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 PDF

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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
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yeast
nematicide
albumen
quantitative
spike
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肖传乐
陈龙
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Guangzhou Weiyin Biology Technology Co ltd
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • C12N1/00Microorganisms, 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
    • C12N1/14Fungi; Culture media therefor
    • C12N1/16Yeasts; Culture media therefor

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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

A kind of N of and spike quantitative for nematode protein15Cold labeling method
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.
CN201611126025.1A 2016-12-09 2016-12-09 A kind of N of and spike quantitative for nematode protein15Cold labeling method Pending CN106538482A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
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

Patent Citations (1)

* Cited by examiner, † Cited by third party
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

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Application publication date: 20170329