CN104672316A - Preparation and identification method of domestic silkworm silk fibroin solution - Google Patents
Preparation and identification method of domestic silkworm silk fibroin solution Download PDFInfo
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- CN104672316A CN104672316A CN201510071886.3A CN201510071886A CN104672316A CN 104672316 A CN104672316 A CN 104672316A CN 201510071886 A CN201510071886 A CN 201510071886A CN 104672316 A CN104672316 A CN 104672316A
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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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention relates to the technical field of biomedicines, and in particular relates to a preparation and identification method of a domestic silkworm silk fibroin solution. The preparation method of the domestic silkworm silk fibroin solution sequentially comprises the following steps: (1) degumming, namely degumming domestic silkworm silks to remove silk sericin on the outer layer to obtain degummed silks; (2) dissolving the silks, namely dissolving the degummed silks by using a ternary solution containing calcium chloride to obtain a fibroin solution; (3) performing renaturation, namely sequentially performing dialysis treatment on the fibroin solution according to a concentration gradient from high to low by using a protein denaturant solution, and then performing water dialysis treatment to obtain a silk fibroin solution; and (4) storing, namely storing the silk fibroin solution. The invention also provides an identification method of the domestic silkworm silk fibroin solution. The method can be used for identifying the quality of the silk fibroin solution. The preparation method of the domestic silkworm silk fibroin solution disclosed by the invention is relatively high in economical performance, ensures that the molecular weight of the domestic silkworm silk fibroin solution is close to that of natural fibroin molecules, and is beneficial to the preparation of subsequent silk fibroin based biomaterials.
Description
Technical field
The present invention relates to field of biomedicine technology, particularly relate to the preparation of a kind of silk fibroin protein solution and authentication method.
Background technology
Silk is the product of Chinese ancient civilization, is widely used in dress material due to characteristics such as its softness, good permeability, quality softnesses from ancient times.In recent decades; because fibroin is nontoxic, nonirritant, there is excellent biocompatibility, the growth of human body cell can be promoted; there is the features such as certain biodegradability, therefore at sciemtifec and technical spheres such as biological medicine, environment protection and daily-use chemical industries also at development.
Silk is primarily of silk gum and fibroin two kinds of protein compositions.Mulberry silk nucleocapsid structure, external sheath sericin (Sericin), internal layer is silk fibroin (Silk Fibroin), wherein silk fibroin content is 70 ~ 80%, silk fibroin is by heavy chain (H chain, molecular weight 350kDa), light chain (L chain, molecular weight 25.8kDa) and glycoprotein P25 (molecular weight 23.55kDa separately adds three oligonucleotide chains) composition.Regenerated silk protein can be prepared into the biomaterial of various ways, as film, gel, support and particle etc., prepares for pharmaceutical carrier, composition engineering reparation and immobilized enzyme.
In prior art, the preparation of regenerated silk protein comprises comes unstuck and molten silk two step: the method for natural silk degumming has summed up five kinds: burning water, soap boiling method, organic acid system, enzyme process and alkaline process, and wherein alkaline process is method the most frequently used in biomaterial preparation silk fibroin process; The method of molten silk has summed up three kinds: strong acid, highly basic and neutral salt solution, and wherein neutral salt comprises one or more in zinc chloride, lithium thiocyanate, nitrocalcite, calcium carbonate, calcium phosphate, lithiumbromide and calcium chloride.Wherein 9.3M lithium bromide water solution is method the most frequently used in biomaterial preparation silk fibroin process, but the expensive investigation and application seriously hindering silk fibroin of lithiumbromide.The shortcoming of above-mentioned three kinds of molten silk methods is as shown in table 1.
For solving the problem, Chinese patent CN1394875A and CN102516777A proposes to use the method for chloride containing calcium ternary solution (being made into by calcium chloride/water/ethanol mol ratio 1:8:2) to carry out molten silk, but indicate the regenerated silk protein structure prepared by chloride containing calcium ternary solution in Chinese patent CN102775465A and be mainly beta sheet lamella, random coil structure reduces, this is unfavorable for the preparation of silk fibroin base biomaterial, silk fibroin base biomaterial especially in drug carrier material preparation process the forming process of beta sheet structure be the fixing committed step of medicine.Add urea destruction fibroin in Chinese patent CN103194068A and carry out stable silk fibroin protein solution, weaken in silk fibroin molecular or the effect of hydrogen bond between several silk fibroin molecular, improve the stability of silk fibroin, but the introducing of urea is unfavorable for that silk fibroin molecular is for vivo drug delivery system or tissue engineering bracket Study and appliance.
The shortcoming of Degumming Procedures for Silkworm Silk in table 1 prior art
Summary of the invention
For solving the problems of the technologies described above, the object of this invention is to provide that a kind of economy is higher, molecular weight close to natural fibroin molecule, be conducive to silk fibroin protein solution manufacturing method prepared by follow-up silk fibroin base biomaterial.
Silk fibroin protein solution manufacturing method of the present invention, comprises the following steps successively:
1) come unstuck: silkworm natural silk degumming is removed outer field sericin, obtains boiled silk;
2) molten silk: use chloride containing calcium ternary solution to dissolve boiled silk, obtain silk fibroin solution;
3) renaturation: use protein denaturation agent solution from high to low successively after silk fibroin solution described in dialysis treatment by concentration gradient, to re-use water dialysis treatment, obtain silk fibroin protein solution;
4) preserve: preserve described silk fibroin protein solution.
Concrete, described protein denaturant is urea, SDS or Guanidinium hydrochloride.
Concrete, described protein denaturant is urea, and the concentration gradient of described protein denaturation agent solution comprises the urea soln that volumetric molar concentration is 4M, 2M and 1M, and the time of silk fibroin solution described in each dialysis treatment is 1 ~ 5 hour.
Concrete, described protein denaturant is SDS, and the concentration gradient of described protein denaturation agent solution comprises the SDS solution that mass fraction is 2% and 1%, and described in each dialysis treatment, the time of silk fibroin solution is 1 ~ 5 hour.
Concrete, described protein denaturant is Guanidinium hydrochloride, and the concentration gradient of described protein denaturation agent solution comprises the guanidine hydrochloride solution that volumetric molar concentration is 2M and 1M, and the time of silk fibroin solution described in each dialysis treatment is 1 ~ 5 hour.
Further, described chloride containing calcium ternary solution comprises calcium chloride, water and the ethanol that mol ratio is 1:8:2, described boiled silk according to bath raio be 1:10 ~ 1:50, temperature is 60 ~ 85 DEG C, the time is within 0.5 ~ 24 hour, carry out molten silk process.
Concrete, described boiled silk is within 0.5 ~ 2 hour, carry out molten silk process according to the molten silk time.
Further, described step 4) in preservation process, after first described silk fibroin protein solution centrifugal treating being removed insolubles, constant temperature is kept in refrigerator.
The silk fibroin protein solution that silk fibroin protein solution manufacturing method of the present invention obtains, can be applied in and prepare on Silk fibroin gel, powder, film frame and film-based fibre, these materials can be widely used in bio-medical, medicinal, makeup, food and field of health care products.
The present invention also provides a kind of silk fibroin protein solution authentication method, for the qualification of silk fibroin protein solution quality, comprises the following steps successively:
1) silk fibroin protein solution is joined 8M urea soln, after hatching for some time in water bath with thermostatic control, collected by centrifugation supernatant liquor;
2) use supernatant liquor described in chromatography column Image processing, wherein, eluent is 4M urea soln;
3) qualification is by the solution under wash-out.
Further, be also added with protein marker in described chromatography column, described protein marker comprises conalbumin, ferritin and thyroglobulin.
By such scheme, the present invention at least has the following advantages: 1) do not use strong acid or highly basic, avoids silk fibroin and is significantly hydrolyzed, and the silk fibroin molecular amount of acquisition is larger; 2) the protein denaturant gradient dialysis such as urea are used to recover silk fibroin secondary structure, by the natural structure that the denatured structure renaturation of beta sheet is alpha-helix and random coil, be beneficial to the preparation of silk fibroin base biomaterial, silk fibroin base biomaterial is especially in drug carrier material preparation process, and beta sheet structure is unfavorable for that medicine is fixed; 3) do not use the molten silk of 9.3M lithium bromide water solution to reduce silk fibroin preparation cost, be conducive to research and the industrialization of silk fibroin; 4) silk fibroin protein solution that obtains of the present invention, outward appearance is clarified, and molecular weight is close to natural fibroin molecule, and in solution, fibroin molecular dispersivity is good; 5) ternary solution be made up of calcium chloride/water/ethanol mol ratio 1:8:2 is used, cheap, in addition this solution coupled columns chromatographic system is not destroyed, and can be used for column chromatography analysis after reducing viscosity, prepares silk fibroin protein solution provide possibility for exploitation column chromatography; 6) protein denaturant especially urea is used, cheap research and the industrialization being conducive to silk fibroin.
Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technique means of the present invention, and can be implemented according to the content of specification sheets, coordinates accompanying drawing to be described in detail as follows below with preferred embodiment of the present invention.
Accompanying drawing explanation
Fig. 1 is the silk fibroin solution observation figure that chloride containing calcium ternary solution dissolves different time;
Fig. 2 is silk fibroin solution renaturation process comparison diagram;
Fig. 3 is the particle diameter that silk fibroin solution dynamic light scattering method records;
Fig. 4 is regenerated silk protein Fourier infrared spectrum figure;
Fig. 5 is regenerated silk protein circular dichroism spectrogram;
Fig. 6 is regenerated silk protein chromatographic elution spectrum;
Fig. 7 is the silk fibroin solution molecular weight distribution comparison diagram that chloride containing calcium ternary solution dissolves different time.
Embodiment
Below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in further detail.Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Embodiment one
The invention provides a kind of silk fibroin protein solution manufacturing method, use the molten silk of chloride containing calcium ternary solution, comprise the following steps:
1) take 110 grams of Calcium Chloride Powder Anhydrouss, be dissolved in 140 grams of pure water, after it fully dissolves and returns to room temperature, add 92 grams of dehydrated alcohols, be prepared into chloride containing calcium ternary liquid as solvent;
2) take 30 grams after being shredded by silkworm silk, be in the aqueous sodium carbonate of 0.02, boil 25 ~ 35 minutes removing silk gum in volumetric molar concentration, then repeatedly wash 3 times by rubbing with the hands with deionized water, after having washed, put into stink cupboard drying, stand-by;
3) be dissolved in by air-dry boiled silk containing in ternary liquid, bath raio is 1:25, dissolves 0.5 ~ 2 hour, as shown in Figure 1, can find out that 2 grams of boiled silks can dissolve by 20 milliliters of chloride containing calcium for 30 minutes completely under 75 ~ 85 DEG C of constant temperatures;
4) silk fibroin solution being loaded molecular weight cut-off is in the dialysis tubing of 8000 ~ 14000, use urea, SDS or guanidine hydrochloride solution by concentration gradient gradient dialysis from high to low, often walk dialysis 1 ~ 5 hour, finally dialyse 30 hours in deionized water, period constantly changes water, obtains dialyzate;
5) proceed in centrifuge tube by dialyzate, centrifugal 20 minutes (repeating 2 times) removing insoluble substance under 9000rpm rotating speed, the solubility of the silk fibroin protein solution obtained is 3 ~ 4% (w/v), preserves in the refrigerator of 4 DEG C.
In above-mentioned steps, gradient dialysis can adopt following concentration gradient:
4M urea, 2M urea, 1M urea, water are dialysed;
4M urea, 2M urea, water are dialysed;
4M urea, 1M urea, water are dialysed;
2M urea or Guanidinium hydrochloride, 1M urea or Guanidinium hydrochloride, water dialysis;
2M urea or Guanidinium hydrochloride, water dialysis;
1M urea or Guanidinium hydrochloride, water dialysis;
2%SDS, 1%SDS, water are dialysed;
1%SDS, water are dialysed;
Water is dialysed.
Embodiment two
The invention provides a kind of silk fibroin protein solution manufacturing method, it come unstuck, substantially identical with described in embodiment one of molten silk, renaturation treatment process, do not repeat them here.
Difference part is: the bath raio of the present embodiment is 1:10, temperature is 60 DEG C, the time is within 0.5 hour, carry out molten silk process, and often walking dialysis is subsequently 1 hour.
The present embodiment through coming unstuck, the silk fibroin solution that obtains of molten silk, renaturation process: the molten silk time is shorter, and molten silk temperature is lower, and silk fibroin molecular amount is comparatively large, and in addition, because dialysis time is shorter, silk fibroin renaturation effect is comparatively general.
Embodiment three
The invention provides a kind of silk fibroin protein solution manufacturing method, it come unstuck, substantially identical with described in embodiment one of molten silk, renaturation treatment process, do not repeat them here.
Difference part is: the bath raio of the present embodiment is 1:50, temperature is 85 DEG C, the time is within 24 hours, carry out molten silk process.
The present embodiment through coming unstuck, the silk fibroin solution that obtains of molten silk, renaturation process: the molten silk time is longer, and molten silk temperature is longer, and silk fibroin molecular amount is less, and in addition, because dialysis time was more than 5 hours, the renaturation effect for silk fibroin is difficult to further raising.
Embodiment four
The invention provides a kind of silk fibroin protein solution manufacturing method, use urea by concentration gradient gradient dialysis from high to low silk fibroin solution, comprise the following steps:
1) to obtain silk fibroin solution with a kind of identical method of embodiment;
2) silk fibroin solution being loaded molecular weight cut-off is in the dialysis tubing of 8000 ~ 14000, use the urea soln that maximum concentration is 4M, 2M, 1M to dialyse step by step respectively, often walk dialysis 3 hours, finally dialyse 30 hours in deionized water, period constantly changes water, obtains dialyzate;
Specifically, gradient dialysis adopts following concentration gradient:
4M urea, 2M urea, 1M urea, water are dialysed;
2M urea, 1M urea, water are dialysed;
1M urea, water are dialysed;
Water is dialysed.
3) pour in centrifuge tube by dialyzate, centrifugal 20 minutes (repeating 2 times) removing insoluble substance under 9000rpm rotating speed, the solubility of the silk fibroin solution obtained is 3 ~ 4% (w/v), preserves in the refrigerator of 4 DEG C.
As shown in Figure 2,4 kinds of schemes are adopted: 1) respectively to 4M urea, 2M urea, 1M urea, water dialysis; 2) respectively to 2M urea, 1M urea, water dialysis; 3) respectively to 1M urea, water dialysis; 4) directly water is dialysed.4-2-1-0,2-1-0,1-0 and 0 in 4 kinds of scheme difference corresponding diagram, can find out different dialysis procedure, silk fibroin solution color is different, and from oyster white to faint yellow, this illustrates that the dispersion state of silk fibroin is different.
As shown in Figure 3,5 kinds of schemes are adopted: 1) respectively to 4M urea, 2M urea, 1M urea, water dialysis; 2) respectively to 2M urea, 1M urea, water dialysis; 3) respectively to 1M urea, water dialysis; 4) directly water is dialysed; 5) silk fibroin protein solution for preparing of 9.3M lithium bromide water solution method.5 kinds of schemes 4-2-1-0,2-1-0 respectively in corresponding diagram, 1-0,0 and LiBr, visible tradition is prepared silk fibroin method and is directly dialysed to water, a large amount of aggregate is there is in solution, and through the sample of gradient dialysis and the result that obtains of the molten silk of LiBr similar, silk fibroin molecular exists with single dispersing form.
Embodiment five
The invention provides a kind of silk fibroin protein solution manufacturing method, use SDS or guanidine hydrochloride solution by concentration gradient gradient dialysis from high to low silk fibroin solution, comprise the following steps:
1) to obtain silk fibroin solution with a kind of identical method of embodiment;
2) silk fibroin solution being loaded molecular weight cut-off is in the dialysis tubing of 8000 ~ 14000, the guanidine hydrochloride solution that uses maximum concentration to be 2M, 1M respectively or 2%, 1% SDS solution dialyse step by step, often walk dialysis 3 hours, last dialysis in deionized water 30 hours, period constantly changes water, obtains dialyzate;
Specifically, gradient dialysis adopts following concentration gradient:
2M Guanidinium hydrochloride, 1M Guanidinium hydrochloride, water are dialysed;
2M Guanidinium hydrochloride, water are dialysed;
1M Guanidinium hydrochloride, water are dialysed;
2%SDS, 1%SDS, water are dialysed;
1%SDS, water are dialysed;
Water is dialysed.
3) pour in centrifuge tube by dialyzate, centrifugal 20 minutes (repeating 2 times) removing insoluble substance under 9000rpm rotating speed, the solubility of the silk fibroin solution obtained is 3 ~ 4% (w/v), preserves in the refrigerator of 4 DEG C.
Use SDS or guanidine hydrochloride solution renaturation silk fibroin solution, the Urea Process adopted in result and embodiment two is more consistent, and most preferred gradient dialysis process is dialyse to 2M Guanidinium hydrochloride, 1M Guanidinium hydrochloride, water and dialyse to 2%SDS, 1%SDS, water.
Embodiment six
Silk fibroin is primarily of 18 seed amino acid composition such as glycine L-Ala, Serine, and its characteristic amino acid, with amino and carboxyl, has amphotericeledrolyte character.Use Fourier infrared spectrum to detect silk fibroin protein solution, absorption peak is at 660cm
-1place is random coil, and absorption peak is at 1655cm
-1, 1546cm
-1, place is mainly α-helixstructure, absorption peak is at 1630cm
-1, 1520cm
-1for beta sheet structure.As shown in Figure 4,5 kinds of schemes are adopted: 1) respectively to 4M urea, 2M urea, 1M urea, water dialysis; 2) respectively to 2M urea, 1M urea, water dialysis; 3) respectively to 1M urea, water dialysis; 4) directly water is dialysed; 5) silk fibroin protein solution for preparing of 9.3M lithium bromide water solution method.5 kinds of schemes 4-2-1-0,2-1-0 respectively in corresponding diagram, 1-0,0 and LiBr, use Fourier infrared spectrum to detect the silk fibroin protein solution obtained in embodiment two, regenerated silk protein is at 1544cm
-1and 1656cm
-1place has obvious absorption peaks.It can thus be appreciated that the silk fibroin of this research preparation is based on α-helixstructure.
Fig. 5 circular dichroism demonstrates above-mentioned conclusion equally, adopts 7 kinds of schemes: 1) respectively to 4M urea, 2M urea, 1M urea, water dialysis; 2) respectively to 2M urea, 1M urea, water dialysis; 3) respectively to 1M urea, water dialysis; 4) directly water is dialysed; 5) silk fibroin protein solution for preparing of 9.3M lithium bromide water solution method; 6) to the fibroin silk fibroin protein solution used after the dialysis of 4M urea, 2M urea, 1M urea, water, methanol modified process is carried out; 7) to the silk fibroin protein solution using the process of 9.3M lithiumbromide to obtain, methanol modified process is carried out.5 kinds of schemes 4-2-1-0,2-1-0 respectively in corresponding diagram, 1-0,0, LiBr, 4-2-1-0-methyl alcohol and LiBr-methyl alcohol.Silk fibroin alpha-helix and random coil structure have stronger circular dichroism signal at about 295nm, and beta sheet structure has stronger signal at 220nm place, as can be seen from the figure the silk fibroin adopting different urea concentration gradients to prepare is alpha-helix and random coil structure, methanol modified process has stronger signal for beta sheet structure afterwards at 220nm place, silk fibroin solution color shown in composition graphs 2, ternary solution dissolves the silk fibroin solution that fibroin obtains that comes unstuck and directly there will be a large amount of aggregate to water dialysis, and after gradient dialysis, solution clarification aggregate-free occurs.
Embodiment seven
The invention provides a kind of silk fibroin protein solution authentication method, comprise the following steps:
1) taking 484 grams of urea is dissolved in the deionized water of 1L, preparation 8M urea soln;
2) silk fibroin protein solution is joined 8M urea soln, bath raio is 1:10, hatches 30 minutes in 37 C water bath, and 10000rpm collects supernatant in centrifugal 20 minutes.
3) elution requirement:
Instrument: use protein chromatographic purifying instrument;
Chromatography column: Superdex 20010/300GL;
Chromatography column balances: 5 times of column volume 4M urea, flow velocity 0.5 ml/min;
Loading ring: 10 ~ 100 microlitre loading rings;
Eluent: 4M urea soln;
Flow velocity: 0.5 ml/min;
Detector detect parameters: UV-A280.
4) protein marker: conalbumin: 75kDa, ferritin: 440kDa, thyroglobulin: 669kDa.
As shown in Figure 6, adopt silk fibroin protein solution, protein marker conalbumin, ferritin and thyroglobulin that in the embodiment of the present invention two, method obtains, after 8M Urea treatment, protein chromatography system elution curve, as can be seen from the figure the larger elution time of molecular weight is shorter.Silk fibroin elution time is the ferritin of 440kDa and molecular weight between molecular weight is the thyroglobulin of 669kDa, can draw according to molecular sieve chromatography separation principle and between 440 to 669kDa, be greater than silk fibroin molecular amount theoretical value 405kDa by the silk fibroin molecular amount prepared by the present invention, this is because silk fibroin is after Urea treatment, intramolecular hydrogen bond is destroyed, molecule becomes wire, causes greatly compared with the sphaeroprotein volume of same molecular amount.Therefore this research can show that silk fibroin molecular prepared by the present invention is nearly natural macromolecular polymkeric substance.Fig. 7 is that ternary solution dissolves boiled silk, the silk fibroin solution that process different time obtains, protein chromatography system elution curve.As can be seen from the figure with molten silk time lengthening, elution time increases gradually, and elution peak broadens gradually, and macromolecule material elutes peak disappears, and small-molecule substance elution peak is obvious gradually, and in other words silk fibroin molecular amount reduces gradually.Therefore, according to the different molten silk time, the present invention can prepare the silk fibroin of different molecular weight.
The above is only the preferred embodiment of the present invention; be not limited to the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
Claims (10)
1. a silk fibroin protein solution manufacturing method, is characterized in that: comprise the following steps successively:
1) come unstuck: silkworm natural silk degumming is removed outer field sericin, obtains boiled silk;
2) molten silk: use chloride containing calcium ternary solution to dissolve boiled silk, obtain silk fibroin solution;
3) renaturation: use protein denaturation agent solution from high to low successively after silk fibroin solution described in dialysis treatment by concentration gradient, to re-use water dialysis treatment, obtain silk fibroin protein solution;
4) preserve: preserve described silk fibroin protein solution.
2. silk fibroin protein solution manufacturing method according to claim 1, is characterized in that: described protein denaturant is urea, SDS or Guanidinium hydrochloride.
3. silk fibroin protein solution manufacturing method according to claim 2, it is characterized in that: described protein denaturant is urea, the concentration gradient of described protein denaturation agent solution comprises the urea soln that volumetric molar concentration is 4M, 2M and 1M, and the time of silk fibroin solution described in each dialysis treatment is 1 ~ 5 hour.
4. silk fibroin protein solution manufacturing method according to claim 2, it is characterized in that: described protein denaturant is SDS, the concentration gradient of described protein denaturation agent solution comprises the SDS solution that mass fraction is 2% and 1%, and described in each dialysis treatment, the time of silk fibroin solution is 1 ~ 5 hour.
5. silk fibroin protein solution manufacturing method according to claim 2, it is characterized in that: described protein denaturant is Guanidinium hydrochloride, the concentration gradient of described protein denaturation agent solution comprises the guanidine hydrochloride solution that volumetric molar concentration is 2M and 1M, and the time of silk fibroin solution described in each dialysis treatment is 1 ~ 5 hour.
6. silk fibroin protein solution manufacturing method according to claim 1, it is characterized in that: described chloride containing calcium ternary solution comprises calcium chloride, water and the ethanol that mol ratio is 1:8:2, described boiled silk according to bath raio be 1:10 ~ 1:50, temperature is 60 ~ 85 DEG C, the time is within 0.5 ~ 24 hour, carry out molten silk process.
7. silk fibroin protein solution manufacturing method according to claim 6, is characterized in that: described boiled silk is within 0.5 ~ 2 hour, carry out molten silk process according to the molten silk time.
8. silk fibroin protein solution manufacturing method according to claim 1, is characterized in that: described step 4) in preservation process, after first described silk fibroin protein solution centrifugal treating being removed insolubles, constant temperature is kept in refrigerator.
9. a silk fibroin protein solution authentication method, is characterized in that: for the qualification of silk fibroin protein solution quality, comprise the following steps successively:
1) silk fibroin protein solution is joined 8M urea soln, after hatching for some time in water bath with thermostatic control, collected by centrifugation supernatant liquor;
2) use supernatant liquor described in chromatography column Image processing, wherein, eluent is 4M urea soln;
3) qualification is by the solution under wash-out.
10. silk fibroin protein solution authentication method according to claim 9, it is characterized in that: be also added with protein marker in described chromatography column, described protein marker comprises conalbumin, ferritin and thyroglobulin.
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| CN201510071886.3A CN104672316B (en) | 2015-02-11 | 2015-02-11 | A kind of silk fibroin protein solution prepares and identification method |
| PCT/CN2015/075019 WO2015144056A1 (en) | 2014-03-27 | 2015-03-25 | Freeze-dried powder of high molecular weight silk fibroin, preparation method therefor and use thereof |
| US15/129,246 US10533037B2 (en) | 2014-03-27 | 2015-03-25 | Freeze-dried powder of high molecular weight silk fibroin, preparation method therefor and use thereof |
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| CN106946987B (en) * | 2017-02-20 | 2020-02-28 | 福建农林大学 | High-concentration acid-soluble collagen solution and preparation method of water-soluble collagen solution |
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| CN108822199A (en) * | 2018-08-02 | 2018-11-16 | 南通纺织丝绸产业技术研究院 | The preparation method of low molecular weight fibroin albumen segment |
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