CN101747409B - Cobra-venom factor and cobra-venom neurotoxin combined separation and purification method - Google Patents
Cobra-venom factor and cobra-venom neurotoxin combined separation and purification method Download PDFInfo
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- CN101747409B CN101747409B CN201010011462.5A CN201010011462A CN101747409B CN 101747409 B CN101747409 B CN 101747409B CN 201010011462 A CN201010011462 A CN 201010011462A CN 101747409 B CN101747409 B CN 101747409B
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Abstract
The invention discloses a cobra-venom factor and cobra-venom neurotoxin combined separation and purification method which includes the following steps: firstly the cobra-venom crude liquid is processed with centrifugation treatment at low temperature and high speed, and then small molecule substances, low molecular peptides and the like contained in the cobra-venom crude liquid are removed through a 3KDa ultrafiltration membrane. The two chromatography processes respectively adopt a CM-Sephrose. FF chromatographic column and a Superdex.200 gel chromatographic column; and cobra-venom neurotoxin and cobra-venom factor which are two active ingredients can be obtained in one production process. The method has the characteristics that the cobra-venom factor and the cobra-venom neurotoxin can be separated, the application efficiency of the cobra-venom is effectively improved, the purities of the products are improved and the method is suitable for industrial production.
Description
Technical field
The invention belongs to biological medicine technology field, especially relate to the combined separation and purification method of a kind of cobra-venom factor and Cobra neurotoxin.
Background technology
Cobra venom is a kind of natural toxalbumin being secreted by elapid poison gland, its chemical composition is very complicated, contain multiple proteins, polypeptide, enzyme and other small-molecule substances, have biologic activity widely, wherein main have neurotoxin, cytotoxin, nerve growth factor and a cobra-venom factor (CVF) etc.Along with the development of modern biotechnology, many components of cobra venom have obtained separation and purification and sequencing, and various compositions are widely used in biological chemistry, molecular biology, toxicology and pharmacology theoretical investigation and clinical application.
From Monaelesser in 1933 and Taguet reported first cobra venom cause the significant curative effect of case of pain to cancerous tissue pressuring nerve since, the analgesic activity of cobra venom has obtained deep research, Cobra neurotoxin (neurotoxin wherein, NTX) shown unique analgesic activity, analgesia mechanism and the morphine of NTX are similar, and analgesic effect is greater than morphine, time length is long, without anesthetic action, without habituation and resistance, but onset is slow compared with morphine; NTX can also give up morphine drug addiction simultaneously, so have unique therapeutic action at aspects such as pain caused by cancer or drug rehabilitations.
At present the extracting method of snake venom neurotoxin is mainly to adopt ion exchange method attached gel chromatography, and the extracting method of prior art exists that step is many, separation cycle is long, extraction cost is high, and snake venom once extracts the problem such as can not reuse afterwards.Such as: the general pearl of Lee etc. disclosed " separation and purification of neurotoxin and analgesic activity research thereof in the cobra-venom of Zhejiang " (Chinese pharmacists, 2004,7,9,659-661), adopt three times column chromatography, many, the consuming time length of step, extraction cost are high, are difficult for as large-scale industrialization separation purifying technique; Disclosed " a kind of short-cut method of separating-purifying Cobratoxin " (1981 such as Gong Chaoliang, 2,4,83～87) in, adopt the absorption of CM-cellulose column linear elution, the linear rate of flow that CM--celluosic resin allows is very low, and very easily compressed, cause technique length consuming time, unstable product quality, belong to should be superseded old technology.
Cobra-venom factor (Cobra venom factor, CVF), is called again Cobraanticomplementary protein (Cobraanticomplementary protein), is a kind of bypass complement activation thing that derives from Elapidae snake venom.Because CVF can finally exhaust complement again by activating complement, and stable in properties, specificity is high, is therefore widely used in the research of preclinical medicine. there is wide potential applicability in clinical practice, and may be for the following aspects: the instrument medicine of Effect of Anti role of complement; For anti-inflammatory and anti-autoimmune disorder; Resisting transplant rejection reaction; Antineoplastic guide medicine etc.
Due to CVF in cobra venom content very as, extract more difficult, so seldom there is the CVF separating and purifying method of open report, Ye Chunling etc. are open the short-cut method of the separating-purifying anticomplement factor " a kind of from snake venom " (Chinese Pharmacological Bulletin, 1997Feb once; 13 (1): 85～87); ion exchange chromatography two-step chromatography is separated again to adopt first gel chromatography; gel chromatography adopts Bio-gel P200 post; ion exchange chromatography adopts DEAE cellulose DE 52 posts, and the method can be prepared sample on a small quantity, but above two kinds of filler rigidity are poor; chromatography column easily compresses; length consuming time, is not suitable for doing scale production process, and most of echidnotoxin fails effectively to utilize.
Summary of the invention
The object of the invention is to improve the deficiency of prior art and provide separated Cobra neurotoxin of a kind of while and cobra-venom factor, substep to obtain major products, reduce phase mutual interference between product, avoided desalination to cause that the solubleness of CVF reduces and the solution that brings is muddy and easy protein-denatured problem, comprehensive utilization ratio and extraction efficiency is high, product purity is high, be applicable to the cobra-venom factor of suitability for industrialized production and the combined separation and purification method of Cobratoxin.
The object of the present invention is achieved like this, the combined separation and purification method of a kind of cobra-venom factor and Cobra neurotoxin, and its feature is that the method includes the steps of:
1), get in the Tris damping fluid that snake venom is dissolved in purified water or pH5-8,10 ℃ of following low-temperature centrifugations, get supernatant liquor, cross millipore filtration;
2), by step 1) resulting supernatant liquor carries out ultrafiltration and is exchanged for dissociating buffer, adopts the ultra-filtration membrane that molecular weight cut-off is 3kDa-5kDa to carry out;
3), by step 2) resulting snake venom carries out respectively ion exchange chromatography and gel chromatography, gets respective components and carries out that desalination is concentrated, lyophilize, obtains respectively cobra-venom factor and the Cobra neurotoxin composition of purifying.
In order further to realize object of the present invention, can be that snake venom used is cobra venom dry powder or lyophilized powder.
In order further to realize object of the present invention, can be described step 1) in, adopting 0-10 ℃ of low-temperature centrifugation, supernatant liquor is crossed 0.22 μ m or 0.44 μ m millipore filtration.
In order further to realize object of the present invention, can be described step 2) in, the damping fluid of exchange is a kind of in Tris-HCl damping fluid between pH5-8 or acetate buffer or PBS damping fluid, and it is the ultrafiltration membrane system between 3-5kDa that ultrafiltration adopts molecular weight cut-off.
In order further to realize object of the present invention, can be described step 3) in comprise the steps:
A, by step 2) supernatant liquor that finally obtains carries out the ultrafiltration that molecular weight cut-off is 3～5kDa;
B, the damping fluid balance cation exchange column that is 5-8 by pH value, after the snake venom loading that step a is obtained, 0-0.5M is containing sodium salt stage gradient or the linear gradient elution of damping fluid;
In c, collection step b, have Cobratoxin activeconstituents peak, carry out desalination concentrate according to step a mode, vacuum lyophilization, obtains Cobra neurotoxin;
D, collection step b Zhong Liuchuan peak component, concentrated according to step a;
The gel chromatography column of the sodium chloride solution balance of e, use 10-100mM, chromatography media comprises Superdex 200, Sephacryl 200 or the isolated molecule amount gel chromatography medium more than 100kDa, gets the concentrated solution upper prop of steps d;
F, collect the cobra-venom factor composition in step e, according to the partial deionization final vacuum lyophilize of step a mode or the direct vacuum lyophilization of desalination not, obtain cobra-venom factor.
The inventive method has the following advantages:
1. simultaneously separated Cobra neurotoxin and cobra-venom factor of the present invention, has improved the utilising efficiency of this rare resources of cobra venom;
2. the present invention gets separated cobra-venom factor composition in the Liu Chuan peak of the separated Cobra neurotoxin of cationic exchange coloum method, in two-step approach, in every step, obtains major products, reduces the phase mutual interference between product;
3. adopt directly band salt freeze-drying CVF, avoided desalination to cause that the solubleness of CVF reduces and solution muddiness and the easy protein-denatured problem brought;
The present invention extracts Cobratoxin and cobra-venom factor simultaneously, and comprehensive utilization ratio and extraction efficiency are high, and product purity is high, and the Cobratoxin purity obtaining is more than 95%, and cobra-venom factor purity is more than 90%.The cobra-venom factor composition that adopts the inventive method to obtain, through the check of SDS-PAGE electrophoresis, non-reduced method is a band, reduction method is three bands.Be applicable to suitability for industrialized production, and the application prospect of these two kinds of products is extensive.
The Cobratoxin that the inventive method obtains, the base polypeptide being formed by 68 amino-acid residues, molecular weight 7000Da left and right.Due to the special construction of its base polypeptide, through the about 12kDa of apparent molecular weight of SDS-PAGE electrophoresis detection, consistent with Cobratide national standard.
The cobra-venom factor that the inventive method obtains (CVF), molecular weight is 130-230kDa, iso-electric point 4.4-6.0, article three, peptide chain composition links by disulfide linkage and non covalent bond, after adding DDT (dithiothreitol (DTT)) reduction, SDS-PAGE verifies as three bands, and molecular weight is followed successively by 30kDa, 50kDa, 66kDa left and right.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the present invention is described in further detail.
Fig. 1 be the inventive method to Cobratoxin with purchased from middle inspection the SDS-PAGE electrophoresis check of Cobratide standard substance Cobratoxin contrast figure.
Fig. 2 be the inventive method to cobra-venom factor add dithiothreitol (DTT) reduction through SDS-polyacrylamide gel electrophoresis check figure.
Embodiment
Embodiment, the combined separation and purification method of a kind of cobra-venom factor and Cobra neurotoxin is to adopt following steps:
1. the processing of the thick poison of sample
Take in the Tris damping fluid that snake venom 20g is dissolved in 200ml purified water or pH5-8,10 ℃ of following 10000rpm low-temperature centrifugation 10min, get supernatant liquor, mistake 0.22 μ m millipore filtration, after film, solution is exchanged for dissociating buffer with the ultra-filtration membrane that molecular weight cut-off is 3kDa-5kDa excessively;
2.CM-Sepharose FF chromatography
Snake venom solution after exchange buffering liquid is loaded to pH 7.5 with peristaltic pump with the flow velocity of 10ml/min, the CM Sepharose FF chromatography column (5.0 * 30cm) of the Tris damping fluid balance of 20mmol/L, and with level pad wash-out Wan Liuchuan peak and collect it, in elution buffer afterwards, add NaCL wash-out respectively, the NaCL concentration adding is 0.1M, 0.2M, 0.4M, institute in steps flow velocity is 10ml/min, collects Cobratoxin;
3. Cobratoxin ultrafiltration desalination, freeze-drying
With the ultra-filtration membrane film bag of 3Ka molecular weight, Cobratoxin solution is removed to buffering salt, become salt-free protein solution, the protein solution after desalination is crossed to 0.22 μ m filter membrane, aseptic subpackaged, after lyophilize, both obtained Cobratoxin bulk drug 1206mg;
4. cobra-venom factor is concentrated
The Liu Chuan peak that step 2 is collected uses the film bag ultrafiltration and concentration of 10KDa molecular weight to 60ml;
5.Superdex 200 chromatographies
60ml cobra-venom factor concentrated solution is loaded to the Superdex200 chromatography column 5.0 * 80cm of 50mM NaCl balance, 10ml/min loading, wash-out, collects cobra-venom factor product peak;
6. desalination, vacuum freezedrying
The cobra-venom factor product peak that step 5 is obtained to 5mM NaCl, is crossed 0.22 μ m filter membrane with the ultrafiltration of 10KDa molecular weight ultra-filtration membrane, aseptic subpackaged, has both obtained cobra-venom factor bulk drug 162mg after lyophilize.Get Cobratoxin that the present embodiment obtains with purchased from middle inspection Cobratide standard substance (Cobratoxin) carry out experiment contrast, SDS-PAGE electrophoresis check figure is shown in Fig. 1, in figure, left side is for product of the present invention, right side be middle inspection Cobratide standard substance.
Get the cobra-venom factor that the present embodiment obtains, add dithiothreitol (DTT) reduction through the check of SDS-polyacrylamide gel electrophoresis, see Fig. 2, as can be seen from Figure, three subunits that CVF is 32000Da, 49000Da, 66000Da by molecular weight form, and meet the feature request of CVF.
Claims (2)
1. a combined separation and purification method for cobra-venom factor and Cobra neurotoxin, is characterized in that the method includes the steps of:
1), get in the Tris damping fluid that snake venom is dissolved in purified water or pH5-8, at 0-10 ℃ of temperature, 10000rpm low-temperature centrifugation 10min, gets supernatant liquor, crosses 0.22 μ m or 0.44 μ m millipore filtration;
2), the resulting supernatant liquor of step 1) is carried out to ultrafiltration and is exchanged for dissociating buffer, adopt the ultra-filtration membrane that molecular weight cut-off is 3kDa-5kDa to carry out, damping fluid is a kind of in Tris-HCl damping fluid between pH5-8 or acetate buffer or PBS damping fluid;
3), by step 2) resulting snake venom successively carries out ion exchange chromatography and gel chromatography, gets respective components desalination, lyophilize, obtains respectively Cobra neurotoxin and the cobra-venom factor composition of purifying, wherein:
A, the snake venom solution after exchange buffering liquid is loaded to pH 7.5 with peristaltic pump with the flow velocity of 10ml/min, the CM Sepharose FF chromatography column (5.0 * 30cm) of the Tris damping fluid balance of 20 mmol/L, and with level pad wash-out Wan Liuchuan peak and collect it, in elution buffer afterwards, add NaCL wash-out respectively, the NaCL concentration adding is 0.1M, 0.2M, 0.4M, institute in steps flow velocity is 10ml/min, collects Cobratoxin;
B, the film bag ultrafiltration and concentration by the Liu Chuan peak of collecting with 10KDa molecular weight, concentrated solution is loaded to the Superdex 200 chromatography column 5.0 * 80cm of 50mM NaCl balance, 10ml/min loading, wash-out, collect cobra-venom factor product peak, the cobra-venom factor product peak obtaining is arrived to 5mM NaCl with the ultrafiltration of 10KDa molecular weight ultra-filtration membrane, cross 0.22 μ m filter membrane, aseptic subpackaged, after lyophilize, both obtained cobra-venom factor composition.
2. the combined separation and purification method of a kind of cobra-venom factor according to claim 1 and Cobra neurotoxin, is characterized in that snake venom used is cobra venom dry powder or lyophilized powder.
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| CN102133232B (en) * | 2011-03-18 | 2013-03-06 | 苏州大学 | Cobra venom physical modification method and application in preparation of analgesia or immunosuppressive drugs |
| CN102351951A (en) * | 2011-10-24 | 2012-02-15 | 贵州益佰制药股份有限公司 | Purification method, extract and preparation of cobra venom neurotoxin |
| CN102526695A (en) * | 2012-01-05 | 2012-07-04 | 苏州人本药业有限公司 | Application of naja atra venin to treatment of diabetes and diabetic nephropathy complicating disease |
| CN112592947A (en) * | 2020-12-08 | 2021-04-02 | 宜昌三峡制药有限公司 | Clean production and fermentation method of neomycin sulfate |
| CN113416242A (en) * | 2021-04-07 | 2021-09-21 | 云南龙凤谷生物药业有限公司 | Preparation method of NGF for cobra venom injection |
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