CN101845093B - Purification method for large-scale production of nerve growth factors in cobra venin - Google Patents

Abstract

The invention discloses a purification method for large-scale production of nerve growth factors in cobra venin, which comprises the following steps of: dissolving freeze-dried cobra venin to perform deposition by using ammonium sulfate solution; then performing centrifugation to extract supernate, and performing ultrafiltration by adopting a membrane bag of an ultrafiltration membrane of which the molecular weight cut-off is 3K; exchanging buffer solution into NaAc buffer solution of which the pH is 5.0 and of which the concentration is 50 mM; and putting samples of the exchanged buffer solution on a CM Sepharose FF chromatographic column to collect target albumen, and putting the target albumen on a Superdex 75 column to collect target albumen so as to prepare nerve growth factor stock solution of the cobra venin. Compared with the prior art, the purification method improves the treatment capacity and yield, and is suitable for the large-scale production.

Description

A kind of purification process that is suitable for the cobra-venom nerve growth factor of large-scale production

Technical field

The invention belongs to biological medicine extractive technique field, especially relate to a kind of method of extracting nerve growth factor (Nerve Growth Factor, NGF) from cobra-venom.

Background technology

Nerve growth factor (Nerve Growth Factor, NGF) is find up to now unique not only to maincenter and the nutritious factor effect of peripheroneural normal neurocyte, and has the bioactive molecules that regulates injured nerve reparation function.Nerve growth factor has great using value clinically, and current indication mainly comprises damage that various peripheral neurophaties, optic nerve injury, spinal cord injury, craniocerebral injury, central nervous system anoxic and ischemic cause and presenile dementia etc.NGF can extract from the submaxillary gland of snake venom, human fetal brains, people's placenta and male mice, also can prepare by engineered method.Wherein, gene engineering expression is the trend of preparation NGF, but because nerve growth factor must just have good biological activity in the eukaryotic cell expression systems such as Chinese hamster ovary celI, technical difficulty is large, and forming suitability for industrialized production still has considerable time.And the tissue-derived difficulties such as human fetal brains, people's placenta, cost are high; High and the risk that has animal tissues to pollute of male mice submaxillary gland raw materials cost.

By propagating poisonous snake artificially, snake venom raw materials enjoy stable sources, cost is low.Poisonous snake of the same race produces snake venom character homogeneous, and the stability of raw material is high.And avoided broken biological tissue to produce the possibility of source of pollution.

The preparation method of original cobra-venom nerve growth factor, that cobra-venom raw material is dissolved in to 50mM Tris-HCL, pH 7.0 is added with in the solution of protease activity inhibitor, centrifugal rear supernatant liquor is loaded to the molecular sieve chromatography with the good Sephadex G-100 of the solution equilibria that dissolves raw material, collect active part, dialysis exchange buffering liquid is to NaAC, the damping fluid of pH 5.0, the sample that has exchanged damping fluid is loaded to the CM-52 post that dialysis buffer liquid balance is good, NaCL solution gradient wash-out, collect active part, active part is loaded to the solution containing NaCL, pH5.0, the good Sephadex G-100 chromatography column of NaAC solution equilibria of 50mmol/L, collect active part, be cobra-venom nerve growth factor.

What this preparation process adopted is carboxymethyl cellulose positively charged ion chromatography medium (CM-52); this medium is in use because flow velocity is too slow; post bed height can change with buffer concentration and pH; and can not bear NaOH cleaning in place more than 0.1M; the weak effect of living again; if for large-scale production, can affect efficiency.The poor rigidity flow velocity of Sephadex G-100 chromatography media is slow, bears after pressure easily distortion and makes flow velocity slower and affect separating effect.Exchange buffering liquid is used dialysis, and speed is slow, dialysis procedure terminal is not easy to grasp, and solution usage is large, is unsuitable for suitability for industrialized production.

Summary of the invention

The object of the invention is to improve the deficiency of prior art and provide a kind of with short production cycle, production efficiency is high, adopt ultrafiltration substitute dialysis and prepare with ion-exchange gel CM Sepharose FF and the high-resolution molecular sieve gel Superdex 75prepgrade of high flow rate, high carrying capacity the method that cobra-venom nerve growth factor is suitable for large-scale production.

The object of the present invention is achieved like this, a kind of purification process of nerve growth factor of the cobra-venom that is suitable for large-scale production, and its feature is:

1) after the cobra-venom dry powder of freeze-drying is water-soluble, add ammonium sulfate, become 50～60% ammoniumsulphate soln, after it fully mixes, standing 30min, the centrifugal 30min of 10000rpm, collects supernatant liquor,

2) selecting molecular weight cut-off is that the ultra-filtration membrane bag of 3k～5k is the 50mMNaAc solution of pH 5.0 by supernatant liquor ultrafiltration exchange buffering liquid;

3) supernatant liquor that completes buffer-exchanged is crossed to 0.22 μ m filter membrane, removed impurity particle and bacterium;

4) supernatant liquor of crossing after film is splined on to the good CM Sepharose FF chromatography column of NaAc buffered soln balance that pH 5.0 concentration are 50mM, flow velocity 5ml/min, loading is complete, in 8 times of column volumes, the NaCL concentration in damping fluid is risen to 0.5M, straight line gradient from 0;

5) albumen with NGF activity in step in collection, with 3K ultra-filtration membrane film bag, protein solution is concentrated;

6) target protein after concentrated is splined on the solution containing 0.15mol/L NaCL, the good Superdex 75prep grade post of Tirs solution equilibria of pH7.520mmol/L, and flow velocity 8ml/min, collects target protein, is cobra-venom nerve growth factor stoste.

The inventive method is compared greatly and has been shortened the production cycle with original method, has improved treatment capacity and yield, is applicable to large-scale production.

With ammoniumsulphate soln, process snake venom, precipitated a large amount of foreign proteins in raw material, for follow-up ion-exchange step eases off the pressure, improved treatment capacity, adopt the advantage of this method to be:

1, the cycle short, as long as whole precipitation process one hour, is significantly less than the needed time of chromatography that adopts.

2, require lowly, in whole process, only need a kind of auxiliary material of ammonium sulfate, and do not need specific installation.

3, effective, can remove at short notice the foreign protein up to about 50%.

Use ultra-filtration membrane film bag, ultra-filtration membrane is wherein the semi-permeable membranes that adopts special process to make with macromolecular material, the molecule that is less than membrane pore size under the effect of pressure sees through film becomes ultrafiltrated, other polymer substance, colloid, ultramicron, bacteriums etc. tunicle stop and become concentrated solution, thereby reach the separation of material, the object of concentrated purification.Ultra-filtration and separation has adaptability widely for separated solute in extremely dilute solution, compares with other sepn process, has following features:

1, ultra-filtration process carries out at normal temperatures, energy consumption is low, do not need heating, without heat effect and phase-state change, not needing to add other material can reach separated, concentrated, purifying is the objects such as classification, therefore be specially adapted to: the 1) concentrating and separating of heat-sensitive substance (biological products, thalline, protein).2) concentration and recovery of extremely dilute solution.3) desalting and purifying under the permanent concentration of constant volume.

2, ultra-filtration membrane resistance to chemical attack, PH wide accommodation.

3, treatment capacity is large, and speed is fast, is suitable for suitability for industrialized production and amplification.

Adopt Ago-Gel medium, have following characteristics:

1, medium carrier is rigid material, granularity 90 μ m, and flow velocity is fast, and binding capacity is large, and linear velocity can reach 300-500cm/h, fast to crude extract processing speed.

2, this cleaning of medium is effective.After active part wash-out, available .0.5M NaOH solution carries out cleaning in place, and wash-out impurity is effective.Avoided tearing open post simultaneously and again filled post, having saved time.

3, this medium filling does not need specific installation, and effect favorable reproducibility, can amplify applicable suitability for industrialized production in proportion.

Adopt ultra-filtration membrane concentrating sample, improve the concentration of sample and can collect more the finished product, improved yield, reduce with chromatography media still less, can complete separation after volume, saved cost.

The present invention is by using ammonium sulfate precipitation foreign protein, and ultrafiltration substitutes dialysis and uses flow velocity chromatography media faster, that resolving power is higher to reach saving production time, production cost, has improved feed throughput, obtains the object of high-quality, high yield product.Be applicable to large-scale production.

Accompanying drawing explanation

Below in conjunction with drawings and Examples, the present invention is described in further detail.

Fig. 1 CM Sepharose FF tomographic map

Fig. 2 Superdex 75prep grade tomographic map

Embodiment

Embodiment, a kind of purification process that is suitable for the cobra-venom nerve growth factor of large-scale production is to comprise the following steps:

(1) ammonium sulfate precipitation raw material:

Qualified freeze-drying cobra-venom dry powder 10g is dissolved in to 200ml water, can agitation as appropriate, but avoid producing too much foam, after snake venom dissolves completely, with the centrifugal 10min of 10000rpm, get supernatant liquor and cross the filter membrane of 0.45 μ m, remove insoluble impurities, add ammonium sulfate 110g, agitation as appropriate, will avoid equally producing too much foam, form after the ammoniumsulphate soln of 55% concentration, standing 30min treats the complete polymerization of the colloid in solution, separate out precipitation, then the centrifugal precipitation that makes of 10000rpm is separated with supernatant, carefully pours out supernatant liquor 120ml, standby;

(2) ultrafiltration exchange buffering liquid:

Pour upper step gained supernatant liquor 120ml into 3KDa molecular weight ultrafiltration system, adjust pressure and flow velocity, after 10 times of volume damping fluid circulations, the damping fluid of supernatant liquor changes the NaAc solution that pH 5.0 concentration are 50mM into, supernatant liquor is poured out after 0.22 μ m filter membrane so that except degerming and impurity particle;

(3) CM Sepharose FF chromatography:

The supernatant soln of crossing after film is loaded to the good CM Sepharose FF chromatography column (5.0 * 30cm) of NaAc solution equilibria that pH 5.0 concentration are 50mM, flow velocity 5ml/min, after Dai Liuchuan peak wash-out, change elution buffer, elution buffer is in level pad, to add NaCl, make NaCl concentration with straight line gradient, rise to 0.5M from 0 equably in 8 times of column volumes, collect active part, see Fig. 1;

(4) ultrafiltration and concentration protein solution:

The active part of collecting is poured ultrafiltration system into, adjusts pressure and flow velocity, during solution 60ml in Storage Cup, pours out, and waits until lower step and uses;

(5) Superdex 75prep grade chromatography:

The protein solution having concentrated is splined on to the NaCl containing 0.15mol/L, pH7.5, the good Superdex 75 prep grade chromatography columns (5.0 * 80cm) of Tirs solution equilibria of 20mmol/L, flow velocity 8ml/min, collect target protein, be cobra-venom nerve growth factor stoste, see Fig. 2;

(6) desalination, vacuum freezedrying

3KDa molecular weight ultra-filtration membrane ultrafiltration desalination for the nerve growth factor product peak that step (5) is obtained, crosses 0.22 μ m filter membrane, aseptic subpackaged, has both obtained nerve growth factor 62mg after lyophilize.

Claims (1)

1. a purification process that is suitable for the cobra-venom nerve growth factor of large-scale production, is characterized in that:

1) after the cobra-venom dry powder of freeze-drying is water-soluble, add ammonium sulfate, become 50 ~ 60% ammoniumsulphate soln, after it fully mixes, standing 30min, the centrifugal 30min of 10000rpm, collects supernatant liquor;

2) selecting molecular weight cut-off is that the ultra-filtration membrane bag of 3KDA is the 50mM NaAc solution of pH 5.0 by supernatant liquor ultrafiltration exchange buffering liquid;

3) supernatant liquor that completes buffer-exchanged is crossed to 0.22 μ m filter membrane, removed impurity particle and bacterium;

4) supernatant liquor of crossing after film is splined on to the good CM Sepharose FF chromatography column of NaAc buffered soln balance that pH 5.0 concentration are 50mM, flow velocity 5ml/min, loading is complete, in 8 times of column volumes, the NaCL concentration in damping fluid is risen to 0.5M, straight line gradient elution from 0;

5) albumen with NGF activity in step in collection, with 3K ultra-filtration membrane film bag, protein solution is concentrated;

6) target protein after concentrated is splined on the solution containing 0.15mol/L NaCL, the good Superdex 75 prep grade chromatography columns of Tirs solution equilibria of pH7.5 20mmol/L, flow velocity 8ml/min, collects target protein, is cobra-venom nerve growth factor stoste.

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