CN108864274A - A kind of purification process of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag - Google Patents
A kind of purification process of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag Download PDFInfo
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- CN108864274A CN108864274A CN201810847748.3A CN201810847748A CN108864274A CN 108864274 A CN108864274 A CN 108864274A CN 201810847748 A CN201810847748 A CN 201810847748A CN 108864274 A CN108864274 A CN 108864274A
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- growth factor
- blood vessel
- cell growth
- endothelial cell
- vegf165b
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/49—Platelet-derived growth factor [PDGF]
Abstract
The present invention relates to the purification process of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag a kind of, belong to biomedicine technical field.There are heparin binding domain, the region and heparin-agarose resin-bondeds using in the 6th and 7 exon of VEGF165b exon by the present invention, by the pH and ion concentration of optimization, the purification process of the VEGF165b of no purification tag of invention a kind of.The VEGF165b albumen of electrophoresis grade purity can be obtained in purification process through the invention, it is possible to increase its clinical application, and reduce production cost.
Description
Technical field
The present invention relates to the purification process of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag a kind of, belong to
Biomedicine technical field.
Background technique
Blood vessel endothelial cell growth factor VEGF (Vascular endothelial growth factor, VEGF) is blood
The most important driver that pipe generates.And the growth of tumour and the nutrition supply that too busy to get away neonate tumour blood vessel offer is provided, institute
To achieve the purpose that inhibit tumour (Tumor by the nutrition supply for inhibiting angiogenesis that can theoretically cut off tumour
angiogenesis:therapeutic implications.N Engl J Med1971；285:1182-86).
Our commonly called VEGF vascular endothelial growth factor families, refer to VEGFA (Vascular
Endothelial growth factor, VEGF-A), text in referred to as VEGF.It has had always been considered as being one since VEGF self-discovery
Class angiogenic factors.VEGF can produce different hypotypes due to the difference of gene cut mode in transcription, and
VEGF165 is to promote the strongest hypotype of angiogenic activities.Research find VEGF there is also it is other it is a kind of inhibit angiogenesis with
VEGF165b is main endogenous isomers.Research shows that VEGF165b and VEGF165 are closely similar on gene structure, only carboxyl
The amino acid difference of end 6 (is CDKPRR in VEGF165, and in VEGF165b is SLTRKD), but function is completely different.
VEGF165b has the function of the angiogenesis that very strong inhibition VEGF165 is mediated.Therefore, VEGF165b has become tumour and eye
Bottom macular degeneration etc., which relies on angiogenesis, leads to the potential treatment drug of disease.
Since VEGF165b and VEGF165 carboxyl terminal there are 6 amino acid differences, cause during VEGF165b tests in vitro
It cannot be combined with heparin (heparin) and heparan sulfate proteoglycan (HSPG), so when purifying 6 need to be added in N-terminal
His purification tag, and the presence of N-terminal additional amino acid hamper its clinical medical value, and remove its purification tag and increase
Production cost.
Application publication number is (CN：Chinese invention patent application 103451220A) discloses a kind of rh- of not tape label
The method of VEGF165 growth factor, it is the rh-VEGF165 recombinant vector of tape label and not carry out inducing expression by constructing,
The rh-VEGF165 recombinated using anion exchange column purification, this method are not particularly suited for the purifying of VEGF165b.
Summary of the invention
To solve the above problems, the present invention provides a kind of vascular endothelial growth factors of no purification tag
The purification process of VEGF165b.
To achieve the goals above, the technical scheme adopted by the invention is that：
A kind of purification process of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag, this method include as follows
Step：
(1) heparin-agarose affinity column is balanced with equilibrating buffer, and the equilibrating buffer includes Tris-
The concentration of HCl and NaCl, Tris-HCl in equilibrating buffer is 10~100mM, and NaCl is dense in equilibrating buffer
Degree is 1~100mM, and the pH of equilibrating buffer is 8.0~8.5；
(2) VEGF165b sample is loaded to heparin-agarose affinity column；
(3) heparin-agarose affinity column is rinsed with the equilibrating buffer；
(4) obtain VEGF165b albumen with elution, be then concentrated to get.
The concentration of Tris-HCl is preferably 10mM in equilibrating buffer described in step (1), and the concentration of NaCl is preferably
10~100mM.
Further, the concentration of NaCl is preferably 100mM in step (1).
Balance in step (1) is carried out using the equilibrating buffer of 5 times of column volumes.
VEGF165b sample in step (2) is obtained by inducing expression.
The flow velocity of sample loading is 1ml/min in step (2).
Flushing in step (3) uses the equilibrating buffer of 10 times of column volumes.
The time rinsed in step (3) is 20min, flow velocity 1ml/min.
Eluent in step (4) includes Tris-HCl and NaCl, and concentration of the Tris-HCl in eluent is 10mM,
Concentration of the NaCl in eluent is 1mM, and the pH of eluent is 8.0~8.5.
Elution in step (4) is carried out using the eluent of 3 times of column volumes.
Elution time described in step (4) is 20min, flow velocity 1ml/min.
Concentration described in step (4) is concentrated using super filter tube.
The aperture of the super filter tube is 10kDa.
Beneficial effects of the present invention：
The present invention is using there are heparin binding domain, the region and heparin in the 6th and 7 exon of exon of VEGF165b
Agarose resin combines, and by the pH and ion concentration of optimization, provides one kind no purification tag easy to operate
The purification process of VEGF165b.The VEGF165b albumen of electrophoresis grade purity can be obtained in purification process through the invention, increases
Its clinical application is possible, and reduces production cost.
Detailed description of the invention
Fig. 1 is the VEGF165b that the SDS-PAGE electrophoresis showed of non denatured state purifies；
Fig. 2 is the western blot qualification figure for purifying VEGF165b；
Fig. 3 is the HUVEC cell Proliferation that the VEGF165b of purifying inhibits VEGF165 to mediate；
Fig. 4 is the VEGF165 and VEGF165b that the SDS-PAGE electrophoresis showed of non denatured state purifies.
Specific embodiment
Embodiments of the present invention are described further with reference to the accompanying drawing.
Bacterial strain, reagent and the instrument used in following example is specific as follows：
(1) bacterial strain：
Pichia pastoris GS115-the VEGF165b of high VEGF expression 165b is that Henan Province of Xinxiang College of Medical Science synthetic biology is real
Room building is tested, strain saves in the laboratory.
(2) reagent：
Reagent agar powder, agarose, glycine, Tris, SDS, acrylamide, the polyacrylamide, nothing used in test
Amino yeast nitrogen (YNB) etc. is purchased from Shanghai Bioisystech Co., Ltd；Biotin is purchased from Genview company of the U.S.；YPD,
BMGY and BMMY is all made of the use of the formula in Pichia anomala expression kits manuals (Catalog no.K1710-01)；
VEGF165b antibody is purchased from R&D company；HUVEC cell and ECM Endothelial cell culture base (Cat No：1001) it is purchased from the U.S.
Sciencell company；Goat anti-mouse secondary antibody, being purchased from Beijing health is ShiJi Co., Ltd；Tryptone, yeast extract and D- sorb
Alcohol is purchased from OXOID Co., Ltd of Britain.
(3) nitrocellulose filter is purchased from Pall Corporation company of the U.S.；Heparin-agarose resin column (HiTrap
It Heparin) is GE Products.
If the buffer, cleaning solution and culture medium in following example are prepared according to a conventional method without specified otherwise.
Embodiment 1
The inducing expression of height copy genotype yeast VEGF165b
The high VEGF expression 165b yeast strain that inoculation experiments room saves in YPD culture solution, stay overnight by 30 DEG C of shaken cultivations,
Next day draws 100 μ l overnight cultures and is inoculated in 25ml BMGY culture solution, 28 DEG C of (28-30 DEG C) continuation shaken cultivations,
Revolving speed is 220r/min, until thalline were collected by centrifugation by 3500g when OD value is 3.6, is resuspended in 200ml BMMY culture solution, is added
Methanol 2ml to concentration be 1%, 29 DEG C of shaken cultivations (28-30 DEG C) (revolving speed 220r/min), inducing expression
VEGF165b adds methanol 2ml in the set time in continuous 4 days, and after fermentation 4 days, 8000r/min is centrifuged 10min, obtains in expression
Clear liquid.
Embodiment 2
Equilibrating buffer is taken, equilibrating buffer includes Tris-HCl and NaCl, and Tris-HCl is in equilibrating buffer
In concentration be 10mM, concentration of the NaCl in equilibrating buffer be 100mM；The pH of equilibrating buffer is 8.0,
VEGF165b's isolates and purifies that specific step is as follows：
(1) the expression supernatant that embodiment 1 obtains uses the membrane filtration in 0.4 μm of aperture first, then uses Vivaflow50
Swirling flow or tangential flow ultrafilter (Vivaflow 50system (30000MWCO, Sartorius Stedim Biotech
S.A it) is concentrated, obtains VEGF165b sample.
(2) heparin-agarose affinity column (HiTrap Heparin, HPGE Healthcare bioscience AB,
Uppsala, Sweden) first with 5 times of column volume displacement equilibrating buffers (pH 8.0,10mM Tris-HCl, 100mM
NaCl it) balances, then by VEGF165b sample with the flow velocity loading heparin-agarose affinity column (HiTrap of 1ml/min
Heparin, HP, GE Healthcare bioscience AB, Uppsala, Sweden).
(3) after loading, then with the equilibrating buffers of 10 times of column volumes (pH 8.0,10mM Tris-HCl,
100mM NaCl) it is rinsed, unbonded foreign protein is washed away, flow velocity 1ml/min elutes 20min.
(4) with the elution of 3 times of column volumes, eluent includes Tris-HCl and NaCl, and Tris-HCl is in eluent
In concentration be 10mM, concentration of the NaCl in eluent is 1M, and the pH of eluent is 8.0, and the flow velocity of elution is 1ml/min,
Elute 20min.
(5) Ultraviolet Detector OD is used280Nm detects appearance situation, collects the last one appearance component.It is obtained with elution
Arrive elution albumen via hole diameter be 10kDa super filter tube concentration to get.
Elution albumen is detected with non denatured SDS-PAGE, the result is shown in Figure 1, and the M of the first swimming lane is standard molecular weight egg in figure
White (kDa), the 4th swimming lane the is equilibrating buffer pH VEGF165b that purifies when being 8.0, purity can reach electrophoresis
Grade.
It to expression supernatant and is purified using VEGF ELISA kit (Wuhan doctor's moral Biotechnology Co., Ltd)
To VEGF165b albumen quantified, can must purify VEGF165b albumen is 1.2mg/L or more, and highest can obtain 2mg/L, yield
70% or more.
Embodiment 3
The difference of the present embodiment and embodiment 2 is only that the pH of equilibration buffer is 8.2, and the ion of equilibrating buffer is dense
For degree to remain as 10mM Tris-HCl, 100mM NaCl isolates and purifies VEGF165b.
The result is shown in Figure 1, the M of the first swimming lane is standard molecular weight albumen (kDa) in figure, and the 5th swimming lane is that equalizing and buffering is molten
The VEGF165b that liquid pH is purified when being 8.2, purity can reach electrophoresis grade.
Embodiment 4
The difference of the present embodiment and embodiment 2 is only that the pH of equilibrating buffer is 8.5, in equilibrating buffer from
Sub- concentration is to remain as 10mM Tris-HCl, and 100mM NaCl isolates and purifies VEGF165b.
The result is shown in Figure 1, the M of the first swimming lane is standard molecular weight albumen (kDa) in figure, and the 6th swimming lane is that equalizing and buffering is molten
The VEGF165b that liquid pH is purified when being 8.5, purity can reach electrophoresis grade.
Embodiment 5
Western Blot identification
(1)SDS-PAGE
1. glue：Separation gel with 5ml 15% adds the concentration glue of 2ml 5% after solidifying completely, after glue makes,
Carefully take out comb；
2. loading：Before loading, sample is diluted with 2 × SDS sample-loading buffer, and sample is added in glue hole；
3. electrophoresis：100V, electrophoresis 2h is arranged in voltage, observes band after electrophoresis, glue dyeing liquor is contaminated 40min, so
It is decolourized afterwards with destainer.
(2)Western blot
1. transferring film：Prepare filter paper and film is put well in order, 50mA transferring film 1h, takes out film after having turned；
2. closing：Film is washed 3 times with 1 × PBS, each 5min；With the skimmed milk power of 25ml or so 5%, close at room temperature
1h, then wash film；
3. primary antibody is incubated for：1:Film and the primary antibody diluted closing (have been avoided gas with plastic bag by 2000 dilution primary antibodies
Bubble), 2h or 4 DEG C is closed at room temperature overnight, recycles primary antibody, film is taken out from sack, cleaning solution PBST is added on the side shaker
Washing 5min is slowly shaken, washes film 3 times altogether, each 5min；
4. secondary antibody is incubated for：1:2000 dilution secondary antibodies, close 2h for film room temperature, discard secondary antibody, and cleaning solution PBST is added in side
Washing 5min is slowly shaken on pendulum shaking table, washes film 3 times altogether, each 5min；
5. DAB is dyed：With DAB colour reagent box, prepares developing solution and be protected from light dyeing in sack, purpose band occur i.e.
Can, avoid dyeing time too long.
Qualification result is shown in Fig. 2, wherein the M of the first swimming lane is standard molecular weight albumen (kDa), 1 indicates VEGF165b.
VEGF165b albumen can show that purifying protein is with one anti-binding of VEGF165b albumen to Fig. 2 as the result is shown
VEGF165b。
Embodiment 6
VEGF165b activity identification
5000 HUVEC cells are added in 96 orifice plates, the complete ECM culture medium that 100 μ l are added (contains 10%FBS and 1%
ECGS) at 37 DEG C, 5%CO26h (6-8h) is cultivated in incubator, culture medium is sucked out and is washed twice with 1 × PBS.
To the incomplete ECM culture medium of every Kong Zhongjia, (the final concentration of 50ng/ml containing 10%FBS and VEGF165 is free of
ECGS), different amounts of VEGF165b solution (concentration 500ng/ml cannots be used up full ECM culture medium and prepares) is sequentially added, is made
The final concentration of 0ng/ml, 10ng/ml of VEGF165b, 25ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 300ng/ml,
500ng/ml, final volume ECM culture medium incomplete less than the addition of 100ul supply 100ul, each concentration gradient sets 6 again
Hole, 37 DEG C, 5%CO2Culture 3 days.
It is added the MTT solution of 10 μ l into each hole culture medium, 37 DEG C, 5%CO23h is cultivated, culture medium is sucked out, 100 μ l is added
DMSO, 37 DEG C, 100rpm shakes and measures light absorption value at 570nm with microplate reader after 10min.
As a result see that Fig. 3, Fig. 3 abscissa are the VEGF165b of various concentration, ordinate is the light absorption value under 570nm.
The HUVEC cell Proliferation that the VEGF165b of Fig. 3 display purifying can inhibit VEGF165 to mediate.
Comparative example 1
The difference of this comparative example and embodiment 2 is only that the pH of equilibrating buffer is 7.4, and equilibrating buffer ion is dense
For degree to remain as 10mM Tris-HCl, 100mM NaCl isolates and purifies VEGF165b.
The result is shown in Figure 1, the M of the first swimming lane is standard molecular weight albumen (kDa) in figure, and the second swimming lane is that equalizing and buffering is molten
The VEGF165b that liquid pH is purified when being 7.4, is not achieved the purity of electrophoresis grade.
Comparative example 2
The difference of this comparative example and embodiment 2 is only that the pH of equilibrating buffer is 7.6, and equilibrating buffer ion is dense
For degree to remain as 10mM Tris-HCl, 100mM NaCl isolates and purifies VEGF165b.
The result is shown in Figure 1, the M of the first swimming lane is standard molecular weight albumen (kDa) in figure, and third swimming lane is that equalizing and buffering is molten
The VEGF165b that liquid pH is purified when being 7.6, is not achieved the purity of electrophoresis grade.
Comparative example 3
Obtain the expression supernatant of VEGF165 and VEGF165b respectively as described in Example 1, as described in Example 2
VEGF165 and VEGF165b are isolated and purified respectively.
The difference of the method isolated and purified and embodiment 2 of this comparative example is only that equilibrating buffer is sodium phosphate,
Ion concentration is 10mM, pH 7.0；Eluent is sodium phosphate and NaCl, and wherein concentration of the sodium phosphate in eluent is 10mM,
Concentration of the NaCl in eluent is 1mM, and the pH of eluent is 7.0.Purification procedures are carried out fully according to embodiment 2, point
As shown in Figure 4 from purification result.
In Fig. 4, the first swimming lane is that M is standard molecular weight albumen (kDa), and the second swimming lane isolates and purifies item for VEGF165's
Band, third swimming lane isolate and purify band for VEGF165b's.VEGF165 can be with heparin-agarose affinity column as the result is shown
In conjunction with, and VEGF165b cannot.
Claims (10)
1. a kind of purification process of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag, it is characterised in that：Mainly
Include the following steps：
(1) heparin-agarose affinity column is balanced with equilibrating buffer, the equilibrating buffer includes Tris-HCl
And the concentration of NaCl, Tris-HCl in equilibrating buffer is 10~100mM, concentration of the NaCl in equilibrating buffer is
1~100mM；The pH of equilibrating buffer is 8.0~8.5；
(2) VEGF165b sample is loaded to heparin-agarose affinity column；
(3) heparin-agarose affinity column is rinsed with the equilibrating buffer；
(4) obtain VEGF165b albumen with elution, be then concentrated to get.
2. the purifying side of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag according to claim 1 a kind of
Method, it is characterised in that：Balance in step (1) is carried out using the equilibrating buffer of 5 times of column volumes.
3. the purifying side of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag according to claim 1 a kind of
Method, it is characterised in that：The flow velocity of sample loading is 1ml/min in step (2).
4. the purifying side of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag according to claim 1 a kind of
Method, it is characterised in that：Flushing in step (3) uses the equilibrating buffer of 10 times of column volumes.
5. a kind of blood vessel endothelial cell growth factor VEGF 165b's of no purification tag according to claim 1 or 4 is pure
Change method, it is characterised in that：The time rinsed in step (3) is 20min, flow velocity 1ml/min.
6. the purifying side of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag according to claim 1 a kind of
Method, it is characterised in that：Eluent in step (4) includes Tris-HCl and NaCl, and concentration of the Tris-HCl in eluent is
The concentration of 10mM, NaCl in eluent is 1mM, and the pH of eluent is 8.0~8.5.
7. a kind of blood vessel endothelial cell growth factor VEGF 165b's of no purification tag according to claim 1 or 6 is pure
Change method, it is characterised in that：Elution in step (4) is carried out using the eluent of 3 times of column volumes.
8. the purifying side of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag according to claim 1 a kind of
Method, it is characterised in that：The time of the elution is 20min, flow velocity 1ml/min.
9. the purifying side of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag according to claim 1 a kind of
Method, it is characterised in that：Concentration described in step (4) is concentrated using super filter tube.
10. the purifying of the blood vessel endothelial cell growth factor VEGF 165b of no purification tag according to claim 9 a kind of
Method, it is characterised in that：The aperture of the super filter tube is 10kDa.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106279435A (en) * | 2016-08-16 | 2017-01-04 | 新乡医学院 | The anti-tumor vaccine of targeting VEGF Yu mucin1, encoding gene, expression vector, expression engineering bacteria and application |
| WO2017141185A1 (en) * | 2016-02-16 | 2017-08-24 | The Regents Of The University Of California | Methods and compositions for treating atherosclerosis |
-
2018
- 2018-07-27 CN CN201810847748.3A patent/CN108864274A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017141185A1 (en) * | 2016-02-16 | 2017-08-24 | The Regents Of The University Of California | Methods and compositions for treating atherosclerosis |
| CN106279435A (en) * | 2016-08-16 | 2017-01-04 | 新乡医学院 | The anti-tumor vaccine of targeting VEGF Yu mucin1, encoding gene, expression vector, expression engineering bacteria and application |
Non-Patent Citations (2)
| Title |
|---|
| DELCOMBEL R ET AL: "New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding, cellular signaling and vessels formation", 《ANGIOGENESIS》 * |
| 张德安等: "《生物大分子实验手册》", 31 December 1991, 吉林大学出版社 * |
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