CN1523337A - Protein and virus quick-speed concentration method - Google Patents

Protein and virus quick-speed concentration method Download PDF

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Publication number
CN1523337A
CN1523337A CNA031294081A CN03129408A CN1523337A CN 1523337 A CN1523337 A CN 1523337A CN A031294081 A CNA031294081 A CN A031294081A CN 03129408 A CN03129408 A CN 03129408A CN 1523337 A CN1523337 A CN 1523337A
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solvent
protein
phase
precipitation
aggegation
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CN1303413C (en
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余伟明
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GENE ERA BIOTECH CO Ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

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Abstract

The invention relates to a protein and virus quickly-enriching method, composed of the steps: 1), flocculating protein: diluting the sample to release protein and virus particles, and adding in agglomerant to make protein and virus particles form agglutinating matter; 2), making separate-phase deposition: adding in phase-separating agent to deposit protein and virus particles and cells between phases; 3), making secondary deposition: adding in depositing solvent to deposit them to the tube bottom; 4), eliminating clear solution and retaining deposit: eliminating top clear solution, washing, centrifuging, re-eliminating top clear solution, re-centrifuging, and then absorbing all the top clear solution to get the enriched solution. It can be finished within 10-20 minutes. It can concentrate by about 100 times, making detection rate and accuracy of protein and virus raised by 100-1000 times. Its enriching speed is specially fast.

Description

Protein, Fast method of virus concentration
Technical field
The present invention relates to a kind of protein, Fast method of virus concentration, be fit to various samples (comprising gargle, culture supernatant, urine, ight soil extract, serum, expectoration extract, nose swab extract, throat swab extract) from the human or animal, and the method for fast enriching and partial purification protein, virion in other animals and plants sample.
Background technology
In the sample that assay office needs, the quantity of protein, virus differs greatly.It is just few to cause the viral load of ill people in clinical sample such as the urine of the different courses of disease, ight soil, serum and latent period patient as poison due to illness.The present method of inspection all has certain restriction to the sample volume size, can not extract protein, viral RNA from the sample of large volume, usually causes false-negative testing result.Chinese patent literature CN1285878 discloses a kind of " method and apparatus that is used for enrichment and searching microbiological specimens ", this method is finished in syringe or coordinator the enrichment culture of microorganism, because conventional cultivation cycle is longer, the needs of incompatibility fast detecting.Protein, virion are carried out concentrating fast and the partial purification technology, and Chinese medicine document and Chinese patent literature are not seen open report.
Summary of the invention
The technical issues that need to address of the present invention are, overcome the existing deficiency of prior art, provide a kind of from bulk sample, protein, virion concentrated and partial purification speed fast, the protein that enrichment factor is high, Fast method of virus concentration improve protein, viral recall rate and accuracy greatly.
Protein of the present invention, Fast method of virus concentration is characterized in that being made of following operation steps:
1) albumen flocculation: dilute sample discharges protein, virion, adds the aggegation solvent, makes protein, virion and cell form agglutinator;
2) phase-splitting precipitation: the bonus point phase solvent, arrive protein, virion and cell precipitation alternate;
3) secondary precipitate: add precipitation solvent, protein, virion and cell precipitation are arrived the pipe end;
4) abandon stay clearly heavy: abandon supernatant, washing, centrifugal, abandon supernatant again, centrifugal again, exhaust supernatant and get pregnant solution.
Finished at 10～20 minutes as the aforesaid operations step 1.
Adding aggegation solvent in the sample that contains protein, virion makes protein, virion be dewatering state or part is saltoutd and is protected.After add dividing phase solvent, form two phase systems immediately, the moisture of following phase by extracting to last phase, and the aggegation solvent of phase almost reaches 100% saturation degree down, protein is all incompatible in two-phase, is formed precipitation and is distributed in alternately and separate out effectively, and is deposited to alternate in centrifugal process.Add precipitation solvent and eliminate two-phase, reclaim protein, virion through centrifugation again.Simultaneously protein, virion also obtain partial purification (as the bacterium in the ight soil, swill and RT-PCR[reverse transcription-polymerize chain reaction style reaction] mortifier all be removed), and detection sensitivity is further improved.Behind the residual salt ion of 50% ethanol (or isopropyl alcohol) washing removal, protein, virion precipitation promptly can be used for the purifying of protein, viral RNA.In addition, divide phase solvent can directly kill virus, reduce the danger of laboratory infection; Aggegation solvent and branch phase solvent all can suppress the RNA enzymatic activity, prevent the degraded of RNA.
Described protein aggegation solvent is: the ammonium sulfate of 90～100% saturation degrees;
Phase solvent was in described minute: isopropyl alcohol: absolute ethyl alcohol=3～10: 1;
Described protein precipitation solvent is: 10～30% Macrogol 6000s;
If in protein aggegation solvent, be added with 80mM NaH 2PO 4With 80mM Na 2HPO 4, then concentration effect is better to be added with glacial acetic acid 2～5ml/L in minute phase solvent.This can reduce pH value, impels coagula consolidation more, is easy to precipitation and reclaims.
Use after described minute phase solvent and the hydro-extractor precooling, its phase-splitting better effects if, precooling is not higher than 10 ℃ usually.
According to different sample volumes, it and three kinds of solvents generally have following proportionate relationship, i.e. sample volume: aggegation solvent volume: phase-splitting solvent volume: precipitation solvent volume=5: 2～3: 6～8: 5～6; Proportionate relationship commonly used is 5: 2: 6.5 in the operation: 5.
For standard operation, each sample volume all needs quantitatively, and meets the aforementioned proportion relation.Aggegation solvent dosage is 2ml among the I class sample 5ml; Dividing the phase solvent dosage is 6.5ml, and the precipitation solvent dosage is 5ml; Aggegation solvent dosage is 0.8ml among the II class sample 2ml; Dividing the phase solvent dosage is 2.5ml, and the precipitation solvent dosage is 2ml.Above-mentioned I class sample is meant: gargle, culture supernatant, urine, ight soil extract; II class sample is meant: serum, expectoration extract, nose swab extract, throat swab extract.
The inventive method reasonable idea, operation is simple, from bulk sample, protein, virion concentrated and partial purification speed fast, can concentrate about 100 times, make protein, viral recall rate, accuracy rate improve 100～1000 times.Particularly enrichment speed is fast, and is significant for control highly infective viral prevalence.
Embodiment
Below by embodiment, technical scheme of the present invention is described in further detail.
One, enrichment experiment is prepared:
Enrichment protein, virion need be done following preparation from all samples:
1. the refuse cylinder that contains thimerosal.
2. suction pipe.
3. 4 ℃ of precoolings divide phase solvent.
4. 50% ethanol, 4 ℃ of precoolings.
5. hydro-extractor is chilled to 4 ℃ in advance.
Enrichment protein, virion are done following preparation respectively from various samples:
1. gargle, culture supernatant, urine: 50ml centrifuge tube.
2. serum: 15ml centrifuge tube.
3. ight soil: 50ml centrifuge tube; Physiological saline or PBS.
4. expectoration: 50ml centrifuge tube; Physiological saline or PBS, adding acetylcysteine (acetylcysteine) to final concentration before the use is 1%.
5. nose swab: 15ml centrifuge tube; Physiological saline or PBS.
6. throat swab: 15ml and 50ml centrifuge tube; Physiological saline or PBS.
Wherein, physiological saline: 0.9%NaCl; PBS:1.44g Na 2HPO 42H 2O (or 1.15g Na 2HPO 4), 0.2g KH 2PO 4, 0.2g KCl, 8.0g NaCl adds water to 1 liter, regulates pH to 7.2, autoclaving.
This routine protein, viral aggegation solvent are the ammonium sulfate of 96% saturation degree; Dividing phase solvent is isopropyl alcohol: absolute ethyl alcohol=3: 1; Protein, viral precipitation solvent are 10% polyglycols 6000.
Two, operation steps
This operation steps is that example designs by the sample with lower volume:
A.5ml gargle, culture supernatant, urine, ight soil extract;
B.2ml serum, expectoration extract, nose swab extract, throat swab extract.
(1) from I class sample---enrichment protein, virion gargle, culture supernatant, urine, the ight soil extract:
1, albumen flocculation: discharge protein, virion, add the aggegation solvent
A. gargle, culture supernatant, urine are got the 5ml sample, change in the 50ml centrifuge tube, add 2ml aggegation solvent.
B. fresh excreta
(1) takes by weighing 2 gram samples, change in the 50ml centrifuge tube.
(2) add 6ml physiological saline or PBS, stir with suction pipe and smash to pieces, piping and druming thoroughly mixes (noticing that it is outer not splash pipe) repeatedly again, fully discharges protein, virion.4 ℃, centrifugal 5 minutes of 5000 * g.
(3) draw the 5ml supernatant, change in the 50ml centrifuge tube, add 2ml aggegation solvent.
2, phase-splitting precipitation: add the branch phase solvent of 4 ℃ of precoolings of 6.5ml, blow and beat mixing repeatedly with suction pipe, room temperature leaves standstill that violent jolting mixes after 5 minutes.4 ℃, centrifugal 5 minutes of 〉=5000 * g arrives protein, virion and cell precipitation alternate.
Attention: divide phase solvent energy kill proteins, virus.For preventing laboratory pollution, acutely do not jolt earlier after adding the aggegation solvent and dividing phase solvent, in order to avoid spill, pollute.Should blow and beat mixing with suction pipe, and violent again jolting mixes after waiting for 5 minutes kill proteins of phase-splitting solvent action, virion.
3, secondary precipitate: add the 5ml precipitation solvent, mix, 4 ℃, centrifugal 3 minutes of 〉=5000 * g arrives the pipe end with protein, virion and cell precipitation.
4, abandon stay clearly heavy: be inverted centrifuge tube and abandon supernatant, add 50% ethanol of 4 ℃ of precoolings of 5ml, rotate centrifuge tube gently and wash the solution (stirring pipe end precipitation) that sticks on the tube wall.4 ℃, centrifugal 3 minutes of 〉=5000 * g.Be inverted centrifuge tube and abandon supernatant, briefly centrifugal, exhaust residual supernatant with water dropper.Note, do not siphon away pipe end precipitation.
Protein, the virion of precipitation should be used for the RNA purifying immediately, or put-20 ℃ of preservations.
(2) from from II class sample---enrichment protein, virion serum, expectoration extract, nose swab extract or the throat swab extract
1, albumen flocculation: discharge protein, virion, add the aggegation solvent
A. serum
Get 2ml serum, add in the 15ml centrifuge tube, add 0.8ml aggegation solvent.
B. expectoration
(1) collects expectoration, change in the 50ml centrifuge tube.
(2) add physiological saline or the PBS that 3ml contains 1% acetylcysteine (acetylcysteine), 3～10 minutes (the vortex duration of oscillation is decided on the viscosity of expectoration) of vortex vibration fully discharges protein, virion.Centrifugal 5 minutes of 〉=5000 * g.
(3) draw the 2ml supernatant, change in the 15ml centrifuge tube, add 0.8ml aggegation solvent.
C. nose swab
(1) nose swab is changed in the 15ml centrifuge tube.
(2) add 3ml physiological saline or PBS, the vortex vibration fully discharged protein, virion in 1 minute.Clamp nose swab with pincers, be pressed in gently and extrude remaining extract on the tube wall, careful taking-up (not being infected with the outlet of pipe) is discarded in the refuse cylinder that contains thimerosal.
(3) draw 2ml solution, change in the 15ml centrifuge tube, add 0.8ml aggegation solvent.
D. throat swab
(1) throat swab is changed in the 50ml centrifuge tube.
(2) add 3ml physiological saline or PBS, stir throat swab repeatedly and fully discharged protein, virion in 1～2 minute.Throat swab is pressed on the tube wall gently, extrudes remaining extract.The taking-up throat swab also is discarded in the refuse cylinder that contains thimerosal.
(3) draw 2ml solution, change in the 15ml centrifuge tube, if the not enough 2ml of liquor capacity, add physiological saline or PBS supplies 2ml; Add 0.8ml aggegation solvent.
2, phase-splitting precipitation: add the branch phase solvent of 4 ℃ of precoolings of 2.5ml, blow and beat mixing repeatedly with suction pipe, room temperature leaves standstill that violent jolting mixes after 5 minutes.4 ℃, centrifugal 5 minutes of 〉=5000 * g arrives protein, virion and cell precipitation alternate.
3, secondary precipitate: add the 2ml precipitation solvent, mix, 4 ℃, centrifugal 3 minutes of 〉=5000 * g arrives the pipe end with protein, virion and cell precipitation.
4, abandon stay clearly heavy: the same.

Claims (8)

1, a kind of protein, Fast method of virus concentration is characterized in that being made of following operation steps:
1) albumen flocculation: dilute sample discharges protein, virion, adds the aggegation solvent, makes protein, virion and cell form agglutinator;
2) phase-splitting precipitation: the bonus point phase solvent, arrive protein, virion and cell precipitation alternate;
3) secondary precipitate: add precipitation solvent, protein, virion and cell precipitation are arrived the pipe end;
4) abandon stay clearly heavy: abandon supernatant, washing, centrifugal, abandon supernatant again, centrifugal again, exhaust supernatant and get pregnant solution.
2, protein according to claim 1, Fast method of virus concentration is characterized in that:
Described protein aggegation solvent is: the ammonium sulfate of 90～100% saturation degrees;
Phase solvent was in described minute: isopropyl alcohol: absolute ethyl alcohol=3～10: 1;
Described protein precipitation solvent is: 10～30% polyglycols 6000;
3, protein according to claim 2, Fast method of virus concentration is characterized in that: also contain 80mM NaH in the described protein aggegation solvent 2PO 4With 80mM Na 2HPO 4Also has glacial acetic acid 2～5ml/L in the described minute phase solvent;
4,, it is characterized in that described minute phase solvent and the equal precooling of hydro-extractor according to claim 2 or 3 described protein, Fast method of virus concentration.
5, according to claim 1 or 2 or 3 described protein, Fast method of virus concentration, it is characterized in that described sample volume: the aggegation solvent volume: the phase-splitting solvent volume: precipitation solvent volume=5: 2～3: 6～8: 5～6.
6, protein according to claim 4, Fast method of virus concentration is characterized in that described sample volume: the aggegation solvent volume: the phase-splitting solvent volume: precipitation solvent volume=5: 2～3: 6～8: 5～6.
7, protein according to claim 5, Fast method of virus concentration is characterized in that: aggegation solvent dosage is 2ml in I class sample 5ml; Dividing the phase solvent dosage is 6.5ml, and the precipitation solvent dosage is 5ml; Aggegation solvent dosage is 0.8ml among the II class sample 2ml; Dividing the phase solvent dosage is 2.5ml, and the precipitation solvent dosage is 2ml.
8, protein according to claim 6, Fast method of virus concentration is characterized in that: aggegation solvent dosage is 2ml in I class sample 5ml; Dividing the phase solvent dosage is 6.5ml, and the precipitation solvent dosage is 5ml; Aggegation solvent dosage is 0.8ml among the II class sample 2ml; Dividing the phase solvent dosage is 2.5ml, and the precipitation solvent dosage is 2ml.
CNB031294081A 2003-06-17 2003-06-17 Protein and virus quick-speed concentration method Expired - Lifetime CN1303413C (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103884574A (en) * 2012-12-19 2014-06-25 中国科学院大连化学物理研究所 Integrated protein C-terminal enrichment method
CN104498445A (en) * 2014-12-17 2015-04-08 广东省微生物研究所 Method for efficiently concentrating virus from water body
CN112014198A (en) * 2020-06-09 2020-12-01 中国科学院生物物理研究所 Kit and method for enriching endogenous low-molecular-weight proteins and polypeptides

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Publication number Priority date Publication date Assignee Title
CN86100551A (en) * 1986-04-02 1987-11-25 交通部石油部海洋水下工程科学研究院 The method of purification of superoxide dismutase (SOD)
JP2595216B2 (en) * 1986-12-12 1997-04-02 日清製粉株式会社 Method for producing crystals of α-amylase inhibitor
AT390801B (en) * 1988-07-28 1990-07-10 Immuno Ag METHOD FOR PRODUCING LYS PLASMINOGEN
EP1023078A4 (en) * 1997-10-17 2004-09-29 Harvest Technologies Corp Precipitation of growth-factor-enriched fibrinogen concentrate from platelet rich plasma
CN1070380C (en) * 1997-12-30 2001-09-05 长春市中心血站 Application and prodn. method of HDL preparation
CN1169836C (en) * 2002-04-11 2004-10-06 中南民族大学 Process for separating and purifying haematoglobin by liquid-solid extracting system

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103884574A (en) * 2012-12-19 2014-06-25 中国科学院大连化学物理研究所 Integrated protein C-terminal enrichment method
CN104498445A (en) * 2014-12-17 2015-04-08 广东省微生物研究所 Method for efficiently concentrating virus from water body
CN112014198A (en) * 2020-06-09 2020-12-01 中国科学院生物物理研究所 Kit and method for enriching endogenous low-molecular-weight proteins and polypeptides

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Assignee: Aisjin Biotechnology (Hangzhou) Co.,Ltd.

Assignor: Yu Weiming

Contract fulfillment period: 2004.11.10 to 2023.6.16

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Denomination of invention: Protein and virus quick-speed concentration method

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Effective date of registration: 20151203

Address after: 310052, room 2, building 256, 102-106 Hung Hong Road, Hangzhou, Zhejiang, Binjiang District

Patentee after: GENE ERA BIOTECH CO.,LTD.

Address before: Stone town of Hangzhou city in Zhejiang province 310022 AFP Village City Industrial Park building B2-1 (Weitejie)

Patentee before: Yu Weiming

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