CN110218693A - For extracting reagent combination, kit and the method for excretion body - Google Patents
For extracting reagent combination, kit and the method for excretion body Download PDFInfo
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Abstract
Reagent combination, kit and the method that the invention discloses a kind of for extracting excretion body, are related to field of biomedicine.Reagent combination disclosed by the invention for extracting excretion body comprising: polybrene, polyethylene glycol and glucan.It is combined using the reagent and extracts excretion body, the extraction efficiency and purity of excretion body are higher, a set of reagent, can be suitable for the extraction of serum or blood plasma simultaneously, and easy to operate, not depend on expensive device or reagent consumptive material.
Description
Technical field
The present invention relates to field of biomedicine, in particular to for extracting excretion body reagent combination, kit and
Method.
Background technique
Excretion body (Exosome) is a kind of diameter 30-150nm, and the extracellular vesica with complete membrane structure participates in a variety of
Physiology and pathologic process.Excretion body is considered as a kind of new mechanism of cell-cell communication, allow cell exchanger matter, lipid and
Inhereditary material.The excretion body in past, cell secretion had once been considered to be only participation waste discharge；With high throughput protein group
With the development of genomics, excretion body be widely regarded as short distance between organism inner cell exchanged with long range it is highly conserved
Approach, this cell-cell communication is all very important normal cell and tumour cell.
All eukaryocyte types can secrete excretion body under culture conditions.Internal all body fluid, including blood, saliva
It can isolated excretion body in liquid, cerebrospinal fluid, ascites or even urine.These excretion bodies transmit multi-signal molecule, including
Protein, mRNA, LncRNA, MicroRNA, DNA etc., the molecule carried can be absorbed by other cells.Therefore, excretion body
Biological information can be transferred to flanking cell.This cell-cell communication is directed not only to physiological function, further relates to some diseases
Pathogenesis, including tumour, cardiovascular disease and neurodegenerative disease etc..It is found in disease model at present, excretion body is logical
Crossing molecular information transmitting plays important role.It is the biomarker of disease and pre- that excretion body, which is also increasingly considered to,
Important indicator afterwards has important clinical diagnosis and therapeutic potential.
Application of the excretion body in disease research field in recent years has been carried out extensively.The also existing diversification of layer of excretion body sample, but
Clinical detection sample mainly includes blood plasma, serum etc..Blood plasma, serum as the most common samples sources of clinical diagnosis, and because
Its excretion body rich content, thus the research in terms of clinical application of blood excretion body is being carried out in a deep going way.Serum, blood plasma
Research can be unfolded for the excretion body of various pathological states in sample.
Currently, excretion body extracting method mainly has ultracentrifugation method, excretion body purity is high obtained, but extract effect
Rate is too low, and disengaging time is long, the high requirements on the equipment；Immunomagnetic beads method, the capture for capableing of specificity have protein tag
Excretion body cause with high costs and extraction efficiency to be only only above ultracentrifugation method due to needing lot of antibodies, magnetic bead etc.；
The kit precipitation method, if the relevant serum excretion body of Thermofisher company and SBI company extracts reagent, outside this method
The extraction efficiency of body is secreted compared to other two methods highest, but the purity of excretion body is relatively low, while capturing excretion body, body
The albumen of high abundance can also precipitate therewith in liquid.Since the research of excretion body requires extracting method quickly and simple, RNA isolation kit point
Demand from excretion body gradually increases on the market, and the quality of excretion body directly influences subsequent excretion body nucleic acid, albumen is caught
It obtains and detects, especially purity, impurity and the rate of recovery can all impact excretion body nucleic acid, albumen research.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The reagent combination that the purpose of the present invention is to provide a kind of for extracting excretion body.It is extracted using reagent combination outer
Body is secreted, the extraction efficiency and purity of excretion body are higher.
Another object of the present invention is to provide a kind of for extracting the kit of excretion body.It is extracted using the kit outer
Body is secreted, extraction efficiency and purity can be improved, it is easy to operate, it is practical, it is applicable to the excretion body of blood plasma or serum sample
It extracts.
Another object of the present invention is to provide a kind of methods for extracting excretion body.This method is easy to operate, and has more
High extraction efficiency, extracted excretion body purity are also higher.
The present invention is implemented as follows:
In a first aspect, the reagent combination that the present invention provides a kind of for extracting excretion body comprising: polybrene, poly- second
Two pure and mild glucans.
Preferably, the reagent combination includes following component:
First component comprising polybrene；
Second component comprising polyethylene glycol and glucan；
Optional third component comprising fibrin ferment；
Preferably, the molecular weight of the polyethylene glycol is 7000-9000；
Preferably, the molecular weight of the glucan is 4000-6000.
Further, in some embodiments of the present invention, the polybrene is solid-state polybrene or concentration >=1mg/
The polybrene solution of mL, preferably powder polybrene, purity >=94%；
Preferably, the molecular weight of the polyethylene glycol is 8000；
Preferably, the molecular weight of the glucan is 4000；
Preferably, the Rate activity of the fibrin ferment is 1000U/mg.
Reagent provided by the invention combination can be used for the extraction of excretion body, be applicable to mentioning for serum or plasma sample
It takes, polybrene can be used for removing the big molecular impurity such as albumen in sample；Polyethylene glycol and glucan are used in mixed way, and being used for will
Precipitating is precipitated in excretion body in sample, and excretion body is isolated from sample.It is combined using the reagent and extracts excretion body with more
Extracted excretion purity can be improved in high extraction efficiency.
Second aspect, the present invention provides a kind of kits for extracting excretion body comprising reagent combination as described above；
Preferably, the kit further includes filter；
Preferably, the kit further includes at least one of 0.22 μm of filter and 0.1 μm of filter；
Preferably, the extraction sample of the kit is serum or blood plasma.
Excretion body is extracted in reagent combination, and the extraction efficiency and purity of excretion body are higher, easy to operate, practical, can
It is suitable for the extraction of blood plasma or serum sample simultaneously.
The third aspect, the present invention provides a kind of methods for extracting excretion body comprising following steps:
Polybrene is added into the sample of excretion body to be extracted, obtains the first solution；
Polyethylene glycol and glucan is added simultaneously into the first solution, obtains the second solution.
Preferably, the method is combined using reagent as described above or kit as described above carries out.
Further, in some embodiments of the present invention, the polyethylene glycol in second solution is final concentration of
6%-10% (mass concentration), the final concentration of 8-40mg/mL of glucan；
Preferably, final concentration of 8% of the polyethylene glycol in the second solution, the final concentration of 8-32mg/mL of glucan.
Preferably, the final concentration of 8-32mg/mL, further preferably 16-32mg/ of the glucan in second solution
ML, more preferably 32mg/mL.
The concentration of polyethylene glycol and glucan is to influence the factor of the extraction effect to excretion body, the poly- second two of suitable concentration
Pure and mild glucan is conducive to improve the extraction efficiency of excretion body, of the invention the study found that the final concentration of polyethylene glycol is controlled
Final concentration control for 6%-10%, glucan is 8-32mg/mL, can be by the recovery rate of raising excretion body, it is preferable that control is poly-
The extraction efficiency tool of the final concentration 8% of ethylene glycol, the final concentration of 16-32mg/mL of glucan, excretion body is significantly improved.
Further, in some embodiments of the present invention, the method also includes: it is poly- polyethylene glycol and Portugal is added
After sugar, 30-90min is reacted under the conditions of second solution is placed in 2-8 DEG C.
Further, in some embodiments of the present invention, the method also includes: after the completion of reaction, will be described
The centrifugation of second solution, takes precipitating, is resuspended, obtains the first re-suspension liquid；
Preferably, the condition of centrifugation is as follows: 10000-14000g, is centrifuged 1-2 minutes；
Preferably, it is resuspended and is precipitated using phosphate buffer.
Further, in some embodiments of the present invention, the method also includes: by the first re-suspension liquid mistake
Polyethylene glycol and glucan is added simultaneously into filtrate in filter, and after mixing, centrifugation takes precipitating, and resuspension obtains the of the body containing excretion
Two re-suspension liquids.
Polyethylene glycol and glucan are added again can be such that excretion body precipitates again from solution, can further improve excretion body
Purity.
Preferably, the condition of centrifugation is as follows: 10000-14000g, is centrifuged 1-2 minutes；
Preferably, it is resuspended and is precipitated using phosphate buffer；
Preferably, first re-suspension liquid is filtered using 0.1 μm of filter.The size of excretion body usually exists
The range of 30-150nm is filtered by 0.1 μm of filter, can filter large granular impurity, is conducive to improve gained excretion body
Purity.
Further, in some embodiments of the present invention, the method also includes:
Before polyethylene glycol and glucan is added, 30-90min is reacted under the conditions of first solution is placed in 2-8 DEG C；
Preferably, the final concentration of 1-4mg/mL of polybrene in first solution；
It is highly preferred that the final concentration of 2mg/mL of the polybrene in first solution.
Further, in some embodiments of the present invention, the method also includes: before polybrene is added, will
The sample filtering；
Preferably, the sample is filtered using 0.22 μm of filter；It is filtered, can be filtered by 0.22 μm of filter
Large granular impurity is conducive to the purity for improving gained excretion body.Preferably, before filtering the sample, the method also includes:
Use sample described in normal saline dilution；
Preferably, the sample is serum or blood plasma；
Preferably, when the sample is blood plasma, the method also includes: it is past filtered after filtering the sample
Fibrin ferment is added in the sample, after mixing, stands 25-35min；
Preferably, the additional amount of fibrin ferment are as follows: sample described in every ml is corresponding to be added 18-22U fibrin ferment.
The method provided by the invention for extracting excretion body is mentioned using the reagent combination of polybrene, polyethylene glycol and glucan
Excretion body is taken, polybrene promotes the polymerization of excretion body, and polyethylene glycol is mixed with glucan promotes excretion body from the precipitation in sample, knot
Large granular impurity in 0.22 μm and 0.1 μm of filter process removal excretion liquid solution is closed, the extraction of excretion body is greatly increased
Efficiency and purity.
Compared to existing supercentrifugation or excretion body extracts kit etc., kit of the invention and method have
Higher extraction efficiency and purity, a set of reagent can be suitable for the extraction of serum or blood plasma simultaneously, and easy to operate, not depend on
Expensive device or reagent consumptive material.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Figure 1A is the excretion bulk concentration and diameter of nano particle follow-up analysis instrument NS300 detection.Glucan working concentration is
8mg/mL, polyethylene glycol working concentration are mass fraction 8%.X-axis represents the range of detection vesica, and from 0-1000nm, Y-axis is represented
The vesica concentration of a certain diameter detected by the sample of detection, unit are number/milliliter, i.e. particles/mL.
Figure 1B is the excretion bulk concentration and diameter of nano particle follow-up analysis instrument NS300 detection.Glucan working concentration is
16mg/mL, polyethylene glycol working concentration are mass fraction 8%.
Fig. 1 C is the excretion bulk concentration and diameter of nano particle follow-up analysis instrument NS300 detection.Glucan working concentration point
Not Wei 24mg/mL, polyethylene glycol working concentration be mass fraction 8%.
Fig. 1 D is the excretion bulk concentration and diameter of nano particle follow-up analysis instrument NS300 detection.Glucan working concentration point
Not Wei 32mg/mL, polyethylene glycol working concentration is mass fraction 8%, Figure 1A-D as the result is shown excretion body quantity with glucan
Working concentration increases and increases.
Fig. 2 is supercentrifugation compared with the method for the present invention is on excretion bulk concentration.Glucan work in the method for the present invention
When making concentration less than 16mg/mL, supercentrifugation is close with the excretion bulk concentration of the method for the present invention, when glucan working concentration exists
When 24-32mg/mL, the excretion bulk concentration that the method for the present invention is extracted is much higher than supercentrifugation.
Fig. 3 is the detection knot for the total serum IgE distribution that Agilent2100 biological analyser obtains different excretion body extracting methods
Fruit is compared.Compared with the method for the present invention, the distribution of nucleic acid fragment is consistent for SBI, QIAGEN RNA isolation kit, is all located at 25-200nt,
But nucleic acid is different.The nucleic acid highest (FU > 8) of the method for the present invention, SBI RNA isolation kit is minimum (FU < 6), QIAGEN
RNA isolation kit is (6 < FU < 8) placed in the middle.
Fig. 4 is the excretion body that the method for the present invention and commercial kit are extracted, and nucleic acid detects actb base by fluorescent PCR
Because obtained Ct value compares, the Ct value average value of the method for the present invention is that the Ct value average value of 23.86, SBI kit is 27.62,
The Ct value average value of QIAGEN kit is 26.28.
Fig. 5 is that protein immunoblot detects excretion body specific proteins CD63, TSG101 and CD9.Supercentrifugation, SBI
The excretion body total protein loading that kit and QIAGEN kit extract is 20mg, and the method for the present invention loading is 10mg.
From the point of view of the result of blot signals quantification of intensities, the excretion body specific proteins total amount of the method for the present invention excretion body enrichment is higher than it
His three kinds of excretion body separating and extracting process.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Reagent and material used by embodiment are as follows:
Glucan: molecular weight 5000 is purchased from Sigma-Aldrich；
Polyethylene glycol: molecular weight 8000 is purchased from Sigma-Aldrich；
Thrombin powder: Rate activity 1000U/mg is purchased from Sigma-Aldrich；
Polybrene powder: purity >=94%.
Embodiment 1
The method provided in this embodiment for extracting excretion body, operates as follows:
1, it is dissolved in PBS phosphate buffer after mixing glucan with polyethylene glycol, is configured to mixed solution；Wherein, it mixes
Closing and controlling the mass concentration of polyethylene glycol in solution is 40%, and glucan concentration is 40mg/mL.
2, the 200 μ L of plasma sample of excretion body to be extracted is settled to 1mL with 0.9% physiological saline.
3, the plasma sample of 1mL constant volume is filtered with 0.22 μm of filter.
4, filtered plasma sample is added 20U fibrin ferment and (uses ddH2O dissolves thrombin powder, and it is molten to be configured to fibrin ferment
Liquid, concentration 1U/ μ L), if serum sample is placed at room temperature for 30min after mixing it is not necessary that fibrin ferment is added.
5, the sample after standing is added polybrene solution and (uses ddH2O dissolves polybrene powder, is configured to storing liquid, concentration
It is 0.2g/mL), make final concentration of 2mg/mL, mixing shakes up, in 4 DEG C of reaction 60min.
6, to after the reaction was completed, the mixed solution of glucan and polyethylene glycol, the volume ratio of sample and mixed solution be added
For 4:1, polyethylene glycol final concentration of 8%, the final concentration of 8mg/mL of glucan；4 DEG C of reaction 60min.
7, to which after the reaction was completed, 12000g, centrifugation 1 minute removes supernatant.
8, gained precipitating is resuspended with 1mL phosphate buffer PBS.
9, acquired solution is filtered with 0.1 μm of filter, and glucan and poly- second is added in filtered solution again
The mixed solution of glycol, mixes well, 12000g, is centrifuged 1 minute, removes supernatant.
10, gained precipitating is resuspended with 200 μ L phosphate buffer PBS, includes excretion body in re-suspension liquid.
11, it detects
The excretion body obtained using nano particle follow-up analysis system NS300 analysis aforesaid operations, the result is shown in Figure 1 A and figure
2。
Embodiment 2
The extracting method of the present embodiment is substantially the same manner as Example 1, and difference is: the glucan concentration in step 1 is
80mg/ml, the final concentration of 16mg/ml of the glucan in step 6.The result is shown in Figure 1 B and Fig. 2.
Embodiment 3
The extracting method of the present embodiment is substantially the same manner as Example 1, and difference is: the glucan concentration in step 1 is
120mg/ml, the final concentration of 24mg/ml of the glucan in step 6.The result is shown in Figure 1 C and Fig. 2.
Embodiment 4
The extracting method of the present embodiment is substantially the same manner as Example 1, and difference is: the glucan concentration in step 1 is
160mg/ml, the final concentration of 32mg/ml of the glucan in step 6.The result is shown in Figure 1 D and Fig. 2.
Comparative example 1
Excretion body is extracted using supercentrifugation, acquired results are shown in Fig. 2.
Supercentrifugation step: by taking 200 microlitres of serum samples as an example.
Step 1: 300g is centrifuged 10min under the conditions of 4 degree.
Step 2: drawing about 190 microlitres of supernatant layer in the first step to new centrifuge tube, under the conditions of 4 degree with pipettor
2000g is centrifuged 10min.
Step 3: drawing about 180 microlitres of supernatant layer in second step to new centrifuge tube, under the conditions of 4 degree with pipettor
10000g is centrifuged 30min.
Step 4: drawing about 170 microlitres of supernatant layer in third step to new centrifuge tube, under the conditions of 4 degree with pipettor
100000g is centrifuged 120min.
Step 5: removing about 160 microlitres of supernatant layer in the 4th step with pipettor, and 90 microlitres of PBS solutions are added again
Suspension excretion body obtains about 100 microlitres of excretion liquid solutions.
Fig. 1 the results show that excretion body obtained by embodiment 1-4 size:
For the excretion body of embodiment 1-4 after NS300 is analyzed, obtained excretion bulk concentration is 10 8-9 number formulary
Magnitude, wherein A is 0.7 × 109, B is 1.1 × 109, C is 3.8 × 109, D is 6.3 × 109, illustrate to work with glucan dense
The increase of degree, the excretion body quantity of separation and concentration dramatically increases from sample, meanwhile, the excretion body diameter average value of A is
The excretion body diameter average value of 131nm, B are 89nm, and the excretion body diameter average value of C is 108nm, and the excretion body diameter of D is average
Value is 108nm, illustrates that polyethylene glycol and glucan mixed liquor can efficiently separate enrichment excretion body from sample.
Fig. 2 the results show that compared to existing supercentrifugation, the extracted excretion bulk concentration of the method for embodiment 1-4 compared with
The extracted excretion body content of height, especially embodiment 3 and 4 is much higher than comparative example 1.It is raw containing nucleic acid, albumen etc. in excretion body
Object macromolecular, excretion body quantity is more, and large biological molecule content wherein included is abundanter, is beneficial to subsequent to holding in excretion body
The analysis of object, and the excretion body total amount that supercentrifugation obtains is lower, causes large biological molecule content therein low, existing section
It grinds instrument and equipment and situations such as error is big, and measurement is inaccurate often occurs to the detection of the lower nucleic acid of content, albumen, pass through raising
Excretion body total amount and its amplifying nucleic acid, Tot Prot are beneficial to effectively analyze the biological significance of excretion body sample.
Experimental example 1
Compare the method for excretion body nucleic acid extraction kit (SBI, QIAGEN) on the market and above-described embodiment 4 to excretion body
The effect of nucleic acid extraction:
1.5mL plasma sample is divided into three parts, every part of 500 μ L, uses the Exo of SBI company respectivelyExosome
Isolation and RNA Purification kit (for Serum&Plasma), the exoRNeasy of QIAGEN company
The method of Serum/Plasma Midi Kit and embodiment 4, and combine the miRNeasy Micro Kit couple of QIAGEN company
The nucleic acid of blood plasma excretion body extracts, and by 2100 biological analyser of Agilent detect that above-mentioned three kinds of methods obtain it is outer
Secrete the content and distribution of body nucleic acid.As a result as shown in Figure 3.
From Fig. 3, it can be found that method either of the invention either SBI, QIAGEN RNA isolation kit is from excretion body
The nucleic acid of extraction has apparent distribution within the scope of 25-500nt.But it is analyzed from ordinate, peak value of the invention (FU >
8) higher compared to SBI (FU > 4) or QIAGEN (FU > 6) kit, it is upper to illustrate that the nucleic acid of the invention that can be captured is higher than
State two kinds of commercial kits.FU full name is fluorescenceunit, represents fluorescence intensity, in 2100 biology point of Agilent
In analyzer detection, nucleic acid is in advance by fluorochrome label, therefore FU numerical value is bigger in testing result, and the nucleic acid for representing the sample is total
It measures higher.
Experimental example 2
Compare of the invention and SBI, QIAGEN kit and difference is detected to the specific expression of nucleic acid amount of excretion body
With reverse transcription reagent box (Takara company: PrimeScriptTMII 1st Strand cDNA Synthesis
Kit), by the excretion body nucleic acid of the excretion body nucleic acid extracted by the present invention and the extraction of SBI, QIAGEN kit in experimental example 1
Reverse transcription is at cDNA.And actb expression quantity in blood plasma excretion body nucleic acid is detected, reaction system is as follows:
Response procedures are as follows: 1:37 DEG C of step, 5min；2:5 DEG C of step, 10min；3:95 DEG C of step, 10sec；60℃,
1min.Wherein step 3: 40cycles is carried out.
The Ct value of each reacting hole in fluorescent PCR instrument ABI7500 is exported after the reaction was completed, as shown in table 1 below:
Table 1
From table 2 and Fig. 4, the present invention detects the Ct that the Average Ct values that actb gene obtains are less than other two kinds of RNA isolation kits
Value, and the standard deviation of Ct is also below SBI kit and QIAGEN kit test result.According to the principle of fluorescent PCR,
Under the premise of cDNA total amount is consistent, the copy number of the gene contained in the smaller representative sample of Ct value is more, therefore explanation is adopted
With in the extracted excretion body of the method for the present invention contain more nucleic acid.Illustrate excretion body quantity and its Nucleic Acid positive
It closes.
Experimental example 3
The method and goldstandard supercentrifugation, SBI and QIAGEN excretion body extracts kit of comparing embodiment 4 are to excretion
Body specific proteins expression quantity detects difference:
There are many most common specific marker proteins, such as CD9, CD63, CD81, TSG101 for excretion body.Select this hair
The method of bright middle embodiment 4 is compared with supercentrifugation and two kinds of commercial kits, is carried out after extracting serum excretion body
Total protein extraction utilizes protein immunoblot experiment after completing protein B CA quantitatively, detects the table of CD9, CD63 and TSG101
Up to amount.
Specifically: according to SBI excretion body extracts kit (ExoQuick-TC), QIAGEN excretion body extracts kit
(miRCURY Exosome Kits) illustrates, while operating according to supercentrifugation, in conjunction with the method for the embodiment of the present invention 4, divides
Indescribably take the 200 total excretion bodies of μ L serum.Each personal 50 μ L excretion body lysates are resuspended excretion body, and with 50 μ L protein lysates
RIPA cracks excretion body, extracts total protein, ice bath 60min, and 12000g is centrifuged 30min under the conditions of subsequent 4 DEG C, honest and upright and thrifty in absorption
100 μ L quantify excretion body protein into new EP pipe, with BCA kit, and dense with microplate reader detection excretion body total protein
Degree.Loading buffer and denatured by boiling, addition 10 μ g albumen progress immunoblot experiment is added in total protein after quantifying.
As a result as shown in Figure 5: the embodiment of the present invention 4 method compared with other methods in, the label protein of excretion body
It can be detected by immunoblot experiment.But in the comparison of distinct methods, the total amount of every kind of label protein is different,
The result of assay is carried out to label protein total amount using imager are as follows: the excretion body CD9 that extracts by the method for the invention and
CD63 total amount is higher than ultracentrifugation and SBI kit, close with QIAGEN kit results (table 2, Fig. 5-B and Fig. 5-D), but from
From the point of view of Blot experiment result, the excretion body that QIAGEN kit extracts, total protein has apparent non-specific band (Fig. 5-
A), illustrate that the extracted excretion body of the kit has pollution, and use the extracted total protein band of the method for the present invention single, it is pure
It spends higher.CD9 and CD63 is excretion body specific proteins, and the excretion body of QIAGEN RNA isolation kit separation and Extraction passes through protein extraction
And immunoblotting, CD9 and CD63 antibody still have non-specific band outside the position that band should occur, illustrate QIAGEN reagent
The foreign protein of non-excretion body is may included in the vesica that box method is extracted, illustrates that its purity extracting method more of the invention is lower.
In addition, the excretion body TSG101 total amount extracted through the invention is higher than ultracentrifugation, SBI and QIAGEN kit
(Fig. 5-C).When every kind of method carries out immunoblot experiment with same protein total amount, the more explanations of excretion body label protein total amount
The excretion body of extraction is more, and method of the invention has optimal excretion body protein enrichment energy under these experimental conditions
Power has higher extraction efficiency.
Table 2
The excretion body protein of 4 extraction through the embodiment of the present invention, immunoblotting loading Tot Prot are ultracentrifugation
In the case where method, SBI RNA isolation kit and QIAGEN RNA isolation kit half, CD63 and CD9 signal strength connects with QIAGEN kit
Closely, TSG101 signal strength is higher than other three kinds of excretion body extracting methods on year-on-year basis.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Shanghai Sheng Ran Biotechnology Co., Ltd
<120>for extracting reagent combination, kit and the method for excretion body
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<400> 1
gtggacatcc gcaaagac 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<400> 2
ccagggtaca tggtggtg 18
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
ctgtacgcca acacagtgct 20
Claims (10)
1. a kind of reagent for extracting excretion body combines, characterized in that it comprises: polybrene, polyethylene glycol and glucan；
Preferably, the reagent combination includes following component:
First component comprising polybrene；
Second component comprising polyethylene glycol and glucan；
Optional third component comprising fibrin ferment；
Preferably, the molecular weight of the polyethylene glycol is 7000-9000；
Preferably, the molecular weight of the glucan is 4000-6000.
2. the reagent combination according to claim 1 for extracting excretion body, which is characterized in that the polybrene is solid-state
Polybrene or concentration >=1mg/mL polybrene solution, preferably powder polybrene, purity >=94%；
Preferably, the molecular weight of the polyethylene glycol is 8000；
Preferably, the molecular weight of the glucan is 4000；
Preferably, the Rate activity of the fibrin ferment is 1000U/mg.
3. a kind of kit for extracting excretion body, which is characterized in that it includes the described in any item reagent sets of claim 1-2
It closes；
Preferably, the kit further includes filter；
Preferably, the kit further includes at least one of 0.22 μm of filter and 0.1 μm of filter；
Preferably, the extraction sample of the kit is serum or blood plasma.
4. a kind of method for extracting excretion body, which is characterized in that it includes the following steps:
Polybrene is added into the sample of excretion body to be extracted, obtains the first solution；
Polyethylene glycol and glucan is added simultaneously into the first solution, obtains the second solution；
Preferably, the method is using the described in any item reagent combinations of claim 1-2 or kit as claimed in claim 3
It carries out.
5. according to the method described in claim 4, it is characterized in that, polyethylene glycol in second solution it is final concentration of
6%-10%, the final concentration of 8-32mg/mL of glucan；
Preferably, final concentration of 8% of the polyethylene glycol in second solution；
Preferably, final concentration of 8-32mg/mL, the further preferably 16-32mg/mL of the glucan in second solution,
More preferably 32mg/mL.
6. according to the method described in claim 5, it is characterized in that, the method also includes: it is poly- polyethylene glycol and Portugal is added
After sugar, 30-90min is reacted under the conditions of second solution is placed in 2-8 DEG C.
7. according to the method described in claim 6, it is characterized in that, the method also includes: after the completion of reaction, by described
The centrifugation of two solution, takes precipitating, is resuspended, obtains the first re-suspension liquid；
Preferably, the condition of centrifugation is as follows: 10000-14000g, is centrifuged 1-2 minutes；
Preferably, it is resuspended and is precipitated using phosphate buffer.
8. the method according to the description of claim 7 is characterized in that the method also includes: by first re-suspension liquid filter,
Polyethylene glycol and glucan is added simultaneously into filtrate, after mixing, centrifugation takes precipitating, is resuspended, obtains the second weight of the body containing excretion
Suspension；
Preferably, the condition of centrifugation is as follows: 10000-14000g, is centrifuged 1-2 minutes；
Preferably, it is resuspended and is precipitated using phosphate buffer；
Preferably, first re-suspension liquid is filtered using 0.1 μm of filter.
9. according to the method described in claim 4, it is characterized in that, the method also includes:
Before polyethylene glycol and glucan is added, 30-90min is reacted under the conditions of first solution is placed in 2-8 DEG C；
Preferably, the final concentration of 1-4mg/mL of polybrene in first solution；
It is highly preferred that the final concentration of 2mg/mL of the polybrene in first solution.
10. according to the method described in claim 4, it is characterized in that, the method also includes: before polybrene is added, will
The sample filtering；
Preferably, the sample is filtered using 0.22 μm of filter；
Preferably, before filtering the sample, the method also includes: use sample described in normal saline dilution；
Preferably, the sample is serum or blood plasma；
Preferably, when the sample is blood plasma, the method also includes: after filtering the sample, described in filtered
Fibrin ferment is added in sample, after mixing, stands 25-35min；
Preferably, the additional amount of fibrin ferment are as follows: sample described in every ml is corresponding to be added 18-22U fibrin ferment.
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| CN106399250A (en) * | 2015-07-31 | 2017-02-15 | 广州市锐博生物科技有限公司 | Method and kit for separating exosome |
| CN107702957A (en) * | 2017-09-22 | 2018-02-16 | 上海华盈生物医药科技有限公司 | The method and reagent of excretion body are extracted in a kind of serum |
| CN108918228A (en) * | 2018-06-04 | 2018-11-30 | 北京全式金生物技术有限公司 | Excretion body in serum or blood plasma prepares kit and excretion preparation |
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| CN106399250A (en) * | 2015-07-31 | 2017-02-15 | 广州市锐博生物科技有限公司 | Method and kit for separating exosome |
| CN107702957A (en) * | 2017-09-22 | 2018-02-16 | 上海华盈生物医药科技有限公司 | The method and reagent of excretion body are extracted in a kind of serum |
| CN108918228A (en) * | 2018-06-04 | 2018-11-30 | 北京全式金生物技术有限公司 | Excretion body in serum or blood plasma prepares kit and excretion preparation |
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