CN104672316B - A kind of silk fibroin protein solution prepares and identification method - Google Patents
A kind of silk fibroin protein solution prepares and identification method Download PDFInfo
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Abstract
The present invention relates to field of biomedicine technology more particularly to a kind of silk fibroin protein solution prepares and identification method.The silk fibroin protein solution manufacturing method of the present invention, includes the following steps successively：1) degumming：Silkworm natural silk degumming is removed to the sericin of outer layer, obtains boiled silk；2) molten silk：Boiled silk is dissolved using chloride containing calcium ternary solution, obtains silk fibroin solution；3) renaturation：After protein denaturation agent solution silk fibroin solution as described in concentration gradient dialysis treatment successively from high to low, water dialysis treatment is reused, obtains silk fibroin protein solution；4) it preserves：Preserve the silk fibroin protein solution.The present invention also provides a kind of silk fibroin protein solution identification method, for the identification of silk fibroin protein solution quality.The silk fibroin protein solution manufacturing method of the present invention, economy is higher, molecular weight close to natural fibroin molecule, be conducive to the preparation of follow-up fibroin albumen base biomaterial.
Description
Technical field
The present invention relates to field of biomedicine technology more particularly to a kind of silk fibroin protein solution prepares and identification side
Method.
Background technology
Silk is the product of Chinese ancient civilization, from ancient times wide due to characteristics such as its softness, good permeability, soft textures
It is general to be applied to dress material.In recent decades, since fibroin is nontoxic, nonirritant, there is excellent biocompatibility, energy
Enough promote the growth of human body cell, there is certain biodegradability, therefore in biological medicine, environmental protection and day
It is also evolving with sciemtifec and technical spheres such as chemical industry.
Silk is mainly made of two kinds of protein of silk gum and fibroin.Mulberry silk nucleocapsid, external sheath sericin
(Sericin), internal layer is fibroin albumen (Silk Fibroin), and wherein fibroin albumen content is 70~80%, and fibroin albumen is
By heavy chain (H chains, molecular weight 350kDa), light chain (L chains, molecular weight 25.8kDa) and glycoprotein P25 (molecular weight 23.55kDa, separately
Add three oligonucleotide chains) composition.Regenerated silk fibroin can be prepared into the biomaterial of diversified forms, as film, gel, stent and
Grain etc., prepared for pharmaceutical carrier, composition engineering reparation and immobilised enzymes etc..
In the prior art, the preparation of regenerated silk fibroin includes two step of degumming and molten silk：The method of natural silk degumming is summarized
There are five types of coming：Burning water, soap boiling method, organic acid system, enzyme process and alkaline process, wherein alkaline process are biological material preparation fibroin albumen mistake
Most common method in journey；There are three types of the method for molten silk sums up：Strong acid, highly basic and neutral salt solution, wherein neutral salt packet
Include one or more of zinc chloride, lithium rhodanate, calcium nitrate, calcium carbonate, calcium phosphate, lithium bromide and calcium chloride.Wherein 9.3M
Lithium bromide water solution is most common method during biological material preparation fibroin albumen, but the expensive serious resistance of lithium bromide
The research and application of fibroin albumen are hindered.The shortcomings that above-mentioned three kinds of molten silk methods, is as shown in table 1.
To solve the above problems, Chinese patent CN1394875A and CN102516777A propose to use chloride containing calcium ternary molten
Liquid is (by calcium chloride/water/ethyl alcohol molar ratio 1：8：2 are made into) method carry out molten silk, but indicated in Chinese patent CN102775465A
The regenerated silk fibroin structure prepared by chloride containing calcium ternary solution is mainly beta sheet lamella, and random coil structure is reduced,
This is unfavorable for the preparation of fibroin albumen base biomaterial, fibroin albumen base biomaterial especially drug carrier material preparation process
The forming process of middle beta sheet structure is the fixed committed step of drug.Urea is added in Chinese patent CN103194068A and destroys silk
Usually stablize silk fibroin protein solution, weaken in silk fibroin molecular or between several silk fibroin moleculars hydrogen bond effect, carry
The stability of high fibroin albumen, however the introducing of urea is unfavorable for silk fibroin molecular for vivo drug delivery system or group
Weaver's engineering support is studied and application.
Table 1 in the prior art silk degumming method the shortcomings that
Invention content
In order to solve the above technical problems, the object of the present invention is to provide a kind of economy is higher, molecular weight is close to natural silk
Plain molecule is conducive to silk fibroin protein solution manufacturing method prepared by follow-up fibroin albumen base biomaterial.
The silk fibroin protein solution manufacturing method of the present invention, includes the following steps successively：
1) degumming：Silkworm natural silk degumming is removed to the sericin of outer layer, obtains boiled silk；
2) molten silk：Boiled silk is dissolved using chloride containing calcium ternary solution, obtains silk fibroin solution；
3) renaturation：Using protein denaturation agent solution as described in concentration gradient dialysis treatment successively from high to low silk fibroin solution
Afterwards, water dialysis treatment is reused, obtains silk fibroin protein solution；
4) it preserves：Preserve the silk fibroin protein solution.
Specifically, the protein denaturant is urea, SDS or guanidine hydrochloride.
Specifically, the protein denaturant is urea, the concentration gradient of the protein denaturation agent solution includes mole
The urea liquid of a concentration of 4M, 2M and 1M, the time of silk fibroin solution described in each dialysis treatment are 1~5 hour.
Specifically, the protein denaturant is SDS, the concentration gradient of the protein denaturation agent solution includes mass parts
Number is 2% and 1% SDS solution, and the time of silk fibroin solution described in each dialysis treatment is 1~5 hour.
Specifically, the protein denaturant is guanidine hydrochloride, the concentration gradient of the protein denaturation agent solution includes rubbing
The guanidine hydrochloride solution of your a concentration of 2M and 1M, the time of silk fibroin solution described in each dialysis treatment are 1~5 hour.
Further, it is 1 that the chloride containing calcium ternary solution, which includes molar ratio,：8：2 calcium chloride, water and ethyl alcohol, institute
It is 1 that boiled silk, which is stated, according to bath raio：10~1：50th, temperature is 60~85 DEG C, the time is to carry out within 0.5~24 hour molten silk processing.
Specifically, the boiled silk carried out molten silk processing according to the molten silk time for 0.5~2 hour.
Further, during the step 4) preserves, the silk fibroin protein solution centrifugal treating is first removed into insoluble matter
Afterwards, constant temperature is stored in refrigerator.
The obtained silk fibroin protein solution of present inventor's fibroin protein solution manufacturing method, can apply and prepare fibroin
On protein gel, powder, film frame and film-based fibre, these materials can be widely used in bio-medical, medicinal, cosmetics,
Food and field of health care products.
The present invention also provides a kind of silk fibroin protein solution identification method, for the identification of silk fibroin protein solution quality,
Include the following steps successively：
1) silk fibroin protein solution is added to 8M urea liquids, after being incubated a period of time in water bath with thermostatic control, be collected by centrifugation
Clear liquid；
2) using supernatant described in chromatographic column Image processing, wherein, eluant, eluent is 4M urea liquids；
3) solution under identification is eluted.
Further, also added with protein marker in the chromatographic column, the protein marker includes conalbumin, iron
Albumen and thyroglobulin.
According to the above aspect of the present invention, the present invention has at least the following advantages：1) without using strong acid or highly basic, fibroin albumen is avoided
It significantly hydrolyzes, the silk fibroin molecular amount of acquisition is larger；2) fibroin has been restored using the dialysis of the protein denaturants such as urea gradient
The denatured structure renaturation of beta sheet is alpha-helix and the natural structure of random coil, conducive to fibroin egg by Protein secondary structure
The preparation of white base biomaterial, in fibroin albumen base biomaterial especially drug carrier material preparation process, beta sheet structure
It is unfavorable for drug to fix；3) fibroin albumen manufacturing cost is reduced without using the molten silk of 9.3M lithium bromide water solutions, is conducive to fibroin
The research and industrialization of albumen；4) silk fibroin protein solution that the present invention is obtained, appearance clarification, molecular weight divide close to natural fibroin
Son, fibroin molecular dispersivity is good in solution；5) use is by calcium chloride/water/ethyl alcohol molar ratio 1：8：The ternary solution of 2 compositions, valency
Lattice are cheap, and the solution does not destroy column chromatography system in addition, column chromatography can be used for analyze after reducing viscosity, to develop column chromatography
Method prepares silk fibroin protein solution and provides possibility；6) it is cheap to be conducive to fibroin using protein denaturant especially urea
The research and industrialization of albumen.
Above description is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after attached drawing is coordinated to be described in detail such as.
Description of the drawings
Fig. 1 is the silk fibroin solution observation figure of chloride containing calcium ternary solution dissolving different time；
Fig. 2 is silk fibroin solution renaturation process comparison diagram；
Fig. 3 is the grain size that silk fibroin solution dynamic light scattering method measures；
Fig. 4 is regenerated silk fibroin Fourier infrared spectrum figure；
Fig. 5 is regenerated silk fibroin circular dichroism spectrogram；
Fig. 6 is regenerated silk fibroin chromatographic elution spectrum；
Fig. 7 is the silk fibroin solution molecular weight distribution comparison diagram of chloride containing calcium ternary solution dissolving different time.
Specific embodiment
With reference to the accompanying drawings and examples, the specific embodiment of the present invention is described in further detail.Implement below
Example is used to illustrate the present invention, but be not limited to the scope of the present invention.
Embodiment one
The present invention provides a kind of silk fibroin protein solution manufacturing method, using the molten silk of chloride containing calcium ternary solution, including
Following steps：
1) 110 grams of anhydrous calcium chlorides are weighed, is dissolved in 140 grams of pure water, is fully dissolved and restore to room temperature after it, add
Enter 92 grams of absolute ethyl alcohols, be prepared into chloride containing calcium ternary liquid as solvent；
2) 30 grams are weighed after silkworm silk is shredded, boils 25~35 in the aqueous sodium carbonate for being 0.02 in molar concentration
Minute removes silk gum, is then washed by rubbing with the hands repeatedly with deionized water 3 times, draught cupboard drying is put into after the completion of washing, for use；
3) air-dried boiled silk is dissolved in liquid containing ternary, bath raio 1：25, it is molten under 75~85 DEG C of constant temperatures
Solution 0.5~2 hour, as shown in Figure 1, it can be seen that 2 grams of boiled silks can be completely dissolved by 20 milliliters of chloride containing calcium for 30 minutes；
4) silk fibroin solution is fitted into the bag filter that molecular cut off is 8000~14000, uses urea, SDS or salt
Sour guanidine solution by concentration gradient, dialyse from high to low by gradient, and often step dialysis 1~5 hour, finally dialysis 30 is small in deionized water
When, water is during which constantly changed, obtains dialyzate；
5) dialyzate is transferred in centrifuge tube, 20 minutes (being repeated 2 times) is centrifuged under 9000rpm rotating speeds and removes insoluble matter
Matter, the solubility of obtained silk fibroin protein solution is 3~4% (w/v), is preserved in 4 DEG C of refrigerator.
In above-mentioned steps, following concentration gradient may be used in gradient dialysis：
4M urea, 2M urea, 1M urea, water dialysis；
4M urea, 2M urea, water dialysis；
4M urea, 1M urea, water dialysis；
2M urea or guanidine hydrochloride, 1M urea or guanidine hydrochloride, water dialysis；
2M urea or guanidine hydrochloride, water dialysis；
1M urea or guanidine hydrochloride, water dialysis；
2%SDS, 1%SDS, water dialysis；
1%SDS, water dialysis；
Water is dialysed.
Embodiment two
The present invention provides a kind of silk fibroin protein solution manufacturing method, degumming, molten silk, renaturation process method and realities
Apply essentially identical described in example one, details are not described herein.
Difference part is：The bath raio of the present embodiment is 1：10th, temperature is 60 DEG C, the time is to carry out at molten silk for 0.5 hour
Reason, then often step dialysis is 1 hour.
The silk fibroin solution that the present embodiment is obtained through degumming, molten silk, renaturation process：The molten silk time is shorter, and molten silk temperature is relatively low,
Silk fibroin molecular amount is larger, in addition, since dialysis time is shorter, fibroin albumen renaturation effect is more general.
Embodiment three
The present invention provides a kind of silk fibroin protein solution manufacturing method, degumming, molten silk, renaturation process method and realities
Apply essentially identical described in example one, details are not described herein.
Difference part is：The bath raio of the present embodiment is 1：50th, temperature is 85 DEG C, the time is to carry out at molten silk for 24 hours
Reason.
The silk fibroin solution that the present embodiment is obtained through degumming, molten silk, renaturation process：The molten silk time is longer, and molten silk temperature is longer,
Silk fibroin molecular amount is smaller, in addition, since dialysis time was more than 5 hours, the renaturation effect of fibroin albumen is difficult into one
Step improves.
Example IV
The present invention provides a kind of silk fibroin protein solution manufacturing method, and concentration gradient gradient from high to low is pressed using urea
Dialysis silk fibroin solution, includes the following steps：
1) to obtain silk fibroin solution with a kind of identical method of embodiment；
2) silk fibroin solution is fitted into the bag filter that molecular cut off is 8000~14000, is using maximum concentration respectively
The urea liquid of 4M, 2M, 1M are dialysed step by step, and often step dialysis 3 hours is finally dialysed 30 hours in deionized water, during which continuous
Water is changed, obtains dialyzate；
Specifically, gradient dialysis uses following concentration gradient：
4M urea, 2M urea, 1M urea, water dialysis；
2M urea, 1M urea, water dialysis；
1M urea, water dialysis；
Water is dialysed.
3) dialyzate is poured into centrifuge tube, 20 minutes (being repeated 2 times) is centrifuged under 9000rpm rotating speeds and removes insoluble matter
Matter, the solubility of obtained silk fibroin solution is 3~4% (w/v), is preserved in 4 DEG C of refrigerator.
As shown in Fig. 2, using 4 kinds of schemes：1) it dialyses respectively to 4M urea, 2M urea, 1M urea, water；2) respectively to 2M
Urea, 1M urea, water dialysis；3) it dialyses respectively to 1M urea, water；4) it directly dialyses to water.In 4 kinds of scheme difference corresponding diagrams
4-2-1-0,2-1-0,1-0 and 0, it can be seen that different dialysis procedures, silk fibroin solution color are different, from milky to yellowish
Color, this illustrates that the dispersity of fibroin albumen is different.
As shown in figure 3, using 5 kinds of schemes：1) it dialyses respectively to 4M urea, 2M urea, 1M urea, water；2) respectively to 2M
Urea, 1M urea, water dialysis；3) it dialyses respectively to 1M urea, water；4) it directly dialyses to water；5) 9.3M lithium bromide water solution sides
The silk fibroin protein solution that method is prepared.4-2-1-0,2-1-0,1-0,0 and LiBr in 5 kinds of scheme difference corresponding diagrams, it is seen that
Tradition prepares fibroin albumen method and directly dialyses to water, there are a large amount of aggregations in solution, and the sample through gradient dialysis with
The result that the molten silks of LiBr obtain is similar, and silk fibroin molecular exists in the form of monodisperse.
Embodiment five
The present invention provides a kind of silk fibroin protein solution manufacturing method, using SDS or guanidine hydrochloride solution by concentration ladder
The degree silk fibroin solution of gradient dialysis from high to low, includes the following steps：
1) to obtain silk fibroin solution with a kind of identical method of embodiment；
2) silk fibroin solution is fitted into the bag filter that molecular cut off is 8000~14000, is using maximum concentration respectively
The guanidine hydrochloride solution of 2M, 1M or 2%, 1% SDS solution are dialysed step by step, often step dialysis 3 hours, finally in deionized water
Dialysis 30 hours, during which constantly changes water, obtains dialyzate；
Specifically, gradient dialysis uses following concentration gradient：
2M guanidine hydrochlorides, 1M guanidine hydrochlorides, water dialysis；
2M guanidine hydrochlorides, water dialysis；
1M guanidine hydrochlorides, water dialysis；
2%SDS, 1%SDS, water dialysis；
1%SDS, water dialysis；
Water is dialysed.
3) dialyzate is poured into centrifuge tube, 20 minutes (being repeated 2 times) is centrifuged under 9000rpm rotating speeds and removes insoluble matter
Matter, the solubility of obtained silk fibroin solution is 3~4% (w/v), is preserved in 4 DEG C of refrigerator.
Using SDS or guanidine hydrochloride solution renaturation silk fibroin solution, as a result with the Urea Process that is used in embodiment two more
Unanimously, most preferred gradient dialysis process be to 2M guanidine hydrochlorides, 1M guanidine hydrochlorides, water dialysis and to 2%SDS, 1%SDS, water it is saturating
Analysis.
Embodiment six
Fibroin albumen is mainly made of 18 kinds of amino acid such as glycine alanine, serine, and characteristic amino acid carries ammonia
Base and carboxyl have ampholytes property.Silk fibroin protein solution is detected using Fourier infrared spectrum, absorption peak is in 660cm-1
Locate as random coil, absorption peak is in 1655cm-1、1546cm-1, place be mainly α-helixstructure, absorption peak is in 1630cm-1、
1520cm-1For beta sheet structure.As shown in figure 4, using 5 kinds of schemes：1) it is saturating to 4M urea, 2M urea, 1M urea, water respectively
Analysis；2) it dialyses respectively to 2M urea, 1M urea, water；3) it dialyses respectively to 1M urea, water；4) it directly dialyses to water；5) 9.3M bromines
Change the silk fibroin protein solution that lithium aqueous process is prepared.4-2-1-0,2-1-0,1-0 in 5 kinds of schemes difference corresponding diagrams, 0
And LiBr, the silk fibroin protein solution obtained in embodiment two is detected using Fourier infrared spectrum, regenerated silk fibroin exists
1544cm-1And 1656cm-1Place has obvious absorption peaks.It can thus be appreciated that this research prepare fibroin albumen using α-helixstructure as
It is main.
Fig. 5 circular dichroisms equally demonstrate above-mentioned conclusion, using 7 kinds of schemes：1) respectively to 4M urea, 2M urea, 1M
Urea, water dialysis；2) it dialyses respectively to 2M urea, 1M urea, water；3) it dialyses respectively to 1M urea, water；4) it is directly saturating to water
Analysis；5) silk fibroin protein solution that 9.3M lithium bromide water solutions method is prepared；6) to use 4M urea, 2M urea, 1M urea,
Fibroin silk fibroin protein solution after water dialysis, carries out methanol modified processing；7) fibroin to 9.3M lithium bromides is used to handle
Protein solution carries out methanol modified processing.4-2-1-0,2-1-0,1-0 in 5 kinds of schemes difference corresponding diagrams, 0, LiBr, 4-2-
1-0- methanol and LiBr- methanol.Fibroin albumen alpha-helix and random coil structure are believed in 295nm or so with stronger circular dichroism
Number, and beta sheet structure has at 220nm compared with strong signal, is as can be seen from the figure prepared using different urea concentration gradients
Fibroin albumen is alpha-helix and random coil structure, after methanol modified processing for beta sheet structure 220nm at compared with
Strong signal, with reference to silk fibroin solution color shown in Fig. 2, the silk fibroin solution that ternary solution dissolving degumming fibroin obtains is directly saturating to water
Analysis will appear a large amount of aggregations, and after gradient is dialysed, solution clarification aggregate-free occurs.
Embodiment seven
The present invention provides a kind of silk fibroin protein solution identification method, includes the following steps：
1) 484 grams of urea are weighed to be dissolved in the deionized water of 1L, prepare 8M urea liquids；
2) silk fibroin protein solution is added to 8M urea liquids, bath raio 1：It is incubated 30 minutes in 10,37 C water baths,
10000rpm is centrifuged 20 minutes and is collected supernatant.
3) elution requirement：
Instrument：Instrument is purified using protein chromatographic；
Chromatographic column：Superdex 20010/300GL；
Chromatograph column equilibration：5 times of column volume 4M urea, 0.5 ml/min of flow velocity；
Loading ring：10~100 microlitres of loading rings；
Eluant, eluent：4M urea liquids；
Flow velocity：0.5 ml/min；
Detector detection parameters：UV-A280.
4) protein marker：Conalbumin：75kDa, ferritin：440kDa, thyroglobulin：669kDa.
As shown in fig. 6, the silk fibroin protein solution obtained using method in the embodiment of the present invention two, protein marker are with clear egg
In vain, ferritin and thyroglobulin, after 8M Urea treatments, protein chromatography system elution curve as can be seen from the figure divides
It is shorter that son measures bigger elution time.Fibroin albumen elution time between molecular weight be 440kDa ferritin and molecular weight be
The thyroglobulin of 669kDa, the fibroin egg according to prepared by being obtained through the present invention molecular sieve chromatography separation principle
White molecular weight between 440 to silk fibroin molecular amount theoretical value 405kDa is more than between 669kDa, this is because fibroin albumen passes through
After Urea treatment, intramolecular hydrogen bond is destroyed, and for molecule into threadiness, the globulin volume of more identical molecular weight is caused greatly.Therefore
This research can show that silk fibroin molecular prepared by the present invention is nearly natural macromolecular polymer.Fig. 7 is dissolved for ternary solution
Boiled silk, the silk fibroin solution that processing different time obtains, protein chromatography system elution curve.As can be seen from the figure with molten silk
Time lengthening, elution time gradually increase, and eluting peak gradually broadens, and macromolecule substance eluting peak disappears, and small-molecule substance
Eluting peak is gradually apparent, and in other words silk fibroin molecular amount continuously decreases.Therefore, according to the different molten silk time, the present invention can
To prepare the fibroin albumen of different molecular weight.
The above is only the preferred embodiment of the present invention, is not intended to restrict the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (6)
1. a kind of silk fibroin protein solution manufacturing method, it is characterised in that：Include the following steps successively：
1) degumming：Silkworm natural silk degumming is removed to the sericin of outer layer, obtains boiled silk；
2) molten silk：Boiled silk is dissolved using chloride containing calcium ternary solution, obtains silk fibroin solution；
3) renaturation：After protein denaturation agent solution silk fibroin solution as described in concentration gradient dialysis treatment successively from high to low,
Water dialysis treatment is reused, obtains silk fibroin protein solution；The protein denaturant is urea, SDS or guanidine hydrochloride；
4) it preserves：Preserve the silk fibroin protein solution.
2. silk fibroin protein solution manufacturing method according to claim 1, it is characterised in that：The protein denaturant
For urea, the concentration gradient of the protein denaturation agent solution includes the urea liquid that molar concentration is 4M, 2M and 1M, every time thoroughly
The time that analysis handles the silk fibroin solution is 1~5 hour.
3. silk fibroin protein solution manufacturing method according to claim 1, it is characterised in that：The protein denaturant
For SDS, the concentration gradient of the protein denaturation agent solution includes the SDS solution that mass fraction is 2% and 1%, dialyses every time
The time for handling the silk fibroin solution is 1~5 hour.
4. silk fibroin protein solution manufacturing method according to claim 1, it is characterised in that：The protein denaturant
For guanidine hydrochloride, the concentration gradient of the protein denaturation agent solution includes the guanidine hydrochloride solution that molar concentration is 2M and 1M, every time
The time of silk fibroin solution described in dialysis treatment is 1~5 hour.
5. silk fibroin protein solution manufacturing method according to claim 1, it is characterised in that：The chloride containing calcium ternary
It is 1 that solution, which includes molar ratio,：8：2 calcium chloride, water and ethyl alcohol, the boiled silk are 1 according to bath raio：10~1：50th, it is warm
It is to carry out within 0.5~24 hour molten silk processing to spend for 60~85 DEG C, time.
6. silk fibroin protein solution manufacturing method according to claim 1, it is characterised in that：The step 4) preserved
Cheng Zhong after the silk fibroin protein solution centrifugal treating first is removed insoluble matter, is preserved in 4 DEG C of refrigerators.
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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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| CN106243186B (en) * | 2015-06-15 | 2020-12-25 | 张鹏 | Circulating operation method capable of being independently used for protein renaturation or used as protein renaturation leading operation |
| CN106047859B (en) * | 2016-06-02 | 2019-03-15 | 苏州大学 | Method and kit for storing DNA by using silk fibroin |
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| CN108822199B (en) * | 2018-08-02 | 2023-06-23 | 南通纺织丝绸产业技术研究院 | Preparation method of low molecular weight silk fibroin chain segment |
| CN109336960B (en) * | 2018-10-22 | 2020-11-20 | 宜宾屏山辉瑞科技有限公司 | Process for preparing catechin fibroin by liquid-solid fluidization method |
| CN109851841A (en) * | 2019-01-31 | 2019-06-07 | 南通纺织丝绸产业技术研究院 | A kind of high resilience fibroin albumen aeroge and preparation method thereof |
| CN110698555A (en) * | 2019-11-04 | 2020-01-17 | 长沙凯泽工程设计有限公司 | Preparation and identification method of silkworm silk fibroin solution |
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| CN1394875A (en) * | 2001-07-11 | 2003-02-05 | 中国科学院化学研究所 | Method for extracting fimbrin |
| CN102516777B (en) * | 2011-11-29 | 2015-02-11 | 苏州大学 | Aqueous solution of silk fibroin and preparation method for aqueous solution |
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