CN106689697A - N15 stable isotope labeling method for experiment mouse protein quantification and tracing - Google Patents
N15 stable isotope labeling method for experiment mouse protein quantification and tracing Download PDFInfo
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- 238000002474 experimental method Methods 0.000 title claims abstract description 13
- 238000011002 quantification Methods 0.000 title claims abstract description 11
- 108090000143 Mouse Proteins Proteins 0.000 title claims abstract description 7
- 238000003141 isotope labeling method Methods 0.000 title abstract 2
- 239000000843 powder Substances 0.000 claims abstract description 36
- 240000002900 Arthrospira platensis Species 0.000 claims abstract description 35
- 235000016425 Arthrospira platensis Nutrition 0.000 claims abstract description 35
- 229940082787 spirulina Drugs 0.000 claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 33
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 33
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 5
- 238000000746 purification Methods 0.000 claims abstract description 3
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 44
- 239000001963 growth medium Substances 0.000 claims description 22
- 238000002372 labelling Methods 0.000 claims description 16
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- 239000002609 medium Substances 0.000 claims description 13
- 150000003839 salts Chemical class 0.000 claims description 13
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 9
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- 230000001954 sterilising effect Effects 0.000 claims description 9
- 239000012153 distilled water Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 230000008859 change Effects 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 241000195493 Cryptophyta Species 0.000 claims description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
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- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000005286 illumination Methods 0.000 claims description 3
- 238000009928 pasteurization Methods 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 241000699670 Mus sp. Species 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 238000006467 substitution reaction Methods 0.000 claims description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims 1
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- 235000014103 egg white Nutrition 0.000 claims 1
- 210000000969 egg white Anatomy 0.000 claims 1
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- 239000006151 minimal media Substances 0.000 claims 1
- 238000004445 quantitative analysis Methods 0.000 abstract description 3
- 238000001948 isotopic labelling Methods 0.000 abstract description 2
- 241001052560 Thallis Species 0.000 abstract 3
- 238000000605 extraction Methods 0.000 abstract 2
- 229910017053 inorganic salt Inorganic materials 0.000 abstract 2
- 108010026552 Proteome Proteins 0.000 abstract 1
- 238000004458 analytical method Methods 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 235000013305 food Nutrition 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 22
- 150000001413 amino acids Chemical class 0.000 description 6
- 229930003231 vitamin Natural products 0.000 description 6
- 239000011782 vitamin Substances 0.000 description 6
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
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- 238000004113 cell culture Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
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- 230000004853 protein function Effects 0.000 description 2
- 230000029983 protein stabilization Effects 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
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- 239000008158 vegetable oil Substances 0.000 description 2
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- 241000700199 Cavia porcellus Species 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
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- 229930003268 Vitamin C Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 230000033228 biological regulation Effects 0.000 description 1
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- 238000009395 breeding Methods 0.000 description 1
- FUFJGUQYACFECW-UHFFFAOYSA-L calcium hydrogenphosphate Chemical compound [Ca+2].OP([O-])([O-])=O FUFJGUQYACFECW-UHFFFAOYSA-L 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 235000021466 carotenoid Nutrition 0.000 description 1
- 150000001747 carotenoids Chemical class 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000001466 metabolic labeling Methods 0.000 description 1
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- 235000019629 palatability Nutrition 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000000575 proteomic method Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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Abstract
The invention discloses an N15 stable isotope labeling method for experiment mouse protein quantification and tracing. According to the method, firstly, yeast is used for converting N15 inorganic salt into bioprotein; when the yeast culture generations reach more than six generations, more than 98 percent of the extraction protein is N15 protein; then, the yeast is ground to realize protein contained in thalli; through extraction and purification, the thalli protein powder containing N15 is prepared; meanwhile, the N15 inorganic salt is used for culturing spirulina; when the culture generations of the spirulina reach more than 6 generations, more than 98 percent of N elements contained in the spirulina is N15; then, the spirulina is harvested and dried to obtain the spirulina dry powder containing N15; the thalli protein powder containing N15 and spirulina dry powder containing N15 are used for replacing conventional N14 raw materials for manufacturing mouse food; the specific N15 isotope labeling is provided for the experiment mouse protein quantification and tracing. The method provided by the invention can be applied to the protein quantitative analysis of the experiment mouse; the operation is simple and convenient; the production cost is low; the applicability is high; the proteome analysis by people on a great number of samples at high efficiency and low cost is facilitated.
Description
Technical field
The present invention relates to the quantitative technical field of protein stabilization isotope marks, refer in particular to a kind of for experimental mouse egg
Quantitative and spike the N of white matter15Cold labeling method.
Background technology
The Protein quantitative analysis of biological specimen or organism are research protein function mechanism and explore protein population
Interior correlation and the important method of binding mode, while also to seek disease protein label and drug targets bring newly
The mode of thinking and research direction.In recent years, with the progress of mass-spectrometric technique and bioinformatics, cold labeling technology
Basis and conventional method in quantitative proteomicses are turned into.Current biological specimen quantification of protein technology mainly has isotope
The SILAC technologies of metabolic marker method.
SILAC (Stable isotope labeling with amino acids in cell culture, SILAC)
It is mammalian cell stable isotope metabolic labeling approaches, its general principle is to adopt heavy external source stable isotope amino acid
(mainly having Lys and Arg) adds medium culture cell, cell to be fitted together to isotope amino acid in the protein of new synthesis,
By (2 after culture 5-6 generations6), the protein of all proteins basic more than 98% of cell is isotopically labeled.Different condition
The protein sample mixed in equal amounts for the treatment of, separates and mass spectral analysis by SDS-PAGE afterwards, can obtain the quantitative result of albumen.
But the cell of in vitro culture is difficult to replicate the interior environment of complexity of living organism, it is impossible to intactly reflect biological growth hair
Educate and regulate and control with the protein function in lysis and physiological change, therefore, the protein stabilization isotope marks skill of animal body
The development of art is particularly important.
Experiment muroid as the most frequently used experimental animal, its physiology course and growth and development process all with the split-phase of the mankind ten
Seemingly, the physiological acoustic signals process of the mankind can truly be simulated.The SILAC experimental mouses for using in the world, mark breeding method be generally
Cold labeling amino acid and mouse grain is made using light, weight, is handed over after raising male and female experimental mouse to the maturity period with SILAC mouse grain
With becoming pregnant, continue to be fed with SILAC mouse grain and become pregnant experimental mouse to producing newborn mouse.It is commissioned to train through two after supporting, can be marked completely
Experimental mouse.
SILAC mouse grains have obvious limitation, and its situation is as follows：
1st, it is cumbersome.SILAC mouse grain preparation flows include：Isotope marks amino acid → complex sign protein → exempt from
Epidemic disease precipitation → electrophoretic separation → dry → prepare mouse grain, complex operation is low using cell culture complex sign protein efficiency ratio, PER, no
It is convenient to implement.
2nd, production cost is high.SILAC reagents are expensive, using SILAC technical marks protein and are made mouse grain, so that
Labelling experiment mouse, reagent therein expends huge, and cultivation several generations experimental mouse demand mouse grain is more, and cost is very high.
3rd, purchase and transport difficult.SILAC mouse grains are produced by offshore company, and country's purchase is cumbersome, and mouse grain is easy
Damage or rotten, it is difficult to undergo long-distance transport.
Spirulina is a kind of conventional microalgae, comprehensive nutrition high with protein content, absorption easy to digest and good palatability
The features such as, and it is safe, have no toxic side effect, it is outstanding feedstuff.In addition, the vitamin content of spirulina also ten
Divide abundant, especially with carotenoid, based on the functional vitamin such as B family vitamin and vitamin C, disclosure satisfy that experimental mouse pair
The demand of vitamin.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided one kind is used for experimental mouse quantification of protein and spike
N15Cold labeling method, the method can be applied to the Protein quantitative analysis of experimental mouse, easy to operate, production cost
It is low, can promote on a large scale, beneficial to people efficiently, low cost Proteomic analysis are carried out to a large amount of samples.
To achieve the above object, technical scheme provided by the present invention is：One kind is for experimental mouse quantification of protein and shows
The N of track15Cold labeling method, first, using yeast by N15Inorganic salts change into bioprotein, when Yeast Cultivation generation
Number reaches 6 more than generation, and it is all N that it extracts albumen more than 98%15Albumen, then yeast is ground, protein contained by release thalline,
Extracted purifying is made containing N15Mycoprotein powder.Utilize N simultaneously15Inorganic salts culture spirulina, when SPIRULINA CULTIVATION algebraically
Reach 6 more than generation, contained N element more than 98% is all N in frond15, then will be obtained containing N after spirulina results, drying15Spiral shell
Rotation algae dry powder.Using containing N15Mycoprotein powder and containing N15The conventional N of spirulina powder substitution14Raw material is fabricated to mouse grain,
For experimental mouse protein labeling is quantitative and spike provides specific N15Isotope marks.
The basic nutrition demand of experimental mouse is including carbohydrate, protein, lipid, vitamin, inorganic salts and moisture etc..In this hair
In bright, containing N15Mycoprotein powder provide sufficient, amino acid is complete and protein source of high-purity, containing N15Spirulina do
Powder provides carbohydrate, good protein, vitamin and trace element.
Protocol step provided by the present invention is as follows：
1) prepared containing only N according to formula as below15Saccharomycete inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
Using high-pressure sterilizing pot, by step 1) in culture medium sterilize at one hundred and twenty degrees centigrade 20 minutes；
2) prepared containing only N according to formula as below15Spirulina inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
Using high-pressure sterilizing pot, by step 2) in culture medium sterilize at one hundred and twenty degrees centigrade 20 minutes；
3) after step 1) and 2) in culture medium be cooled to room temperature after, it is inorganic that picking single bacterium colony saccharomycete is seeded in saccharomycete
In culture medium, culture in incubator is placed on；Choose a small amount of algae kind, be seeded in spirulina inorganic medium, regulation temperature and
Illumination, is passed through air and is placed at the uniform velocity oscillator and cultivate, and reaches uniform effect；
4) treat that saccharomycete culture algebraically reaches 6 more than generation, take out culture medium and be centrifuged, it is clear with the physiological saline after sterilizing
Wash, obtain yeast thalline；
6) after the thalline being collected into is weighed, with sodium chloride solution with 1:1~1.5 ratio is mixed into new bacterium solution, places
In under 50~60 DEG C of environment, timing is shaken, and makes saccharomycete self-dissolving discharge protein；
7) after self-dissolving, inactivated with the enzyme in pasteurization decree bacterium solution, collected after centrifugation liquid；
8) by step 7) in liquid freezing dry after, obtain containing N15Mycoprotein powder；
9) treat that SPIRULINA CULTIVATION algebraically reaches 6 more than generation, take out culture medium and filter, collect frond；
10) with sterilized water flush clean frond reduction salinity repeatedly, frond after cleaning is evenly laid out to be opened wide in wide-mouth
In container, refrigerator freezing is put into；
11) the good spirulina of pre-freeze is put into hot air drier and is dried, obtained containing N15Spirulina powder；
12)N15Test the preparation of mouse feed：By step 8) in the mycoprotein powder that is made and step 11) in the spiral shell that is made
Rotation algae dry powder replaces conventional N as the basic nitrogen source and primary carbon source of feed14Raw material is used to cultivate, and adds without N element
Other raw materials, be made final N15Experiment mouse feed.
In step 3) in, yeast is by the N in culture medium15Inorganic salts change into bioprotein, and spirulina is by culture medium
N15Inorganic salts are converted into bioprotein and other materials containing N, and when Yeast Cultivation algebraically reaches 6 more than generation, it extracts albumen 98%
Below all it is N15Albumen；When SPIRULINA CULTIVATION algebraically reaches 6 more than generation, contained N element more than 98% is all N in frond15,
And can be according to the different saccharomycete of the final experimental mouse Feed selection prepared, spirulina and containing only N in practical operation15It is inorganic
Culture medium prescription.
In step 12) in, N can be prepared according to situation15Experiment mice feed, N15Experimental rat feed, N15Experimental guinea pig
Feed etc..N element is all from step 1 in the final feed for preparing) to step 12) in extracted mycoprotein powder after purification
End and the N of spirulina powder15, for experimental mouse protein labeling is quantitative and spike provides specific N15Isotope marks, and nothing
Depending on the specific manufacturing conditions of machine salt and feed will be according to the growth characteristics of experimental mouse.
The present invention compared with prior art, has the following advantages that and beneficial effect：
1st, it is simple to operate.The operation being related in the present invention belongs to basic experiment operation, and experimenter only needs simple training
Just it is independently operable afterwards and complete whole preparation flow.
2nd, low production cost.Agents useful for same of the present invention and material are mostly general reagent and material, cheap；The present invention
The equipment for being used is simple and easy to get, without special messenger's operation, saves high cost of equipment and cost of labor.
3rd, strong adaptability.Scheme proposed by the invention, in common lab or can reach and push away in the workshop of production standard
Extensively, the mouse grain for being produced using this programme is applied to the raising and research of kinds of experiments mouse, can substantially reduce and use stable isotope
Calibration experiment mouse studies the difficulty of proteomics.
Specific embodiment
With reference to multiple specific embodiments, the invention will be further described.
(the N of embodiment 115The preparation of mouse feed)
Described in the present embodiment for experimental mouse quantification of protein and the N of spike15Cold labeling method, specifically
N15The preparation method of cold labeling mouse feed, comprises the following steps：
1) prepared containing only N according to formula as below15Saccharomycete inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
Using high-pressure sterilizing pot, by step 1) in culture medium sterilize at one hundred and twenty degrees centigrade 20 minutes；
2) prepared containing only N according to formula as below15Spirulina inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
Using high-pressure sterilizing pot, by step 2) in culture medium sterilize at one hundred and twenty degrees centigrade 20 minutes；
3) after step 1) and 2) in culture medium be cooled to room temperature after, it is inorganic that picking single bacterium colony saccharomycete is seeded in saccharomycete
In culture medium, culture in the environment of proper temperature is placed on；A small amount of algae kind is chosen, is seeded in spirulina inorganic medium, adjusted
The appropriate temperature of section and illumination, are passed through air and are placed at the uniform velocity oscillator and cultivate, and reach uniform effect；
4) treat that saccharomycete culture algebraically reaches 6 more than generation, take out culture medium and be centrifuged, it is clear with the physiological saline after sterilizing
Wash, obtain yeast thalline；
6) after the thalline being collected into is weighed, with sodium chloride solution with 1:1~1.5 ratio is mixed into new bacterium solution, places
In under 50~60 DEG C of environment, timing is shaken, and makes saccharomycete self-dissolving discharge protein；
7) after self-dissolving, inactivated with the enzyme in pasteurization decree bacterium solution, collected after centrifugation liquid；
8) by step 7) in liquid freezing dry after, obtain containing N15Mycoprotein powder；
9) treat that SPIRULINA CULTIVATION algebraically reaches 6 more than generation, take out culture medium and filter, collect frond；
10) with sterilized water flush clean frond reduction salinity repeatedly, frond after cleaning is evenly laid out to be opened wide in wide-mouth
In container, refrigerator freezing is put into；
11) the good spirulina of pre-freeze is put into hot air drier and is dried, obtained containing N15Spirulina powder；
12)N15The preparation of mouse feed：
| Feed ingredient | Consumption (g) |
| Containing N15Spirulina powder | 400~500 |
| Containing N15Mycoprotein powder | 40~50 |
| Starch | 100~150 |
| Vegetable oil | 100~150 |
| Salt | 10~15 |
| Calcium monohydrogen phosphate | 10~15 |
| Ironic citrate | 1~1.5 |
Explanation：Raw material needed for being weighed according to formula, adds suitable quantity of water to mix well after mixing, rubbed to dough with hand and filled and powder
Untill not scattering.Wet feed is uniformly layered in the open shallow bottom container at rectangle bottom, such as lunch box, after compacting left-hand thread be covered with it is fresh-keeping
The operating table surface of film, with nylon wire cut growth cube particle.Wet feed after cutting is emitted on pallet and is put into drying in baking oven.
Feed after drying should after cooling be distributed into pouch and seal, irradiated thereafter sterilized.
(the N of embodiment 215The preparation of rat feed)
The present embodiment makes N as different from Example 115Rat feed, two kinds are prepared containing only N according to formula as below15's
Inorganic medium：
Saccharomycete inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
Spirulina inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
Thereafter obtained containing N according to technique productions same as Example 115Mycoprotein powder and containing N15Spirulina powder
End.
N15Rat feed is formulated
| Feed ingredient | Consumption (g) |
| Containing N15Spirulina powder | 400~500 |
| Containing N15Mycoprotein powder | 50~60 |
| Starch | 100~150 |
| Vegetable oil | 100~150 |
| Salt | 10~15 |
| Calcium phosphate | 10~15 |
Explanation：Raw material needed for being weighed according to formula, adds suitable quantity of water to mix well after mixing, rubbed to dough with hand and filled and powder
Untill not scattering.Fetch bigbore syringe, remove syringe needle, during wet feed inserted into syringe inner chamber, be inserted in piston.With injection
Device squeezes out the cylindrical particle being of convenient length, and is neatly thrown on pallet, is put into drying in baking oven.Feed after drying should
Pouch is distributed into after cooling and is sealed, it is irradiated thereafter sterilized.
Embodiment described above is only the preferred embodiments of the invention, not limits practical range of the invention with this, therefore
The change that all shapes according to the present invention, principle are made, all should cover within the scope of the present invention.
Claims (3)
1. a kind of for experimental mouse quantification of protein and the N of spike15Cold labeling method, it is characterised in that：First, profit
With yeast by N15Inorganic salts change into bioprotein, and when Yeast Cultivation algebraically reaches 6 more than generation, it extracts albumen more than 98% all
It is N15Albumen, then yeast is ground, protein contained by release thalline, extracted purifying is made containing N15Mycoprotein powder,
Utilize N simultaneously15Inorganic salts culture spirulina, when SPIRULINA CULTIVATION algebraically reaches 6 more than generation, in frond contained N element 98% with
On all be N15, then will be obtained containing N after spirulina results, drying15Spirulina powder, using containing N15Mycoprotein powder and
Containing N15The conventional N of spirulina powder substitution14Raw material is fabricated to mouse grain, for experimental mouse protein labeling is quantitative and spike is provided
Specific N15Isotope marks, it specifically includes following steps：
1) prepared containing only N according to formula as below15Saccharomycete inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
Using high-pressure sterilizing pot, by step 1) in culture medium sterilize at one hundred and twenty degrees centigrade 20 minutes；
2) prepared containing only N according to formula as below15Spirulina inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
Using high-pressure sterilizing pot, by step 2) in culture medium sterilize at one hundred and twenty degrees centigrade 20 minutes；
3) after step 1) and 2) in culture medium be cooled to room temperature after, picking single bacterium colony saccharomycete is seeded in saccharomycete minimal media
Cultivated in base；Algae kind, is seeded in spirulina inorganic medium needed for choosing, and adjusts temperature and illumination, is passed through air and is placed on
Cultivated at the uniform velocity on oscillator, reach uniform effect；
4) treat that saccharomycete culture algebraically reaches 6 more than generation, take out culture medium and be centrifuged, cleaned with the physiological saline after sterilizing, obtain
To yeast thalline；
6) after the thalline being collected into is weighed, with sodium chloride solution with 1:1~1.5 ratio is mixed into new bacterium solution, is positioned over 50
Under~60 DEG C of environment, timing is shaken, and makes saccharomycete self-dissolving discharge protein；
7) after self-dissolving, inactivated with the enzyme in pasteurization decree bacterium solution, collected after centrifugation liquid；
8) by step 7) in liquid freezing dry after, obtain containing N15Mycoprotein powder；
9) treat that SPIRULINA CULTIVATION algebraically reaches 6 more than generation, take out culture medium and filter, collect frond；
10) with sterilized water, flush clean frond reduces salinity repeatedly, the evenly laid out container opened wide in wide-mouth of the frond after cleaning
In, it is put into refrigerator freezing；
11) the good spirulina of pre-freeze is put into hot air drier and is dried, obtained containing N15Spirulina powder；
12)N15Test the preparation of mouse feed：By step 8) in the mycoprotein powder that is made and step 11) in the spirulina that is made
Dry powder replaces conventional N as the basic nitrogen source and primary carbon source of feed14Raw material is used to cultivate, and adds its without N element
His raw material, is made final N15Experiment mouse feed.
2. according to claim 1 a kind of for experimental mouse quantification of protein and the N of spike15Cold labeling side
Method, it is characterised in that：In step 3) in, yeast is by the N in culture medium15Inorganic salts change into bioprotein, and spirulina will be cultivated
N in base15Inorganic salts are converted into bioprotein and other materials containing N, and when Yeast Cultivation algebraically reaches 6 more than generation, it extracts egg
White more than 98% is all N15Albumen；When SPIRULINA CULTIVATION algebraically reaches 6 more than generation, in frond, contained N element more than 98% is all
It is N15, and according to the different saccharomycete of the final experimental mouse Feed selection prepared, spirulina and containing only N in practical operation15Nothing
Machine culture medium prescription.
3. according to claim 1 a kind of for experimental mouse quantification of protein and the N of spike15Cold labeling side
Method, it is characterised in that：In step 12) in, N can be prepared according to situation15Experiment mice feed, N15Experimental rat feed, N15It is real
Test cavy feed, N element is all from step 1 in the final feed for preparing) to step 12) in extracted thalline egg after purification
White powder end and the N of spirulina powder15, for experimental mouse protein labeling is quantitative and spike provides specific N15Isotope marks,
And depending on the specific manufacturing conditions of inorganic salts and feed will be according to the growth characteristics of experimental mouse.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810978A (en) * | 2005-01-27 | 2006-08-02 | 深圳市东西方生物技术有限公司 | Process of culturing micro algae to produce isotope glucose |
| CN106119321A (en) * | 2016-06-30 | 2016-11-16 | 肖传乐 | A kind of for bioprotein quantitatively and the N of spike15cold labeling method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810978A (en) * | 2005-01-27 | 2006-08-02 | 深圳市东西方生物技术有限公司 | Process of culturing micro algae to produce isotope glucose |
| CN106119321A (en) * | 2016-06-30 | 2016-11-16 | 肖传乐 | A kind of for bioprotein quantitatively and the N of spike15cold labeling method |
Non-Patent Citations (1)
| Title |
|---|
| 任征，等: "稳定同位素15N标记螺旋藻的生物合成", 《同位素》 * |
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