CN106538482A - A kind of N of and spike quantitative for nematode protein15Cold labeling method - Google Patents
A kind of N of and spike quantitative for nematode protein15Cold labeling method Download PDFInfo
- Publication number
- CN106538482A CN106538482A CN201611126025.1A CN201611126025A CN106538482A CN 106538482 A CN106538482 A CN 106538482A CN 201611126025 A CN201611126025 A CN 201611126025A CN 106538482 A CN106538482 A CN 106538482A
- Authority
- CN
- China
- Prior art keywords
- yeast
- nematicide
- albumen
- quantitative
- spike
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000002372 labelling Methods 0.000 title claims abstract description 27
- 241000244206 Nematoda Species 0.000 title claims abstract description 18
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 47
- 239000005645 nematicide Substances 0.000 claims abstract description 34
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 32
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 32
- 239000000843 powder Substances 0.000 claims abstract description 18
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims abstract description 9
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 7
- 239000000284 extract Substances 0.000 claims abstract description 5
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 claims abstract description 5
- 239000001963 growth medium Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 12
- 239000002609 medium Substances 0.000 claims description 11
- 239000012153 distilled water Substances 0.000 claims description 9
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 210000004027 cell Anatomy 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 7
- 238000005119 centrifugation Methods 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 4
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 3
- 238000002604 ultrasonography Methods 0.000 claims description 3
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 239000006151 minimal media Substances 0.000 claims description 2
- 235000016709 nutrition Nutrition 0.000 claims description 2
- 108010058643 Fungal Proteins Proteins 0.000 claims 1
- 108091005804 Peptidases Proteins 0.000 claims 1
- 239000004365 Protease Substances 0.000 claims 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 238000011081 inoculation Methods 0.000 claims 1
- 239000002994 raw material Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 14
- 238000012360 testing method Methods 0.000 abstract description 8
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000000575 proteomic method Methods 0.000 abstract description 2
- 238000004445 quantitative analysis Methods 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 23
- 241000588724 Escherichia coli Species 0.000 description 8
- 235000001727 glucose Nutrition 0.000 description 7
- 241000244203 Caenorhabditis elegans Species 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 6
- 239000008103 glucose Substances 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000002689 soil Substances 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 4
- LQJAALCCPOTJGB-YUMQZZPRSA-N Arg-Pro Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(O)=O LQJAALCCPOTJGB-YUMQZZPRSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 108010060035 arginylproline Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 102000009027 Albumins Human genes 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 210000002429 large intestine Anatomy 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 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 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 206010013786 Dry skin Diseases 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 101150010952 OAT gene Proteins 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 150000002304 glucoses Chemical class 0.000 description 1
- 238000001948 isotopic labelling Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 238000001466 metabolic labeling Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000029983 protein stabilization Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
Classifications
-
- 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
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Environmental Sciences (AREA)
- Mycology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Animal Husbandry (AREA)
- Virology (AREA)
- Botany (AREA)
- Biomedical Technology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Microbiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of N of and spike quantitative for nematode protein15Cold labeling method, first, using yeast by N15Inorganic salt changes into bioprotein, and when Yeast Cultivation algebraically reaches 6 more than generation, it is all N which extracts albumen more than 98%15Albumen, then yeast is ground, is made containing N15Tropina powder, using containing N15Tropina powder replace conventional N14Albumen, for cultivating the symbiosis thalline of various nematicides or nematicide, for nematode protein labelling is quantitative and spike provides the N of specificity15Isotope marks.The inventive method is applied to the Protein quantitative analysis of nematicide, and easy to operate, experimentation cost is low, and the test period is short, beneficial to people efficiently, low cost Proteomic analysis are carried out to a large amount of samples.
Description
Technical field
The present invention relates to the quantitative technical field of protein stabilization isotope marks, refers in particular to a kind of for elegans proteins
Matter is quantitative and the N of spike15Cold labeling method.
Background technology
Biological vivo protein quantitative analyses become research protein function and seek disease protein label and medicine
The important channel of target.Nematicide (Caenorhabditis Elegans) as one of important model organism, its vivo protein
Matter quantitative analyses are significant to life science.Quantification of protein technology is mainly in Isotope metabolism labeling method at present
SILAC technologies.
SILAC (Stable isotope labeling with amino acids in cell culture, SILAC)
It is mammalian cell stable isotope metabolic labeling approaches, its ultimate principle is respectively with natural isotope (light-duty) or stable
The essential amino acids of isotope (heavy type) labelling replace corresponding aminoacid in cell culture medium, cell Jing after 5-6 multiplication cycle,
The aminoacid of cold labeling instead of original aminoacid in being completely incorporated into the protein that cell newly synthesizes.Not isolabeling
The crack protein of cell is pressed cell number or protein content equal proportion and is mixed, separated, carry out Mass Spectrometric Identification after purification.
SILAC is typically used for tissue culture cellss, and the application on nematicide starts from the Mark of University of Dundee of Britain
Larance et al., they have modified SILAC methods, by using a kind of heavy lysine and the large intestine of heavy arginine labelling
Bacillus feeds nematicide, and so as to both aminoacid with specific marker are brought in nematicide body, they are also using mass spectrum point
Analysis, has mixed heavy arginine and lysine in as a result showing F1 offsprings.Meanwhile, in order to avoid the conversion of arginine-general propylhomoserin
The SILAC labelling peptides segment signal for causing is reduced, and they select Arg-Pro and turn also using the method for RNAi feeding
Required OAT gene orn-1 by its silence during change, so as to reduce the conversion of Arg-Pro.Matter
Spectrum result shows that this method has almost blocked the conversion of Arg-Pro completely, and testing follow-up SILAC can be more efficient
Carry out on ground.
But the SILAC technologies of above-mentioned improvement also have obvious limitation, its situation is as follows：
1st, it is cumbersome.The quantitative flow processs of SILAC of Mark Larance include：Simultaneously labelling → albumen is carried for escherichia coli culture
Take → immunoprecipitation → electrophoretic separation → digestions → Mass Spectrometer Method → determine that escherichia coli are fully labeled after, using labelling
Escherichia coli culture nematicide → elegans proteins extraction → immunoprecipitation → electrophoretic separation → digestions → Mass Spectrometer Method afterwards →
Detection by quantitative, complex operation, labeling effciency are low, it has not been convenient to implement.
2nd, experimentation cost is high.SILAC reagents are expensive, will obtain enough nematicides, just need substantial amounts of SILAC examinations
Agent, reagent cost are very high；And the technology such as electrophoresis required in test and Mass Spectrometer Method, either purchase of equipment still purchase
Related service is bought, the expense of great number is required for.
3rd, the test period is long.As SILAC needs to add labelling during being cultivated, so needing long
Time could compare thoroughly labelling, and in general, SILAC is tested from start to end, including data analysiss, takes around 20
By 25 days.And above-mentioned test or multiple labelling, it is meant that to repeatedly test, process step is very loaded down with trivial details, be taken
Between be also multiplied.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, there is provided a kind of quantitative and spike for nematode protein
N15Cold labeling method, the method can be applicable to the Protein quantitative analysis of nematicide, and easy to operate, experimentation cost is low,
Test period is short, beneficial to people efficiently, low cost Proteomic analysis are carried out to a large amount of samples.
For achieving the above object, technical scheme provided by the present invention is：It is a kind of to be used for nematode protein quantitatively and spike
N15Cold labeling method, first, using yeast by N15Inorganic salt changes into bioprotein, when Yeast Cultivation algebraically
Reach 6 more than generation, it is all N which extracts albumen more than 98%15Albumen, then yeast is ground, protein contained by release thalline, Jing
Extraction purification is made containing N15Tropina powder, using containing N15Tropina powder replace conventional N14Albumen, for training
The symbiosis thalline of various nematicides or nematicide is supported, for nematode protein labelling is quantitative and spike provides the N of specificity15Isotope mark
Note.
Protocol step provided by the present invention is as follows：
1) prepare containing only N according to formula as below15N is not contained (14) inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
2) use high-pressure sterilizing pot, by step 1) in medium sterilization；
3) after step 2) in culture medium be cooled to room temperature after, picking single bacterium colony yeast be inoculated with the medium, place
Cultivate in the environment of proper temperature；
4) treat that yeast culture algebraically reaches 6 more than generation, take out culture medium and be centrifuged.Cast out supernatant after centrifugation, protect
Stay bacterial sediment；
5) pour appropriate physiological saline solution cleaning thalline into, centrifugation is cast out supernatant, obtains yeast paste；
6) after yeast paste is weighed, yeast mixture is configured to sterilized water so as to self-dissolving；
7), after self-dissolving terminates, using Ultrasound Instrument smudge cellses, then in yeast mixture trace albumin enzyme is added to be digested；
8), after enzymolysis terminates, yeast mixture heating enzyme denaturing is lived；
9) yeast mixture recentrifuge is separated, obtains supernatant, cast out precipitation；
10) yeast supernatants elder generation pre-freeze, after condensing into solid, is dried to obtain N in placing into freezer dryer15Yeast protein
Powder；
11)N15The preparation of nematicide feedstuff：According to the nutritional mode of nematicide, different feed formulas are selected.If cultivating
Nematicide directly can absorb nourishment from environment material, can be by step 10) in made by N15Yeast protein powder is used as feedstuff
Base stock, replace conventional N14Albumen is used to cultivate, and adds the inorganic salt material without N, makes final N15Nematicide
Feedstuff；If nematicide to be cultivated needs symbiosis thalline or microorganism of ingesting, available step 10) in made by N15Yeast protein
Powder makes microbiological culture media, turns out containing N15The microorganism of specific marker, then it is quantitative as nematode protein labelling
The N of specificity is provided with spike15Isotope marks.
In step 3) in, yeast is by the N in culture medium15Inorganic salt changes into bioprotein, when Yeast Cultivation algebraically reaches 6
More than generation, it is all N which extracts albumen more than 98%15Albumen, and according to the final nematicide Feed selection prepared in practical operation
Different yeast and containing only N15Minimal media based formulas.
The present invention compared with prior art, has the advantage that and beneficial effect：
1st, it is simple to operate.The operation being related in the present invention is all relatively easy and easy left-hand seat, and operator Jing simple trainings are just
Whole flow process can be completed.
2nd, experimentation cost is low.Agents useful for same of the present invention and material are general reagent and material mostly, low cost；Institute of the present invention
The equipment for using is simple and easy to get, operates without the need for special messenger, saves high cost of equipment and cost of labor.
3rd, the test period is short.The method labeling effciency that refers in the present invention is high, and can be carried with multiple batches of while carry out significantly
High efficiency, time-consuming, in general, experiment from start to end, including data analysiss, takes around 8 to 15 days.
Specific embodiment
With reference to multiple specific embodiments, the invention will be further described.
1 (N of embodiment15Around the preparation of nematicide feedstuff)
The N of the and spike quantitative for nematode protein described in the present embodiment15Cold labeling method, specifically
N15The preparation method of cold labeling nematicide feedstuff, comprises the following steps：
1) prepare containing only N according to formula as below15N is not contained (14) inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
2) use high-pressure sterilizing pot, by step 1) in medium sterilization；
3) after step 2) in culture medium be cooled to room temperature after, picking single bacterium colony yeast be inoculated with the medium, place
Cultivate in the environment of proper temperature；
4) treat that yeast culture algebraically reaches 6 more than generation, take out culture medium and be centrifuged.Cast out supernatant after centrifugation, protect
Stay bacterial sediment；
5) pour appropriate physiological saline solution cleaning thalline into, centrifugation is cast out supernatant, obtains yeast paste；
6) after yeast paste is weighed, yeast mixture is configured to sterilized water so as to self-dissolving；
7), after self-dissolving terminates, using Ultrasound Instrument smudge cellses, then in yeast mixture trace albumin enzyme is added to be digested；
8), after enzymolysis terminates, yeast mixture heating enzyme denaturing is lived；
9) yeast mixture recentrifuge is separated, obtains supernatant, cast out precipitation；
10) yeast supernatants elder generation pre-freeze, after condensing into solid, is dried to obtain N in placing into freezer dryer15Yeast protein
Powder；
N1Around the preparation of nematicide feedstuff：Directly can absorb nourishment from environment material around nematicide, using glucose yeast egg
White culture medium, preparation method are as follows：
The glucose of 1～3g is weighed, and water dissolution is distilled with 20～40ml；The N of 1～2g is taken in addition15Yeast protein powder, uses
20～40ml distills water dissolution.After the sterilization of liquids after dissolving, two kinds of liquid equal-volume mixing are protected in being put into refrigerator (4 DEG C)
Deposit, processed 5 minutes with ultrasonic oscillator using front.
Culture medium is poured in culture dish, liquid level is about 1cm, add around nematicide, culture dish is placed in into constant temperature culture
Cultivate in case around nematicide, growing state and number change of the timing observation daily around nematicide, per 3 days the culture medium of supplementary 1ml with
Ensure around nematicide normal growth.
2 (N of embodiment15The preparation of Caenorhabditis elegans feedstuff)
The present embodiment makes culture N as different from Example 115Caenorhabditis elegans feedstuff, due to Caenorhabditis elegans
By escherichia coli job of ingesting, accordingly, it would be desirable to use N15Yeast protein powder culture escherichia coli, as Caenorhabditis elegans
Food.Wherein, prepare containing only N according to formula as below15N is not contained (14) inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
Thereafter N is obtained according to technique productions same as Example 115Yeast protein powder.
Using N15Yeast protein powder prepares colibacillary culture medium, cultivates escherichia coli more than 6 more than generation, for feeding
Foster Caenorhabditis elegans.
Comprise the following steps that：
Escherichia coli culture medium：Weigh 2～3gNaCl, 2～3gN15Yeast protein powder, 15～17g agar is dissolved in 500～
In the distilled water of 600ml, separately take 10g glucoses and be dissolved in 500～600ml distilled water, after pressure sterilizing, two kinds of liquid are mixed
Close, make Escherichia coli culture medium.
M9 buffer：Contain 13～15gNa in every liter of buffer2HPO4·12H2O (or 6g Na2HPO4), 2～3g
KH2PO4, 4～5g NaCl, 0.2～0.3gMgSO4·7H2O, suitable matching while using.
Caenorhabditis elegans are cleaned with M9 buffer, 8～10s is centrifuged with the rotating speed of 8000～10000r/min, abandons
Clear liquid, precipitate with M9 buffer it is resuspended after be centrifuged again, abandon supernatant.200 μ l precipitate are inoculated into respectively and scribble large intestine bar
In the culture medium of bacterium, after putting 14～16 DEG C of biochemical cultivation case culture culture about 72h, i.e., in visible culture medium, there are a large amount of adults and children
Worm.
3 (N of embodiment15The preparation of soil nematodess feedstuff)
The present embodiment makes culture N as different from Example 115Soil nematodess feedstuff, soil nematodess can be ingested yeast,
Wherein, according to according to formula as below prepare containing only N15N is not contained (14) inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
Thereafter according to technique same as Example 1, to step 5) when obtain N15Yeast, subsequently by formula as below
Prepare culture medium culturing, the feedstuff as soil nematodess.
Comprise the following steps that：
Glucose yeast bacterium culture medium is prepared：0.8～1g of glucose is weighed, is put in the conical flask of 50mL dryings, is added
15～20mL distilled water, vibration, promotes glucose to be dissolved completely in distilled water, after high pressure steam sterilization, is cooled to room temperature, then
0.1～0.3g of active yeast is added in conical flask, in putting 18～22 DEG C of calorstats, about 24h is cultivated.
Glucose yeast 13～15mL of bacterium culture medium is measured with graduated cylinder, is placed in 90mm (25mL) culture dish, is divided thereto
Not Jia Ru 4～5mL sterilized water, to reduce the average density of nematicide in culture fluid.Last each addition soil nematodess about 200, use
Culture dish is sealed by preservative film, is cultivated under the conditions of being placed in 23～25 DEG C.
Embodiment described above is only the preferred embodiments of the invention, not limits the practical range of the present invention with this, therefore
The change made by all shapes according to the present invention, principle, all should cover within the scope of the present invention.
Claims (2)
1. a kind of N of and spike quantitative for nematode protein15Cold labeling method, it is characterised in that：First, utilize
Yeast is by N15Inorganic salt changes into bioprotein, and when Yeast Cultivation algebraically reaches 6 more than generation, which extracts albumen more than 98% is all
N15Albumen, then yeast is ground, is made containing N15Tropina powder, using containing N15Tropina powder replace it is conventional
N14Albumen, for cultivating the symbiosis thalline of various nematicides or nematicide, quantitatively provides specifically with spike for nematode protein labelling
The N of property15Isotope marks；Which comprises the following steps：
1) prepare containing only N according to formula as below15Inorganic medium：
In 1 liter of distilled water, comprising following component by mass percentage：
2) use high-pressure sterilizing pot, by step 1) in medium sterilization；
3) after step 2) in culture medium be cooled to room temperature after, picking single bacterium colony yeast inoculation cultivate in the medium；
4) treat that yeast culture algebraically reaches 6 more than generation, take out culture medium and be centrifuged, after centrifugation, cast out supernatant, retain bacterium
Body is precipitated；
5) pour physiological saline solution cleaning thalline into, centrifugation is cast out supernatant, obtains yeast paste；
6) after yeast paste is weighed, yeast mixture is configured to sterilized water so as to self-dissolving；
7), after self-dissolving terminates, using Ultrasound Instrument smudge cellses, then in yeast mixture protease is added to be digested；
8), after enzymolysis terminates, yeast mixture heating enzyme denaturing is lived；
9) yeast mixture recentrifuge is separated, obtains supernatant, cast out precipitation；
10) yeast supernatants elder generation pre-freeze, after condensing into solid, is dried to obtain N in placing into freezer dryer15Yeast protein powder
End；
11)N15The preparation of nematicide feedstuff：According to the nutritional mode of nematicide, different feed formulas are selected, if line to be cultivated
Worm directly can absorb nourishment from environment material, then by step 10) in made by N15Base of the yeast protein powder as feedstuff
This raw material, replaces conventional N14Albumen is used to cultivate, and adds the inorganic salt material without N, makes final N15Nematicide is raised
Material；If nematicide to be cultivated needs symbiosis thalline or microorganism of ingesting, step 10 can be used) in made by N15Yeast protein
Powder makes microbiological culture media, turns out containing N15The microorganism of specific marker, then it is quantitative as nematode protein labelling
The N of specificity is provided with spike15Isotope marks.
2. the N of a kind of and spike quantitative for nematode protein according to claim 115Cold labeling method,
It is characterized in that：In step 3) in, yeast is by the N in culture medium15Inorganic salt changes into bioprotein, when Yeast Cultivation algebraically reaches
To 6 more than generation, it is all N which extracts albumen more than 98%15Albumen, and according to the final nematicide feedstuff choosing prepared in practical operation
Select different yeast and containing only N15Minimal media based formulas.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611126025.1A CN106538482A (en) | 2016-12-09 | 2016-12-09 | A kind of N of and spike quantitative for nematode protein15Cold labeling method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611126025.1A CN106538482A (en) | 2016-12-09 | 2016-12-09 | A kind of N of and spike quantitative for nematode protein15Cold labeling method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106538482A true CN106538482A (en) | 2017-03-29 |
Family
ID=58396603
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611126025.1A Pending CN106538482A (en) | 2016-12-09 | 2016-12-09 | A kind of N of and spike quantitative for nematode protein15Cold labeling method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106538482A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119321A (en) * | 2016-06-30 | 2016-11-16 | 肖传乐 | A kind of for bioprotein quantitatively and the N of spike15cold labeling method |
-
2016
- 2016-12-09 CN CN201611126025.1A patent/CN106538482A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119321A (en) * | 2016-06-30 | 2016-11-16 | 肖传乐 | A kind of for bioprotein quantitatively and the N of spike15cold labeling method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101402923A (en) | Plant lactobacillus M1-UVs29 and uses thereof | |
| CN101240016A (en) | Shark protein antihypertensive peptide and preparation method and application thereof | |
| CN107568434A (en) | Bei Laisi bacillus produces the preparation method and applications of protease fermented liquid | |
| CN103467584B (en) | The acquisition of a kind of prokaryotic gene engineering heterozygosis cationic antibacterial peptide CC and fermentation process thereof | |
| CN107095312A (en) | A kind of krill polypeptide formulations with reducing blood lipid ability and preparation method thereof | |
| CN106479944B (en) | A kind of bacillus amyloliquefaciens and its application | |
| CN114164145B (en) | Brevibacillus borstelensis, neutral protease and application thereof | |
| Fu et al. | Flocculation activity of carp protamine in microalgal cells | |
| CN101974460B (en) | Ocean source Bacillus barbaricus SCSIO 02429 and method for preparing squid small peptide by using same | |
| Wang et al. | The optimization of fermentation conditions and enzyme properties of Stenotrophomonas maltophilia for protease production | |
| CN104711217B (en) | A kind of biologically active peptide of new promotion lactobacter growth | |
| CN108410767A (en) | Bacillus natto and its application in fermentation Ruditapes philippinarum prepares active polysaccharide | |
| CN106538482A (en) | A kind of N of and spike quantitative for nematode protein15Cold labeling method | |
| CN106119321A (en) | A kind of for bioprotein quantitatively and the N of spike15cold labeling method | |
| CN103283943B (en) | Compound microbiological preparation for fermenting soybean meal and applications of preparation | |
| WO2016161549A1 (en) | Method for producing aerobic single cell protein by using autolysis process | |
| CN106480091B (en) | A kind of method that PEP-1 peptide concatermer mediates green fluorescent protein transduction buffalo embryo | |
| CN108715828A (en) | Vomitoxin induces the method for building up of the procedural Necrosis Model of chitterlings epithelial cell | |
| CN105483201B (en) | Fermentation preparation method of whey antioxidant peptide | |
| CN105200107B (en) | The extracting method of Onchidium struma muscle crude protein | |
| CN105200102B (en) | Method for extracting glutathione from candida utilis fermentation liquor | |
| CN108660086A (en) | A kind of fish blood agar culture-medium and preparation method thereof | |
| CN102928581B (en) | External measurement method for transgenic plant material digestibility | |
| CN103468770A (en) | Preparation method of lactobacillus growth promoting peptide in broad bean protein hydrolysate | |
| RU2245362C2 (en) | Nutrient medium for culturing plague microorganism vaccine strain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170329 |