CN102424701A - Method for purifying human growth hormone-like protein in silkworm pupa - Google Patents
Method for purifying human growth hormone-like protein in silkworm pupa Download PDFInfo
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- CN102424701A CN102424701A CN201110309518XA CN201110309518A CN102424701A CN 102424701 A CN102424701 A CN 102424701A CN 201110309518X A CN201110309518X A CN 201110309518XA CN 201110309518 A CN201110309518 A CN 201110309518A CN 102424701 A CN102424701 A CN 102424701A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 36
- 241000255789 Bombyx mori Species 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title abstract description 9
- 241000382353 Pupa Species 0.000 title abstract description 6
- 230000003054 hormonal effect Effects 0.000 title abstract 3
- 238000000746 purification Methods 0.000 claims abstract description 29
- 238000010521 absorption reaction Methods 0.000 claims abstract description 25
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 10
- 102000002265 Human Growth Hormone Human genes 0.000 claims description 26
- 108010000521 Human Growth Hormone Proteins 0.000 claims description 26
- 239000000854 Human Growth Hormone Substances 0.000 claims description 26
- 102000004895 Lipoproteins Human genes 0.000 claims description 24
- 108090001030 Lipoproteins Proteins 0.000 claims description 24
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- 239000000872 buffer Substances 0.000 claims description 15
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- 238000010168 coupling process Methods 0.000 claims description 12
- 238000005859 coupling reaction Methods 0.000 claims description 12
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000012528 membrane Substances 0.000 claims description 9
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 7
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
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- 238000001262 western blot Methods 0.000 claims description 4
- 238000001261 affinity purification Methods 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
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- 239000012148 binding buffer Substances 0.000 claims description 3
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- 101150038956 cup-4 gene Proteins 0.000 claims description 3
- 230000008021 deposition Effects 0.000 claims description 3
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- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 3
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 3
- 238000003828 vacuum filtration Methods 0.000 claims description 3
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- 239000008280 blood Substances 0.000 abstract description 11
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- 241000238631 Hexapoda Species 0.000 description 1
- 235000008098 Oxalis acetosella Nutrition 0.000 description 1
- 241001119522 Paullinia pinnata Species 0.000 description 1
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- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention belongs to a method for purifying human growth hormone-like protein in silkworm pupa. The method comprises the following steps of: pretreating the silkworm pupa; primarily separating the human growth hormone-like protein; performing affinity chromatography purification and ultrafiltration and concentration by using a product of a No.4 protein absorption peak, and the like. The silkworm pupa is used as a raw material, and is separated and purified to form natural medicinal protein for reducing blood viscosity and improving blood microcirculation; the method is easy to implement, the yield is high, and the method is suitable for large-scale production; and the purified natural protein can serve as a healthcare medicine without toxic or side effect.
Description
Technical field
The invention belongs to the purification process of the similar lipoprotein of human growth hormone in a kind of silkworm chrysalis.
Background technology
Silkworm chrysalis is human a kind of new nutrition source, be Ministry of Health's approval as unique insects food in the new resource for food list of bread and cheese management.Silkworm chrysalis has high nutritive value, contains rich in protein, lipid acid, VITAMINs.Edible silkworm chrysalis reduces blood cholesterol and triglyceride level to the SUV esterification; Blood viscosity lowering, improving blood microcirculation has certain effect.But at present, basically also do not occur on the market with the product of effective constituent in the silkworm chrysalis as drug development, lack these natural components purification process.
Summary of the invention
The objective of the invention is to design the purification process of the similar lipoprotein of human growth hormone in a kind of silkworm chrysalis, can utilize silkworm chrysalis, obtain to have blood viscosity lowering through separation and purification as raw material; Improve the natural pharmaceutical protein of blood microcirculation; This method is easy and simple to handle, and output is high, is applicable to scale operation; The native protein of purifying can be used as health medicine, has no side effect.
For this reason, the step down that comprises of the present invention:
(1) silkworm chrysalis pre-treatment, with the PBS liquid of fresh silkworm chrysalis and precooling, 1～5 ℃ of temperature, pH value were 7.4, joined homogenate 30sec in the juicer by weight 1: 1, at ice bath 4～8min on ice, continued homogenate, triplicate fully dissolves the albumen in the silkworm chrysalis,
The gained article are transferred in the Centrifuge Cup 4 ℃, and the centrifugal 20min of 12000rpm gets supernatant and removes the insoluble impurity of macrobead through nine layers of filtered through gauze, and the recentrifuge of will filtrating filters, triplicate,
Collect 4 ℃ of filtrate filtered, 60% ammonium sulfate precipitated protein is removed grease, and the centrifugal 30min of 8000rpm abandons supernatant, and with the resuspended deposition of PBS liquid of precooling, 0.45 μ m filter membrane vacuum filtration gets the pre-treatment supernatant, 4 ℃ of preservations;
(2) initial gross separation of the similar lipoprotein of human growth hormone, the Size Exclusion among the selection AKTA explorer10 separates the pre-treatment supernatant and sloughs pigment, and moving phase is PBS liquid, and the pH value is 7.4, pressure 0.5Mpa, flow velocity 1.5ml/min; After the EP, occur 6 albumen absorption peaks altogether, judge that according to the purpose appearance time the 5th and No. 6 albumen absorption peak is pigment and small-molecule substance below the 17kDa; The similar lipoprotein of human growth hormone, molecular weight 30kDa should appear at the peak No. 4; Therefore collect 4 the albumen absorption peaks in front; And carry out SDS-PAGE electrophoresis and Western Blot and detect, the result show the similar lipoprotein of human growth hormone deposit with No. 4 protein peak in, carry out subsequent purification with the gained article of No. 4 albumen absorption peaks;
(3) affinitive layer purification carries out affinitive layer purification with the gained article of No. 4 albumen absorption peaks,
The Plus coupling Resin that 2mL 50% is got in the preparation of affinity column packs in the post shell, and balance is emitted residual liquid to room temperature; Add 3 times of column volume Coupling Buffer, 0.1M Sodium phosphate, 0.15M NaCl; The pH value is 7.2, and the balance pillar adds 2mL hGH standard substance 1mg/ml and 40 μ L Cyanoborohy dride afterwards; Shaking table is hatched 4h or 4 ℃ of maintenance 7～9h under the room temperature, and uncap in stink cupboard is collected the liquid of emitting; Use the BCA protein quantification, calculate coupling efficiency; Add 5 times of column volume Quenching Buffer again, 1M Tris-HCl, the pH value is 7.4,0.05%NaN
3, wash filler, add 2mL Quenching Buffer and 40 μ L Cyanoborohy dride afterwards, shaking table is hatched 30min under the room temperature; Emit the gas and the solution of reaction at the stink cupboard uncap, add 5 times of volume Wash Solution, 1M NaCl, 0.05%NaN
3Wash pillar; Use Coupling Buffer balance pillar again, 4 ℃ of preservations;
The similar lipoprotein of affinity purification human growth hormone, the equilibrium at room temperature pillar, uncap discards protection liquid afterwards, with the Binding Buffer of 5 times of column volumes, 0.15M PBS, the pH value is 7.4, once more the balance pillar; The gained article through 0.45 μ m membrane filtration after upper prop, the room temperature shaking table is hatched 1h, emits solution, uses Wash Buffer; 0.15M PBS, pH7.4, flush away does not combine sample, adds Elution Buffer (0.1M Glycine-HCl at last; The pH value is 2.5, and the albumen of elution of bound on pillar is used Neutralization Buffer; 0.5M Tris-HCl, the pH value is 8.5, and adjust pH is to neutral; Collect sample, carry out the SDS-PAGE electrophoresis detection behind the BCA protein quantification;
(4) ultrafiltration and concentration uses hollow-fibre membrane to carry out ultrafiltration, selects suitable hollow-fibre membrane specification, and molecular weight cut-off is 3kDa; Clean the back application of sample, with an amount of aseptic PBS, the pH value is 7.4, ultrafiltration and concentration; Aseptic subpackaged, lyophilized powder is processed in-50 ℃ of lyophilizes, gets finished product of the present invention.
Described affinitive layer purification is that the gained article with No. 4 albumen absorption peaks carry out affinitive layer purification.
Aforesaid method has been realized the object of the invention.
Advantage of the present invention is to utilize silkworm chrysalis as raw material, obtains to have blood viscosity lowering through separation and purification, improves the natural pharmaceutical protein of blood microcirculation; This method is easy and simple to handle, and output is high, is applicable to scale operation; The native protein of purifying can be used as health medicine, has no side effect.
Description of drawings
Fig. 1 (1), (2) and Fig. 2 are the initial gross separation figure of the similar lipoprotein of human growth hormone in the silkworm chrysalis.
Wherein M. is low molecular mass protein standard; 1. total pupa albumen sample; 2.1
#The absorption peak protein sample; 3.2
#The absorption peak protein sample; 4.3
#The absorption peak protein sample; 5.4
#The absorption peak protein sample.
Fig. 3 is the Western Blot detection figure of the similar lipoprotein of human growth hormone in the silkworm chrysalis.
Wherein M. is low molecular mass protein standard; 1.4
#The absorption peak protein sample; 3. purification of samples.
Fig. 4 and Fig. 5 are that the K562 cells in vitro detects the similar lipoprotein growth promoting function of human growth hormone comparison diagram.
Embodiment
Extremely shown in Figure 5 like Fig. 1, the purification process of the similar lipoprotein of human growth hormone in a kind of silkworm chrysalis.Comprise step down: (1) silkworm chrysalis pre-treatment, with the PBS liquid of fresh silkworm chrysalis and precooling, 1～5 ℃ of temperature; The pH value was 7.4, joined homogenate 30sec in the juicer by weight 1: 1, at ice bath 4～8min on ice; Continue homogenate, triplicate fully dissolves the albumen in the silkworm chrysalis.The gained article are transferred in the Centrifuge Cup 4 ℃, and the centrifugal 20min of 12000rpm gets supernatant and removes the insoluble impurity of macrobead through nine layers of filtered through gauze, and the recentrifuge of will filtrating filters, triplicate.Collect 4 ℃ of filtrate filtered, (weight ratio) 60% ammonium sulfate precipitated protein is removed grease, and the centrifugal 30min of 8000rpm abandons supernatant, and with the resuspended deposition of PBS liquid of precooling, 0.45 μ m filter membrane vacuum filtration gets the pre-treatment supernatant, 4 ℃ of preservations.(2) initial gross separation of the similar lipoprotein of human growth hormone, the Size Exclusion among the selection AKTA explorer10 separates the pre-treatment supernatant and sloughs pigment, and moving phase is PBS liquid, and the pH value is 7.4, pressure 0.5Mpa, flow velocity 1.5ml/min; After the EP, occur 6 albumen absorption peaks altogether, judge that according to the purpose appearance time the 5th and No. 6 albumen absorption peak is pigment and small-molecule substance below the 17kDa; The similar lipoprotein of human growth hormone, molecular weight 30kDa should appear at the peak No. 4; Therefore collect 4 the albumen absorption peaks in front; And carry out SDS-PAGE electrophoresis and Western Blot and detect, the result show the similar lipoprotein of human growth hormone deposit with No. 4 protein peak in, carry out subsequent purification with the gained article of No. 4 albumen absorption peaks.(3) affinitive layer purification carries out affinitive layer purification with the gained article of No. 4 albumen absorption peaks.The Plus coupling Resin that 2mL 50% is got in the preparation of affinity column packs in the post shell, and balance is emitted residual liquid to room temperature; Add 3 times of column volume Coupling Buffer, 0.1M Sodium phosphate, 0.15M NaCl; The pH value is 7.2, and the balance pillar adds 2mL hGH standard substance 1mg/ml and 40 μ L Cyanoborohy dride afterwards; Shaking table is hatched 4h or 4 ℃ of maintenance 7～9h under the room temperature, and uncap in stink cupboard is collected the liquid of emitting; Use the BCA protein quantification, calculate coupling efficiency; Add 5 times of column volume Quenching Buffer again, 1M Tris-HCl, the pH value is 7.4,0.05%NaN
3, wash filler, add 2mL Quenching Buffer and 40 μ L Cyanoborohy dride afterwards, shaking table is hatched 30min under the room temperature; Emit the gas and the solution of reaction at the stink cupboard uncap, add 5 times of volume Wash Solution, 1MNaCl, 0.05%NaN
3Wash pillar; Use Coupling Buffer balance pillar again, 4 ℃ of preservations.The similar lipoprotein of affinity purification human growth hormone, the equilibrium at room temperature pillar, uncap discards protection liquid afterwards, with the Binding Buffer of 5 times of column volumes, 0.15M PBS, the pH value is 7.4, once more the balance pillar; The gained article through 0.45 μ m membrane filtration after upper prop, the room temperature shaking table is hatched 1h, emits solution, uses Wash Buffer; 0.15M PBS, pH7.4, flush away does not combine sample, adds Elution Buffer (0.1M Glycine-HCl at last; The pH value is 2.5, and the albumen of elution of bound on pillar is used Neutralization Buffer; 0.5M Tris-HCl, the pH value is 8.5, and adjust pH is to neutral; Collect sample, carry out the SDS-PAGE electrophoresis detection behind the BCA protein quantification.(4) ultrafiltration and concentration uses hollow-fibre membrane to carry out ultrafiltration, selects suitable hollow-fibre membrane specification, and molecular weight cut-off is 3kDa; Clean the back application of sample, with an amount of aseptic PBS, the pH value is 7.4, ultrafiltration and concentration; Aseptic subpackaged, lyophilized powder is processed in-50 ℃ of lyophilizes, gets finished product of the present invention.Described affinitive layer purification is that the gained article with No. 4 albumen absorption peaks carry out affinitive layer purification.
Preservation of the present invention and detection are that the gained article are aseptic subpackaged, and lyophilized powder is processed in-50 ℃ of lyophilizes.12%SDS-PAGE leakage of electricity swimming, test sample has the band of target protein at the 30kDa place.
Biological activity of the present invention identifies it is to get 0.5mg sample after the freeze-drying to be dissolved in that (0.5mg/ml) uses the Cy5 labelled protein in the 1mlPBS damping fluid, and the tail vein injection mouse detects lipids contents behind the 24h.Detect its growth promoting function through the K562 cells in vitro.
Can find out that from Fig. 1 wherein Fig. 1 (1) is reference curve figure, Fig. 1 (2) is an experimental data figure; The pupa albumen component is after protein purification system is handled; Obtained good separating effect, obtained 6 albumen absorption peaks altogether, judged according to reference curve known molecular amount sample appearance time; No. 4 peak molecular weight analyte sizes are between 17-43kDa in the experimental group, and No. 5 No. 6 peaks are the small-molecule substance below the 17kDa.
Can find out that from Fig. 2 molecular weight is that albumen crowd about 30kDa has obtained good separation, mainly concentrate on No. 4 protein peaks.
Can find out that from Fig. 3 the similar lipoprotein of human growth hormone is distributed in No. 4 protein peaks, and molecular weight being 30kDa, is to exist with monomeric form under native state.
Can find out through behind the affinitive layer purification from Fig. 4, can obtain the similar lipoprotein of highly purified human growth hormone
Can find out from Fig. 5; Select for use K562 cells in vitro detection system to detect the growth promoting function of the similar lipoprotein of human growth hormone; With human growth hormone as positive control; The K562 cell obviously increases when recording dosage after 3 days and being 200ng/ml concentration, and its effect is the half the of human growth hormone control group.
In a word, the present invention can utilize silkworm chrysalis as raw material, obtains to have blood viscosity lowering through separation and purification; Improve blood microcirculation, somatotrophic natural pharmaceutical protein, this method is easy and simple to handle; And output is high, is applicable to scale operation, and the native protein of purification has no side effect; Can be used as the health medicine exploitation, remedy the blank in this field, market.
Claims (2)
1. the purification process of the similar lipoprotein of human growth hormone in the silkworm chrysalis is characterized in that: comprise step down,
(1) silkworm chrysalis pre-treatment, with the PBS liquid of fresh silkworm chrysalis and precooling, 1～5 ℃ of temperature, pH value were 7.4, joined homogenate 30sec in the juicer by weight 1: 1, at ice bath 4～8min on ice, continued homogenate, triplicate fully dissolves the albumen in the silkworm chrysalis,
The gained article are transferred in the Centrifuge Cup 4 ℃, and the centrifugal 20min of 12000rpm gets supernatant and removes the insoluble impurity of macrobead through nine layers of filtered through gauze, and the recentrifuge of will filtrating filters, triplicate,
Collect 4 ℃ of filtrate filtered, 60% ammonium sulfate precipitated protein is removed grease, and the centrifugal 30min of 8000rpm abandons supernatant, and with the resuspended deposition of PBS liquid of precooling, 0.45 μ m filter membrane vacuum filtration gets the pre-treatment supernatant, 4 ℃ of preservations;
(2) initial gross separation of the similar lipoprotein of human growth hormone, the Size Exclusion among the selection AKTA explorer10 separates the pre-treatment supernatant and sloughs pigment, and moving phase is PBS liquid, and the pH value is 7.4, pressure 0.5Mpa, flow velocity 1.5ml/min; After the EP, occur 6 albumen absorption peaks altogether, judge that according to the purpose appearance time the 5th and No. 6 albumen absorption peak is pigment and small-molecule substance below the 17kDa; The similar lipoprotein of human growth hormone, molecular weight 30kDa should appear at the peak No. 4; Therefore collect 4 the albumen absorption peaks in front; And carry out SDS-PAGE electrophoresis and Western Blot and detect, the result show the similar lipoprotein of human growth hormone deposit with No. 4 protein peak in, carry out subsequent purification with the gained article of No. 4 albumen absorption peaks;
(3) affinitive layer purification carries out affinitive layer purification with the gained article of No. 4 albumen absorption peaks, and the Plus coupling Resin that 2mL 50% is got in the preparation of affinity column packs in the post shell, and balance is to room temperature; Emit residual liquid, add 3 times of column volume Coupling Buffer, 0.1M Sodium phosphate; 0.15M NaCl, the pH value is 7.2, the balance pillar; Add 2mL hGH standard substance 1mg/ml and 40 μ L Cyanoborohy dride afterwards, shaking table is hatched 4h or 4 ℃ of maintenance 7～9h, uncap in stink cupboard under the room temperature; The liquid that collection is emitted is used the BCA protein quantification, calculates coupling efficiency; Add 5 times of column volume Quenching Buffer again, 1M Tris-HCl, the pH value is 7.4,0.05%NaN
3, wash filler, add 2mL Quenching Buffer and 40 μ L Cyanoborohy dride afterwards, shaking table is hatched 30min under the room temperature; Emit the gas and the solution of reaction at the stink cupboard uncap, add 5 times of volume Wash Solution, 1M NaCl, 0.05%NaN
3Wash pillar; Use Coupling Buffer balance pillar again, 4 ℃ of preservations;
The similar lipoprotein of affinity purification human growth hormone, the equilibrium at room temperature pillar, uncap discards protection liquid afterwards, with the Binding Buffer of 5 times of column volumes, 0.15M PBS, the pH value is 7.4, once more the balance pillar; The gained article through 0.45 μ m membrane filtration after upper prop, the room temperature shaking table is hatched 1h, emits solution, uses Wash Buffer; 0.15M PBS, pH7.4, flush away does not combine sample, adds Elution Buffer (0.1MGlycine-HCl at last; The pH value is 2.5, and the albumen of elution of bound on pillar is used Neutralization Buffer; 0.5M Tris-HCl, the pH value is 8.5, and adjust pH is to neutral; Collect sample, carry out the SDS-PAGE electrophoresis detection behind the BCA protein quantification;
(4) ultrafiltration and concentration uses hollow-fibre membrane to carry out ultrafiltration, selects suitable hollow-fibre membrane specification, and molecular weight cut-off is 3kDa; Clean the back application of sample, with an amount of aseptic PBS, the pH value is 7.4, ultrafiltration and concentration; Aseptic subpackaged, lyophilized powder is processed in-50 ℃ of lyophilizes, gets finished product of the present invention.
2. by the purification process of the similar lipoprotein of human growth hormone in the described silkworm chrysalis of claim 1, it is characterized in that: described affinitive layer purification is that the gained article with No. 4 albumen absorption peaks carry out affinitive layer purification.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110309518.XA CN102424701B (en) | 2011-10-13 | The purification process of human growth hormone-like protein in silkworm pupa |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110309518.XA CN102424701B (en) | 2011-10-13 | The purification process of human growth hormone-like protein in silkworm pupa |
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| Publication Number | Publication Date |
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| CN102424701A true CN102424701A (en) | 2012-04-25 |
| CN102424701B CN102424701B (en) | 2016-12-14 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014075443A1 (en) * | 2012-11-13 | 2014-05-22 | 天津耀宇生物技术有限公司 | Purification method of protein group of 30 kd in silkworm pupa |
| CN105203677A (en) * | 2015-10-16 | 2015-12-30 | 江苏大学 | Method for quickly detecting proteins in bombyx batryticatus according to biological mass spectrometry technology |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1412222A (en) * | 2001-10-16 | 2003-04-23 | 上海丝绸（集团）有限公司 | Silkworm chrysalis protein decolouring and deodorizing refining process |
| CN101343624A (en) * | 2008-06-25 | 2009-01-14 | 浙江中奇生物药业股份有限公司 | Recombined human growth hormone gene bacilliform virus, preparation and application thereof |
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1412222A (en) * | 2001-10-16 | 2003-04-23 | 上海丝绸（集团）有限公司 | Silkworm chrysalis protein decolouring and deodorizing refining process |
| CN101343624A (en) * | 2008-06-25 | 2009-01-14 | 浙江中奇生物药业股份有限公司 | Recombined human growth hormone gene bacilliform virus, preparation and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014075443A1 (en) * | 2012-11-13 | 2014-05-22 | 天津耀宇生物技术有限公司 | Purification method of protein group of 30 kd in silkworm pupa |
| CN105203677A (en) * | 2015-10-16 | 2015-12-30 | 江苏大学 | Method for quickly detecting proteins in bombyx batryticatus according to biological mass spectrometry technology |
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