the PBS homogenate of 1000mL precooling; Homogenate is in 0 ~ 2 DEG C, 12000rpm, and centrifugal 30min, collects supernatant.
2. ammonium sulfate precipitation
(1) supernatant constant volume, according to ammonium sulfate saturation computation device, adds a certain amount of solid (NH in supernatant
4)
2sO
4, to solution (NH
4)
2sO
4saturation ratio is to stir 30min on 25%, 4 DEG C of magnetic stirring apparatus, 4 DEG C of static 90min.4 DEG C, 12000rpm, centrifugal 30min, collects supernatant.
(2) supernatant constant volume, according to ammonium sulfate saturation computation device, takes a certain amount of solid (NH
4)
2sO
4, to solution (NH
4)
2sO
4saturation ratio is to stir 30min on 65%, 4 DEG C of magnetic stirring apparatus, 4 DEG C of static 90min.4 DEG C, 12000rpm, centrifugal 30min, collecting precipitation, the PBS of for example 1g precipitation 5mL of the PBS(of 5 times of volumes is resuspended), on 4 DEG C of magnetic stirring apparatuss, stir after 30min, 4 DEG C, 12000rpm, centrifugal 60min, abandons precipitation and stays supernatant.
3. ultra-filtration membrane bag ultrafiltration
According to ultra-filtration membrane bag Sartocube
?︱ Sartocon
?︱ Sartocon
?slice Sartocon Slice 200 Cassettes operational manuals (being purchased from Sartorius Stedim Biotech company) install ultra-filtration membrane bag, supernatant is crossed 80kD ultra-filtration membrane bag, repeatedly rinse (for example precooling PBS of 100mL supernatant 500mL volume) with the precooling PBS of 3 times of volumes, collect effluent liquid.
50kD ultra-filtration membrane bag ultrafiltration effluent liquid is 1/8th (it is 100mL that for example 800mL effluent liquid is concentrated into trapped fluid volume) of original volume to trapped fluid volume.In gained trapped fluid, be 30kD albumen group, trapped fluid is sent to freeze-drying and is made lyophilized powder.Purifying protein electrophoresis result is shown in Fig. 1.
The present embodiment arranges three different ammonium sulfate precipitation saturation ratios and carries out three groups of simultaneous tests, and following operation is carried out respectively in every group of test:
PBS formula: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4, pH7.4 ~ 7.6.
1. homogenate and centrifugal
Get 100g silkworm chrysalis lyophilized powder (Zhejiang Zhong Qi Biomedics Inc. provides), the PBS homogenate of 1000mL precooling; Homogenate is in 0 ~ 2 DEG C, 12000rpm, and centrifugal 30min, collects supernatant.
2. ammonium sulfate precipitation
(1) supernatant constant volume, according to ammonium sulfate saturation computation device, adds a certain amount of solid (NH in supernatant
4)
2sO
4, to solution (NH
4)
2sO
4saturation ratio is to stir 30min on 25%, 4 DEG C of magnetic stirring apparatus, 4 DEG C of static 90min.4 DEG C, 12000rpm, centrifugal 30min, collects supernatant.
(2) supernatant constant volume, according to ammonium sulfate saturation computation device, takes a certain amount of solid (NH
4)
2sO
4, to solution (NH
4)
2sO
4saturation ratio is that other two groups of 45%(is adjusted into respectively 65% and 80%), on 4 DEG C of magnetic stirring apparatuss, stir 30min, 4 DEG C of static 90min.4 DEG C, 12000rpm, centrifugal 30min, collecting precipitation, the PBS of for example 1g precipitation 5mL of the PBS(of 5 times of volumes is resuspended), on 4 DEG C of magnetic stirring apparatuss, stir after 30min, 4 DEG C, 12000rpm, centrifugal 60min, abandons precipitation and stays supernatant.
3. ultra-filtration membrane bag ultrafiltration
According to ultra-filtration membrane bag Sartocube
?︱ Sartocon
?︱ Sartocon
?slice Sartocon Slice 200 Cassettes operational manuals (being purchased from Sartorius Stedim Biotech company) install ultra-filtration membrane bag, supernatant is crossed 80kD ultra-filtration membrane bag, repeatedly rinse (for example precooling PBS of 100mL supernatant 500mL volume) with the precooling PBS of 10 times of volumes, collect effluent liquid.
50kD ultra-filtration membrane bag ultrafiltration effluent liquid is 1/8th (it is 100mL that for example 800mL effluent liquid is concentrated into trapped fluid volume) of original volume to trapped fluid volume.In gained trapped fluid, be 30kD albumen group, trapped fluid is sent to freeze-drying and is made lyophilized powder.Three groups of purifying protein electrophoresis result are shown in Fig. 2.
PBS formula: 137mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 2mM KH
2pO
4, pH7.4 ~ 7.6.
1. homogenate and centrifugal
Get 100g silkworm chrysalis lyophilized powder (Zhejiang Zhong Qi Biomedics Inc. provides), the PBS homogenate of 1000mL precooling (4 DEG C of Temperature drop in refrigerators) three times, each 1min, the middle homogenate cup 1min that lowers the temperature on ice; Homogenate is in 4 DEG C, 8000rpm, and centrifugal 30min, 9 layers of filtered through gauze for supernatant liquor, in triplicate, collect supernatant.
2. ammonium sulfate precipitation
(1) supernatant constant volume, according to ammonium sulfate saturation computation device, adds a certain amount of solid (NH in supernatant
4)
2sO
4, to solution (NH
4)
2sO
4saturation ratio is to stir 30min on 25%, 4 DEG C of magnetic stirring apparatus, 4 DEG C of static 90min.4 DEG C, 8000rpm, centrifugal 30min, 9 layers of filtered through gauze, collect supernatant.
(2) supernatant constant volume, according to ammonium sulfate saturation computation device, takes a certain amount of solid (NH
4)
2sO
4, to solution (NH
4)
2sO
4saturation ratio is to stir 30min on 65%, 4 DEG C of magnetic stirring apparatus, 4 DEG C of static 90min.4 DEG C, 8000rpm, centrifugal 30min, collecting precipitation, the PBS of for example 1g precipitation 5mL of the PBS(of 5 times of volumes is resuspended), on 4 DEG C of magnetic stirring apparatuss, stir after 30min, 4 DEG C, 8000rpm, centrifugal 60min, abandons precipitation and stays supernatant.
3. ultra-filtration membrane bag ultrafiltration
According to Sartocube
?︱ Sartocon
?︱ Sartocon
?slice Sartocon Slice 200 Cassettes operational manuals (being purchased from Sartorius Stedim Biotech company) install ultra-filtration membrane bag, supernatant is crossed 100kD ultra-filtration membrane bag, repeatedly rinse (for example precooling PBS of 100mL supernatant 500mL volume) with the precooling PBS of 5 times of volumes, collect effluent liquid.
30kD ultra-filtration membrane bag ultrafiltration effluent liquid is 1/10th (it is 100mL that for example 1000mL effluent liquid is concentrated into trapped fluid volume) of original volume to trapped fluid volume.In gained trapped fluid, be 30kD albumen group, trapped fluid is sent to freeze-drying and is made lyophilized powder.The electrophoresis result of each step is shown in Fig. 3.
The above embodiment is only the preferred embodiment for absolutely proving that the present invention lifts, and protection scope of the present invention is not limited to this.What those skilled in the art did on basis of the present invention is equal to alternative or conversion, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
Claims (1)
1. 30kD albumen group's purification process in a silkworm pupa, it is characterized in that, purification step is: the PBS and silkworm pupa mixing homogenate for method that use homogenate, with the cell of broken silkworm pupa, after the speed of cytoclasis liquid more than 8000rpm is centrifugal by 9 layers of filtered through gauze for the supernatant after centrifugal cytoclasis liquid, get afterwards supernatant and carry out ammonium sulfate precipitation, precipitate resuspended after through the ultrafiltration of 80kD ~ 100kD ultra-filtration membrane, effluent liquid passes through the ultrafiltration of 30kD ~ 50kD ultra-filtration membrane again, use the PBS of 3 ~ 10 times of trapped fluid volumes repeatedly to rinse ultrafiltration system, collect elutant, final acquisition 30kD albumen group,
Wherein, purifying carries out under the temperature condition of 0 ~ 4 DEG C;
Described ammonium sulfate precipitation saturation ratio is 25% (NH
4)
2sO
4salt is molten, (the NH that is 65% by saturation ratio
4)
2sO
4saltout;
Precipitate resuspended after through the ultrafiltration of 100kD ultra-filtration membrane, effluent liquid passes through the ultrafiltration of 30kD ultra-filtration membrane again.
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| PCT/CN2013/077522 WO2014075443A1 (en) | 2012-11-13 | 2013-06-20 | Purification method of protein group of 30 kd in silkworm pupa |
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| CN102516381B (en) * | 2011-12-19 | 2013-10-30 | 天津耀宇生物技术有限公司 | Natural sex storage protein-2 as well as preparation and use for same |
| CN102977184B (en) * | 2012-11-13 | 2014-07-09 | 天津耀宇生物技术有限公司 | Purification method of protein group of 30 kD in silkworm pupa |
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| CN1403471A (en) * | 2002-07-12 | 2003-03-19 | 成都心友生物技术有限责任公司 | Stepped prepn of natural active protein and peptide |
| CN1430975A (en) * | 2003-01-23 | 2003-07-23 | 上海澳博海洋生物技术开发有限公司 | Extractive of ocean star worm, its preparing method and application |
| CN101418287A (en) * | 2008-11-28 | 2009-04-29 | 天津科技大学 | Aspergillus niger liquid state fermentation pectic enzyme and control to colloidal matter in white water and paper stuff |
| CN101717446A (en) * | 2009-11-17 | 2010-06-02 | 浙江大学 | Preparation for PRRSV-N-IgY antibody and application thereof in detecting PRRSV |
| CN102161699A (en) * | 2011-01-31 | 2011-08-24 | 中国科学院海洋研究所 | Clam polypeptide and preparation method thereof |
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| CN102977184A (en) | 2013-03-20 |
| WO2014075443A1 (en) | 2014-05-22 |
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