CN104672316B - A kind of silk fibroin protein solution prepares and identification method - Google Patents

Abstract

The invention relates to the technical field of biomedicine, in particular to a method for preparing and identifying silkworm silk fibroin solution. The preparation method of the silkworm silk fibroin protein solution of the present invention comprises the following steps in turn: 1) degumming: degumming the silkworm silk to remove the sericin protein in the outer layer to obtain degummed silk; 2) dissolving the silk: dissolving it with a ternary solution containing calcium chloride Degumming silk to obtain a silk fibroin solution; 3) renaturation: use protein denaturant solution to dialyze the silk fibroin solution according to the concentration gradient from high to low, and then use water dialysis to obtain a silk fibroin solution; 4) preserve : Preserve the silk fibroin solution. The invention also provides a method for identifying the silk fibroin solution, which is used for identifying the quality of the silk fibroin solution. The preparation method of the silkworm silk fibroin protein solution of the present invention has high economic efficiency, molecular weight close to that of natural silk fibroin molecules, and is beneficial to the preparation of subsequent silk fibroin-based biological materials.

Description

A kind of silk fibroin protein solution preparation and identification method

technical field

The invention relates to the technical field of biomedicine, in particular to a method for preparing and identifying silkworm silk fibroin solution.

Background technique

Silk is a product of ancient Chinese civilization. It has been widely used as clothing material since ancient times due to its softness, good air permeability and soft texture. In recent decades, because silk protein is non-toxic, non-irritating, has excellent biocompatibility, can promote the growth of human cells, and has certain biodegradability, it has been widely used in biomedicine, environmental protection and daily use. Technological fields such as chemical engineering are also developing continuously.

Silk is mainly composed of two proteins, sericin and silk fibroin. Mulberry silk core-shell structure, the outer layer is coated with sericin (Sericin), and the inner layer is silk fibroin (Silk Fibroin), in which the silk fibroin content is 70-80%. Silk fibroin is composed of heavy chain (H chain, Molecular weight 350kDa), light chain (L chain, molecular weight 25.8kDa) and glycoprotein P25 (molecular weight 23.55kDa, plus three oligosaccharide chains). Regenerated silk fibroin can be prepared into various forms of biomaterials, such as films, gels, scaffolds, and particles, for drug carriers, composition engineering repairs, and immobilized enzyme preparations.

In the prior art, the preparation of regenerated silk fibroin includes two steps of degumming and silk melting: there are five methods for silk degumming: boiling water method, soap boiling method, organic acid method, enzymatic method and alkali method, wherein the alkali method is The most commonly used method in the process of preparing biological materials with silk fibroin; there are three methods for dissolving silk: strong acid, strong alkali and neutral salt solution, wherein the neutral salt includes zinc chloride, lithium thiocyanate, calcium nitrate, One or more of calcium carbonate, calcium phosphate, lithium bromide and calcium chloride. Among them, 9.3M lithium bromide aqueous solution is the most commonly used method in the process of preparing silk fibroin from biological materials, but the high price of lithium bromide seriously hinders the research and application of silk fibroin. The disadvantages of the above three methods of melting silk are shown in Table 1.

In order to solve the above problems, Chinese patents CN1394875A and CN102516777A propose to use the method of ternary solution containing calcium chloride (made up of calcium chloride/water/ethanol molar ratio 1:8:2) to dissolve silk, but Chinese patent CN102775465A specifies The structure of the regenerated silk fibroin prepared by the ternary solution containing calcium chloride is mainly β-sheet, and the random coil structure is reduced, which is not conducive to the preparation of silk fibroin-based biomaterials, especially for pharmaceuticals. The formation of β-sheet structure in the preparation of carrier materials is a key step for drug immobilization. In Chinese patent CN103194068A, urea is added to destroy silk fibroin to stabilize silk fibroin solution, which weakens the effect of hydrogen bonds in silk fibroin molecules or between several silk fibroin molecules, and improves the stability of silk fibroin. However, the introduction of urea is not conducive to silk fibroin. Peptide molecules are used in the research and application of in vivo drug delivery systems, or tissue engineering scaffolds.

The shortcoming of silk degumming method in the prior art of table 1

Contents of the invention

In order to solve the above-mentioned technical problems, the purpose of the present invention is to provide a method for preparing silkworm silk fibroin solution with high economy, molecular weight close to that of natural silk fibroin, which is beneficial to the preparation of subsequent silk fibroin-based biological materials.

Silkworm silk fibroin protein solution preparation method of the present invention, comprises the following steps successively:

1) degumming: degumming silkworm silk to remove the sericin in the outer layer to obtain degummed silk;

2) Dissolving silk: using a ternary solution containing calcium chloride to dissolve the degummed silk to obtain a silk fibroin solution;

3) Refolding: use protein denaturant solution to dialyze the silk fibroin solution sequentially according to the concentration gradient from high to low, and then use water to dialyze to obtain a silk fibroin solution;

4) Preservation: Preserve the silk fibroin solution.

Specifically, the protein denaturant is urea, SDS or guanidine hydrochloride.

Specifically, the protein denaturant is urea, the concentration gradient of the protein denaturant solution includes urea solutions with molar concentrations of 4M, 2M and 1M, and the time for each dialysis treatment of the silk fibroin solution is 1-5 hours.

Specifically, the protein denaturant is SDS, the concentration gradient of the protein denaturant solution includes SDS solutions with a mass fraction of 2% and 1%, and the time for each dialysis treatment of the silk fibroin solution is 1 to 5 hours .

Specifically, the protein denaturant is guanidine hydrochloride, the concentration gradient of the protein denaturant solution includes guanidine hydrochloride solutions with molar concentrations of 2M and 1M, and the time for each dialysis treatment of the silk fibroin solution is 1-5 hours.

Further, the calcium chloride-containing ternary solution includes calcium chloride, water and ethanol at a molar ratio of 1:8:2, and the degummed silk is at a bath ratio of 1:10 to 1:50 at a temperature of 60 ~85°C, the time is 0.5 ~ 24 hours for silk-melting treatment.

Specifically, the degummed silk is subjected to a silk-melting treatment according to a silk-melting time of 0.5 to 2 hours.

Further, during the preservation process of step 4), the silk fibroin solution is first centrifuged to remove insoluble matter, and then stored in a refrigerator at a constant temperature.

The silk fibroin solution obtained by the silkworm silk fibroin solution preparation method of the present invention can be applied to the preparation of silk fibroin gel, powder, film support and film fiber, and these materials can be widely used in biomedicine, medicine, cosmetics, Food and health products field.

The present invention also provides a silk fibroin protein solution identification method, which is used to identify the quality of the silk fibroin solution, comprising the following steps in sequence:

1) Add the silk fibroin solution to 8M urea solution, incubate in a constant temperature water bath for a period of time, and collect the supernatant by centrifugation;

2) using column chromatography to process the supernatant, wherein the eluent is 4M urea solution;

3) Identification of the eluted solution.

Further, protein markers are added to the chromatographic column, and the protein markers include conalbumin, ferritin and thyroglobulin.

By means of the above scheme, the present invention has at least the following advantages: 1) without using strong acid or strong alkali, the silk fibroin is avoided from being greatly hydrolyzed, and the obtained silk fibroin has a relatively large molecular weight; 2) the protein denaturant such as urea is used to restore The secondary structure of silk fibroin is refolded, and the denatured structure of β-sheet is refolded into the natural structure of α-helix and random coil, which is beneficial to the preparation of silk fibroin-based biomaterials, especially drug carrier materials. In the preparation process, the β-fold structure is not conducive to drug immobilization; 3) the silk fibroin preparation cost is reduced without using 9.3M lithium bromide aqueous solution to dissolve the silk, which is beneficial to the research and industrialization of silk fibroin; 4) the silk fibroin obtained by the present invention Fibroin solution, clear appearance, molecular weight close to natural silk molecules, good dispersion of silk molecules in the solution; 5) using a ternary solution composed of calcium chloride/water/ethanol molar ratio 1:8:2, the price is low, In addition, the solution does not damage the column chromatography system, and can be used for column chromatography analysis after reducing the viscosity, which provides the possibility of preparing silk fibroin solution for the development of column chromatography; 6) the use of protein denaturants, especially urea, is beneficial to Research and industrialization of silk fibroin.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention and accompanying drawings are described in detail below.

Description of drawings

Fig. 1 is the observation figure of the silk fibroin solution that contains calcium chloride ternary solution dissolving different times;

Fig. 2 is a comparison diagram of the renaturation process of silk fibroin solution;

Fig. 3 is the particle diameter that silk fibroin solution dynamic light scattering method records;

Fig. 4 is regenerated silk fibroin Fourier transform infrared spectrogram;

Figure 5 is a circular dichroism chromatogram of regenerated silk fibroin;

Fig. 6 is regenerated silk fibroin chromatographic elution profile;

Fig. 7 is a comparison chart of molecular weight distribution of silk fibroin solution dissolved in calcium chloride-containing ternary solution at different times.

Detailed ways

The specific implementation manners of the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Embodiment one

The invention provides a method for preparing silkworm silk fibroin protein solution, which uses a ternary solution containing calcium chloride to dissolve the silk, comprising the following steps:

1) Weigh 110 grams of anhydrous calcium chloride, dissolve it in 140 grams of pure water, and after it is fully dissolved and returned to room temperature, add 92 grams of absolute ethanol to prepare a calcium chloride-containing ternary solution as a solvent;

2) Cut silkworm cocoons into pieces and weigh 30 grams, boil them in an aqueous solution of sodium carbonate with a molar concentration of 0.02 for 25-35 minutes to remove sericin, then wash them repeatedly with deionized water for 3 times, and put them in a fume hood after washing dry, ready to use;

3) Dissolve the air-dried degummed silk in the ternary solution with a bath ratio of 1:25, and dissolve it at a constant temperature of 75-85°C for 0.5-2 hours. As shown in Figure 1, it can be seen that 20 ml of chlorinated Calcium can completely dissolve 2 grams of degummed silk in 30 minutes;

4) Put the silk fibroin solution into a dialysis bag with a molecular weight cut-off of 8000-14000, use urea, SDS or guanidine hydrochloride solution to dialyze from high to low according to the concentration gradient, each step of dialysis for 1-5 hours, and finally in deionized water Dialysis for 30 hours, during which the water was changed continuously to obtain dialysate;

5) Transfer the dialysate into a centrifuge tube, centrifuge at 9000rpm for 20 minutes (repeat 2 times) to remove insoluble matter, and obtain a silk fibroin solution with a solubility of 3 to 4% (w/v). Store in the refrigerator.

In the above steps, gradient dialysis can use the following concentration gradient:

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 dialysis.

Embodiment two

The present invention provides a method for preparing silkworm silk fibroin protein solution. The degumming, silk dissolving and renaturation treatment methods are basically the same as those described in Example 1, and will not be repeated here.

The difference is that in this example, the bath ratio is 1:10, the temperature is 60°C, and the time is 0.5 hours for silk-melting treatment, and then each step of dialysis is 1 hour.

The silk fibroin solution obtained through degumming, silk melting and renaturation treatment in this example: the silk melting time is shorter, the silk melting temperature is lower, and the molecular weight of silk fibroin is larger. In addition, due to the shorter dialysis time, the silk fibroin renaturation The effect is more general.

Embodiment three

The present invention provides a method for preparing silkworm silk fibroin protein solution. The degumming, silk dissolving and renaturation treatment methods are basically the same as those described in Example 1, and will not be repeated here.

The difference is that in this embodiment, the bath ratio is 1:50, the temperature is 85° C., and the time is 24 hours for silk-melting treatment.

The silk fibroin solution obtained by degumming, silk melting and renaturation treatment in this example: the time of silk dissolution is longer, the temperature of silk dissolution is longer, and the molecular weight of silk fibroin is smaller. In addition, since the dialysis time exceeds 5 hours, the silk fibroin It is difficult to further improve the refolding effect.

Embodiment Four

The invention provides a method for preparing silkworm silk fibroin protein solution, which uses urea to dialyze the silk fibroin solution according to the concentration gradient from high to low, comprising the following steps:

1) obtain silk fibroin solution with a kind of method identical with embodiment;

2) Put the silk fibroin solution into a dialysis bag with a molecular weight cut-off of 8,000 to 14,000, and use urea solutions with the highest concentrations of 4M, 2M, and 1M to dialyze step by step, each step for 3 hours, and finally dialyze in deionized water for 30 hours , during which the water is constantly changed to obtain dialysate;

Specifically, gradient dialysis employs the following concentration gradients:

4M urea, 2M urea, 1M urea, water dialysis;

2M urea, 1M urea, water dialysis;

1M urea, water dialysis;

Water dialysis.

3) Pour the dialysate into a centrifuge tube, and centrifuge at 9000rpm for 20 minutes (repeated twice) to remove insoluble matter. The resulting silk fibroin solution has a solubility of 3 to 4% (w/v). Store in the refrigerator.

As shown in Figure 2, four schemes are adopted: 1) dialysis to 4M urea, 2M urea, 1M urea, and water respectively; 2) dialysis to 2M urea, 1M urea, and water respectively; 3) dialysis to 1M urea, water respectively; 4) Dialyze directly against water. The four schemes correspond to 4-2-1-0, 2-1-0, 1-0 and 0 in the figure respectively. It can be seen that the different dialysis processes have different colors of silk fibroin solution, from milky white to light yellow, which shows that The dispersion state of silk fibroin is different.

As shown in Figure 3, five schemes are adopted: 1) dialysis to 4M urea, 2M urea, 1M urea, and water respectively; 2) dialysis to 2M urea, 1M urea, and water respectively; 3) dialysis to 1M urea, water respectively; 4) direct water dialysis; 5) silk fibroin solution prepared by 9.3M lithium bromide aqueous solution method. The five schemes correspond to 4-2-1-0, 2-1-0, 1-0, 0 and LiBr respectively in the figure. It can be seen that the traditional method of preparing silk fibroin directly dialyzes water, and there are a large number of aggregates in the solution, while The sample obtained by gradient dialysis was similar to the result obtained by LiBr silk dissolution, and the silk fibroin molecules existed in a monodisperse form.

Embodiment five

The invention provides a method for preparing silkworm silk fibroin protein solution, using SDS or guanidine hydrochloride solution to dialyze the silk fibroin solution according to the concentration gradient from high to low, comprising the following steps:

1) obtain silk fibroin solution with a kind of method identical with embodiment;

2) Put the silk fibroin solution into a dialysis bag with a molecular weight cut-off of 8,000 to 14,000, and use the highest concentration of 2M, 1M guanidine hydrochloride solution or 2%, 1% SDS solution for step-by-step dialysis, each step of dialysis for 3 hours, Finally, dialyze in deionized water for 30 hours, during which the water is changed continuously to obtain dialysate;

Specifically, gradient dialysis employs the following concentration gradients:

2M guanidine hydrochloride, 1M guanidine hydrochloride, water dialysis;

2M guanidine hydrochloride, water dialysis;

1M guanidine hydrochloride, water dialysis;

2% SDS, 1% SDS, water dialysis;

1% SDS, water dialysis;

Water dialysis.

3) Pour the dialysate into a centrifuge tube, and centrifuge at 9000rpm for 20 minutes (repeated twice) to remove insoluble matter. The resulting silk fibroin solution has a solubility of 3 to 4% (w/v). Store in the refrigerator.

Use SDS or guanidine hydrochloride solution refolded silk fibroin solution, the result is relatively consistent with the urea method adopted in embodiment two, the most preferred gradient dialysis method is to 2M guanidine hydrochloride, 1M guanidine hydrochloride, water dialysis and to 2%SDS, 1 %SDS, water dialysis.

Embodiment six

Silk fibroin is mainly composed of 18 kinds of amino acids such as glycine, alanine, and serine. The characteristic amino acids have amino and carboxyl groups, and have ampholyte properties. Using Fourier transform infrared spectroscopy to detect silk fibroin solution, the absorption peaks are random coils at 660cm ^-1 , the main absorption peaks are α-helical structures at 1655cm ^-1 and 1546cm ^-1 , and the absorption peaks are at 1630cm ^-1 and 1520cm ^{-1 1} is the β-sheet structure. As shown in Figure 4, five schemes are adopted: 1) dialysis to 4M urea, 2M urea, 1M urea, and water respectively; 2) dialysis to 2M urea, 1M urea, and water respectively; 3) dialysis to 1M urea, water respectively; 4) direct water dialysis; 5) silk fibroin solution prepared by 9.3M lithium bromide aqueous solution method. The five schemes respectively correspond to 4-2-1-0, 2-1-0, 1-0, 0 and LiBr in the figure, use Fourier transform infrared spectroscopy to detect the silk fibroin solution obtained in Example 2, and regenerate silk fibroin There are obvious absorption peaks at 1544cm ^-1 and 1656cm ^-1 . It can be seen that the silk fibroin prepared in this study is dominated by α-helical structure.

The circular dichroism spectrum in Figure 5 also verifies the above conclusions, using 7 schemes: 1) dialysis against 4M urea, 2M urea, 1M urea, and water respectively; 2) dialysis against 2M urea, 1M urea, and water respectively; 3) dialysis against 1M urea, water dialysis; 4) direct water dialysis; 5) silk fibroin solution prepared by 9.3M lithium bromide aqueous solution method; 6) use 4M urea, 2M urea, 1M urea, water after dialysis The solution was subjected to methanol denaturation treatment; 7) The silk fibroin solution obtained by treatment with 9.3M lithium bromide was subjected to methanol denaturation treatment. The five schemes correspond to 4-2-1-0, 2-1-0, 1-0, 0, LiBr, 4-2-1-0-methanol and LiBr-methanol in the figure respectively. The α-helix and random coil structure of silk fibroin have strong circular dichroism signals at around 295nm, while the β-sheet structure has a strong signal at 220nm. It can be seen from the figure that silk fibroin prepared with different urea concentration gradients The proteins are all α-helical and random coil structures. After methanol denaturation treatment, the β-sheet structure has a strong signal at 220nm. Combined with the color of the silk fibroin solution shown in Figure 2, the silk obtained by dissolving the degummed silk in the ternary solution A large number of aggregates will appear when the plain solution is directly dialyzed against water, but after gradient dialysis, the solution is clear and no aggregates appear.

Embodiment seven

The invention provides a silk fibroin protein solution identification method, comprising the following steps:

1) Weigh 484 grams of urea and dissolve it in 1L of deionized water to prepare an 8M urea solution;

2) Add silk fibroin solution to 8M urea solution with a bath ratio of 1:10, incubate in a water bath at 37 degrees Celsius for 30 minutes, and centrifuge at 10,000 rpm for 20 minutes to collect the supernatant.

3) Elution conditions:

Instrument: use protein chromatography purifier;

Chromatographic column: Superdex 20010/300GL;

Chromatographic column balance: 5 times column volume 4M urea, flow rate 0.5 ml/min;

Sample loop: 10-100 microliter sample loop;

Eluent: 4M urea solution;

Flow rate: 0.5ml/min;

Detector detection parameters: UV-A280.

4) Protein markers: conalbumin: 75kDa, ferritin: 440kDa, thyroglobulin: 669kDa.

As shown in Figure 6, the silk fibroin solution, protein markers conalbumin, ferritin and thyroglobulin obtained by the method in Example 2 of the present invention, after being treated with 8M urea, the elution curve of the protein chromatography system, from It can be seen from the figure that the larger the molecular weight, the shorter the elution time. The elution time of silk fibroin is between ferritin with a molecular weight of 440kDa and thyroglobulin with a molecular weight of 669kDa. According to the separation principle of molecular sieve chromatography column, it can be concluded that the molecular weight of silk fibroin prepared by the present invention is between 440 and 669kDa It is greater than the theoretical value of silk fibroin molecular weight of 405kDa. This is because after the silk fibroin is treated with urea, the hydrogen bonds in the molecule are destroyed, and the molecule becomes linear, which is larger than the globulin with the same molecular weight. Therefore, this study can conclude that the silk fibroin molecule prepared by the present invention is a near-natural macromolecular polymer. Fig. 7 is the elution curve of the silk fibroin solution obtained by dissolving the degummed silk in the ternary solution and treating it for different times, and the protein chromatography system. It can be seen from the figure that with the prolongation of silk melting time, the elution time gradually increases, the elution peak gradually becomes wider, the elution peak of large molecular weight substances disappears, and the elution peak of small molecular substances gradually becomes obvious, in other words, the molecular weight of silk fibroin gradually decreases . Therefore, according to different silk-melting times, the present invention can prepare silk fibroin with different molecular weights.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements can be made without departing from the technical principle of the present invention. and modifications, these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (6)

1. a silk fibroin protein solution preparation method, is characterized in that: comprises the following steps successively: 1) degumming: degumming silkworm silk to remove the sericin in the outer layer to obtain degummed silk; 2) Dissolving silk: using a ternary solution containing calcium chloride to dissolve the degummed silk to obtain a silk fibroin solution; 3) Refolding: using a protein denaturant solution according to the concentration gradient from high to low to dialyze the silk fibroin solution sequentially, and then use water dialysis to obtain a silk fibroin solution; the protein denaturant is urea, SDS or hydrochloric acid guanidine; 4) Preservation: Preserve the silk fibroin solution. 2. the silk fibroin protein solution preparation method according to claim 1, is characterized in that: described protein denaturant is urea, and the concentration gradient of described protein denaturant solution comprises the urea solution that molar concentration is 4M, 2M and 1M , the time for each dialysis treatment of the silk fibroin solution is 1-5 hours. 3. The silkworm silk fibroin protein solution preparation method according to claim 1, characterized in that: the protein denaturant is SDS, and the concentration gradient of the protein denaturant solution includes 2% and 1% SDS in parts by mass solution, the time for each dialysis treatment of the silk fibroin solution is 1 to 5 hours. 4. The silkworm silk fibroin protein solution preparation method according to claim 1, characterized in that: the protein denaturant is guanidine hydrochloride, and the concentration gradient of the protein denaturant solution comprises molar concentrations of 2M and 1M guanidine hydrochloride solutions , the time for each dialysis treatment of the silk fibroin solution is 1-5 hours. 5. The silkworm silk fibroin protein solution preparation method according to claim 1, characterized in that: the calcium chloride-containing ternary solution includes calcium chloride, water and ethanol in a molar ratio of 1:8:2, the The above-mentioned degummed silk is subjected to silk-melting treatment according to a bath ratio of 1:10-1:50, a temperature of 60-85° C., and a time of 0.5-24 hours. 6. The silk fibroin protein solution preparation method according to claim 1, characterized in that: in the step 4) during preservation, the silk fibroin solution is first centrifuged to remove insoluble matter, and then placed in a refrigerator at 4°C save.