CN115819541A - Recombinant spider silk strengthened by cysteine and its preparation method and application - Google Patents

Abstract

The invention provides a cysteine-strengthened recombinant spider silk, a preparation method and application thereof. The amino acid sequence of the recombinant spider silk protein has the sequence shown in SEQ ID NO:6. The polynucleotide encoding the amino acid sequence of the protein has the following structure: the 5' end of the parental web spider major ampullate gland silk MaSp2 gene improved by cysteine is connected with an N-domain, and the 3' end is connected with a C-domain; The gene was inserted into the pET‑28a plasmid vector to obtain a recombinant plasmid, which was then introduced into Escherichia coli for expression to obtain a recombinant spider silk protein. The recombinant spider silk prepared by wet spinning using the recombinant spider silk protein has a dense β-sheet crystal structure, excellent mechanical properties and wet strength, which opens up prospects for the industrial application of recombinant spider silk.

Description

Recombinant spider silk strengthened by cysteine and its preparation method and application

technical field

The invention belongs to the field of biopolymer fiber materials, and in particular relates to a cysteine-enhanced recombinant spider silk and its preparation method and application.

Background technique

Spider traction silk, that is, the major ampullate silk, exhibits excellent mechanical properties such as high strength and high toughness, and its mechanical properties are even better than some current artificial high-performance fibers. Its excellent mechanical properties benefit from the delicate microstructure in the fiber, in which β-sheet crystals are responsible for having a very high elastic modulus, which provides extremely high tensile strength for spider silk, while the amorphous α-helix structure is The main source of high tenacity and ductility of spider silk. In addition to mechanical properties, spider silk also has excellent biocompatibility and biodegradability, which greatly expands the application of spider silk in the field of biomaterials. Therefore, spider silk materials have become a research hotspot in academia and industry today. .

Restricted by the nature of spiders' aggressiveness, self-protection awareness and regional aggressiveness, it is impossible to collect spider silk in large quantities through large-scale farming like harvesting silk. Moreover, the structure and function of spider silk glands are extremely complex, and the aggregation and self-assembly behavior of silk protein molecular chains in the spinning process has not yet been fully understood. Therefore, spider silk proteins were expressed in bioreactors such as E. coli and yeast by genetic engineering (Whittall et al. 2020), and high-performance recombinant spider silk was prepared by artificial spinning (Venkatesan, Chen, and Hu 2019) become a research hotspot in recent years. The researchers hope to simulate the performance of the spinning stock solution and the spinning environment in the spider silk gland through amino acid sequence optimization and bionic spinning, and to explore the protein molecular chains in the natural spinning process by studying the physical and chemical properties of the spider silk protein molecular chain. Chain aggregation and self-assembly behavior, and provide a reference for subsequent optimization and simulation. Oktaviani et al. explored the dynamic behavior of the Random Coil in the spider spinning solution and the formation process of the β-sheet structure (Oktaviani et al.2018); Malay and Saric et al. studied the spinning environment in the silk gland, N- The /C-domain guides the self-assembly behavior of protein molecular chains (Saric et al.2021; Malay et al.2020).

In addition to the above-mentioned fields, in the field of textiles, recombinant spider silk still has broad application prospects due to its excellent performance, and some companies and research teams have used recombinant spider silk to trial-produce textiles, such as the Biofabric running shoes that the German start-up company AMSilk cooperated with Adidas, The MoonParka down jacket jointly developed by The North Face Company and Japan’s Spiber Company, etc. However, these products and fabrics still face problems such as low wet strength, poor durability, and irreversible shrinkage when exposed to water, resulting in high product costs and substandard quality. Obviously, these problems have seriously affected the wide application of recombinant spider silk in the textile field.

Contents of the invention

Aiming at the problems of low wet strength, poor durability, and irreversible shrinkage when exposed to water in the prior art, the first object of the present invention is to provide a recombinant spider silk protein; the second object of the present invention is to provide a A polynucleotide encoding the amino acid sequence of the protein; the third purpose of the present invention is to provide a recombinant plasmid comprising the polynucleotide DNA sequence; the fourth purpose of the present invention is to provide a host bacterium that has been transformed into the recombinant plasmid; The fifth object of the invention is to provide a method for preparing recombinant spider silk protein; the sixth object of the present invention is to provide a method for preparing recombinant spider silk; the seventh object of the present invention is to provide recombinant spider silk protein obtained by wet spinning The recombinant spider silk prepared by the preparation method of spider silk or recombinant spider silk; the eighth object of the present invention is to provide the application of the recombinant spider silk in the field of biological materials, high-performance fiber materials or textile materials. In the present invention, by using the gene encoding cysteine to transform the MaSp2 gene of the Euprosthenops australis major ampullate silk, a stronger disulfide bond is formed between the protein molecular chains to optimize the crystal structure in the fiber, thereby strengthening the fiber Excellent mechanical properties and waterproof properties; the recombinant spider silk protein gene can be efficiently expressed in Escherichia coli, and the obtained recombinant spider silk fiber has uniform shape and excellent mechanical strength, which opens up prospects for the industrial application of recombinant spider silk.

The purpose of the present invention is achieved by the following technical means:

In a first aspect, the present invention provides a recombinant spider silk protein, the amino acid sequence of which has the sequence shown in SEQ ID NO:6. The sequence of SEQ ID NO: 6 is as follows:

SHTTPWTNPGLAENFMNSFMQGLSSMPGFTASQLDDMSTIAQSMVQSIQSLAAQGRTSPNKLQALNMAFASSMAEIAASEEGGGSLSTKTSSIASAMSNAFLQTTGVVNQPFINEITQLVSMFAQAGMNDVSAQGGFGQGAGGNAAACAAAAAACAAAQQGGQGGFGGQGQGGFGPGAGSSAACAAAACAAGQGGQGRGGFGQGVTSGGYGYGTSAAAGAGVAAGSYAGAVNRLSSAEAASRVSSNIAAIASGGASALPSVISNIYSGVVASGVSSNEALIQALLELLSALVHVLSSASIGNVSSVGVDSTLNVVQDSVGQYVG

In the second aspect, the present invention also provides a polynucleotide encoding the amino acid sequence of recombinant spider silk protein, the polynucleotide has the following structure:

The 5' end of the MaSp2 gene of the parental web spider's major ampullate gland silk with improved cysteine is connected with the N-domain and the 3' end is connected with the C-domain.

Among the above-mentioned polynucleotides, preferably, cysteine modification is carried out using the Euprosthenops australis major ampullate silk MaSp2 gene as a template, and the MaSp2 gene has the DNA sequence shown in SEQ ID NO: 1, A total of 213 base pairs, the SEQ ID NO: 1 sequence is as follows:

5'- CAAGGAGGATTTGGTCAAGGTGCTGGAGGTAATGCCGCAGCC GCT GCAGCAGCCGCCGCAGCA GC A GCAGCAGCTCAACAAGGTGGTCAAGGTGGTTTTGGAGGACAAGGTCAAGGAGGATTTGGACCTGGAGCAGGAAGTTCTGCAGCT GCA GCCGCTGCAGCA GCA GCAGCTGGTCAAGGTGGACAAGGAAGAGGAGGATTCG'

Among the above polynucleotides, preferably, the DNA sequence of the cysteine-improved brood spider major ampullate silk MaSp2 gene has the sequence shown in SEQ ID NO:2. It is an improved DNA sequence obtained by substituting a certain amount of cysteine gene in the polyalanine DNA sequence of SEQ ID NO: 1 sequence, with a total of 213 base pairs. The sequence of SEQ ID NO: 2 is as follows:

5'-CAAGGAGGATTTGGTCAAGGTGCTGGAGGTAATGCCGCAGCC TGT GCAGCAGCCGCCGCAGCA TGT GCAGCAGCTCAACAAGGTGGTCAAGGTGGTTTTGGAGGACAAGGTCAAGGAGGATTTGGACCTGGAGCAGGAAGTTCTGCAGCT TGT GCCGCTGCAGCA TGT GCAGCTGGTCAAGGTGGACAAGGAAGAGGAGGATTCG'

Among the above polynucleotides, preferably, the N-domain is selected from the Euprosthepsthepsaustralis MaSp1 gene, the DNA sequence of the MaSp1 gene has the sequence shown in SEQ ID NO: 3, with a total of 399 bases Yes, the sequence of SEQ ID NO: 3 is as follows:

5’-TCACACACTACACCATGGACAAACCCAGGACTCGCAGAAAACTTCATGAACAGTTTCATGCAAGGCCTGAGCTCGATGCCAGGTTTCACGGCAAGCCAATTGGATGATATGTCAACCATCGCACAATCCATGGTACAGTCAATACAATCCTTGGCGGCACAAGGCAGGACATCACCGAATAAGCTGCAGGCCCTTAACATGGCTTTTGCATCTTCGATGGCAGAAATCGCGGCATCCGAAGAAGGAGGGGGAAGCCTTTCCACCAAAACTAGCTCTATAGCCAGTGCAATGTCCAACGCGTTTCTGCAAACAACTGGAGTGGTAAACCAACCGTTCATAAATGAAATAACTCAGCTCGTTAGCATGTTTGCTCAAGCAGGTATGAATGATGTCAGTGCT-3’

Among the above polynucleotides, preferably, the C-domain is selected from the MiSp gene of Araneus ventricosus, the DNA sequence of the MiSp gene has the sequence shown in SEQ ID NO: 4, and there are 360 in total In base pairs, the sequence of SEQ ID NO: 4 is as follows:

5’-GTTACATCTGGAGGTTACGGATATGGAACCAGTGCAGCTGCAGGAGCTGGAGTTGCAGCAGGTTCATATGCAGGTGCTGTCAATCGCTTGTCTAGTGCTGAAGCTGCCAGTAGAGTATCCTCTAATATTGCAGCTATTGCATCTGGTGGTGCTTCCGCCCTCCCCAGTGTTATTTCAAATATTTACTCAGGTGTCGTTGCTTCTGGTGTTTCTTCTAATGAAGCTCTGATTCAAGCTCTGTTGGAACTCCTTTCCGCACTTGTTCATGTTTTAAGCAGTGCCTCTATCGGTAATGTTAGCTCAGTAGGAGTAGATAGTACATTGAATGTTGTTCAGGATTCAGTAGGCCAATATGTAGGT-3’

Among the above polynucleotides, preferably, the DNA sequence of the polynucleotide has the sequence shown in SEQ ID NO: 5, that is, the polynucleotide sequence encoding the amino acid sequence of the recombinant spider silk protein, the polynucleotide (ie: The target gene M.NT2RepCT) has a total of 972 base pairs, and the sequence of SEQ ID NO: 5 is as follows:

5’-TCACACACTACACCATGGACAAACCCAGGACTCGCAGAAAACTTCATGAACAGTTTCATGCAAGGCCTGAGCTCGATGCCAGGTTTCACGGCAAGCCAATTGGATGATATGTCAACCATCGCACAATCCATGGTACAGTCAATACAATCCTTGGCGGCACAAGGCAGGACATCACCGAATAAGCTGCAGGCCCTTAACATGGCTTTTGCATCTTCGATGGCAGAAATCGCGGCATCCGAAGAAGGAGGGGGAAGCCTTTCCACCAAAACTAGCTCTATAGCCAGTGCAATGTCCAACGCGTTTCTGCAAACAACTGGAGTGGTAAACCAACCGTTCATAAATGAAATAACTCAGCTCGTTAGCATGTTTGCTCAAGCAGGTATGAATGATGTCAGTGCTCAAGGAGGATTTGGTCAAGGTGCTGGAGGTAATGCCGCAGCCTGTGCAGCAGCCGCCGCAGCATGTGCAGCAGCTCAACAAGGTGGTCAAGGTGGTTTTGGAGGACAAGGTCAAGGAGGATTTGGACCTGGAGCAGGAAGTTCTGCAGCTTGTGCCGCTGCAGCATGTGCAGCTGGTCAAGGTGGACAAGGAAGAGGAGGATTCGGTCAAGGTGTTACATCTGGAGGTTACGGATATGGAACCAGTGCAGCTGCAGGAGCTGGAGTTGCAGCAGGTTCATATGCAGGTGCTGTCAATCGCTTGTCTAGTGCTGAAGCTGCCAGTAGAGTATCCTCTAATATTGCAGCTATTGCATCTGGTGGTGCTTCCGCCCTCCCCAGTGTTATTTCAAATATTTACTCAGGTGTCGTTGCTTCTGGTGTTTCTTCTAATGAAGCTCTGATTCAAGCTCTGTTGGAACTCCTTTCCGCACTTGTTCATGTTTTAAGCAGTGCCTCTATCGGTAATGTTAGCTCAGTAGGAGTAGATAGTACATTGAATGTTGTTCAGGATTCAGTAGGCCAATATGTAGGT-3’

In the third aspect, the present invention also provides a recombinant plasmid, the DNA sequence of which comprises the above-mentioned polynucleotide sequence.

In the fourth aspect, the present invention also provides a host bacterium transformed with the above-mentioned recombinant plasmid.

In the fifth aspect, the present invention also provides a method for preparing the above-mentioned recombinant spider silk protein, which includes the following steps:

The above polynucleotide DNA sequence is amplified by primers to obtain the target gene, and then the target gene is inserted into a plasmid vector to obtain a recombinant plasmid, and the recombinant plasmid or the above-mentioned recombinant plasmid is introduced into a host bacterium for expression to obtain recombinant spider silk protein.

In the above preparation method, preferably, the DNA sequence of the upstream primer of the primer has the sequence shown in SEQ ID NO: 7; the DNA sequence of the downstream primer of the primer has the sequence shown in SEQ ID NO: 8.

The DNA sequence (SEQ ID NO: 7) of the upstream primer is:

5'-CGCGGATCCGCGATGTCACACACTACACCATGGACAAACC-3'

The DNA sequence (SEQ ID NO: 8) of the downstream primer is:

5'-CCCAAGCTTGGGACCTACATATTGGCCTACTGAATCCTGA-3'

In the above method for preparing recombinant spider silk protein, preferably, the plasmid vector includes pET-28a plasmid, but not limited thereto.

In the above method for preparing recombinant spider silk protein, preferably, the host bacteria include Escherichia coli, but not limited thereto.

In the sixth aspect, the present invention also provides a method for preparing recombinant spider silk, which includes the following steps:

Purifying the recombinant spider silk protein prepared by the above preparation method, and concentrating by centrifugal filtration to obtain a stock solution of recombinant spider silk spinning protein;

A wet spinning machine is used to extrude the recombinant spider silk spinning protein stock solution, and the extruded fibers are solidified in a coagulation bath, followed by hot steam annealing and post-stretching treatment, and dried at room temperature to obtain the recombinant spider silk.

In the above method for preparing recombinant spider silk, preferably, the eluent is used to purify the recombinant spider silk protein, and the eluent contains: 50-80 mM Tris-HCl, the pH value is 7.5, 100-120 mM NaCl, 1-1.5mM EDTA and 1-2mM DTT (dithiothreitol); the mass concentration of the purified recombinant spider silk protein is 2%-3%.

In the above method for preparing recombinant spider silk, preferably, the recombinant spider silk protein solution is concentrated with a 10,000 MWCO (molecular weight cut-off) centrifugal filter, and the mass concentration of the obtained recombinant spider silk spinning stock solution is 20%-25%.

In the above method for preparing recombinant spider silk, preferably, the specification of the extrusion needle of the wet spinning machine is 30G (30G is the type of spinning needle, and the specific specification is the inner diameter of the needle is 0.16mm, and the outer diameter is 0.19mm); The extrusion speed of the recombinant spider silk spinning protein stock solution is 10-30 μL/min.

In the above method for preparing recombinant spider silk, preferably, the coagulation bath uses DMSO (dimethyl sulfoxide); the time for solidification by using the coagulation bath is 1-2 hours. The use of DMSO can not only promote protein coagulation to form fibers, but also provide a weak oxidative environment for proteins to promote the formation of disulfide bonds between β-sheet layers.

In the above method for preparing recombinant spider silk, preferably, the temperature of the hot steam for the hot steam annealing step is 80-120° C., and the annealing time is 30-90 s.

In the above method for preparing recombinant spider silk, preferably, the post-stretching process is performed at a stretching rate of 1-2 mm/s, and a stretching ratio of 0.5-0.8.

In the seventh aspect, the present invention also provides a recombinant spider silk, which has a β-sheet crystal structure (compactness); the diameter of its fibers is 50-60 μm, and the average fracture stress of the fibers is 30-40 MPa. The average elongation at break is 10% to 20%.

In the above recombinant spider silk, preferably, the recombinant spider silk is prepared from the above recombinant spider silk protein by wet spinning or prepared by the above recombinant spider silk preparation method.

The β-sheet crystals of recombinant spider silk are usually formed by stacking multiple layers of polyalanine molecules, and the layers are tightly combined by hydrogen bonds. However, in the process of artificially simulating the spinning process, there are often defects in the crystal structure due to the low concentration of the spinning solution and the inability to completely simulate the spinning process, which further leads to a decrease in the strength of the filament. The present invention inserts a certain amount of cysteine at a specific position in the polyalanine sequence, aiming to form an anchoring effect through the disulfide bond formed in the coagulation bath to stabilize the β-sheet crystal. At the same time, because cysteine is Hydrophilic amino acid can inhibit the self-assembly behavior of polyalanine segment in aqueous solution to increase the concentration of spinning solution.

In an eighth aspect, the present invention also provides the application of the above-mentioned recombinant spider silk in the field of biological materials, fiber materials or textile materials.

Beneficial effects of the present invention:

The recombinant spider silk protein gene of the present invention can be efficiently expressed in Escherichia coli, and the fiber of the prepared recombinant spider silk has a dense β-sheet crystal structure, excellent mechanical properties and wet strength, and the diameter of the fiber is 50-60 μm. The average breaking stress of the fiber can reach 30-40MPa, and the average breaking elongation is 10%-20%, which opens up prospects for the industrial application of the recombinant spider silk.

Description of drawings

Fig. 1 is a flowchart of the recombinant plasmid prepared in Example 1 of the present invention.

Fig. 2 is an electron microscope image of the surface of the recombinant spider silk fiber prepared in Example 1 of the present invention and its cross section.

Fig. 3 is a graph showing molecular dynamics simulation results of recombinant spider silk prepared in Example 1 of the present invention.

Detailed ways

In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is described in detail below, but it should not be construed as limiting the scope of implementation of the present invention. The raw materials used in the following examples are conventionally commercially available in the art unless otherwise specified; the methods used are conventional methods in the art unless otherwise specified.

Example 1:

This embodiment provides a method for preparing recombinant spider silk, which includes the following steps:

1. The design of the recombinant spider silk protein gene (the specific sequence of the design is entrusted to a gene synthesis company):

(1) Cysteine improvement was carried out using the Euprosthenops australis major ampullate silk MaSp2 gene as a template. The Euprosthenops australis major ampullate silk MaSp2 gene has a total of 213 base pairs, and its DNA sequence is as follows: Shown in SEQ ID NO:1.

(2) Substituting a certain amount of cysteine gene in the polyalanine DNA sequence of SEQ ID NO: 1 sequence to obtain cysteine-improved MaSp2 gene of parental web spider major ampullate gland silk, with a total of 213 base pairs , whose DNA sequence is shown in SEQ ID NO:2.

(3) connecting the N-structural domain at the 5' end and the C-structural domain at the 3' end of the sequence of SEQ ID NO: 2 to obtain a recombinant spider silk protein gene.

The N-domain is selected from the MaSp1 gene of Euprosthenops australis, with a total of 399 base pairs, and its DNA sequence is shown in SEQ ID NO:3.

The C-domain is selected from the MiSp gene of Araneus ventricosus, with a total of 360 base pairs, and its DNA sequence is shown in SEQ ID NO:4.

The finally obtained recombinant spider silk protein gene (M.NT2RepCT) has a total of 972 base pairs, and its DNA sequence is shown in SEQ ID NO:5.

2. Preparation of recombinant plasmid and recombinant spider silk protein:

(1) The sequence of SEQ ID NO: 5 was amplified by primers to obtain the target gene, and the pET-28a plasmid vector was digested with HindIII and BamHI restriction endonucleases to prepare the digestion vector. The buffer was BamHI buffer, and the digestion temperature was 37°C. Purify the digested vector and gene of interest using agarose gel electrophoresis and measure their concentrations using a spectrophotometer. Add a certain amount of digestion vector and target gene to the microcentrifuge tube, so that the ratio of DNA fragments is 3:1, add 1 μL BamHI buffer (10x), 1 μL 10mM ATP, 1 μL DNA ligase, add sterile water Make up to 10μL. Shake the centrifuge tube and incubate the microcentrifuge tube at 20°C for 4 hours to obtain the recombinant plasmid containing the target gene. The DNA sequence of the upstream primer of the primer has the sequence shown in SEQ ID NO: 7; the DNA sequence of the downstream primer of the primer has the sequence shown in SEQ ID NO: 8; the flow chart of preparing the recombinant plasmid is shown in Figure 1 .

(2) The recombinant plasmid was introduced into Escherichia coli for expression to obtain the recombinant spider silk protein. Specifically:

Thaw 100 μL Escherichia coli BL21(DE3) competent cells on ice for 10 minutes, add 2 μL of recombinant plasmid, and then ice-bath for 30 minutes, heat shock at 42°C for 60 seconds, and then ice-bath for 2 minutes. Add 900 μL of LB liquid medium preheated at 37°C to Escherichia coli, shake and incubate at 37°C for 45 minutes on a constant temperature shaker at 180 rpm; coli strains containing the gene of interest were obtained by culturing overnight at 37°C on LB agar plates.

3. Preparation of recombinant spider silk:

(1) Purify the obtained recombinant spider silk protein, the eluent used in the purification process contains 50mM Tris-HCl (pH7.5), 100mM NaCl, 1mM EDTA and 1mM DTT, the obtained recombinant spider silk The mass concentration of the protein solution is 3%; then the protein solution is concentrated to 20wt% by using a 10000MWCO centrifugal filter to obtain a high-concentration recombinant spider silk spinning protein stock solution.

(2) Pour the recombinant spider silk spinning protein stock solution into the raw material tank of the wet spinning machine, the extrusion needle specification is 30G, the extrusion speed is 10μL/min, and the coagulation bath uses DMSO; the recombinant spider silk fiber is extruded Cured in the coagulation bath for 1 hour. After curing, the fiber was taken out from the coagulation bath, and after 30 seconds of hot steam annealing at 80°C, it entered the post-drawing step. The fiber was stretched at a rate of 1 mm/s and stretched by 0.5 times. After stretching and drying at room temperature, the recombinant spider silk was prepared.

The average diameter of the recombinant spider silk fiber prepared in this example is 60 μm, the surface of the fiber is smooth (as shown in FIG. 2 ), the average breaking stress of the fiber reaches 30 MPa, and the average breaking elongation is 10%.

Molecular dynamics simulation was carried out on the recombinant spider silk, and the simulation results are shown in Fig. 3 . The yellow part (that is, the dark part of the black and white picture) is the optimized β-sheet crystal part, and the gray part (that is: the light part in the black and white picture RH 0%) is the amorphous region structure such as α-helix, random coil, etc. The turquoise part (that is, the light-colored part in the black-and-white image RH 25% to 100%) is water molecules. It can be seen from Figure 3 that in a humid environment, water molecules invade the amorphous region structure to a limited extent, and as the humidity gradually increases, water molecules begin to invade the β-sheet crystal structure, but due to the protection of interlayer disulfide bonds, even In the high humidity environment of RH100%, the β-sheet structure can still maintain most of its integrity.

sequence listing

<110> City University of Hong Kong

<120> Recombinant spider silk strengthened by cysteine and its preparation method and application

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<213> Euprosthenops australis

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Glu Gly Gly Gly Ser Leu Ser Thr Lys Thr Ser Ser Ile Ala Ser Ala

85 90 95

Met Ser Asn Ala Phe Leu Gln Thr Thr Gly Val Val Val Asn Gln Pro Phe

100 105 110

Ile Asn Glu Ile Thr Gln Leu Val Ser Met Phe Ala Gln Ala Gly Met

115 120 125

Asn Asp Val Ser Ala Gln Gly Gly Phe Gly Gln Gly Ala Gly Gly Asn

130 135 140

Ala Ala Ala Cys Ala Ala Ala Ala Ala Ala Ala Cys Ala Ala Ala Gln Gln

145 150 155 160

Gly Gly Gln Gly Gly Phe Gly Gly Gln Gly Gln Gly Gly Phe Gly Pro

165 170 175

Gly Ala Gly Ser Ser Ala Ala Cys Ala Ala Ala Ala Cys Ala Ala Gly

180 185 190

Gln Gly Gly Gln Gly Arg Gly Gly Gly Phe Gly Gln Gly Val Thr Ser Gly

195 200 205

Gly Tyr Gly Tyr Gly Thr Ser Ala Ala Ala Gly Ala Gly Val Ala Ala

210 215 220

Gly Ser Tyr Ala Gly Ala Val Asn Arg Leu Ser Ser Ala Glu Ala Ala

225 230 235 240

Ser Arg Val Ser Ser Asn Ile Ala Ala Ile Ala Ser Gly Gly Ala Ser

245 250 255

Ala Leu Pro Ser Val Ile Ser Asn Ile Tyr Ser Gly Val Val Ala Ser

260 265 270

Gly Val Ser Ser Asn Glu Ala Leu Ile Gln Ala Leu Leu Glu Leu Leu

275 280 285

Ser Ala Leu Val His Val Leu Ser Ser Ser Ala Ser Ile Gly Asn Val Ser

290 295 300

Ser Val Gly Val Asp Ser Thr Leu Asn Val Val Gln Asp Ser Val Gly

305 310 315 320

Gln Tyr Val Gly

<210> 7

<211> 40

<212>DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 7

cgcggatccg cgatgtcaca cactacacca tggacaaacc 40

<210> 8

<211> 40

<212>DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 8

cccaagcttg ggaccctacat attggcctac tgaatcctga 40

Claims (22)

1. A recombinant spider silk protein, the amino acid sequence of the recombinant spider silk protein has the sequence shown in SEQ ID NO:6. 2. A polynucleotide encoding the amino acid sequence of the recombinant spider silk protein of claim 1, the polynucleotide has the following structure: The 5' end of the MaSp2 gene of the parental web spider's major ampullate gland silk with improved cysteine is connected with the N-domain and the 3' end is connected with the C-domain. 3. The polynucleotide according to claim 2, wherein the DNA sequence of the cysteine-improved brood web spider's major ampullate silk MaSp2 gene has the sequence shown in SEQ ID NO:2. 4. The polynucleotide according to claim 2, wherein the N-domain is selected from the MaSp1 gene of the nursery web spider, and the DNA sequence of the MaSp1 gene has the sequence shown in SEQ ID NO:3. 5 . The polynucleotide according to claim 2 , wherein the C-domain is selected from the MiSp gene of E. magna, and the DNA sequence of the MiSp gene has the sequence shown in SEQ ID NO:4. 6. The polynucleotide according to any one of claims 2-5, wherein the DNA sequence of the polynucleotide has the sequence shown in SEQ ID NO:5. 7. A recombinant plasmid, the DNA sequence of which comprises the sequence of the polynucleotide according to any one of claims 2-6. 8. A host bacterium transformed into the recombinant plasmid according to claim 7. 9. The preparation method of recombinant spider silk protein described in claim 1, it comprises the following steps: The DNA sequence of the polynucleotide described in any one of claims 2 to 6 is amplified by primers to obtain the target gene, and then the target gene is inserted into a plasmid vector to obtain a recombinant plasmid, and the recombinant plasmid or the polynucleotide described in claim 7 The recombinant plasmid is introduced into the host bacteria for expression to obtain the recombinant spider silk protein. 10. The preparation method according to claim 9, wherein the upstream primer DNA sequence of the primer has the sequence shown in SEQ ID NO: 7; the DNA sequence of the downstream primer of the primer has the sequence shown in SEQ ID NO: 8 sequence. 11. The preparation method according to claim 9, wherein the plasmid vector comprises pET-28a plasmid. 12. The preparation method according to claim 9, wherein the host bacteria comprise Escherichia coli. 13. A method for preparing recombinant spider silk, comprising the following steps: Purifying the recombinant spider silk protein prepared by the preparation method described in any one of claims 9 to 12, and concentrating by centrifugal filtration to obtain a stock solution of recombinant spider silk spinning protein; A wet spinning machine is used to extrude the recombinant spider silk spinning protein stock solution, and the extruded fibers are solidified in a coagulation bath, followed by hot steam annealing and post-stretching treatment, and dried at room temperature to obtain the recombinant spider silk. 14. The preparation method according to claim 13, wherein the recombinant spider silk protein is purified using an eluent, the composition of the eluent contains: 50-80 mM Tris-HCl, the pH value is 7.5, 100- 120mM NaCl, 1-1.5mM EDTA and 1-2mM DTT; the mass concentration of the purified recombinant spider silk protein is 2%-3%. 15. The preparation method according to claim 13, wherein the recombinant spider silk protein solution is concentrated by using a 10000MWCO centrifugal filter, and the obtained recombinant spider silk spinning stock solution has a mass concentration of 20%-25%. 16. The preparation method according to claim 13, wherein the specification of the extrusion needle of the wet spinning machine is 30G; the extrusion speed of the recombinant spider silk spinning protein stock solution is 10-30 μL/min. 17. The preparation method according to claim 13, wherein the coagulation bath adopts DMSO; the time for solidification by the coagulation bath is 1-2 hours. 18. The preparation method according to claim 13, wherein the temperature of the hot steam for the hot steam annealing step is 80-120°C, and the annealing time is 30-90s. 19. The preparation method according to claim 13, wherein the stretching rate in the post-stretching process is 1-2 mm/s, and the stretching ratio is 0.5-0.8. 20. A recombinant spider silk, the recombinant spider silk has a β-sheet crystal structure; the diameter of its fiber is 50-60 μm, the average breaking stress of the fiber is 30-40 MPa, and the average breaking elongation is 10%-20 %. 21. The recombinant spider silk according to claim 20, wherein the recombinant spider silk is prepared from the recombinant spider silk protein according to claim 1 by wet spinning or obtained from any one of claims 13-19. prepared by the preparation method. 22. The application of the recombinant spider silk according to claim 20 or 21 in the field of biomaterials, fiber materials or textile materials.

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