Cysteine-enhanced recombinant spider silk and methods of making and using same

Abstract

The application provides a cysteine-strengthened recombinant spider silk and a preparation method and application thereof. An amino acid sequence of the recombinant spider silk protein has the sequence shown in SEQ ID NO: 6. A polynucleotide encoding the amino acid sequence of the protein has the following structure: a 5' end of a cysteine-modified MaSp2 gene of the major ampullate gland of the spider Argiope bruennichi is connected with an N-domain, and a 3' end is connected with a C-domain; the gene is inserted into a pET-28a plasmid vector to obtain a recombinant plasmid, and the recombinant plasmid is introduced into E. coli to express the recombinant spider silk protein. The recombinant spider silk prepared by wet spinning using the recombinant spider silk protein has a dense beta-sheet crystal structure, excellent mechanical properties and wet strength, and opens up a prospect for industrial application of the recombinant spider silk.

Description

Technical Field

[0001] This invention belongs to the field of biopolymer fiber materials, and specifically relates to a cysteine-reinforced recombinant spider silk, its preparation method, and its application. Background Technology

[0002] Spider silk, also known as large-budded glandular silk, exhibits excellent mechanical properties such as high strength and high toughness, even surpassing some current man-made high-performance fibers. Its superior mechanical properties are attributed to the intricate microstructure within the fiber. β-sheet crystals contribute to its extremely high elastic modulus, providing exceptional tensile strength, while the amorphous α-helix structure is the primary source of its high toughness and ductility. In addition to its mechanical properties, spider silk also possesses excellent biocompatibility and biodegradability, greatly expanding its applications in biomaterials. Therefore, spider silk materials have become a research hotspot in both academia and industry.

[0003] Due to spiders' inherent aggressiveness, self-preservation instincts, and territorial aggression, large-scale harvesting of spider silk is not possible through methods like silkworm farming. Furthermore, the structure and function of spider silk glands are extremely complex, and the aggregation and self-assembly behavior of silk protein chains during the spinning process is not fully understood. Therefore, expressing spider silk proteins in bioreactors such as *E. coli* and yeast using genetic engineering methods (Whittall et al. 2020) and preparing high-performance recombinant spider silk through artificial spinning methods (Venkatesan, Chen, and Hu 2019) has become a research hotspot in recent years. Researchers hope to simulate the spinning solution properties and spinning environment within spider silk glands through amino acid sequence optimization and biomimetic spinning methods, and to explore the aggregation and self-assembly behavior of protein chains during natural spinning by studying the physicochemical properties of spider silk protein chains, providing a reference for subsequent optimization and simulation. Oktaviani et al. investigated the kinetics of Random Coil and the formation of β-sheet structures in spider spinning solutions (Oktaviani et al. 2018); Malay and Saric et al. studied the guiding role of N- / C-domains on protein chain self-assembly behavior in the spinning environment within the silk gland (Saric et al. 2021; Malay et al. 2020).

[0004] Besides the aforementioned fields, reconstituted spider silk still holds broad application prospects in the textile industry due to its superior performance. Companies and research teams have already used reconstituted spider silk to trial-produce textiles, such as the Biofabric running shoes developed by the German startup AMSilk in collaboration with Adidas, and the MoonParka down jacket jointly developed by The North Face and Japan's Spiber. However, these products and fabrics still face problems such as low wet strength, poor durability, and irreversible shrinkage upon contact with water, leading to high product costs, substandard quality, and delayed market launches. Clearly, these issues severely hinder the widespread application of reconstituted spider silk in the textile industry. Summary of the Invention

[0005] To address the problems of low wet strength, poor durability, and irreversible shrinkage upon contact with water in existing recombinant spider silk technologies, the first objective of this invention is to provide a recombinant spider silk protein; the second objective is to provide a polynucleotide encoding the amino acid sequence of this protein; the third objective is to provide a recombinant plasmid containing the DNA sequence of this polynucleotide; the fourth objective is to provide a host bacterium transformed with the recombinant plasmid; the fifth objective is to provide a method for preparing the recombinant spider silk protein; the sixth objective is to provide a method for preparing recombinant spider silk; the seventh objective is to provide recombinant spider silk obtained by wet spinning of recombinant spider silk protein or recombinant spider silk prepared by the same method; and the eighth objective is to provide applications of this recombinant spider silk in the fields of biomaterials, high-performance fiber materials, or textile materials. This invention utilizes the gene encoding cysteine ​​to modify the MaSp2 gene of the large ampulla gland silk of the brooding spider (Euprosthenops australis), thereby optimizing the crystal structure of the fiber by forming stronger disulfide bonds between protein molecular chains, thus enhancing the mechanical and waterproof properties of the fiber. This recombinant spider silk protein gene can be efficiently expressed in Escherichia coli, and the resulting recombinant spider silk fibers have uniform morphology and excellent mechanical strength, opening up prospects for the industrial application of recombinant spider silk.

[0006] The objective of this invention is achieved through the following technical means:

[0007] In a first aspect, the present invention provides a recombinant spider silk protein having the amino acid sequence shown in SEQ ID NO: 6. The sequence of SEQ ID NO: 6 is as follows:

[0008] SHTTPWTNPGLAENFMNSFMQGLSSMPGFTASQLDDMSTIAQSMVQSIQSLAAQGRTSPNKLQALNMAFASSMAEIAASEEGGGSLSTKTSSIASAMSNAFLQTTGVVNQPFINEITQLVSMFAQAGMNDVSAQGGFGQGAGGNAAACAAAAAACAAAQQGG QGGFGGQGQGGFGPGAGSSAACAAAACAAGQGGQGRGGFGQGVTSGGYGYGTSAAAGAGVAAGSYAGAVNRLSSAEAASRVSSNIAAIASGGASALPSVISNIYSGVVASGVSSNEALIQALLELLSALVHVLSSASIGNVSSVGVDSTLNVVQDSVGQYVG

[0009] Secondly, the present invention also provides a polynucleotide encoding a recombinant spider silk protein amino acid sequence, the polynucleotide having the following structure:

[0010] The cysteine-modified MaSp2 gene of the bursa-tuber filament of the brooding spider has an N-domain at its 5' end and a C-domain at its 3' end.

[0011] Of the aforementioned polynucleotides, preferably, the cysteine ​​residues are modified using the MaSp2 gene of the large ampullae gland filament of the brooding spider (Euprosthenops australis) as a template. The MaSp2 gene has the DNA sequence shown in SEQ ID NO: 1, consisting of 213 base pairs. The SEQ ID NO: 1 sequence is as follows:

[0012] 5'-CAAGGAGGATTTGGTCAAGGTGCTGGAGGTAATGCCGCAGCC GCT GCAGCAGCCGCCGCAGCA GC A GCAGCAGCTCAACAAGGTGGTCAAGGTGGTTTTGGAGGACAAGGTCAAGGAGGATTTGGACCTGGAGCAGGAAGTTCTGCAGCT GCA GCCGCTGCAGCA GCA GCAGCTGGTCAAGGTGGACAAGGAAGAGGAGGATTCGGTCAAGGT-3'

[0013] Preferably, among the aforementioned polynucleotides, the DNA sequence of the cysteine-modified MaSp2 gene from the brooding spider's large ampulla gland silk has the sequence shown in SEQ ID NO: 2. This modified DNA sequence is obtained by replacing a certain amount of cysteine ​​in the polyalanine DNA sequence of SEQ ID NO: 1, resulting in a total of 213 base pairs. The SEQ ID NO: 2 sequence is as follows:

[0014] 5'-CAAGGAGGATTTGGTCAAGGTGCTGGAGGTAATGCCGCAGCC TGT GCAGCAGCCGCCGCAGCA TG T GCAGCAGCTCAACAAGGTGGTCAAGGTGGTTTTGGAGGACAAGGTCAAGGAGGATTTGGACCTGGAGCAGGAAGTTCTGCAGCT TGT GCCGCTGCAGCA TGT GCAGCTGGTCAAGGTGGACAAGGAAGAGGAGGATTCGGTCAAGGT-3'

[0015] Preferably, in the aforementioned polynucleotides, the N-domain is selected from the MaSp1 gene of the brooding spider (Euprosthenopsaustralis), and the DNA sequence of the MaSp1 gene has the sequence shown in SEQ ID NO: 3, which contains 399 base pairs. The SEQ ID NO: 3 sequence is as follows:

[0016] 5'-TCACACACTACACCATGGACAAACCCAGGACTCGCAGAAAACTTCATGAACAGTTTCATGCAAGGCCTGAGCTCGATGCCAGGTTTCACGGCAAGCCAATTGGATGATATGTCAACCATCGCACAATCCATGGTACAGTCAATACAATCCTTGGCGGCACAAGGCAGGACATCACCGAATAAGCTGCAGGCCCTTAACA TGGCTTTTGCATCTTCGATGGCAGAAATCGCGGCATCCGAAGAAGGAGGGGGAAGCCTTTCCACCAAAACTAGCTCTATAGCCAGTGCAATGTCCAACGCGTTTCTGCAAACAACTGGAGTGGTAAAACCAACCGTTCATAAATGAAATAACTCAGCTCGTTAGCATGTTTGCTCAAGCAGGTATGAATGATGTCAGTGCT-3'

[0017] Preferably, in the above-mentioned polynucleotides, the C-domain is selected from the MiSp gene of the orb-weaver spider (Araneus ventricosus), and the DNA sequence of the MiSp gene has the sequence shown in SEQ ID NO: 4, which has a total of 360 base pairs. The sequence of SEQ ID NO: 4 is as follows:

[0018] 5'-GTTACATCTGGAGGTTACGGATATGGAACCAGTGCAGCTGCAGGAGCTGGAGTTGCAGCAGGTTCATATGCAGGTGCTGTCAATCGCTTGTCTAGTGCTGAAGCTGCCAGTAGAGTATCCTCTAATATTGCAGCTATTGCATCTGGTGGTGCTTCCGCCCTCCCCAGTGTTATTTCAAAT ATTTACTCAGGTGTCGTTGCTTCTGGTGTTTCTTCTAATGAAGCTTCTGATTCAAGCTCTGTTGGAACTCCTTTCCGCACTTGTTCATGTTTTAAGCAGTGCCTCTATCGGTAATGTTAGCTCAGTAGGAGTAGATAGTACATTGAATGTTGTTCAGGATTCAGTAGGCCAATATGTAGGT-3'

[0019] Preferably, the DNA sequence of the aforementioned 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. This polynucleotide (i.e., the target gene M.NT2RepCT) has 972 base pairs. The sequence in SEQ ID NO: 5 is as follows:

[0020] 5’-TCACACACTACACCATGGACAAACCCAGGACTCGCAGAAAACTTCATGAACAGTTTCATGCAAGGCCTGAGCTCGATGCCAGGTTTCACGGCAAGCCAATTGGATGATATGTCAACCATCGCACAATCCATGGTACAGTCAATACAATCCTTGGCGGCACAAGGCAGGACATCACCGAATAAGCTGCAGGCCCTTAACATGGCTTTTGCATCTTCGATGGCAGAAATCGCGGCATCCGAAGAAGGAGGGGGAAGCCTTTCCACCAAAACTAGCTCTATAGCCAGTGCAATGTCCAACGCGTTTCTGCAAACAACTGGAGTGGTAAACCAACCGTTCATAAATGAAATAACTCAGCTCGTTAGCATGTTTGCTCAAGCAGGTATGAATGATGTCAGTGCTCAAGGAGGATTTGGTCAAGGTGCTGGAGGTAATGCCGCAGCCTGTGCAGCAGCCGCCGCAGCATGTGCAGCAGCTCAACAAGGTGGTCAAGGTGGTTTTGGAGGACAAGGTCAAGGAGGATTTGGACCTGGAGCAGGAAGTTCTGCAGCTTGTGCCGCTGCAGCATGTGCAGCTGGTCAAGGTGGACAAGGAAGAGGAGGATTCGGTCAAGGTGTTACATCTGGAGGTTACGGATATGGAACCAGTGCAGCTGCAGGAGCTGGAGTTGCAGCAGGTTCATATGCAGGTGCTGTCAATCGCTTGTCTAGTGCTGAAGCTGCCAGTAGAGTATCCTCTAATATTGCAGCTATTGCATCTGGTGGTGCTTCCGCCCTCCCCAGTGTTATTTCAAATATTTACTCAGGTGTCGTTGCTTCTGGTGTTTCTTCTAATGAAGCTCTGATTCAAGCTCTGTTGGAACTCCTTTCCGCACTTGTTCATGTTTTAAGCAGTGCCTCTATCGGTAATGTTAGCTCAGTAGGAGTAGATAGTACATTGAATGTTGTTCAGGATTCAGTAGGCCAATATGTAGGT-3’

[0021] Thirdly, the present invention also provides a recombinant plasmid whose DNA sequence comprises the aforementioned polynucleotide sequence.

[0022] Fourthly, the present invention also provides a host bacterium that has been transformed with the above-mentioned recombinant plasmid.

[0023] Fifthly, the present invention also provides a method for preparing the above-mentioned recombinant spider silk protein, which includes the following steps:

[0024] The target gene is obtained by amplifying the above-mentioned polynucleotide DNA sequence using primers. The target gene is then inserted into a plasmid vector to obtain a recombinant plasmid. The recombinant plasmid or the above-mentioned recombinant plasmid is introduced into a host bacterium for expression to obtain recombinant spider silk protein.

[0025] In the above preparation method, preferably, the upstream primer DNA sequence has the sequence shown in SEQ ID NO: 7; and the downstream primer DNA sequence has the sequence shown in SEQ ID NO: 8.

[0026] The DNA sequence of the upstream primer (SEQ ID NO: 7) is as follows:

[0027] 5'-CGCGGATCCGCGATGTCACACACTACACCATGGACAAACC-3'

[0028] The DNA sequence of the downstream primer (SEQ ID NO: 8) is as follows:

[0029] 5'-CCCAAGCTTGGGACCTACATATTGGCCTACTGAATCCTGA-3'

[0030] In the above-described method for preparing recombinant spider silk protein, preferably, the plasmid vector includes pET-28a plasmid, but is not limited thereto.

[0031] In the above-described method for preparing recombinant spider silk protein, preferably, the host bacterium includes Escherichia coli, but is not limited thereto.

[0032] Sixthly, the present invention also provides a method for preparing recombinant spider silk, which includes the following steps:

[0033] The recombinant spider silk protein obtained by the above preparation method was purified, centrifuged, filtered and concentrated to obtain the recombinant spider silk spinning protein stock solution.

[0034] The recombinant spider silk spinning protein solution was extruded using a wet spinning machine. The extruded fibers were solidified in a coagulation bath, followed by hot steam annealing and post-stretching treatment, and then dried at room temperature to obtain recombinant spider silk.

[0035] In the above-mentioned method for preparing recombinant spider silk, preferably, the recombinant spider silk protein is purified using an eluent containing: 50-80 mM Tris-HCl, pH 7.5, 100-120 mM NaCl, 1-1.5 mM EDTA, and 1-2 mM DTT (dithiothreitol); the mass concentration of the purified recombinant spider silk protein is 2%-3%.

[0036] In the above-mentioned method for preparing recombinant spider silk, preferably, the recombinant spider silk protein solution is concentrated using a 10000MWCO (molecular weight cutoff) centrifugal filter, and the mass concentration of the recombinant spider silk spinning solution obtained is 20% to 25%.

[0037] In the above-mentioned method for preparing recombinant spider silk, preferably, the specification of the extrusion needle of the wet spinning machine is 30G (30G is the model of the spinning needle, and the specific specifications are an inner diameter of 0.16mm and an outer diameter of 0.19mm); the extrusion speed of the recombinant spider silk spinning protein stock solution is 10-30μL / min.

[0038] In the above-described method for preparing recombinant spider silk, preferably, the coagulation bath uses DMSO (dimethyl sulfoxide); the solidification time in the coagulation bath is 1-2 hours. The use of DMSO not only promotes protein coagulation to form fibers, but also provides a weak oxidizing environment for the protein to promote the formation of disulfide bonds between β-sheet layers.

[0039] In the above-mentioned method for preparing recombinant spider silk, preferably, the hot steam temperature for the hot steam annealing process is 80-120°C, and the annealing time is 30-90s.

[0040] In the above-mentioned method for preparing reconstituted spider silk, preferably, the stretching rate of the post-stretching process is 1-2 mm / s, and the stretching ratio is 0.5-0.8.

[0041] In a seventh aspect, the present invention also provides a reconstituted spider silk having a β-sheet crystal structure (with density); the diameter of its fibers is 50-60 μm, the average breaking stress of the fibers is 30-40 MPa, and the average breaking elongation is 10%-20%.

[0042] Preferably, the recombinant spider silk described above is prepared by wet spinning of the recombinant spider silk protein or by the recombinant spider silk preparation method described above.

[0043] The β-sheet crystals of reconstituted spider silk are typically formed by the stacking of multiple layers of polyalanine molecules, tightly bound together by hydrogen bonds. However, during artificial spinning simulations, defects in the crystal structure often arise due to low spinning solution concentration and the inability to fully simulate the spinning process, leading to a further reduction in filament strength. This invention inserts a specific amount of cysteine ​​at a specific position within the polyalanine sequence. This aims to stabilize the β-sheet crystals by forming disulfide bonds in the coagulation bath, while simultaneously, because cysteine ​​is a hydrophilic amino acid, it can inhibit the self-assembly behavior of the polyalanine segment in aqueous solution, thereby increasing the spinning solution concentration.

[0044] Eighthly, the present invention also provides the application of the above-mentioned recombinant spider silk in the fields of biomaterials, fiber materials or textile materials.

[0045] The beneficial effects of this invention are:

[0046] The recombinant spider silk protein gene of this invention can be efficiently expressed in Escherichia coli. The recombinant spider silk fibers obtained have a dense β-sheet crystal structure, excellent mechanical properties and wet strength. The fiber diameter is 50-60 μm, the average breaking stress of the fiber can reach 30-40 MPa, and the average breaking elongation is 10%-20%, which opens up prospects for the industrial application of recombinant spider silk. Attached Figure Description

[0047] Figure 1 This is a flowchart of the recombinant plasmid prepared in Example 1 of the present invention.

[0048] Figure 2 The image shows an electron microscope (EM) image of the surface and cross-section of the recombinant spider silk fiber prepared in Example 1 of this invention.

[0049] Figure 3 This is a graph showing the molecular dynamics simulation results of the recombinant spider silk prepared in Example 1 of this invention. Detailed Implementation

[0050] To provide a clearer understanding of the technical features, objectives, and beneficial effects of this invention, the technical solution of this invention is described in detail below, but this should not be construed as limiting the scope of implementation of this invention. Unless otherwise specified, the raw materials used in the following embodiments are all commercially available in the art; and unless otherwise specified, the methods used are conventional methods in the art.

[0051] Example 1:

[0052] This embodiment provides a method for preparing recombinant spider silk, which includes the following steps:

[0053] 1. Design of recombinant spider silk protein gene (the specific sequence design was outsourced to a gene synthesis company):

[0054] (1) Cysteine ​​modification was performed using the MaSp2 gene of the large ampullae gland filament of Eurosthenops australis as a template. The MaSp2 gene of the large ampullae gland filament of Eurosthenops australis has 213 base pairs and its DNA sequence is shown in SEQ ID NO: 1.

[0055] (2) A certain amount of cysteine ​​gene was replaced in the polyalanine DNA sequence of SEQ ID NO:1 to obtain the cysteine-modified MaSp2 gene of the burrowing spider's large ampulla gland silk, which has a total of 213 base pairs and its DNA sequence is shown in SEQ ID NO:2.

[0056] (3) Connect the N-domain to the 5' end and the C-domain to the 3' end of the SEQ ID NO: 2 sequence to obtain the recombinant spider silk protein gene.

[0057] The N-domain is selected from the MaSp1 gene of Eurosthenops australis, which contains 399 base pairs. Its DNA sequence is shown in SEQ ID NO: 3.

[0058] The C-domain is selected from the MiSp gene of Araneus ventricosus, and consists of 360 base pairs. Its DNA sequence is shown in SEQ ID NO: 4.

[0059] The recombinant spider silk protein gene (M.NT2RepCT) obtained at the end has 972 base pairs, and its DNA sequence is shown in SEQ ID NO: 5.

[0060] 2. Preparation of recombinant plasmids and recombinant spider silk proteins:

[0061] (1) The target gene was obtained by amplifying the sequence of SEQ ID NO: 5 using primers. 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℃. The digestion vector and target gene were purified by agarose gel electrophoresis, and their concentrations were determined by spectrophotometry. A certain amount of digestion vector and target gene were added to a microcentrifuge tube to make the DNA fragment ratio 3:1. 1 μL of BamHI buffer (10x), 1 μL of 10mM ATP, and 1 μL of DNA ligase were added, and sterile water was added to a final volume of 10 μL. After shaking the microcentrifuge tube, it was incubated at 20℃ for 4 h to obtain the recombinant plasmid containing the target gene. The upstream primer DNA sequence of the primer has the sequence shown in SEQ ID NO: 7; the downstream primer DNA sequence of the primer has the sequence shown in SEQ ID NO: 8; the flowchart for preparing the recombinant plasmid is shown below. Figure 1 As shown.

[0062] (2) The recombinant plasmid was introduced into *E. coli* for expression to obtain recombinant spider silk protein. Specifically:

[0063] Thaw 100 μL of Escherichia coli BL21(DE3) competent cells on ice for 10 min, add 2 μL of recombinant plasmid, incubate on ice for 30 min, then heat shock at 42℃ for 60 s, followed by incubation on ice for 2 min. Add 900 μL of antibiotic-free LB liquid medium preheated to 37℃ to the E. coli, and incubate at 37℃ with shaking at 180 rpm for 45 min. Spread 100 μL of the culture medium evenly on an LB agar plate with a concentration of 50 μg / mL kanamycin, and incubate overnight at 37℃ to obtain an E. coli strain containing the target gene.

[0064] 3. Preparation of reconstituted spider silk:

[0065] (1) The obtained recombinant spider silk protein was purified. The eluent used in the purification process contained 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1 mM EDTA and 1 mM DTT. The mass concentration of the obtained recombinant spider silk protein solution was 3%. Then, the protein solution was concentrated to 20 wt% using a 10000 MCO centrifugal filter to obtain a high-concentration recombinant spider silk spinning protein stock solution.

[0066] (2) The recombinant spider silk spinning protein solution was poured into the raw material tank of a wet spinning machine. The extrusion needle specification was 30G, the extrusion speed was 10μL / min, and the coagulation bath was DMSO. After extrusion, the recombinant spider silk fiber was cured in the coagulation bath for 1 hour. After curing, the fiber was taken out of the coagulation bath and annealed with hot steam at 80℃ for 30 seconds before entering the post-stretching step. The fiber was stretched at a stretching rate of 1mm / s and a stretching ratio of 0.5 times and then dried at room temperature to prepare recombinant spider silk.

[0067] The recombinant spider silk fibers prepared in this embodiment have an average diameter of 60 μm and a smooth surface (as shown in the image). Figure 2 As shown in the figure, the average breaking stress of the fiber reaches 30 MPa, and the average breaking elongation is 10%.

[0068] Molecular dynamics simulations were performed on the recombinant spider silk, and the simulation results are as follows: Figure 3 As shown. The yellow areas (i.e., the dark areas in the black and white image) represent the optimized β-sheet crystal portion; the gray areas (i.e., the light-colored areas in the black and white image at RH 0%) represent amorphous regions such as α-helix and random coils; and the cyan-green areas (i.e., the light-colored areas in the black and white image at RH 25%–100%) represent water molecules. Figure 3 It can be seen that under humid conditions, water molecules have limited intrusion into the amorphous region structure. As the humidity gradually increases, water molecules begin to intrude into the β-sheet crystal structure. However, due to the protection of interlayer disulfide bonds, the β-sheet structure can still maintain most of its integrity even under high humidity conditions of RH100%. sequence list <110> City University of Hong Kong <120> Cysteine-enhanced recombinant spider silk, its preparation method and application <130> GAI21CN4819 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 213 <212> DNA <213> Euprosthenops australis <220> <223> MaSp2 gene <400> 1 caaggaggat ttggtcaagg tgctggaggt aatgccgcag ccgctgcagc agccgccgca 60 gcagcagcag cagctcaaca aggtggtcaa ggtggttttg gaggacaagg tcaaggagga tttggacctg gagcaggag ttctgcagct gcagccgctg cagcagcagc agctggtcaa ggtggacaag gaagaggagg attcggtcaa ggt <210> 2 <211> 213 <212> DNA <213> Artificial Sequence <220> <223> Enjoy the high-quality MaSp2 insert <400> 2 caaggaggat ttggtcaagg tgctggaggt aatgccgcag cctgtgcagc agccgccgca gcatgtgcag cagctcaaca aggtggtcaa ggtggttttg gaggacaagg tcaaggagga tttggacctg gagcaggaag ttctgcagct tgtgccgctg cagcatgtgc agctggtcaa ggtggacaag gaagaggagg attcggtcaa ggt <210> 3 <211> 399 <212> DNA <213> Euprosthenops australis <220> <223> MaSp1 insert <400> 3 tcacacacta caccatggac aaacccagga ctcgcagaaa acttcatgaa cagtttcatg caaggcctga gctcgatgcc aggtttcacg gcaagccaat tggatgatat gtcaaccatc 120 gcacaatcca tggtacagtc aatacaatcc ttggcggcac aaggcaggac atcaccgaat 180 aagctgcagg cccttaacat ggcttttgca tcttcgatgg cagaaatcgc ggcatccgaa 240 gaaggagggg gaagcctttc caccaaaact agctctatag ccagtgcaat gtccaacgcg 300 tttctgcaaa caactggagt ggtaaaccaa ccgttcataa atgaaataac tcagctcgtt 360 agcatgtttg ctcaagcagg tatgaatgat gtcagtgct 399 <210> 4 <211> 360 <212> DNA <213> Araneus ventricosus <220> <223> MiSp gene <400> 4 gttacatctg gaggttacgg atatggaacc agtgcagctg caggagctgg agttgcagca 60 ggttcatatg caggtgctgt caatcgcttg tctagtgctg aagctgccag tagagtatcc 120 tctaatattg cagctattgc atctggtggt gcttccgccc tccccagtgt tatttcaaat 180 atttactcag gtgtcgttgc ttctggtgtt tcttctaatg aagctctgat tcaagctctg 240 ttggaactcc tttccgcact tgttcatgtt ttaagcagtg cctctatcgg taatgttagc 300 tcagtaggag tagatagtac attgaatgtt gttcaggatt cagtaggcca atatgtaggt 360 <210> 5 <211> 972 <212> DNA <213> Artificial Sequence <220> <223> M. NT2RepCT gene <220> <221> CDS <222> (1)..(972) <400> 5 tca cac act aca cca tgg aca aac cca gga ctc gca gaa aac ttc atg 48 Ser His Thr Thr Pro Trp Thr Asn Pro Gly Leu Ala Glu Asn Phe Met 1 5 10 15 aac agt ttc atg caa ggc ctg agc tcg atg cca ggt ttc acg gca agc 96 Asn Ser Phe Met Gln Gly Leu Ser Ser Met Pro Gly Phe Thr Ala Ser 20 25 30 caa ttg gat gat atg tca acc atc gca caa tcc atg gta cag tca ata 144 Gln Leu Asp Asp Met Ser Thr Ile Ala Gln Ser Met Val Gln Ser Ile 35 40 45 caa tcc ttg gcg gca caa ggc agg aca tca ccg aat aag ctg cag gcc 192 Gln Ser Leu Ala Ala Gln Gly Arg Thr Ser Pro Asn Lys Leu Gln Ala 50 55 60 ctt aac atg gct ttt gca tct tcg atg gca gaa atc gcg gca tcc gaa 240 Leu Asn Met Ala Phe Ala Ser Ser Met Ala Glu Ile Ala Ala Ser Glu 65 70 75 80 gaa gga ggg gga agc ctt tcc acc aaa act agc tct ata gcc agt gca 288 Glu Gly Gly Gly Ser Leu Ser Thr Lys Thr Ser Ser Ile Ala Ser Ala 85 90 95 atg tcc aac gcg ttt ctg caa aca act gga gtg gta aac caa ccg ttc 336 Met Ser Asn Ala Phe Leu Gln Thr Thr Gly Val Val Asn Gln Pro Phe 100 105 110 ata aat gaa ata act cag ctc gtt agc atg ttt gct caa gca ggt atg 384 Ile Asn Glu Ile Thr Gln Leu Val Ser Met Phe Ala Gln Ala Gly Met 115 120 125 aat gat gtc agt gct caa gga gga ttt ggt caa ggt gct gga ggt aat 432 Asn Asp Val Ser Ala Gln Gly Gly Phe Gly Gln Gly Ala Gly Gly Asn 130 135 140 gcc gca gcc tgt gca gca gcc gcc gca gca tgt gca gca gct caa caa 480 Ala Ala Ala Cys Ala Ala Ala Ala Ala Ala Cys Ala Ala Ala Gln Gln 145 150 155 160 ggt ggt caa ggt ggt ttt gga gga caa ggt caa gga gga ttt gga cct 528 Gly Gly Gln Gly Gly Phe Gly Gly Gln Gly Gln Gly Gly Phe Gly Pro 165 170 175 gga gca gga agt tct gca gct tgt gcc gct gca gca tgt gca gct ggt 576 Gly Ala Gly Ser Ser Ala Ala Cys Ala Ala Ala Ala Cys Ala Ala Gly 180 185 190 caa ggt gga caa gga aga gga gga ttc ggt caa ggt gtt aca tct gga 624 Gln Gly Gly Gln Gly Arg Gly Gly Phe Gly Gln Gly Val Thr Ser Gly 195 200 205 ggt tac gga tat gga acc agt gca gct gca gga gct gga gtt gca gca 672 Gly Tyr Gly Tyr Gly Thr Ser Ala Ala Ala Gly Ala Gly Val Ala Ala 210 215 220 ggt tca tat gca ggt gct gtc aat cgc tct agt gct gaa gct gcc 720 Gly Ser Tyr Ala Gly Ala Val Asn Arg Leu Ser Ser Glu Ala Ala Ala 225 230 235 240 agt aga gta tcc tct aat att gca gct att gca tct ggt ggt gct tcc Being Arg Will Be Being Asn Ile Wing Ile Wing Being Gly Gly Only Being 245 250 255 gcc ctc ccc agt gtt att tca aat att tac tca gtc gtt gct tct 816 Ala Leu Pro Ser Val Ile Ser Asn Ile Tyr Ser Gly Val Val Ala Ser 260 265 270 ggt gtt tct tct aat gaa gct ctg att caa gct ctg tg gaa ctc ctt 864 Gly Val Ser Ser Asn Horse Glu Gly Ile Gln Horse Glu Horse Glu 275 280 285 tcc gca ctt gtt cat gtt tta agc agt gcc tct atc ggt aat gtt agc 912 To Be Wing Leu Will His Will Be Leu To Be Wing To Be Ile Gly Asn Will Be 290,295,300 tca gta gga gta gat agt aca ttg aat gtt gtt cag gat tca gta ggc 960 Ser Val Gly Val Asp Ser Thr Leu Asn Val Val Gln Asp Ser Val Gly 305 310 315 320 caa tat gta ggt 972 Gln Tyr Val Gly <210> 6 <211> 324 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 Ser His Thr Thr Pro Trp Thr Asn Pro Gly Leu Ala Glu Asn Phe Met 1 5 10 15 Asn Ser Phe Met Gln Gly Leu Ser Ser Met Pro Gly Phe Thr Ala Ser 20 25 30 Gln Leu Asp Asp Met Ser Thr Ile Ala Gln Ser Met Val Gln Ser Ile 35 40 45 Gln Ser Leu Ala Ala Gln Gly Arg Thr Ser Pro Asn Lys Leu Gln Ala 50 55 60 Leu Asn Met Ala Phe Ala Ser Ser Met Ala Glu Ile Ala Ala Ser Glu 65 70 75 80 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 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 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 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 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 ggacctacat attggcctac tgaatcctga 40

Claims

1. A recombinant spider silk protein, the amino acid sequence of which is 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 having the following structure: The cysteine-modified MaSp2 gene of the bursa-tuber filament of the brooding spider has an N-domain at its 5' end and a C-domain at its 3' end.

3. The polynucleotide according to claim 2, wherein, The DNA sequence of the cysteine-modified MaSp2 gene of the bursa-tuber filament in the maternal web spider 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 brooding 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 Orb-weaver spider, 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 to 5, wherein, The DNA sequence of this polynucleotide has the sequence shown in SEQ ID NO:

5.

7. A recombinant plasmid whose DNA sequence comprises the sequence of the polynucleotide as described in any one of claims 2 to 6.

8. A host bacterium that has been transformed with the recombinant plasmid of claim 7.

9. The method for preparing the recombinant spider silk protein according to claim 1, comprising the following steps: The target gene is obtained by amplifying the DNA sequence of the polynucleotide as described in any one of claims 2 to 6 using primers. The target gene is then inserted into a plasmid vector to obtain a recombinant plasmid. The recombinant plasmid or the recombinant plasmid as described in claim 7 is introduced into a host bacterium for expression to obtain 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 downstream primer DNA sequence of the primer has the sequence shown in SEQ ID NO:

8.

11. The preparation method according to claim 9, wherein, The plasmid vector includes the pET-28a plasmid.

12. The preparation method according to claim 9, wherein, The host bacteria include Escherichia coli.

13. A method for preparing reconstituted spider silk, comprising the following steps: The recombinant spider silk protein prepared by the preparation method according to any one of claims 9 to 12 is purified, and then concentrated by centrifugation and filtration to obtain the recombinant spider silk spinning protein stock solution. The recombinant spider silk spinning protein solution was extruded using a wet spinning machine. The extruded fibers were solidified in a coagulation bath, followed by hot steam annealing and post-stretching treatment, and then dried at room temperature to obtain recombinant spider silk.

14. The preparation method according to claim 13, wherein, The recombinant spider silk protein was purified using an eluent containing: 50-80 mM Tris-HCl, pH 7.5, 100-120 mM NaCl, 1-1.5 mM EDTA, and 1-2 mM DTT; the mass concentration of the purified recombinant spider silk protein was 2%-3%.

15. The preparation method according to claim 13, wherein, The recombinant spider silk protein solution was concentrated using a 10000MWCO centrifugal filter, and the mass concentration of the recombinant spider silk spinning solution was 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 solution is 10~30μL / min.

17. The preparation method according to claim 13, wherein, The coagulation bath uses DMSO; the solidification time using the coagulation bath is 1~2 hours.

18. The preparation method according to claim 13, wherein, The hot steam temperature for the hot steam annealing process is 80~120℃, and the annealing time is 30~90s.

19. The preparation method according to claim 13, wherein, The stretching rate for the post-stretching process is 1~2 mm / s, and the stretching ratio is 0.5~0.

8.

20. A reconstituted spider silk having a β-sheet crystal structure; the fiber diameter is 50-60 μm, the average breaking stress of the fiber is 30-40 MPa, and the average elongation at break is 10%-20%; The recombinant spider silk is prepared by wet spinning of the recombinant spider silk protein according to claim 1 or by the preparation method according to any one of claims 13 to 19.

21. The application of the recombinant spider silk of claim 20 in the fields of biomaterials, fiber materials or textile materials.

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