CN105732796A - Cobra venom nerve growth factor purification and preparation method - Google Patents
Cobra venom nerve growth factor purification and preparation method Download PDFInfo
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Abstract
The invention discloses a cobra venom nerve growth factor purification and preparation method. Firstly, a DEAE CL-6B anion exchange column is adopted and then a Sephadex G-50 gel chromatography column, a Macro-prep High S cation exchange column is utilized for separation in sequence, a PC12 cell test method is adopted to dilute collected peaks with ultra-pure water after each time of separation, and then testing is performed to select the peaks having the highest cobra venom nerve growth factor activity as samples for subsequent separation, wherein the minimum concentration of NGF induced PC12 cell differentiation is determined through dilution. An inventor finds peaks having the highest NGF activity in the cobra venom separation process and determines that the minimum concentration of the NGF induced PC12 cell differentiation is up to 0.1 micrograms/ml. Tests show that electrophoresed pure NGF can be obtained by applying the method and have high purity and high activity.
Description
Technical field
The invention belongs to snake venom nerve growth factor preparation field, particularly relate to a kind of Recombinant Naja naja atra NGF purification system
Preparation Method.
Background technology
Nerve growth factor (Nerve growth factor, NGF) is the nutritional labeling that nervous system is important, is ensureing god
Play an important role in the normal function of system, it addition, NGF also with endocrine, immunity, reproductive system, old age
Dementias etc. have close association.Its Main Function has four aspects:<1>promotes nervous system to grow during body development
Grow；<2>protective effect nutritious to Normal neuronal cells after body maturation；<3>nerve injury (such as cerebrovas-cularaccident and
Cerebral trauma) after can promote neuron regeneration and functional rehabilitation；<4>learning and memory function is improved.
Recombinant Naja naja atra NGF is as the main active of medicine, its good effect, rapid-action, safe and effective, malicious
Side effect is little, is prevention and the ideal medicament for the treatment of nervous system disease.Existing cobra venom NGF is isolated and purified mainly to be adopted
With the combination of gel filtration chromatography method Yu ion exchange chromatography, and after isolated and purified, use PC12 cell method, two-way immunity
Institute's separating resulting is verified by the methods such as diffusion method or SDS-PAGE electrophoresis method, but owing to the influence factor of post effect is various,
And be difficult to be controlled by, therefore the poor repeatability of this type of method, the activity of the NGF obtained is relatively low.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of simple, efficient, controlled Recombinant Naja naja atra NGF purification
Preparation method.
For solve above-mentioned technical problem, the present invention by the following technical solutions:
Recombinant Naja naja atra NGF method for preparing purified, first uses DEAE CL-6B anion-exchange column then to recycle
Sephadex G-50 gel chromatography column, Macro-prep High S cation exchange column separate successively, separate every time
After choose the sample that separates as lower step of the highest peak of snake venom nerve growth factor activity.
Choose the highest peak of snake venom nerve growth factor activity and use PC12 cellular assay method, with ultra-pure water to collected each
Test after peak dilution.
Extension rate is 10 times, 100 times, 1000 times, 10000 times.
Above-mentioned Recombinant Naja naja atra NGF method for preparing purified, comprises the following steps:
(1) DEAE CL-6B anion-exchange column eluting separates；
(2) Sephadex G-50 gel chromatography column eluting separates；
(3) Macro-prep High S cation exchange column eluting separates.
Step (1) is carried out by following operation: DEAE CL-6B post buffer A is balanced 8h, then the sample that will prepare
It is splined on the pillar balanced, uses buffer B to carry out eluting by the elution program arranged, collect each peak respectively, use PC12
Each peak snake venom nerve growth factor activity is measured by cellular assay method, chooses the peak lyophilizing that activity is the highest, standby；Buffering
Liquid A is the Tris-HCl buffer of 0.05M, pH8.5；Buffer B is the NaCl solution of 0.5M, pH8.5.
The preparation of sample is carried out by following operation: weigh cobra venom lyophilized powder 1g, adds 10ml level pad, and 4 DEG C molten
Solve 4h；To be dissolved completely after, 8000rpm, 4 DEG C of centrifugal 15min；Take supernatant, first cross 0.45 μm filter membrane, after 0.22 μm
After filter membrane, standby；Level pad is the Tris-HCl buffer of 0.05M, pH8.5.
Step (2) is carried out by following operation: by the lyophilized powder of activity summit in step (1), add 5ml PB buffer
After fully dissolving, it is splined on the G-50 gel column that PB buffer balances, uses PB buffer by the elution program arranged
Carry out eluting, collect each peak respectively, use PC12 cellular assay method that each peak snake venom nerve growth factor activity is measured,
Choose the peak lyophilizing that activity is the highest, standby；PB buffer be the sodium dihydrogen phosphate of 0.02M and the disodium hydrogen phosphate of 0.02M by
The buffer of ratio 137: 63 mixed preparing, after mixing, pH of buffer is 6.5.
Step (3) is carried out by following operation: by the lyophilized powder of activity summit in step (2), add 1ml PB buffer
After fully dissolving, 10000rpm is centrifuged 15min, takes supernatant, after crossing 0.22 μm filter membrane, is splined on PB buffer and puts down
The Macro-prep High S post weighed, uses buffer B to carry out eluting by the elution program arranged, collects each peak respectively,
Use PC12 cellular assay method that each peak snake venom nerve growth factor activity is measured, choose the peak lyophilizing that activity is the highest,
Preserve in-20 DEG C of refrigerators；The PB buffer dissolved is 0.02M, pH4.0；The PB buffer of balance is 0.02M,
pH6.5；Buffer B is the NaCl solution of 0.5M, pH6.5.(note: the PB buffer dissolved is by balance use
PB buffer adds a small amount of acetic acid, regulates pH to 4.0；Balance the sodium dihydrogen phosphate that PB buffer is 0.02M and
The buffer of the disodium hydrogen phosphate of 0.02M 137: 63 mixed preparing in proportion, after mixing, pH of buffer is 6.5.)
PC12 cellular assay method is carried out by following operation: with ultra-pure water, collected each peak is diluted 10 times respectively, 100 times,
1000 times, after 10000 times, add 48 orifice plates containing PC12 cell, observation of cell state after 72h.
The problem existed for the preparation of current Recombinant Naja naja atra NGF, it is raw that inventor establishes a kind of cobra neurotoxin
Long factor method for preparing purified, first uses DEAE CL-6B anion-exchange column then to recycle Sephadex G-50 gel
Chromatographic column, Macro-prep High S cation exchange column separate successively, use PC 12 cellular assay after separating every time
Method ultra-pure water choose testing after the dilution of collected each peak the highest peak of snake venom nerve growth factor activity as under
The sample that step separates.Wherein, by the Cmin of dilution metering NGF PC 12 cells Induced by differentiation, inventor have found
The most highly active peak of NGF in snake venom separation process, and determine that the Cmin of NGF PC 12 cells Induced by differentiation reaches 0.1
μg/ml.Test shows, the application present invention can get electrophoretically pure NGF, and its purity is high, activity is high.
Accompanying drawing explanation
Fig. 1 is G-50 post separating resulting.
Fig. 2 is CM CL-6B anion column separating resulting.
Fig. 3 is DEAE CL-6B anion column separating resulting.
Fig. 4 is G-50 gel column separating resulting.
Fig. 5 is High S cation seperation column separating resulting.
Fig. 6 is PC12 cellular identification result (light microscopic 10 ×).
Fig. 7 is cell state (light microscopic 20 ×) after NGF effect 72h.
Fig. 8 is the isolated and purified result of SDS-PAGE electroresis appraisal.
Detailed description of the invention
1 materials and methods
(Guangxi Medical University's snake venom institute provides, and Naja is Reptilia for 1.1 instruments and reagent Guangxi Cobra Venom lyophilized powder
Squamata Ophidia Elapidae Elaps, naja atra Cantor, Latin entitled naja atra), electronic analytical balance (English
Sartorius company of state), L18-4584-01 system (Pharmacia Biotech company of the U.S.), BioLogic DuoFlow
System (BIO-RAD company of the U.S.), CO2Incubator (Thermo Forma company of the U.S.), the 1640 culture medium (U.S.
Gbico company), hyclone (Wisnt company of Canada), horse serum (Hyclone company of the U.S.), other examinations
Agent is domestic analytical pure.
1.2 experimental technique
1.2.1PC12 cell is cultivated the PC12 cell 1640 culture medium containing 10% horse serum and 5% hyclone, in
5%CO2, the incubator of 37 DEG C is cultivated.Take the good PC12 cell of growth conditions and be inoculated in 48 orifice plates, stand-by.
1.2.2 sample preparation weighs cobra venom lyophilized powder 1g, adds 10ml buffer (putting down of the chromatographic column that will use
Weighing apparatus buffer), 4 DEG C dissolve 4h.To be dissolved completely after, 8000rpm, 4 DEG C of centrifugal 15min.Take supernatant, first cross 0.45 μm
Filter membrane, after 0.22 μm filter membrane, standby.(note: original method first step is G-50 gel column, buffering used
Liquid is 1%HAc；The improved method first step is DEAE CL-6B ion exchange column, and buffer used is the (Tris-HCl of 0.05M
Buffer, pH8.5.)
((1st) step specimen in use is the 10ml sample in 1.2.2 to the most original method, (2nd) step specimen in use
It is the Peak Activity sample that obtains of (1st) step)
(1) after G-50 gel column is balanced 8h with the HAc buffer of 1% by G-50 gel column, then the sample that will have configured
Being splined on pillar, arrange elution program as shown in table 1, eluent used is 1%HAc.Collect each peak respectively, and join
Containing 48 orifice plates of PC12 cell, observation of cell state after 72h, by Peak Activity lyophilizing, standby.
(2) the NaAc-HAc buffer (pH5.8) of CM CL-6B post 0.025M is put down by CM CL-B6 cation seperation column
After weighing apparatus 8h, then the Peak Activity of G-50 being splined on pillar, arrange elution program as shown in table 2, wherein buffer B is 1M
NaCl solution, pH5.8.Collect each peak respectively, and join 48 orifice plates containing PC12 cell, observe after 72h
Cell state, by Peak Activity lyophilizing, standby.
1.2.4 ((1st) step specimen in use is the 10ml sample in 1.2.2 to improved method, (2nd) step specimen in use
Being the most highly active peak sample that obtains of (1st) step, (3rd) step specimen in use is the most highly active peak arrived of (2nd) step
Sample)
(1) DEAE CL-6B anion column by DEAE CL-6B post with buffer A (the Tris-HCl buffer of 0.05M,
PH8.5) balance 8h, then the sample prepared is splined on the pillar balanced, it is as shown in table 3 that elution program is set, wherein
Buffer B is the NaCl solution of 0.5M, pH8.5.Collect each peak respectively, and dilute 10 times with ultra-pure water respectively, 100
Times, 1000 times, after 10000 times, add 48 orifice plates containing PC12 cell, observation of cell state after 72h.By NGF
The peak lyophilizing that activity is the highest, standby.
(2) G-50 gel column is by the lyophilized powder of NGF activity summit in DEAE CL-6B, add 5ml PB buffer (0.02M,
PH6.5), after fully dissolving, it is splined on the G-50 gel column that PB buffer balances.Elution program is as shown in table 4.Point
Do not collect each peak, and dilute 10 times with ultra-pure water respectively, 100 times, 1000 times, add containing PC12 thin after 10000 times
48 orifice plates of born of the same parents, observation of cell state after 72h.By peak lyophilizing the highest for NGF activity, standby.
(3) Macro-prep High S cation seperation column takes NGF activity summit lyophilized powder in G-50, adds 1ml PB and delays
Rush liquid (0.02M, pH4.0) fully to dissolve.To be dissolved completely after, 10000rpm, centrifugal 15min.Take supernatant, mistake
After 0.22 μm filter membrane, it is splined on the Macro-prep High S post that PB buffer (0.02M, pH6.5) has balanced.Wash
De-program is as shown in table 5.Wherein buffer B is the NaCl solution of 0.5M, pH6.5.Collect each peak respectively, and with super
Pure water dilutes 10 times respectively, 100 times, 1000 times, adds 48 orifice plates containing PC12 cell, in 72h after 10000 times
Rear observation of cell state.By peak lyophilizing the highest for NGF activity, in-20 DEG C of preservations.
1.2.5SDS-PAGE electrophoresis takes High S post isolated Peak Activity lyophilized powder aqueous solution (concentration is 4mg/ml)
16 μ l, add 4 μ l albumen sample-loading buffer (producers: the green skies；Article No.: P0015L), mixing is placed on boiling water bath
Middle 3min, is splined on the SDS-PAGE glue of 12%.Wherein concentrating glue part applied voltage is 60V, the electricity consumption of separation gel part institute
Pressure is 120V, when the blue bands of albumen sample-loading buffer runs to time at 1cm at the bottom of separation gel, stops electrophoresis.Coomassie is bright
Blue R-250 dyeing 4h rear decoloring.
1.2.6SDS-PAGE non denatured electrophoresis takes mgh S post isolated Peak Activity lyophilized powder aqueous solution (concentration is
4mg/ml) 16 μ l, adds 4 μ l non-denatured protein sample-loading buffer (producers: the green skies；Article No.: P0016N), mixed
Directly on PAGE gel, non denatured electrophoresis is carried out, without β-mercapto in non-denatured protein sample-loading buffer therein after even
Base ethanol etc. can make the composition that protein disulfide bond destroys.Voltage and the same 1.2.4 of dyeing time.
1.2.7 the minimum NGF concentration of PC 12 cells Induced by differentiation is by the isolated and purified pure NGF of the electrophoresis obtained, the dilutest
Releasing to protein concentration is 0.1 μ g/ml, after 0.5 μ g/ml, 1 μ g/ml, 2 μ g/ml and 4 μ g/ml, adds containing PC12
24 orifice plates of cell, each concentration arranges 3 multiple holes, observation of cell state after 72 hours.
2 results
2.1 former methodical separating resulting G-50 post separating resultings are as it is shown in figure 1, arrow indication is to reflect through PC12 cell
Fixed Peak Activity.CM CL-6B post separating resulting is as in figure 2 it is shown, arrow indication is the Peak Activity through PC12 cellular identification.
The separating resulting DEAE CL-6B anion column separating resulting of 2.2 improved methods is as it is shown on figure 3, arrow indication is warp
The active summit (45-55 pipe) of PC12 cellular identification.G-50 gel column separating resulting as shown in Figure 4, arrow institute
Refer to as the active summit (6-21 pipe) through PC12 cellular identification.High S cation seperation column separating resulting as it is shown in figure 5,
Arrow indication is the active summit (26-30 pipe) through PC12 cellular identification.It is computed, 2g cobra venom lyophilized powder
Isolated and purified yield is 0.24%.
The 2.3 PC12 cells passing through PC12 cellular identification most highly active peak result regular culture conditions (blank group) are circle
Shape, ellipse or some irregular shapes, there is no obvious projection, such as A in Fig. 6；In the presence of having NGF, after 72h, can
See that almost each cell all grows raised from cell space to surrounding, can form network-like structure, such as B in Fig. 6；And deposit without NGF
Time, substantially without significant change, such as C in Fig. 6.
As shown in table 6 by the NGF activity result of each detached peaks of PC12 cell detection difference extension rate, wherein "-" generation
Table without NGF activity, "+" represent have NGF activity.
The minimum NGF concentration of 2.4 PC 12 cells Induced by differentiations is by Macro-prep High S post the highest isolated work
Property peak press pipe collect, often pipe is diluted to protein concentration respectively is 0.1 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 2 μ g/ml
After 4 μ g/ml, adding 48 orifice plates containing PC12 cell, each concentration of each sample arranges 3 multiple holes, in 72
Observation of cell state after hour, and record each activity.
After the NGF activity peak effect PC12 cell 72h of variable concentrations, cell substantially has three shown in Fig. 7 kind state.
The relative activity of Macro-prep High S post isolated most highly active peak different parts is as shown in table 7, and draw can
The Cmin making PC12 cell break up is 0.1 μ g/ml.
2.5SDS-PGAE electrophoresis result through SDS-PAGE electroresis appraisal, Macro-prep High S post gained through PC12
It is pure that the NGF activity peak of cellular identification has reached electrophoresis.Wherein the band of Denatured protein is below 15kDa, sees A in Fig. 8.Non-
Denaturing protein electrophoresis result is also single band, and as shown in B in Fig. 8, band is below 25kDa.Known albumen mobility
The logarithm of X and molecular weight Mr is linear, meets logMr=aX+b, and wherein a, b are constant.With X as abscissa,
LogMr is that vertical coordinate draws standard curve, and the formula obtaining Fig. 8 A is logMr=-1.3768X+5.186, R2=0.963, through meter
Calculating, the molecular weight of degeneration NGF is about 14kDa.The formula of Fig. 8 B is logMr=-1.2684X+5.1201, R2=0.951,
Being computed, the molecular weight of non denatured NGF is about 23.3kDa.
The contrast of 2.6 original methods and improved method by buffer used, the isolated and purified time, Peak Activity number and
The Cmin that final separation result can make PC12 break up makes contrast, and comparing result is shown in Table 8.Can obtain from table, improvement side
Method required time is shorter, and the Peak Activity of gained is simple spike, and the NGF activity obtained is relatively in the past higher.
Table 1G-50 elution program
Table 2CM CL-6B elution program
DEAE CL-6B elution program in table 3 present invention
G-50 elution program in table 4 present invention
Macro-prep High S elution program in table 5 present invention
NGF activity after the dilution of table 6 improved method each detached peaks
Table 7Macro-prep High S most highly active peak
The original method of table 8 and the contrast of improved method
Claims (9)
1. a Recombinant Naja naja atra NGF method for preparing purified, it is characterised in that: first use DEAE CL-6B anion to hand over
Change that then post recycles Sephadex G-50 gel chromatography column, Macro-prep High S cation exchange column enters successively
Row separates, and chooses the sample that the highest peak of snake venom nerve growth factor activity separates as lower step after separating every time.
Recombinant Naja naja atra NGF method for preparing purified the most according to claim 1, it is characterised in that described in choose Serpentis
The peak that poison Nerve Growth Factor Activity is the highest uses PC12 cellular assay method, after diluting collected each peak with ultra-pure water
Test.
Recombinant Naja naja atra NGF method for preparing purified the most according to claim 3, it is characterised in that described dilution times
Number is 10 times, 100 times, 1000 times, 10000 times.
Recombinant Naja naja atra NGF method for preparing purified the most according to claim 1, it is characterised in that include following step
Rapid:
(1) DEAE CL-6B anion-exchange column eluting separates；
(2) Sephadex G-50 gel chromatography column eluting separates；
(3) Macro-prep High S cation exchange column eluting separates.
Recombinant Naja naja atra NGF method for preparing purified the most according to claim 1, it is characterised in that step (1) is pressed
Hereinafter operation is carried out: DEAE CL-6B post buffer A balances 8h, then is splined on by the sample prepared and balances
Pillar, use buffer B to carry out eluting by the elution program arranged, collect each peak respectively, use the inspection of PC12 cell
Each peak snake venom nerve growth factor activity is measured by method of testing, and chooses the peak lyophilizing that activity is the highest, standby；Described slow
Rush the Tris-HCl buffer that liquid A is 0.05M, pH8.5；Described buffer B is the NaCl solution of 0.5M, pH8.5.
Recombinant Naja naja atra NGF method for preparing purified the most according to claim 1, it is characterised in that described sample
Preparation is carried out by following operation: weigh cobra venom lyophilized powder 1g, adds 10ml level pad, and 4 DEG C dissolve 4h；
To be dissolved completely after, 8000rpm, 4 DEG C of centrifugal 15min；Take supernatant, first cross 0.45 μm filter membrane, after 0.22 μm
After filter membrane, standby；Described level pad is the Tris-HCl buffer of 0.05M, pH8.5.
Recombinant Naja naja atra NGF method for preparing purified the most according to claim 1, it is characterised in that step (2) is pressed
Hereinafter operation is carried out: by the lyophilized powder of activity summit in step (1), adds after 5ml PB buffer fully dissolves,
It is splined on the G-50 gel column that PB buffer balances, uses PB buffer to carry out eluting by the elution program arranged,
Collect each peak respectively, use PC12 cellular assay method that each peak snake venom nerve growth factor activity is measured, choose work
The peak lyophilizing that property is the highest, standby；Described PB buffer is sodium dihydrogen phosphate and the disodium hydrogen phosphate of 0.02M of 0.02M
The buffer of 137: 63 mixed preparing in proportion, after mixing, pH of buffer is 6.5.
Recombinant Naja naja atra NGF method for preparing purified the most according to claim 1, it is characterised in that step (3) is pressed
Hereinafter operation is carried out: by the lyophilized powder of activity summit in step (2), adds after 1ml PB buffer fully dissolves,
10000rpm is centrifuged 15min, takes supernatant, after crossing 0.22 μm filter membrane, is splined on what PB buffer balanced
Macro-prep High S post, uses buffer B to carry out eluting by the elution program arranged, collects each peak respectively, adopt
By PC12 cellular assay method, each peak snake venom nerve growth factor activity is measured, chooses the peak lyophilizing that activity is the highest,
Preserve in-20 DEG C of refrigerators；The PB buffer of described dissolving is 0.02M, pH4.0；The PB buffering of described balance
Liquid is 0.02M, pH6.5；Described buffer B is the NaCl solution of 0.5M, pH6.5.
9. according to the Recombinant Naja naja atra NGF method for preparing purified described according to claim 5 to 7, it is characterised in that PC12
Cellular assay method is carried out by following operation: with ultra-pure water, collected each peak is diluted 10 times respectively, 100 times, and 1000
Times, add 48 orifice plates containing PC12 cell, observation of cell state after 72h after 10000 times.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107098956A (en) * | 2017-03-13 | 2017-08-29 | 广西医科大学 | Cytotoxin 4N method for preparing purified and its application |
| CN113214379A (en) * | 2021-04-07 | 2021-08-06 | 云南龙凤谷生物药业有限公司 | Preparation method of cobra venom nerve growth factor |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1255503A (en) * | 1999-11-09 | 2000-06-07 | 边六交 | Method for purifying nerve growth factor from snake venom |
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2016
- 2016-04-27 CN CN201610268411.8A patent/CN105732796A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1255503A (en) * | 1999-11-09 | 2000-06-07 | 边六交 | Method for purifying nerve growth factor from snake venom |
Non-Patent Citations (2)
| Title |
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| 候瑞雪等: "广西眼镜蛇毒神经生长因子分离纯化方法", 《基因组学与应用生物学》 * |
| 吴鹏: "从中华眼镜蛇蛇毒中分离纯化神经生长因子的色谱工艺研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107098956A (en) * | 2017-03-13 | 2017-08-29 | 广西医科大学 | Cytotoxin 4N method for preparing purified and its application |
| CN107098956B (en) * | 2017-03-13 | 2021-02-09 | 广西医科大学 | Purification preparation method and application of cobra venom cytotoxin-4N |
| CN113214379A (en) * | 2021-04-07 | 2021-08-06 | 云南龙凤谷生物药业有限公司 | Preparation method of cobra venom nerve growth factor |
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