CN106279397A - A kind of extracting method of nerve growth factor - Google Patents
A kind of extracting method of nerve growth factor Download PDFInfo
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- CN106279397A CN106279397A CN201510313567.9A CN201510313567A CN106279397A CN 106279397 A CN106279397 A CN 106279397A CN 201510313567 A CN201510313567 A CN 201510313567A CN 106279397 A CN106279397 A CN 106279397A
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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
- C07K14/48—Nerve growth factor [NGF]
Abstract
The invention provides the extracting method of a kind of nerve growth factor, described method includes: collect homogenate supernatant by centrifugal after mouse submandibular gland tissue homogenate；Collect after described homogenate supernatant is carried out the attached cation-exchange chromatography of CM back suction and flow through liquid；Carry out being acidified collected after centrifugation acidolysis supernatant to the described liquid that flows through；Described acidolysis supernatant is carried out Liquidity limit displacement chromatography and collects the destination protein liquid that target protein peak is corresponding；Described destination protein liquid is carried out hydrophobic chromatography, it is thus achieved that the destination protein liquid after hydrophobic chromatography.The technique of the present invention simplifies processing step, and flow process is simple, not only substantially reduces the time used by preparation, improves homogeneity and the activity of product simultaneously, and sample purity have also been obtained further raising, and isolated and purified for NGF provides new approach.
Description
Technical field
The present invention relates to biological technical field, be specifically related to the extraction of a kind of nerve growth factor
Method.
Background technology
Mouse nerve growth factor (mouse nerve growth factor, mNGF) is to send out the earliest
Existing NGF, is also to study the most thorough NGF.Complete mNGF is by α, β, γ tri-kinds
Subunit is with α2β2γ2Form composition.Wherein, two β subunits are the biological active center of mNGF,
There is all biological function of mNGF；α and γ subunit can protect β subunit to avoid mice jaw
The shear action of protease and the combination of suppression β subunit and NGF receptor in lower gland.Two β
Subunit forms dimer with non-covalent bond, i.e. β-NGF.
The separation purifying technique setting up β-NGF is the basis of its application and development.Nineteen sixty Cohen
First propose and establish the isolation and purification method of β-NGF in mouse submandibular gland.Subsequently, many
Document reports the improved method of isolated and purified NGF from Mus submaxillary gland.Strategy is separated according to it
Difference, these methods can be divided into two classes: a class is the 7S NGF that first isolated is complete,
Depolymerization 7S NGF discharges and has bioactive β subunit dimer (β-NGF) again, then enters
One step isolated β-NGF；Another kind of is that the β being directly separated and having bioactive NGF is sub-
Base dimer, i.e. 2.5S NGF.
The most domestic have four injection Mus NGF listing, the side of each company purification β-mNGF
Method is mostly based on two step cationes (CM sepharose FF) displacement chromatography, the main stream of method
Journey includes: historrhexis, and------the anti-adsorption chromatography of-CM---is fully dialysed or ultrafiltration---in fully dialysis
Acidifying is dissociated---CM adsorption chromatography exclusion chromatography.Detailed process can be found in appoints fine jade in evening etc. to deliver
Article " lyophilizing mouse nerve growth factor pilot scale preparation and the discussion of some problem " in introduce
Method, is specially mouse submandibular gland tissue homogenate, must be homogenized supernatant after being centrifuged, will homogenate
After supernatant is fully dialysed, carry out the attached ion-exchange chromatography of back suction, collect and flow through liquid；Flow through liquid
Again dialyse or after ultrafiltration, carry out acidifying and dissociate, be centrifuged, obtain acidolysis supernatant；Acidolysis supernatant
Carrying out adion displacement chromatography, collecting target protein peak, destination protein liquid is through exclusion chromatography
Obtain NGF extracting solution.
But comprising two steps dialysis in this technique, dialysis step is time-consuming and complex operation.Often step is thoroughly
Analysis is time-consuming up to more than 20 hours, and dialysis volume seepage the most easily occurs and ruptures time bigger.Additionally
Due in mouse submandibular gland rich in β-NGF endopeptidase and class protaminase protease, both enzymes
β-NGF aminoterminal or the aminoacid of c-terminus can be sheared, so the time of dialysis step is the longest,
The probability that NGF is sheared is the biggest, easily causes NGF two end portions amino acid whose disappearance ratio and increases
Adding, affect homogeneity and the activity of product, the existence of the most multiple shear pattern is unfavorable for product
Quality control.
It is therefore desirable to provide a kind of quick, easy nerve growth factor extracting method, to obtain
Obtain homogeneity strong, the measured nerve growth factor of matter high, active.
Summary of the invention
It is an object of the invention to overcome drawbacks described above present in prior art, it is provided that a kind of Mus
The preparation method of NGF.
The discovery that the present inventor is very surprised in research process, in NGF extraction step
Add hydrophobic chromatography process, can reach on the premise of not using in conventional method two steps dialysis with
The close even preferably extraction effect of general extraction methods, can be greatly saved extraction and be taken
Between, the homogeneity of product is also improved with activity simultaneously.
Based on the above extracting method finding to the invention provides a kind of nerve growth factor, described
Method includes: collect homogenate supernatant by centrifugal after mouse submandibular gland tissue homogenate；To described even
Slurry supernatant is collected after carrying out the attached cation-exchange chromatography of CM back suction and is flowed through liquid；Flow through described
Liquid carries out being acidified collected after centrifugation acidolysis supernatant；Described acidolysis supernatant is carried out absorption sun from
Sub-displacement chromatography collects the destination protein liquid that target protein peak is corresponding；Described destination protein liquid is entered
Row hydrophobic chromatography, it is thus achieved that the destination protein liquid after hydrophobic chromatography.
Optionally, the balance liquid of the drainage column used in described hydrophobic chromatography is that salt concentration is
The pH value of 0.5-3.0mol/L is the buffer of 6.0-9.0.
Optionally, the balance liquid of the drainage column used in described hydrophobic chromatography is by selected from chlorination
A kind of salt in sodium, ammonium sulfate and sodium sulfate is dissolved in phosphate buffer or Tris-HCl buffering
Liquid obtains, and the final concentration of 0.5-3.0mol/L of salt, the pH value that dissolve in balance liquid are
6.0-9.0。
Optionally, when described salt is sodium chloride, the final concentration of 0.5-3.0mol/L of sodium chloride；
When described salt is ammonium sulfate, the final concentration of 0.5-2.0mol/L of ammonium sulfate；When described salt is
During sodium sulfate, the final concentration of 0.5-2.0mol/L of sodium sulfate.
Optionally, the pH value of described phosphate buffer is 6.0-8.0, and described Tris-HCl buffers
The pH value of liquid is 7.1-9.0.
Optionally, the drainage column medium in described hydrophobic chromatography be Butyl-S-FF, Phenyl FF,
Phenyl HP, Butyl FF, OctylFFButyl HP, Phenyl HS FF or Fractogel
EMD Phenyl(S)。
Optionally, during hydrophobic chromatography, loading flow velocity is 5-300cm/hr, is preferably
80-200cm/hr。
Optionally, the column temperature of hydrophobic chromatography process is 2～8 DEG C.
Optional described method also includes carrying out exclusion chromatography step.
Optionally, the pH value of described homogenate supernatant is 6.0-7.0.
Optionally, the preparation method of homogenate supernatant comprises the following steps:
Fully it is homogenized after mouse submandibular gland is mixed with purified water, centrifugal collection supernatant, uses pH
The concentration of 6.0～7.0 be 0.5mol/L phosphate buffer regulation supernatant pH after obtain even
Slurry supernatant.
Optionally, the balance liquid used during the attached cation-exchange chromatography of CM back suction is concentration
The phosphate buffer of 0.01-0.1mol/L, pH6.0-7.0；Loading flow velocity is 5-700cm/h.
Optionally, described acidifying is centrifugal comprises the following steps: to flowing through addition acidic buffer in liquid
Liquid regulation pH value, to 3.5-4.5, adds NaCl, makes that NaCl's in system is final concentration of
0.1-0.5mol/L, stands centrifugal acquisition acidolysis supernatant.
Optionally, flow through in liquid add acidic buffer be pH be the acetate buffer of 3.5-4.5
Liquid or citrate buffer.
Optionally, the step of described Liquidity limit displacement chromatography includes:
With Liquidity limit displacement chromatography balance liquid, filler is balanced, by acidolysis supernatant
After sample, first with cation-exchange chromatography balance liquid eluting, the protein of absorption is used
PH8.5～9.5, the Tris-HC1 of concentration 0.01～0.10mol/L wash miscellaneous buffer and wash miscellaneous, then use
0.01～the 0.10mol/L Tris-HC1-0.2 of pH8.5～9.5～0.8mol/L NaCl elution buffer
Carry out gradient elution；Wherein, described Liquidity limit displacement chromatography balance liquid containing concentration is
The acetate of 0.01-0.1mol/L and the NaCl of 0.1-0.5mol/L, pH value is 3.5-4.5.This
Bright provided method has the advantage that
1, from technological process, the present invention directly carries out loading after being homogenized by lower jaw glandular tissue,
Directly it is acidified after CM back suction attached salt ion displacement chromatography, is not related to dialysis solution displacement or ultrafiltration
Step, the production operation time greatly reduces, saves 14～48 hours compared with conventional method.
2, extracting the complete type β-NGF ratio obtained to significantly improve, sample uniformity carries significantly
High: to judge from isoelectric point, IP, from before three generations be reduced to two bands；In terms of mass spectral results,
It is reduced to two peaks from four previous peaks；Isoelectric point, IP and mass spectroscopy molecular component analysis result show this
The technique of invention can suppress the carboxypeptidase Degradation to the C terminal arginine of NGF effectively,
It also is able to effectively reduce N end AA and is sheared the ratio of form.
3, the purity of the destination protein liquid obtained after hydrophobic chromatography can reach more than 99% even
100%.
To sum up, the technique of the present invention simplifies processing step, and flow process is simple, is not only greatly shortened
Time used by preparation, improve homogeneity and the activity of product, and sample purity simultaneously
Have also been obtained further raising, isolated and purified for NGF provides new approach.
Accompanying drawing explanation
Fig. 1 is HPLC purity detecting result figure in embodiment 1；
Fig. 2 is HPLC purity detecting result figure in comparative example 1；
Fig. 3 is SDS-PAGE purity detecting result figure in embodiment 1；
Fig. 4 is SDS-PAGE molecular weight detection result figure in embodiment 1；
Fig. 5 is isoelectric focusing electrophoresis comparison diagram in embodiment 1 and comparative example；
Fig. 6 is mass spectroscopy molecular amount testing result figure in embodiment 1；
Fig. 7 is mass spectroscopy molecular amount testing result figure in comparative example 1；
Fig. 8 is isoelectric focusing electrophoresis result.
Detailed description of the invention
Below will the present invention is described in detail by detailed description of the invention.It will be appreciated that
Being given merely to play descriptive purpose of following example, is not used to the present invention's
Scope limits.Those skilled in the art is in the feelings without departing substantially from spirit of the invention and spirit
Under condition, the present invention can be carried out various amendment and replacement.
The invention provides the extracting method of a kind of nerve growth factor, by mouse submandibular gland tissue
After homogenate, centrifugal collection is homogenized supernatant；Described homogenate supernatant is carried out the attached sun of CM back suction from
Collect after sub-displacement chromatography and flow through liquid；Carry out being acidified in collected after centrifugation acidolysis to the described liquid that flows through
Clear liquid；Described acidolysis supernatant is carried out Liquidity limit displacement chromatography and collects target protein peak pair
The destination protein liquid answered；Described destination protein liquid is carried out hydrophobic chromatography, it is thus achieved that after hydrophobic chromatography
Destination protein liquid.
In one embodiment of the invention, described method also includes regulating described homogenate supernatant
The pH value of liquid carries out the attached cation-exchange chromatography of CM back suction to 6.0-7.0 again.In described homogenate
The preparation method of clear liquid comprises the following steps: by mouse submandibular gland and the purified water being cooled to 4 DEG C in advance
Carry out pressure homogenizing after mixing according to the mass volume ratio of 1:1-20, the component after homogenizing is existed
Under 8000-12000g, centrifugal 20-60min collects supernatant, by the concentration of pH 6.0～7.0 is
The pH of described homogenate supernatant is adjusted by the phosphate solution of 0.5mol/L.Wherein, inciting somebody to action
Mouse submandibular gland can first tentatively crush with tissue pulverizer, then after mixing with purified water
Pressure homogenizing is carried out with high pressure homogenizer.
In embodiments of the present invention, the method for cation-exchange chromatography attached for CM back suction does not has
Having particularly restriction, the method that this area can be used conventional is carried out, such as, can use
CM32, CM Sepharose FF or other EFFECTIVE MEDIUM.The anti-Liquidity limit of described CM is handed over
Change the phosphate-buffered that balance liquid is 0.01-0.1mol/L, pH6.0-7.0 used in chromatography process
Liquid；Loading flow velocity is 5-700cm/h.The present invention one preferred embodiment in,
After being balanced with balance liquid can by CM-32 post on described homogenate supernatant (4.5 ×
15cm), then with the phosphate buffer that concentration is 0.02mol/L, pH6.8 with 300ml/h's
Flow velocity eluting, the non-adsorbable protein peak of collection is for flowing through liquid.
In the present invention, described acidifying is centrifugal may comprise steps of: add to flowing through in liquid
Acidic buffer regulation pH value, to 3.5-4.5, adds NaCl, makes the end of NaCl in system dense
Degree is 0.1-0.5mol/L, is centrifuged 20-60min in 8000-16000g and obtains acidolysis supernatant after standing
Liquid.Preferably, flow through in liquid add acidic buffer be pH be the acetate buffer of 3.5-4.5
Liquid or citrate buffer.Concrete, preferably it is acidified centrifugal effect to obtain, permissible
Liquid is directly added into the acetate buffer that pH4.0 concentration is 0.5mol/L makes system to flowing through
PH is down to less than 4.5, adds NaCl, makes the final concentration of 0.4mol/L of NaCl in system,
After standing 10min, it is centrifuged 40min in 4 DEG C of 16000g, collects supernatant and obtain acidolysis supernatant.
In one embodiment of the invention, Liquidity limit displacement chromatography, often can use
Rule method is carried out.Will described acidolysis supernatant loading to the cation exchange layer after fully balance
Analysis post, rinses to baseline, with washing the miscellaneous peak of miscellaneous buffer solution elution to base with balance liquid after completion of the sample
Line, then carry out gradient elution with elution buffer, on Ultraviolet Detector, display digit is from baseline
Collect when beginning to ramp up, reduce to stop collecting target protein peak during baseline.It is for instance possible to use
Article " lyophilizing mouse nerve growth factor pilot scale preparation and the spy of some problem that Ren Wanqiong etc. deliver
Beg for " in introduce method carry out described Liquidity limit displacement chromatography step.
Wherein, described Liquidity limit displacement chromatography filler includes but not limited to CM Sepharose
FF, SP Sepharose HP, Capto MMC and Fractogel EMD SO3-650.
In one embodiment of the invention, described Liquidity limit displacement chromatography can pass through
Following steps are carried out: be balanced filler, by acid with Liquidity limit displacement chromatography balance liquid
After solution supernatant is with the flow velocity loading of 70-400cm/hr, first use cation-exchange chromatography balance liquid
Eluting, the protein pH8.5～9.5 of absorption, the Tris-HC1 of concentration 0.01～0.10mol/L wash
Miscellaneous buffer is washed miscellaneous, then with the 0.01～0.10mol/L of pH8.5～9.5
Tris-HC1-0.2～0.8mol/LNaCl elution buffer carries out gradient elution；Wherein, described suction
In attached cation-exchange chromatography balance liquid containing concentration be 0.01-0.1mol/L acetate and
The NaCl of 0.1-0.5mol/L, pH value is 3.5-4.5.
In one embodiment of the invention, putting down of the drainage column used in described hydrophobic chromatography
Weighing apparatus liquid is that a kind of salt in sodium chloride, ammonium sulfate and sodium sulfate is dissolved in phosphate-buffered
Liquid or Tris-HCl buffer obtain, the final concentration of the salt dissolved in the balance liquid of drainage column
PH value for 0.5-3.0mol/L, the balance liquid of drainage column is 6.0-9.0.
In one embodiment of the invention, sodium chloride is dissolved in phosphate buffer or
Tris-HCl buffer obtains the balance liquid of drainage column, sodium chloride final concentration of
0.5-3.0mol/L, preferably 1-2mol/L.
In another embodiment of the invention, ammonium sulfate is dissolved in phosphate buffer or
Tris-HCl buffer obtains the balance liquid of drainage column, ammonium sulfate final concentration of
0.5-2.0mol/L, preferably 0.8-1.6mol/L
In another embodiment of the present invention, sodium sulfate is dissolved in phosphate buffer or
Tris-HCl buffer obtains the balance liquid of drainage column, sodium sulfate final concentration of
0.5-2.0mol/L, preferably 0.8-1.6mol/L.
Wherein, the pH value of described phosphate buffer is 6.0-8.0, and concentration is 0.01-0.1mol/L,
The pH value of described Tris-HCl buffer is 7.1-9.0, and concentration is 0.01-0.1mol/L.
Before hydrophobic chromatography, first the concentration of salt ion in described destination protein liquid is adjusted
Whole, the method for adjustment can be to make described purpose egg by the way of adding identical solid salt
Ion concentration in the balance liquid of the kind of white liquor intermediate ion and concentration and drainage column and kind one
Cause.
Rinse to baseline with the balance liquid of drainage column after completion of the sample, then with salt-free accordingly
Buffer carries out eluting, and on Ultraviolet Detector, display digit starts when beginning to ramp up to collect, fall
For stopping during baseline collecting target protein peak.
Wherein, the drainage column medium in described hydrophobic chromatography be Butyl-S-FF, Phenyl FF,
Phenyl HP, Butyl FF, OctylFF, Butyl HP, Phenyl HS FF or Fractogel
EMD Phenyl(S).In the case of You Xuan, the drainage column medium in described hydrophobic chromatography is
ButylFF。
In the present invention, in hydrophobic chromatography, loading flow velocity is 5-300cm/h, is preferably
80-200cm/h, column temperature is 2～8 DEG C.
Preferably, exclusion chromatography is carried out after method provided by the present invention is additionally included in hydrophobic chromatography
Obtain NGF extracting solution.
Exclusion chromatography can use conventional method, and the balance liquid selected during exclusion chromatography can be common
Exclusion chromatography buffer, the phosphate of preferably 0.01～0.1mol/L is (containing 0.1～0.2mol/L
Sodium chloride, pH6.5～7.0) buffer, on Ultraviolet Detector, display digit is from the beginning of baseline
Collect target protein peak during rising, reduce to stop collecting target protein peak during baseline.
Exclusion chromatographic media can use Superdex 75 prep grade chromatography, G25 chromatography etc. its
Its EFFECTIVE MEDIUM.As used article " the lyophilizing mouse nerve growth factor pilot scale appointing fine jade in evening etc. to deliver
Preparation and the discussion of some problem " the middle method introduced, Sephadex G-75 column chromatography post 2.6
× 110cm, with 0.05mol/L, pH4.0 acetate eluting, flow velocity 78ml/h, collects eluting peak
Main peak, is purification circuit B gained 2.5SNGF.
Preferably, it is thus achieved that NGF extracting solution carry out pre-filtering with 0.1 μm film after, in addition it is also necessary to use
Cross except viromembrane and filter virus, collect filtered solution, extract through being product NGF after the assay was approved
Liquid.
Chromatography specifically described herein is all carried out in Conventional chromatography device, modal chromatographic column circle
Cylindricality or be similar to cylinder hollow pipe, typically make it upright during use.Can be according to concrete raw
Produce scale selection and select height and the diameter of chromatographic column.The medium filled in chromatographic column is as required
Select.
Method provided by the present invention can also include that the conventional step of medicine is prepared in some extractions
Suddenly, such as inactivation of virus, except steps such as virus filtrations.
The virus inactivating method that described virus inactivating method can use this area conventional enters
OK, low ph value viral inactivation method, S/D viral inactivation method and sodium caprylate virus can such as be used
Inactivation method.The present invention, can be at this for having no particular limits the opportunity carrying out inactivation of virus
Any stage inventing described method is carried out.
Described can use this area customary filtration methods except virus filtration step, use filter membrane mistakes more
Filter, typically finally filters after extraction step completes.
Method provided by the present invention is by adding hydrophobic chromatography step in NGF preparation process
Mode obtains the beneficial effect of time-consuming, to improve product homogeneity and activity, so that
Whole extracting method forms an efficient entirety, preferably realizes the goal of the invention of the present invention,
Inventor to homogenate the collection method of supernatant, CM back suction attached cation-exchange chromatography method,
Acidifying centrifugal method, Liquidity limit displacement chromatography step have all carried out as above substantial amounts of
Adjust so that whole preparation system forms, with hydrophobic chromatography step, the entirety cooperated, it is possible to
Preferably it is applicable to be extracted the technical scheme of nerve growth factor by hydrophobic chromatography step.But
It should be appreciated that above-mentioned specific embodiments is only the preferable case of the present invention and and not exclusive
Situation, those skilled in the art can reasonably predict detailed description of the invention that description is given it
Other outer equivalents or substantially mode of texturing are possible to.
The present invention is described further for the following examples.
Embodiment 1
1) homogenate: take mouse submandibular gland, adds pre-cooling purified water and is fully homogenized, homogenate warp
11000 × g, 1hr, 4 DEG C of pelleted by centrifugation, 4 DEG C are centrifuged, and collect centrifuged supernatant, add appropriate
0.5mol/L phosphate solution (pH 6.5) regulation centrifuged supernatant pH is stand-by to 6.8 ± 0.2.
2) CM back suction is attached: by step 1) centrifuged supernatant that obtains carries out CM Sepharose
Fast Flow cation-exchange chromatography, CM Sepharose Fast Flow chromatographic column is used
0.02mol/L, pH6.8 phosphate buffer fully balance after loading, after completion of the sample with balance
Liquid rinses, and collects albumen eluting peak, and flow velocity is 150cm/h.
3) acidifying: add 1mol/L acetate buffer (pH4.0) in flowing through liquid and reduce rapidly
PH to 4.0, then adds NaCl and makes the final concentration of the NaCl in system reach 0.4mol/L,
It is centrifuged 30min in 4 DEG C of 10000g after standing about 5min, takes acidolysis supernatant.
4) CM2 post: CM Sepharose FF chromatographic column 0.05mol/L acetate buffer
Loading after liquid (pH4.0) fully balance, after completion of the sample, rinses to baseline with balance liquid,
With the miscellaneous peak of 0.05mol/L Tris-HC1 (pH9.0) buffer solution elution to baseline, then use
0.05mol/LTris-HC1 and 0.05mol/L Tris-HC1-0.4mol/L NaCl (pH9.0) ladder
Degree eluting, collects target peak, display digit on Ultraviolet Detector according to uv absorption situation
Start when beginning to ramp up to collect, reduce to stop collecting target protein peak during baseline.
5) hydrophobic chromatography: Butyl Sepharose 4 FF chromatographic column is through 0.02mol/L phosphate
(pH6.8)-1.5mol/L sodium chloride buffer fully balances.In step 4) feed liquid that obtains
Middle addition solid sodium chloride makes the final concentration of 1.5mol/L of sodium chloride, sodium to be chlorinated in feed liquid
Loading after fully dissolving, speed is 120cm/h, rinses to baseline with balance liquid after completion of the sample,
Then target peak is collected with 0.02mol/L phosphate (pH6.8) eluting.
6) except virus: by 5) in hydrophobic chromatography eluting peak feed liquid with after 0.1 μm membrane filtration,
Filter virus with aperture 20nm except viromembrane is crossed again, collect filtered solution, be NGF and extract
Liquid.
Comparative example 1
1) homogenate: take submaxillary gland, adds pre-cooling purified water and is fully homogenized, homogenate through 11000 × g,
1hr, 4 DEG C of pelleted by centrifugation, collect centrifuged supernatant.
2) dialysis: centrifuged supernatant is abundant in 20mol/L, pH6.8 phosphate buffer
Dialysis 24hr.
3) the anti-adsorption chromatography of CM: CM Sepharose FF chromatographic column 20mmol/L, pH6.8
Phosphate buffer fully balance after loading.Rinse with balance liquid after completion of the sample, collect egg
White eluting peak.
4) dialysis: albumen flows through liquid and sets to 0 in .25mmol/L, pH6.8 phosphate buffer saturating
Analysis 24h, therebetween displacement dialysis solution 2 times
5) acidifying: after dialysis, liquid adds 1mol/L acetate buffer (pH4.0) and reduces rapidly pH
To 4.0, then adding NaCl and make into 0.4mol/L, after standing about 5min, 10000g is centrifuged
30min, takes supernatant.
6) CM2 post: CM Sepharose FF chromatographic column 50mmol/L acetate buffer
(pH4.0) loading after fully balancing, after completion of the sample, rinses to baseline with balance liquid, uses
The miscellaneous peak of 50mmol/L Tris-HC1 (pH9.0) buffer solution elution is to baseline, then uses
50mmol/LTris-HC1 and 50mmol/L Tris-HC1-0.4mol/L NaCl (pH9.0) ladder
Degree eluting, collects target peak, display digit on Ultraviolet Detector according to uv absorption situation
Start when beginning to ramp up to collect, reduce to stop collecting target protein peak during baseline.
7) Superdex 75 prep grade chromatographic column pH6.8,0.05mol/L phosphate
-0.15mol/L sodium chloride buffer fully balance after loading, Ultraviolet Detector shows number
Word started to collect when baseline begins to ramp up, reduced to stop collecting target protein peak during baseline.
8) target protein liquid is with after 0.1 μm film pre-filtering, then with aperture 20nm except viromembrane mistake
Filter virus, collect filtered solution, be NGF extracting solution.
Test case 1 embodiment 1 detects with the indices of the NGF extracting solution of comparative example 1
1, the purity of HPLC detection NGF extracting solution
The NGF extracting solution that embodiment 1 and comparative example 1 prepare is carried out HPLC detection, detection side
Method is with reference to patent ZL200510130348.3, to the NGF extracting solution of embodiment 1 and comparative example 1
Testing result is shown in Fig. 1 and Fig. 2 respectively.
From HPLC testing result figure it is known that the sample purity of embodiment 1 is 100%, right
The sample purity of ratio 1 is 93.29%.
2, the molecular weight of SDS-PAGE electrophoresis detection NGF and purity
Utilize SDS-PAGE electrophoresis sample-loading buffer, in the situation adding or being added without mercaptoethanol
Under, the mNGF that the embodiment 1 of low-molecular-weight Marker and 10 μ g is obtained loading respectively, enter
Row electrophoresis, deposition condition is 200V constant voltage, 45 minutes.Carry out with Coomassie brilliant blue G-25
Dyeing, detection NGF molecular weight and purity, result as Fig. 3, shown in 4.
From Fig. 3,4, mNGF molecular weight is about 13kDa, illustrate to be obtained purpose
Albumen is correct, and only one band is without other miscellaneous bands, and purity can reach 100%.
3, isoelectric focusing electrophoresis
According to the article appointing fine jade in evening etc. to deliver, " lyophilizing mouse nerve growth factor pilot scale is prepared and some
The discussion of problem " in introduce method carry out isoelectric focusing electrophoresis, result as it is shown in figure 5, etc.
Electric focusing interpretation of result is as shown in table 1.
Table 1
From the result of Fig. 5 and Biao 1,1 swimming lane is the NGF using embodiment 1 method to obtain,
Isoelectric point, IP is 9.1,8.9, predominantly two bands, the content (isoelectric point, IP is 9.1 bands) of total length NGF
Reach 77.8%；4 swimming lanes are the NGF that comparative example 1 method obtains, isoelectric point, IP is 9.1,8.9,
8.6, isoelectric point, IP is 3 bands, and homogeneity can only achieve 51.5%, and wherein total length NGF (etc.
Electricity point be 9.1 band) content can only achieve 13.5%, illustrate what the present invention prepared
MNGF homogeneity is higher.
4, molecular weight determination and the homogeneity checking thereof of mNGF is carried out with LC-MS mass spectrography
Chromatograph of liquid uses Agilent 1290 Infinity LC reversed phase chromatography system, and chromatographic column makes
By Poroshell 300 SB-C8,2.1mm × 75mm, 5 μm, column temperature is room temperature.Flowing phase
Composition: A:0.1% formic acid/water, B:0.1% formic acid/acetonitrile.Sample size: 0.1mg/ml, 20 μ l.
This post 5min is balanced, with 30 minutes from 5%B linear elution to 95%B with 5%B.Mass spectrograph
Use Agilent 6530 ESI-Q-TOF MS.Sweep limits: m/z 100～3200.Ultraviolet can
See a length of A280nm of light wave.
Destination protein mNGF that embodiment 1 and comparative example 1 are obtained measure total ion maps and
Ultraviolet spectra, result is the most as shown in Figure 6 and Figure 7.As can be seen from Figure 6, it comprises two peaks,
Wherein: it is 12357.18 that molecular weight is surveyed at peak 1, theoretical molecular is 12358.04, corresponding list
Bodily form formula is the NGF that N end shears 8 AA.It is 13251.78 that molecular weight is surveyed at peak 2, theoretical point
Son amount is 13251.01, and reply monomeric form is the NGF of total length 118AA.As can be seen from Figure 7,
It comprises 4 peaks, wherein: except the N end corresponding with Fig. 6 shears the NGF (peak 2) of 8 AA
With the NGF (peak 4) of total length 118AA, the monomer of the most other two kinds of forms.
From the foregoing, it will be observed that the content of complete type NGF is remote in the destination protein NGF of embodiment 1 acquisition
Far above in comparative example 1, the content of complete type NGF, utilizes method provided by the present invention to extract
NGF in product homogeneity and integrity, be better than the NGF in comparative example 1.
5, chick embryonic dorsal root ganglion method and TF-1 cell method measure mNGF activity
1) chick embryonic dorsal root ganglion method measures mNGF activity
NGF sample is diluted: the mNGF sample that A liquid: 6ng extracts adds 1ml serum-free
DMEM culture fluid dissolves；B liquid: take A liquid 50 μ l and add serum-free DMEM culture fluid 4.95ml；
C liquid: take B liquid 60 μ l and add serum-free DMEM culture fluid 2.94ml (total amount 3ml) and make final concentration
For (3AU/ml).A, B liquid is diluted in centrifuge tube, and C liquid, in cell bottle, is made by C liquid
For headpin, then 3 times of gradient dilutions are: No. 2, No. 3, No. 4, No. 5, No. 6 liquid to be measured.Often
Individual liquid to be measured adds 1 culture bottle, 2ml/ bottle.Simultaneously with serum-free DMEM culture fluid as sky
White comparison, with the standard substance purchased from Zhong Jian institute as positive control (reference material).Add 8 ages in days
Chick embryonic dorsal root ganglion is placed on 5%CO2, in 37 DEG C of saturated humidity incubators, after 24 hours
Observed result.
During to grow preferably in every milliliter of testing sample the content of NGF as 1 active unit
(AU).Several 3rd and the 4th two dilution is started from the dilution factor that negative control result occurs
Degree takes the best conduct of growth and judges endpoint calculation titer.Reference material is the mark purchased from Zhong Jian institute
Quasi-product, every loading amount is 1000AU.
NGF than the computing formula lived is:
The ratio of testing sample is lived, and (AU/ml) × [sample is pre-dilute for (AU/mg)=reference material activity
Release the activity (AU/ml) at multiple × correspondence reference material dilution point/reference material actual measurement activity
(AU/ml)]
Make to carry out embodiment 1 the most simultaneously and comparative example 1 extracts the destination protein obtained
Determination of activity.As a result, the NGF activity of embodiment 1 and comparative example 1 has all reached 500,000
AU/mg, the results are shown in Table 2.
2) TF-1 cell method measures mNGF activity
Detailed method of operation is CN103376248A according to patent publication No., and patent name is " god
Through growth factor activity method for quantitatively determining " in method in embodiment 1 operate, than work
Property testing result is shown in Table 2.
Table 2
| Lot number | Chick embryo method is than living | TF-1 cell method is than living |
| Embodiment 1 | 5×105AU/mg | 11.7×105U/mg |
| Comparative example 1 | 5×105AU/mg | 5.0×105U/mg |
As shown in Table 2, owing to dorsal root ganglion method sensitivity is relatively low, so two embodiments
MNGF does not has difference than living, but is lived by highly sensitive TF-1 cell method detection ratio, energy
Find out that the specific activity of embodiment 1 has significant improvement.
6, target protein peak purity contrast before and after hydrophobic chromatography
Use target protein peak purity before and after aforementioned HPLC detection hydrophobic chromatography step, data statistics
See table 3, purity brings up to 100% from about 70%, and hydrophobic chromatography step effect is obvious.
Table 3
| Feed liquid | Purity |
| The target protein peak collected before hydrophobic step | 72.7% |
| The target protein peak collected after hydrophobic chromatography | 100% |
7, embodiment 1 and the overall testing result contrast table of comparative example 1 see table 4
Table 4
Embodiment 2
Preparing mNGF according to method same as in Example 1, difference is except hydrophobic chromatography walks
Outside Zhou, the attached cation-exchange chromatography of CM back suction, acidifying are centrifuged, Liquidity limit displacement chromatography
With exclusion chromatography step all with reference to the article appointing fine jade etc. in evening to deliver " in lyophilizing mouse nerve growth factor
Trial-production is standby and the discussion of some problem " in route B in the method introduced prepare mNGF.
Test case 2
Test case 2 is for illustrating the indices of the mNGF extracting solution of embodiment 2, detection method
Identical with test case 1, each detection purpose result is listed in table 5 and table 6, and (table 6 is homogeneity detection
Result).
Table 5
Table 6
From result above, use the NGF that embodiment 2 method obtains, isoelectric point, IP is 9.1,
8.9, predominantly two bands, homogeneity has reached 69.4%, illustrates what the present invention prepared
MNGF homogeneity height and predominantly integrity NGF.
Embodiment 3
Preparing mNGF according to method same as in Example 1, difference is to walk at hydrophobic chromatography
Insert after Zhou following steps: Superdex 75 prep grade chromatographic column pH6.8,
0.05mol/L phosphate-0.15mol/L sodium chloride buffer fully balance after loading, in ultraviolet
On detector, display digit starts when beginning to ramp up to collect, and reduces to stop collecting target during baseline
Protein peak, it is thus achieved that gel permeation chromatography sample.
Test case 3
Test case 3 is for illustrating the indices detection of the NGF extracting solution of embodiment 3.Right
Gel permeation chromatography sample detects, and compares with comparative example 1 result, and result is added up such as
Table 7 below.
Table 7
| Detection project | Embodiment 3 | Embodiment 1 | Comparative example 1 |
| HPLC purity | 100.0% | 100.0% | 93.00% |
| Isoelectric point, IP | 8.9、9.1 | 8.9、9.1 | 8.6、8.9、9.1 |
| Molecular weight (kD) | 13.0 | 13.0 | 12.1 |
| Remaining host protein (ng/mg) | 80 | 250 | 300 |
| DNA remains (ng/mg) | 8 | 100 | 310 |
From data in table, the technique of embodiment 3 compared with Example 1, product residual
Remaining host protein and DNA residual significantly reduce；Compared with comparative example 1, not only product is equal
One property significantly improves, and remaining host protein and DNA residual are significantly reduced, and patent technique is excellent
Gesture is obvious, especially embody gel permeation chromatography can significantly reduce in product endotoxin and
DNA residual quantity.
Embodiment 4-9
Preparing mNGF according to method same as in Example 1, difference is to utilize in table 8
The parameter be given carries out hydrophobic chromatography step:
Table 8
(2) experimental result
1, each hydrophobic sample testing acquisition is carried out purity, isoelectric point, IP and cell to live than method alive
Property detection, result see table 9.
Table 9
| Embodiment | HPLC purity | Isoelectric point, IP | TF-1 cell method activity (ten thousand U/mg) |
| 4 | 99.5% | 9.1,8.9 | 9.9×105 |
| 5 | 100.0% | 9.1,8.9 | 10.8×105 |
| 6 | 99.6% | 9.1,8.9 | 9.7×105 |
| 7 | 100.0% | 9.1,8.9 | 10.0×105 |
| 8 | 100.0% | 9.1,8.9 | 10.2×105 |
| 9 | 99.7% | 9.1,8.9 | 9.9×105 |
From data in table, the sample purity of each hydrophobic chromatography experiment has all reached 99.0%
Above, activity is all 9.5 × 105More than U/mg, isoelectric analysis all only has two bands,
Product homogeneity relatively old technology is obviously improved.Isoelectric focusing electrophoresis result is shown in Fig. 8, wherein
In A figure and B figure, right side has the swimming lane of three bands to be the sample in comparative example 1.
2, homogeneity test result
From Fig. 8 (Fig. 8 A and Fig. 8 B), experiment 1-6 is respectively this use embodiment
The NGF that method in 4-9 obtains, isoelectric point, IP is 9.1,8.9, predominantly two bands, total length
The content (isoelectric point, IP is 9.1 bands) of NGF has reached more than 65%；Comparative example 1 method obtains
The NGF arrived, isoelectric point, IP is 9.1,8.9,8.6, and isoelectric point, IP is 3 bands, and homogeneity is poor,
And wherein the content of total length NGF (isoelectric point, IP is the band of 9.1) can only achieve 13.5%,
Illustrate that the mNGF homogeneity that the present invention prepares is higher.
Although, the most with a general description of the specific embodiments the present invention is made
Detailed description, but on the basis of the present invention, it can be made some modifications or improvements, this
Will be apparent to those skilled in the art.Therefore, without departing from present invention spirit
On the basis of these modifications or improvements, belong to the scope of protection of present invention.
Claims (15)
1. the extracting method of a nerve growth factor, it is characterised in that described method includes:
Homogenate supernatant is collected by centrifugal after mouse submandibular gland tissue homogenate；Described homogenate supernatant is entered
Collect after the attached cation-exchange chromatography of row CM back suction and flow through liquid；The described liquid that flows through is acidified
Collected after centrifugation acidolysis supernatant；Described acidolysis supernatant is carried out Liquidity limit displacement chromatography
Collect the destination protein liquid that target protein peak is corresponding；Described destination protein liquid is carried out hydrophobic layer
Analysis, it is thus achieved that the destination protein liquid after hydrophobic chromatography.
Extracting method the most according to claim 1, it is characterised in that described hydrophobic chromatography
The balance liquid of the drainage column of middle use be salt concentration be the pH value of 0.5-3.0mol/L be 6.0-9.0
Buffer.
Extracting method the most according to claim 2, it is characterised in that described hydrophobic chromatography
The balance liquid of the drainage column of middle use is by the one in sodium chloride, ammonium sulfate and sodium sulfate
Salt is dissolved in what phosphate buffer or Tris-HCl buffer obtained, dissolves in balance liquid
The final concentration of 0.5-3.0mol/L of salt, pH value are 6.0-9.0.
Extracting method the most according to claim 3, it is characterised in that when described salt is chlorine
When changing sodium, the final concentration of 0.5-3.0mol/L of sodium chloride；When described salt is ammonium sulfate, sulfur
The final concentration of 0.5-2.0mol/L of acid ammonium；When described salt is sodium sulfate, the end of sodium sulfate is dense
Degree is 0.5-2.0mol/L.
5. according to the extracting method described in claim 3 or 4, it is characterised in that described phosphoric acid
The pH value of salt buffer is 6.0-8.0, and the pH value of described Tris-HCl buffer is 7.1-9.0.
6. according to the extracting method described in any one in claim 1-5, it is characterised in that
Drainage column medium in described hydrophobic chromatography be Butyl-S-FF, Phenyl FF, Phenyl HP,
Butyl FF, OctylFFButyl HP, Phenyl HS FF or Fractogel EMD Phenyl (S).
7. according to the extracting method described in any one in claim 1-6, it is characterised in that
During hydrophobic chromatography, loading flow velocity is 5～300cm/hr, preferably 80～200cm/hr.
Extracting method the most according to claim 7, it is characterised in that hydrophobic chromatography process
Column temperature be 2～8 DEG C.
9. according to the extracting method described in any one in claim 1-8, it is characterised in that
Described method also includes exclusion chromatography step.
10. according to the extracting method described in any one in claim 1～9, it is characterised in that
The pH value of described homogenate supernatant is 6.0-7.0.
11. extracting method according to claim 10, it is characterised in that homogenate supernatant
Preparation method comprise the following steps:
Fully it is homogenized after mouse submandibular gland is mixed with purified water, centrifugal collection supernatant, uses pH
The concentration of 6.0～7.0 be 0.5mol/L phosphate buffer regulation supernatant pH after obtain even
Slurry supernatant.
12. according to the extracting method described in any one in claim 1-11, it is characterised in that
The balance liquid used during the attached cation-exchange chromatography of CM back suction is concentration
The phosphate buffer of 0.01-0.1mol/L, pH6.0-7.0.
13. according to the extracting method described in any one in claim 1-12, it is characterised in that
Described acidifying is centrifugal to be comprised the following steps: add acidic buffer regulation pH value in liquid to flowing through
To 3.5-4.5, add NaCl, make the final concentration of 0.1-0.5mol/L of NaCl in system, quiet
Put centrifugal acquisition acidolysis supernatant.
14. extracting method according to claim 13, it is characterised in that flow through in liquid and add
The acidic buffer entered be pH be acetate buffer or the citrate buffer of 3.5-4.5.
15. according to the extracting method described in any one in claim 1-14, it is characterised in that
The step of described Liquidity limit displacement chromatography includes:
With Liquidity limit displacement chromatography balance liquid, filler is balanced, by acidolysis supernatant
Sample, first with cation-exchange chromatography balance liquid eluting, the protein pH8.5～9.5 of absorption,
The Tris-HC1 of concentration 0.01～0.10mol/L washes miscellaneous buffer and washes miscellaneous, then with pH8.5's～9.5
0.01～0.10mol/L Tris-HC1-0.2～0.8mol/L NaCl elution buffer carry out gradient and wash
De-；Wherein, described Liquidity limit displacement chromatography balance liquid containing concentration is
The acetate of 0.01-0.1mol/L and the NaCl of 0.1-0.5mol/L, pH value is 3.5-4.5.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048477A (en) * | 2017-12-15 | 2018-05-18 | 南京理工大学 | The method for preparing polypeptide based on escherichia expression system |
| CN109762056A (en) * | 2017-11-10 | 2019-05-17 | 舒泰神（北京）生物制药股份有限公司 | A kind of extracting method of nerve growth factor |
| CN114252519A (en) * | 2020-09-23 | 2022-03-29 | 舒泰神（北京）生物制药股份有限公司 | Method for determining purity of nerve growth factor |
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| CN1752102A (en) * | 2005-09-05 | 2006-03-29 | 武汉海特生物制药股份有限公司 | Technology of preparing rat nerve growth factor using organic solvent virus deactivation method |
| CN101613394A (en) * | 2008-06-27 | 2009-12-30 | 熊玲媛 | The preparation method of the preparation method of mouse nerve growth factor and injection mouse nerve growth factor |
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Cited By (4)
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| CN109762056A (en) * | 2017-11-10 | 2019-05-17 | 舒泰神（北京）生物制药股份有限公司 | A kind of extracting method of nerve growth factor |
| CN108048477A (en) * | 2017-12-15 | 2018-05-18 | 南京理工大学 | The method for preparing polypeptide based on escherichia expression system |
| CN114252519A (en) * | 2020-09-23 | 2022-03-29 | 舒泰神（北京）生物制药股份有限公司 | Method for determining purity of nerve growth factor |
| CN114252519B (en) * | 2020-09-23 | 2023-11-24 | 舒泰神（北京）生物制药股份有限公司 | Method for determining purity of nerve growth factor |
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