CN108822199A - The preparation method of low molecular weight fibroin albumen segment - Google Patents
The preparation method of low molecular weight fibroin albumen segment Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of low molecular weight fibroin albumen polypeptide chain, after the dissolution of silk cocoon case degumming, cystine in reducing agent reduction fibroin albumen are added, by the H chain in fibroin albumen（390kDa）With L chain（25kDa）Between disulfide bond open, and under no oxygen existence condition, using the method for bilayer dialysis, dialysis 5 days or more, the fibroin polypeptide chain of different molecular weight size was separated in silk fibroin protein solution.The present invention is compared with the methods of tradition change Degumming method, dissolution mechanism, chromatography filtering, this method separative efficiency is high, it will not influence the bioactivity of fibroin albumen polypeptide, it is used as a kind of bio-medical material, soluble carrier or peptide modified dose have huge market prospects and development potentiality.
Description
Technical field
The present invention relates to protein purification fields, are a kind of extraction of low molecular weight fibroin albumen segment and preparation particularly
Effective ways.
Background technique
Fibroin albumen is a kind of subunit structure, includes the long-chain of an entitled H chain (390kDa), a name in composition
For the short chain and glycoprotein chains P25 of L chain (25kDa)（30kDa）, wherein H chain is connected by disulfide bond with L chain.By hydrophobic work
With the set of every 6 H-L chains combines to form a basic unit, ratio H with a P25 glycoprotein:L:P25 = 6:6:1.
It include in composition 18 kinds of amino acid, wherein with glycine, alanine based on serine, accounts for about the 80% of total content.Fibroin egg
It is white to be used as a kind of natural macromolecular material, there is very excellent performance.Such as fibroin albumen have good biocompatibility,
Biological degradability, good mechanical property etc. can be widely used in cultivating cell, in organizational project, to maintain to have
There is the Mechanical Form of the cambium of preformed shape.In three polypeptide chains of fibroin albumen, H chain hydrophobicity is stronger, easy to form
Beta sheet structure, crystallinity are stronger；And then hydrophily is stronger for other two polypeptide chains, hydrogel easy to form.If can divide
From both polypeptides, small-molecular-weight polypeptide portion is obtained, then can obtain hydrophilic-silk fibroin polypeptide chain, be used to prepare hydrophilic
Property material.
It is less for the isolated report of fibroin albumen difference segment at present.Method is substantially：（1）Change Degumming method：
Increase Na2CO3Dosage, interrupt molecular force between silk fibroin molecular segment（2）Dissolving method CaCl2Ethyl alcohol ternary is molten
Liquid system dissolution, acid, alkali, enzyme hydrolysis；（3）Chromatography technology：It is sieved using the dextran molecule of different molecular weight to purpose egg
It is white to be screened；Although above method has certain feasibility, limitation is also bigger.Such as：1, change degumming work
The method of skill and dissolution mechanism breaks up silk fibroin molecular segment, that is to say, that H segment, L segment and P25 segment
It has broken apart and, become no longer complete, molecular weight distribution is wider, does not carry out isolating and purifying truly, acquired
Different molecular weight segment, can not also know which segment molecule chain it belongs to originally in.2, the method filtered using gel chromatography, for
Laboratory technician operates level requirement height, and sephadex molecular sieve is easy blocking, and low output, separative efficiency is slow, furthermore says, if not
The disulfide bonds that cysteine is formed are opened, the heavy chain and light chain in fibroin albumen will not separate, in chromatography process just not
Speed difference can be generated, also results in heavy chain that can connect light chain and chromatograph together and comes out, be not up to separation truly.
Before making the present invention, many researchers are to increase Na2CO3Dosage or use CaCL2Ethyl alcohol ternary is molten
Liquid system breaks up fibroin albumen, obtains the wide continuous mixed solution of molecular weight distribution, separates to solution, greatly broken
It is broken the integrality of silk fibroin molecular segment.Such as：If face sunlight et al.《A kind of system of middle-molecular-weihydroxyethyl water-soluble fibroin albumen
It is standby》, utilize CaCL2Ethanol-water system degrades to fibroin albumen, then the left side 50 kDa is obtained by gel permeation chromatography method
Right silk fibroin molecular segment, due to CaCL2In ethanol-water system course of dissolution, fibroin molecule segment is broken up, then
The silk fibroin molecular of 50 kDa or so is obtained using the method for chromatographic column, the method destroys silk fibroin molecular chain completely
Integrality.Publication No. CN107722116A Chinese invention patent provides a kind of separation method of the silk peptide of small-molecular-weight,
It is sieved using the dextran molecule of plurality of classes, establishes molecular sieve column and gradient separations, but the method operation step are carried out to fibroin polypeptide
It is rapid cumbersome, height is required for operator, the low molecular weight content of acquisition is few.
Summary of the invention
For above-mentioned deficiency, it is an object of the invention to provide a kind of preparation sides of low molecular weight fibroin albumen segment
Method can guarantee the integrality of fibroin albumen small molecule segment and obtain the fibroin albumen low molecular weight polypeptide of more amount.
A kind of preparation method of low molecular weight fibroin albumen polypeptide chain, includes the following steps：
（1）Degumming：Pure silk cellulose fiber will be obtained after the drying of raw material silk cocoon case degumming；Wherein, with the Degumming method of conventional silkworm cocoon
It is identical, use concentration for 0.01-0.1mol/L, the sodium bicarbonate-carbonate buffer solution degumming of pH=9.5, degumming temperature 100
DEG C, slightly boiled 30min is kept, silk is taken out, is cleaned up with deionized water, degumming is repeated three times, sloughs silk gum, then take out and set
Drying obtains pure silk cellulose fiber in 60 DEG C of baking oven；
（2）Dissolution：By the step（1）The pure silk cellulose fiber of middle acquisition is dissolved with lithium-bromide solution, obtains fibroin albumen bromination
Lithium solution, and the fibroin albumen lithium-bromide solution is cooled to room temperature；Wherein, molten in lithium bromide with conventional fibroin albumen
The method dissolved in liquid is identical, and the concentration of lithium-bromide solution is 8.5-9.5 mol/L, and solution temperature is 58-62 DEG C, dissolution time
For 0.5-1h, the amount for the pure silk cellulose fiber being added in lithium-bromide solution is 50-150g/L；
（3）Reduction：First reducing agent is added to the step（2）In in the fibroin albumen lithium-bromide solution that is cooled to room temperature,
Normal-temperature reaction 1-20h；
（4）Dialysis：By the step（3）Fibroin albumen lithium-bromide solution after middle reduction pours into pair of lamina dialysis apparatus, will fill
The double-deck dialysis apparatus of fibroin albumen lithium-bromide solution after having the reduction is placed in dialysis 36h or more in the first dialyzate, often
First dialyzate is updated every 2-6h, then the double-deck dialysis apparatus is placed in the second dialyzate again and continues dialysis 24
Hour or more, second dialyzate is updated every 2-6h, the second dialyzate is deionized water, wherein the first dialyzate and second
The temperature of dialyzate is 4-8 DEG C；
Wherein, the double-deck dialysis apparatus includes that internal layer bag filter and the outer layer covered outside the internal layer bag filter are dialysed
Bag, forms middle layer between the internal layer bag filter and outer layer bag filter, deionized water, institute is filled in the middle layer
The internal layer bag filter and outer layer bag filter stated are made of semi-permeable membrane, wherein the retention of the semi-permeable membrane of the outer layer bag filter point
Son amount is 5-15 kDa, and the molecular cut off of the semi-permeable membrane of the internal layer bag filter is 50-300 kDa, the silk after the reduction
Fibroin lithium-bromide solution is filling in the internal layer bag filter；
Wherein, first dialyzate is formulated with the following method：Into deionized water, first leads to and gone described in nitrogen removal
Then dissolved oxygen in ionized water is charged with the second reducing agent, then adjusting pH value to pH is 7-9, is made weakly alkaline the
One dialyzate.
（5）It extracts：Complete the step（4）Dialysis after, extract the solution in the middle layer of the double-deck dialysis apparatus,
The aqueous solution of the as described low molecular weight fibroin albumen polypeptide chain.
In above-mentioned technical proposal, it is preferable that in the step（3）In, first reducing agent is mercaptoethanol, second two
One of mercaptan, propanethiol, dimercaptopropane, dithiothreitol (DTT), butanethiol, cysteine, glutathione.
In above-mentioned technical proposal, it is preferable that in the step（3）In, it is added in the fibroin albumen lithium-bromide solution
The amount of first reducing agent is 10-200mmol/L.
In above-mentioned technical proposal, it is preferable that when preparing first dialyzate, it is described to remove first to lead to nitrogen 0.5-1h
Dissolved oxygen in deionized water.
In above-mentioned technical proposal, it is preferable that when preparing first dialyzate, second reducing agent of addition is Asia
One of sodium sulphate, sodium thiosulfate, sodium dithionate, sodium pyrosulfite.
In above-mentioned technical proposal, it is preferable that when preparing first dialyzate, the amount of second reducing agent of addition
For 0.001-0.01mol/L.
In above-mentioned technical proposal, it is preferable that when preparing first dialyzate, by being added described in sodium bicarbonate adjusting
PH value so that first dialyzate is in alkalescent.
In above-mentioned technical proposal, it is preferable that the diameter of the internal layer bag filter is 6-36mm.
In above-mentioned technical proposal, it is preferable that the diameter of the outer layer bag filter is 22-56mm.
The principle of the invention is：
The present invention uses the buffer solution degumming of alkalescent sodium bicarbonate-carbonate, dissolves fibroin using lithium-bromide solution, reduces de-
The degradation of glue, course of dissolution for fibroin albumen, to obtain relatively complete fibroin albumen；Use step（3）In first also
Disulfide bond is formed by by cysteine in former agent reduction fibroin albumen, with this by the H chain in fibroin albumen（390kDa）With L chain
（25kDa）Between disulfide bond open；It is dialysed repeatedly under no oxygen existence condition using the double-deck dialysis apparatus, it will be also with this
Different molecular weight fibroin polypeptide chain in fibroin albumen lithium-bromide solution after original（H chain（390kDa）With L chain（25kDa）Separation
It comes.Disulfide bond recombines in dialysis procedure in order to prevent, when preparing the first dialyzate, first leads to nitrogen in deionized water
Gas removes dissolved oxygen, the second reducing agent is then added, to guarantee the first dialysis environment as reproducibility environment.Due to fibroin albumen
Middle disulfide bond has been opened, after dialysis, because fibroin albumen segment low molecular weight part polypeptide chain is L segment and P25
The molecular weight of segment can be accelerated low in 25kDa-30kDa using internal layer bag filter made of the high semi-permeable membrane of molecular cut off
The dialysis of molecular weight fractions segment, therefore small molecular mass moieties can be given using the internal layer bag filter of 50 kDa-300 kDa
And retain high molecular weight moieties；The fibroin polypeptide chain of silk fibroin water solution small molecular amount part passes through in semi-permeable membrane entrance
In solution outside layer bag filter；The smaller polypeptide chain segment being degraded of molecular weight is lesser by the molecular cut off of outer layer
Outer layer bag filter（Molecular cut off is 5 kDa-15 kDa）It is discharged in dialyzate, stays in the double-deck dialysis apparatus middle layer
As L chain and P25 chain part.
The present invention obtains following beneficial effect compared with prior art：
（1）By the present invention in that being broken the disulfide bond between H chain and L chain with the first reducing agent, other segments are not destroyed, guarantee silk
The integrality of fibroin small molecule segment.
（2）Compared with the separation of gel chromatography column, the fibroin albumen low molecular weight polypeptide amount that this method obtains is more, separation
It is high-efficient.
Detailed description of the invention
Attached drawing 1 is the photo of gel electrophoresis test silk fibroin molecular amount；
Attached drawing 2 is the surface tension figure of silk fibroin water solution；
Attached drawing 3 is contact angle figure of the silk fibroin water solution on glass.
Specific embodiment
By the technology contents of invention are described in detail, construction feature, are reached purpose and efficacy, simultaneously below in conjunction with embodiment
Cooperation attached drawing is described in detail.
Embodiment one：
（1）The preparation of boiled silk：Weigh 80 g Cocoon shells, the carbon of compound concentration 0.01mol/L, pH=9.5 in the balance
Sodium bicarbonate-carbonate buffer solution is heated to be added after boiling weighed by sour hydrogen sodium -4000 milliliters of sodium carbonate buffer
80g cocoon shell continues slightly boiled 30min, takes out silk and is cleaned up with deionized water, repeats the above test three times, sloughs silk gum,
It then takes out drying in the baking oven for be placed in 60 DEG C and obtains pure silk cellulose fiber.
（2）Dissolution：Take 15g step（1）Obtained in pure silk cellulose fiber be dissolved in 150ml concentration be 9.3 mol/L LiBr
In solution, 45min is dissolved at 60 DEG C, obtains fibroin albumen lithium-bromide solution, and fibroin albumen lithium-bromide solution is cooled to
Room temperature.
（3）Reduction：First reducing agent mercaptoethanol is added to step（2）In the fibroin albumen lithium bromide that is cooled to room temperature
In solution, the concentration of mercaptoethanol is 50mmol/L, after leading to nitrogen 10min, sealing, and normal-temperature reaction 5h.
（4）Prepare the first dialyzate：First lead to the dissolved oxygen in nitrogen 35min removal deionized water into deionized water, so
After be charged with the second reducing agent sodium thiosulfate, the concentration of sodium thiosulfate is 0.005mol/L, sodium bicarbonate 1g is added,
Make the first dialyzate in alkalescent.
（5）Prepare the double-deck dialysis apparatus：Inside choose molecular cut off be 50kDa, the internal layer bag filter that diameter is 16mm,
Outside choose molecular cut off be 8kDa, diameter 24mm outer layer bag filter, between internal layer bag filter and outer layer bag filter in
Interbed fills deionized water, forms the double-deck dialysis apparatus.
（6）Dialysis：By step（3）Fibroin albumen lithium-bromide solution after middle reduction pours into step（5）In the double-deck dialysis
In the internal layer bag filter of device, the double-deck dialysis apparatus is put into step（4）In dialyse 3 days in obtained first dialyzate, wherein
First dialyzate is changed every 3h, the temperature of the first dialyzate is 4 DEG C；The double-deck dialysis apparatus is then put into the second dialyzate
In continue dialysis 2 days, change second dialyzate every 3h, wherein the second dialyzate be deionized water, the temperature of the second dialyzate
Degree is 4 DEG C.
（7）It extracts：Complete step（6）Dialysis after, extract the solution in the middle layer of the double-deck dialysis apparatus, i.e. acquisition silk
The aqueous solution of fibroin low molecular weight polypeptide segment.
Embodiment two：
（1）The preparation of boiled silk：Weigh 80 g Cocoon shells, the carbon of compound concentration 0.01mol/L, pH=9.5 in the balance
Sodium bicarbonate-carbonate buffer solution is heated to be added after boiling weighed by sour hydrogen sodium -4000 milliliters of sodium carbonate buffer
80g cocoon shell continues slightly boiled 30min, takes out silk and is cleaned up with deionized water, repeats the above test three times, sloughs silk gum,
It then takes out drying in the baking oven for be placed in 60 DEG C and obtains pure silk cellulose fiber.
（2）Dissolution：Take 10g step（1）Obtained in pure silk cellulose fiber be dissolved in 140ml concentration be 8.8 mol/L LiBr
In solution, 40min is dissolved at 60 DEG C, obtains fibroin albumen lithium-bromide solution, and fibroin albumen lithium-bromide solution is cooled to
Room temperature.
（3）Reduction：First reducing agent dithioglycol is added to step（2）In the fibroin albumen lithium bromide that is cooled to room temperature
In solution, the concentration of dithioglycol is 70mmol/L, after leading to nitrogen 15min, sealing, and normal-temperature reaction 6h.
（4）Prepare the first dialyzate：First lead to the dissolved oxygen in nitrogen 45min removal deionized water into deionized water, so
After be charged with the second reducing agent sodium dithionate, the concentration of sodium dithionate is 0.007mol/L, and sodium bicarbonate is added
0.8g makes the first dialyzate in alkalescent.
（5）Prepare the double-deck dialysis apparatus：Choose the internal layer dialysis that molecular cut off is 100kDa, diameter is 20mm in inside
Bag, the outer layer bag filter that molecular cut off is 10kDa, diameter 30mm is chosen in outside, between internal layer bag filter and outer layer bag filter
Middle layer fill deionized water, form the double-deck dialysis apparatus.
（6）Dialysis：By step（3）Fibroin albumen lithium-bromide solution after middle reduction pours into step（5）In the double-deck dialysis
In the internal layer bag filter of device, the double-deck dialysis apparatus is put into step（4）In dialyse 3 days in obtained first dialyzate, wherein
First dialyzate is changed every 3.5h, the temperature of the first dialyzate is 5 DEG C；The double-deck dialysis apparatus is then put into the second dialysis
Continue dialysis 2 days in liquid, change second dialyzate every 3.5h, wherein the second dialyzate is deionized water, the second dialyzate
Temperature be 5 DEG C.
（7）It extracts：Complete step（6）Dialysis after, extract the solution in the middle layer of the double-deck dialysis apparatus, i.e. acquisition silk
The aqueous solution of fibroin low molecular weight polypeptide segment.
Embodiment three：
（1）The preparation of boiled silk：Weigh 70 g Cocoon shells, the carbon of compound concentration 0.03mol/L, pH=9.5 in the balance
Sodium bicarbonate-carbonate buffer solution is heated to be added after boiling weighed by sour hydrogen sodium -4000 milliliters of sodium carbonate buffer
70g cocoon shell continues slightly boiled 30min, takes out silk and is cleaned up with deionized water, repeats the above test three times, sloughs silk gum,
It then takes out drying in the baking oven for be placed in 60 DEG C and obtains pure silk cellulose fiber.
（2）Dissolution：Take 12g step（1）Obtained in pure silk cellulose fiber be dissolved in 150ml concentration be 9 mol/L LiBr it is molten
In liquid, 40min is dissolved at 60 DEG C, obtains fibroin albumen lithium-bromide solution, and fibroin albumen lithium-bromide solution is cooled to room
Temperature.
（3）Reduction：First reducing agent dithiothreitol is added to step（2）In the fibroin albumen bromination that is cooled to room temperature
In lithium solution, the concentration of dithiothreitol (DTT) is 100mmol/L, after leading to nitrogen 17min, sealing, and normal-temperature reaction 10h.
（4）Prepare the first dialyzate：First lead to the dissolved oxygen in nitrogen 55min removal deionized water into deionized water, so
After be charged with the second reducing agent sodium sulfite, the concentration of sodium sulfite is 0.01mol/L, and sodium bicarbonate 1.1g is added, makes
First dialyzate is in alkalescent.
（5）Prepare the double-deck dialysis apparatus：Choose the internal layer dialysis that molecular cut off is 200kDa, diameter is 24mm in inside
Bag, the outer layer bag filter that molecular cut off is 12kDa, diameter 36mm is chosen in outside, between internal layer bag filter and outer layer bag filter
Middle layer fill deionized water, form the double-deck dialysis apparatus.
（6）Dialysis：By step（3）Fibroin albumen lithium-bromide solution after middle reduction pours into step（5）In the double-deck dialysis
In the internal layer bag filter of device, the double-deck dialysis apparatus is put into step（4）In dialyse 3 days in obtained first dialyzate, wherein
First dialyzate is changed every 4h, the temperature of the first dialyzate is 6 DEG C；The double-deck dialysis apparatus is then put into the second dialyzate
In continue dialysis 2 days, change second dialyzate every 4h, wherein the second dialyzate be deionized water, the temperature of the second dialyzate
Degree is 6 DEG C.
（7）It extracts：Complete step（6）Dialysis after, extract the solution in the middle layer of the double-deck dialysis apparatus, i.e. acquisition silk
The aqueous solution of fibroin low molecular weight polypeptide segment.
Example IV：
（1）The preparation of boiled silk：Weigh 85 g Cocoon shells, the carbon of compound concentration 0.015mol/L, pH=9.5 in the balance
Sodium bicarbonate-carbonate buffer solution is heated to be added after boiling weighed by sour hydrogen sodium -4000 milliliters of sodium carbonate buffer
85g cocoon shell continues slightly boiled 30min, takes out silk and is cleaned up with deionized water, repeats the above test three times, sloughs silk gum,
It then takes out drying in the baking oven for be placed in 60 DEG C and obtains pure silk cellulose fiber.
（2）Dissolution：Take 16g step（1）Obtained in pure silk cellulose fiber be dissolved in 150ml concentration be 9.2 mol/L LiBr
In solution, 40min is dissolved at 60 DEG C, obtains fibroin albumen lithium-bromide solution, and fibroin albumen lithium-bromide solution is cooled to
Room temperature.
（3）Reduction：First reducing agent glutathione is added to step（2）In the fibroin albumen lithium bromide that is cooled to room temperature
In solution, the concentration of glutathione is 150mmol/L, after leading to nitrogen 20min, sealing, and normal-temperature reaction 15h.
（4）Prepare the first dialyzate：First lead to the dissolved oxygen in nitrogen 50min removal deionized water into deionized water, so
After be charged with the second reducing agent sodium pyrosulfite, the concentration of sodium pyrosulfite is 0.008mol/L, sodium bicarbonate 1g is added,
Make the first dialyzate in alkalescent.
（5）Prepare the double-deck dialysis apparatus：Choose the internal layer dialysis that molecular cut off is 150kDa, diameter is 26mm in inside
Bag, the outer layer bag filter that molecular cut off is 15kDa, diameter 44mm is chosen in outside, between internal layer bag filter and outer layer bag filter
Middle layer fill deionized water, form the double-deck dialysis apparatus.
（6）Dialysis：By step（3）Fibroin albumen lithium-bromide solution after middle reduction pours into step（5）In the double-deck dialysis
In the internal layer bag filter of device, the double-deck dialysis apparatus is put into step（4）In dialyse 3 days in obtained first dialyzate, wherein
First dialyzate is changed every 4.5h, the temperature of the first dialyzate is 5 DEG C；The double-deck dialysis apparatus is then put into the second dialysis
Continue dialysis 2 days in liquid, change second dialyzate every 4.5h, wherein the second dialyzate is deionized water, the second dialyzate
Temperature be 5 DEG C.
（7）It extracts：Complete step（6）Dialysis after, extract the solution in the middle layer of the double-deck dialysis apparatus, i.e. acquisition silk
The aqueous solution of fibroin low molecular weight polypeptide segment.
Fibroin albumen produced by the present invention and common fibroin albumen are passed through into gel electrophoresis test molecule amount respectively, obtained
To gel electrophoresis test photo as described in Figure 1, fibroin albumen (SF) is in high molecular weight, low molecular weight as can see from Figure 1
All it is distributed, and silk fibroin molecular amount produced by the present invention concentrates on 25kDa-30kDa or so；
Silk fibroin water solution produced by the present invention and common silk fibroin water solution are made into surface tension of liquid experiment respectively,
As shown in Fig. 2, from figure 2 it can be seen that, under same concentrations, fibroin albumen low molecular weight polypeptide of the invention（L-SF）Surface
The surface tension of the common fibroin albumen of warp tension ratio (SF) is high, illustrates that the hydrophily of fibroin albumen low molecular weight polypeptide compares fibroin albumen
Height illustrates that silk fibroin water solution made from the embodiment of the present invention is the water-soluble of hydrophilic low molecule fibroin albumen polypeptide chain
Liquid；
Silk fibroin water solution produced by the present invention and common silk fibroin water solution are made into glass surface contact angle respectively
Measurement, as shown in figure 3, it can be seen in figure 3 that under same concentrations, fibroin albumen low molecular weight polypeptide of the invention（L-SF）
Contact angle it is smaller than the contact angle of fibroin albumen (SF), it is almost nil, illustrate the hydrophily of fibroin albumen low molecular weight polypeptide very
It is good, illustrate that silk fibroin water solution made from the embodiment of the present invention is the water-soluble of hydrophilic low molecule fibroin albumen polypeptide chain
Liquid；
The basic principles, main features and advantages of the present invention have been shown and described above.The technical staff of the industry
It should be appreciated that the present invention is not limited to the above embodiments, the above embodiments and description only describe the present invention
Principle, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention claims
Protection scope is delineated by the appended claims, the specification and equivalents thereof from the appended claims.
Claims (9)
1. a kind of preparation method of low molecular weight fibroin albumen polypeptide chain, which is characterized in that include the following steps：
（1）Degumming：Pure silk cellulose fiber will be obtained after the drying of raw material silk cocoon case degumming；
（2）Dissolution：By the step（1）The pure silk cellulose fiber of middle acquisition is dissolved with lithium-bromide solution, obtains fibroin albumen bromination
Lithium solution, and the fibroin albumen lithium-bromide solution is cooled to room temperature；
（3）Reduction：First reducing agent is added to the step（2）In in the fibroin albumen lithium-bromide solution that is cooled to room temperature,
Normal-temperature reaction 1-20h；
（4）Dialysis：By the step（3）Fibroin albumen lithium-bromide solution after middle reduction pours into pair of lamina dialysis apparatus, will fill
The double-deck dialysis apparatus of fibroin albumen lithium-bromide solution after having the reduction is placed in dialysis 36h or more in the first dialyzate, often
First dialyzate is updated every 2-6h, then the double-deck dialysis apparatus is placed in the second dialyzate again and continues dialysis 24
Hour or more, second dialyzate is updated every 2-6h, the second dialyzate is deionized water, wherein the first dialyzate and second
The temperature of dialyzate is 4-8 DEG C；
Wherein, the double-deck dialysis apparatus includes that internal layer bag filter and the outer layer covered outside the internal layer bag filter are dialysed
Bag, forms middle layer between the internal layer bag filter and outer layer bag filter, deionized water, institute is filled in the middle layer
The internal layer bag filter and outer layer bag filter stated are made of semi-permeable membrane, wherein the retention of the semi-permeable membrane of the outer layer bag filter point
Son amount is 5-15 kDa, and the molecular cut off of the semi-permeable membrane of the internal layer bag filter is 50-300 kDa, the silk after the reduction
Fibroin lithium-bromide solution is filling in the internal layer bag filter；
Wherein, first dialyzate is formulated with the following method：Into deionized water, first leads to and gone described in nitrogen removal
Then dissolved oxygen in ionized water is charged with the second reducing agent, then adjusting pH value to pH is 7-9, is made weakly alkaline the
One dialyzate,
（5）It extracts：Complete the step（4）Dialysis after, extract the solution in the middle layer of the double-deck dialysis apparatus, as
The aqueous solution of the low molecular weight fibroin albumen polypeptide chain.
2. the preparation method of low molecular weight fibroin albumen polypeptide chain according to claim 1, which is characterized in that described
Step（3）In, first reducing agent is mercaptoethanol, dithioglycol, propanethiol, dimercaptopropane, dithiothreitol (DTT), fourth sulphur
One of alcohol, cysteine, glutathione.
3. the preparation method of low molecular weight fibroin albumen polypeptide chain according to claim 1, which is characterized in that described
Step（3）In, the amount for first reducing agent being added in the fibroin albumen lithium-bromide solution is 10-200mmol/L.
4. the preparation method of low molecular weight fibroin albumen polypeptide chain according to claim 1, which is characterized in that prepare institute
When the first dialyzate stated, lead to nitrogen 0.5-1h first to remove the dissolved oxygen in the deionized water.
5. the preparation method of low molecular weight fibroin albumen polypeptide chain according to claim 1, which is characterized in that prepare institute
When the first dialyzate stated, second reducing agent of addition is sodium sulfite, sodium thiosulfate, sodium dithionate, burnt sulfurous
One of sour sodium.
6. the preparation method of low molecular weight fibroin albumen polypeptide chain according to claim 1, which is characterized in that prepare institute
When the first dialyzate stated, the amount of second reducing agent of addition is 0.001-0.01mol/L.
7. the preparation method of low molecular weight fibroin albumen polypeptide chain according to claim 1, which is characterized in that prepare institute
When the first dialyzate stated, pH value is adjusted by the way that sodium bicarbonate is added.
8. the preparation method of low molecular weight fibroin albumen polypeptide chain according to claim 1, which is characterized in that in described
The diameter of layer bag filter is 6-36mm.
9. the preparation method of low molecular weight fibroin albumen polypeptide chain according to claim 1, which is characterized in that described outer
The diameter of layer bag filter is 22-56mm.
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