Recombinant spider silk protein fiber based on SpiCE-DS8 protein and preparation method thereof

Abstract

The invention discloses a recombinant spider silk protein fiber based on SpiCE-DS8 protein and a preparation method of the recombinant spider silk protein fiber, and belongs to the technical field of polymer fiber materials. The invention discloses a key functional domain SpiCE-DS8-S (KGGLDILGGSLKLSKAGKDGKNFF) of a SpiCE-DS8 protein, and the functional domain can be used for promoting the self-assembly of silk protein and the formation of fibers. The SpiCE-DS8 protein is used as a raw material of a spinning solution, the strength, toughness and elongation of the prepared recombinant spider silk protein fiber are remarkably improved, the possibility is provided for improving the mechanical property of in-vitro recombinant silk, the spinning process is simplified, continuous spinning can be achieved, and industrial mass production is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of polymer fiber materials, and in particular relates to a recombinant spider silk protein fiber based on SpiCE-DS8 protein and a preparation method thereof. Background Art

[0002] As one of the strongest biomaterials, spider dragline silk has super toughness that is superior to most synthetic materials due to its high strength and good ductility. The preparation of high-concentration protein spinning solution is one of the important prerequisites for recombinant spider silk protein bionic spinning. Spiders in the superfamily Theridiidae are all web-building spiders, and their dragline silk has super strong comprehensive mechanical properties. Its application prospects are very broad, but due to the spider's natural aggressiveness, self-protection awareness and territorial aggressiveness, it is impossible to collect spider silk in large quantities through large-scale breeding like harvesting silkworms. Therefore, spiders cannot produce silk in large quantities like silkworms and be used by people in the form of cocoons.

[0003] Recombinant spider silk still has broad application prospects due to its excellent properties. Various experimental methods (such as wet and dry spinning) have been used to mimic the natural spinning process of silkworms to produce silk fibers from regenerated silk fibroin (RSF), some of which induce excellent mechanical properties. However, most of these methods do not allow continuous production because the spinning usually requires several hours of treatment in a coagulation bath or the fibers must be stretched manually. Therefore, this method is not suitable for industrial production and can only provide a small amount of fiber samples for laboratory use. Even if a few wet spinning methods can establish a continuous spinning process for RSF, the mechanical properties of silk fibers are far behind those of natural silk. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a recombinant spider silk protein fiber based on SpiCE-DS8 protein and a preparation method thereof, so as to solve the technical problems that wet spinning is difficult for continuous spinning and the mechanical properties of silk fibers are poor.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a SpiCE-DS8 protein, whose amino acid sequence is shown in SEQ ID NO: 1.

[0006] The present invention also discloses a gene encoding the SpiCE-DS8 protein, and the nucleotide sequence thereof is shown in SEQ ID NO:2.

[0007] The invention also discloses a recombinant plasmid containing the gene.

[0008] The present invention also discloses a method for preparing recombinant spider silk protein fibers, comprising the following steps:

[0009] S1. Cut the silk cocoons into pieces, and then boil them in alkaline solution for 20-40 minutes, take out the silk, rinse, and dry them to obtain silk fibroin crude protein; alkaline conditions can effectively promote the swelling, dissolution and hydrolysis of sericin, and remove part of the wax by saponification;

[0010] S2, dissolving the silk fibroin crude protein, heating at 50-60°C for 3-5h, then dialyzing for 46-50h, and finally freeze-drying to obtain silk fibroin;

[0011] S3, dissolving silk fibroin in a polar solvent, then adding SpiCE-DS8 protein and incubating for 10-14 hours to obtain a recombinant silk fibroin spinning solution; the ratio of silk fibroin, polar solvent and SpiCE-DS8 protein is 0.3-0.4 g: 8-12 mL: 0.03-0.07 g;

[0012] S4. Wet spinning is adopted to extrude the recombinant silk protein spinning solution into the coagulation solution, and the wet-spun fibers are collected by pulling at a speed of 5-7 cm / s to finally obtain recombinant spider silk protein fibers.

[0013] On the basis of the above technical solution, the present invention can also be improved as follows:

[0014] Furthermore, the alkali solution is sodium carbonate, sodium bicarbonate or urea solution.

[0015] Further, the concentration of the alkali solution is 0.01-0.03M.

[0016] Furthermore, the drying temperature is 55-65°C and the drying time is 8-12h.

[0017] Furthermore, the solvent used for dissolving in S2 is LiBr or LiSCN solution. Cellulose can be partially dissolved by LiBr or LiSCN solution to form cellulose nanofibrils, thereby preparing a porous silk fibroin / cellulose sponge material with a micro-nano hierarchical structure.

[0018] Furthermore, the concentration of the solvent used for dissolving S2 is 9-10M.

[0019] Furthermore, the molecular weight cut-off of the dialysis membrane used for dialysis is 13,000-15,000 Daltons.

[0020] Furthermore, the freeze-drying temperature is -30 to -10°C and the time is 22 to 26 hours.

[0021] Furthermore, the polar solvent is hexafluoroisopropanol (HFIP). Hexafluoroisopropanol (HFIP) is a strong polar solvent that can cause changes in the internal conformation of protein molecules, thereby enhancing their solubility, and is used to dissolve proteins containing a high content of β-structures.

[0022] Furthermore, the extrusion speed of the recombinant silk protein spinning solution is 45-55 μL / min.

[0023] Furthermore, the coagulation solution comprises 75-85 vol% EtOH solution, 0.5-0.7 M ammonium acetate and 0.5-1.5 wt% PEG4000, with a pH of 5-6.

[0024] The invention also discloses recombinant spider silk protein fibers prepared by the preparation method.

[0025] The beneficial effects of the present invention are as follows: the SpiCE-DS8 protein disclosed in the present invention exists both in the spider's large ampullate gland and on the spider's dragline silk, which is related to the super-strong comprehensive mechanical properties of the Theridioidea spiders. The SpiCE-DS8 protein can promote the mechanical properties of silk fibers in vitro spinning, and its strain and toughness are significantly improved, and the key functional domain of the SpiCE-DS8 protein, SpiCE-DS8-S (KGGLDILGGLLKLSKAGKDGKNFF), can promote the self-assembly and fiber formation of silk protein. By using the SpiCE-DS8 protein as the raw material of the spinning solution, the obtained recombinant spider silk protein fibers are significantly improved in strength, toughness, and elongation, which provides the possibility of improving the mechanical properties of in vitro recombinant silk, and the method of the present invention simplifies the spinning process, can achieve continuous spinning, and is conducive to industrial mass production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a flow chart of the method of the present invention;

[0027] Figure 2 The stress-strain curve of the practical species of silk fibroin (Chuanshan × Shushui);

[0028] Figure 3 This is the stress-strain curve of the transgenic spider silkworm (SP2-KI). DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

[0030] The amino acid sequence of SpiCE-DS8 protein (SEQ ID NO: 1) is as follows:

[0031] MLTCRNLCVVLVLTVVLLSAIPCDAKGGLDILGGLLKLSKAGKDGKNFFQDLGKGLK DEFKGFFKRI.

[0032] The nucleotide sequence of the gene encoding the SpiCE-DS8 protein (SEQ ID NO: 2) is as follows:

[0033] ATGTTGACTTGCAGAAATTTATGTGTAGTATTGGTACTGACCGTTGTTCTCCTGTCAGCGATTCCATGTGATGCAAAAGGTGGTTTAGATATATTAGGTGGACTTCTAAAATTGTCGAAAGCCGGTAAAGATGGCAAAAACTTTTTTCAAGATTTGGGCAAGGGATTGAAAGACGAATTTAAAGGATTCTTTAAAAGGATTTGA.

[0034] Example

[0035] A method for preparing recombinant spider silk protein fibers, the process is as follows Figure 1 As shown, the following steps are included:

[0036] S1. Remove the signal peptide, synthesize SpiCE-DS8-L protein (KGGLDILGGLLKLSKAGKDGKNFFQDLGKGLKDEFKGFFKRI, SEQ ID NO: 3) and synthesize SpiCE-DS8-S protein (KGGLDILGGLLKLSKA GKDGKNFF, SEQ ID NO: 4), use a peptide synthesizer to synthesize, purify the peptides, and obtain SpiCE-DS8 protein with a purity greater than 95%;

[0037] S2, take 5g of silk cocoons, cut them into pieces, and then boil them in a 0.02M Na2CO3 solution for 30 minutes, take out the degummed silk, wash it with ultrapure water for 3 times, and dry it at 60℃ for 10 hours to obtain silk fibroin crude protein;

[0038] S3, dissolving 3 g of silk fibroin crude protein in 300 mL of 9.3 M LiBr solution, heating in a water bath at 55° C. for 4 h to completely dissolve the silk fibroin, then dialyzing the solution for 48 h using a dialysis membrane with a water cutoff molecular weight of 14,000 Daltons until the conductivity is less than 10 μS, freeze-drying the desalted silk fibroin crude protein at -20° C. for 24 h to obtain silk fibroin protein with impurities removed;

[0039] S4, dissolving 0.35 g of silk fibroin in 10 mL of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), then adding 0.05 g of SpiCE-DS8 protein and incubating for 12 h to obtain a light yellow, viscous, transparent recombinant silk protein spinning solution;

[0040] S5. Wet spinning was performed to extrude the recombinant silk protein spinning solution into a coagulation solution (80 vol% EtOH solution, 0.6 M ammonium acetate and 1 wt% PEG4000, pH 5.3, temperature 23°C) at a speed of 50 μL / min. After the spinning solution came into contact with the coagulation solution, clearly visible fibers were immediately formed. The fibers were placed on a spinning wheel and the wet-spun fibers were collected by pulling at a speed of 6 cm / s to finally obtain recombinant spider silk protein fibers.

[0041] Experimental example

[0042] The silk fibroin practical species (Chuanshan × Shushui) and the transgenic spider silkworm (SP2-KI, the silk contains 80% spider protein Masp2) were used as the control group, and the two different silk cocoons were used as the source of silk fibroin crude protein in step S2, and the recombinant spider silk protein fiber was prepared by the method of Example 1. The mechanical properties of the silk fiber samples were measured:

[0043] (1) The diameters of different parts (n=3) of the fiber sample were measured by microscopic observation, and the cross-sectional areas of adjacent fiber sections were calculated.

[0044] (2) The strain rate was 10 mm / min (0.033 s) at 25 °C and 48% relative humidity using a dynamic mechanical analyzer (DMA Q800) and a 1 N load cell. -1 ) conditions, each fiber was attached to a rectangular cardboard with a hole diameter of 1 cm using epoxy resin.

[0045] (3) For each sample, 30 silk fibers were tested and the average value was taken.

[0046] The measurement results are shown in Table 1. Figure 2 and Figure 3 shown.

[0047] Table 1 Mechanical properties data

[0048]

[0049] For materials that do not yield during the stretching process, the values ​​of elongation at break and tensile strain at break are the same, and the average strain is the average tensile strain at break. As shown in Table 1, the mechanical properties of silk fibers of the practical silk fibroin species (Chuanshan × Shushui) and transgenic spider silkworm (SP2-KI) were improved in terms of elongation and toughness after the addition of SpiCE-DS8-L protein. The elongation, toughness and breaking strength were all improved after the addition of SpiCE-DS8-S protein, especially in terms of toughness, which was improved by more than 120% for the practical species and nearly 20% for the transgenic spider silkworm. This shows that the addition of SpiCE-DS8-L protein can improve the mechanical properties of wet spinning in vitro and contribute to the industrial production of wet spinning, while the effect of SpiCE-DS8-S protein is better, indicating that SpiCE-DS8-S protein belongs to a protein functional domain that plays a key role. When the same mass of protein is added, the number of protein functional domains contained in the truncated SpiCE-DS8-S protein is larger, so the effect of promoting fiber bundling in the filament formation is greater, and thus the mechanical properties are stronger.

[0050] Figure 2 The stress-strain curve of the practical species of silk fibroin (Chuanshan × Shushui) of Bombyx mori is shown in Figure 2. Figure 3 This is the stress-strain curve of the transgenic spider silkworm (SP2-KI). It can be seen from the figure that the strain is significantly increased after adding SpiCE-DS8-L protein, and the strain and stress are also increased after adding SpiCE-DS8-S protein. A similar effect is also seen in the transgenic spider silkworm SP2-KI.

Claims

1. A SpiCE-DS8 protein, characterized in that The amino acid sequence of the SpiCE-DS8 protein is shown in SEQ ID NO:

1.

2. A gene encoding the SpiCE-DS8 protein according to claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

3. A recombinant plasmid, characterized in that: Comprising the gene according to claim 2.

4. A method for preparing recombinant spider silk protein fibers, characterized in that: The following steps are involved: S1. Cut the silk cocoons into pieces, boil them in alkali solution for 20-40 minutes, take out the silk, rinse and dry them to obtain silk fibroin crude protein; S2, dissolving the silk fibroin crude protein, heating at 50-60°C for 3-5h, then dialyzing for 46-50h, and finally freeze-drying to obtain silk fibroin; S3, dissolving the silk fibroin in a polar solvent, then adding the SpiCE-DS8 protein according to claim 1, and incubating for 10-14 hours to obtain a recombinant silk fibroin spinning solution; the ratio of the silk fibroin, polar solvent and SpiCE-DS8 protein is 0.3-0.4 g: 8-12 mL: 0.03-0.07 g; S4. Wet spinning is adopted to extrude the recombinant silk protein spinning solution into the coagulation solution, and the wet-spun fibers are collected by pulling at a speed of 5-7 cm / s to finally obtain recombinant spider silk protein fibers.

5. The method for preparing recombinant spider silk protein fibers according to claim 4, characterized in that: The solvent used for dissolving S2 is LiBr or LiSCN solution.

6. The method for preparing recombinant spider silk protein fibers according to claim 4, characterized in that: The molecular weight cut-off of the dialysis membrane used for the dialysis is 13000-15000 Daltons.

7. The method for preparing recombinant spider silk protein fibers according to claim 4, characterized in that: The freeze drying temperature is -30 to -10°C and the time is 22 to 26 hours.

8. The method for preparing recombinant spider silk protein fibers according to claim 4, characterized in that: The extrusion speed of the recombinant silk protein spinning solution is 45-55 μL / min.

9. The method for preparing recombinant spider silk protein fibers according to claim 4, characterized in that: The coagulation solution comprises 75-85 vol% EtOH solution, 0.5-0.7 M ammonium acetate and 0.5-1.5 wt% PEG4000, and the pH value is 5-6.

10. A recombinant spider silk protein fiber, characterized in that: The method is prepared by any one of claims 4 to 9.

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