CN115819541A

CN115819541A - Cysteine-reinforced recombinant spider silk and preparation method and application thereof

Info

Publication number: CN115819541A
Application number: CN202111085698.8A
Authority: CN
Inventors: 胡金莲; 张琦
Original assignee: City University of Hong Kong CityU
Current assignee: City University of Hong Kong CityU
Priority date: 2021-09-16
Filing date: 2021-09-16
Publication date: 2023-03-21

Abstract

The invention provides a cysteine-reinforced recombinant spider silk and a preparation method and application thereof. The amino acid sequence of the recombinant spider silk protein has the amino acid sequence of SEQ ID NO: 6. The polynucleotide for coding the protein amino acid sequence has the following structure: the 5 'end of the cysteine-modified major ampullate spidroin MaSp2 gene of the web spider is connected with an N-structural domain, and the 3' end is connected with a C-structural domain; the gene is inserted into a pET-28a plasmid vector to obtain a recombinant plasmid, and the recombinant plasmid is introduced into escherichia coli to be expressed to obtain the recombinant spider silk protein. The recombinant spider silk prepared by the recombinant spider silk protein through wet spinning has a compact beta-sheet crystal structure, excellent mechanical properties and wet strength, and develops prospects for industrial application of the recombinant spider silk.

Description

Cysteine-reinforced recombinant spider silk and preparation method and application thereof

Technical Field

The invention belongs to the field of biopolymer fiber materials, and particularly relates to cysteine-reinforced recombinant spider silk and a preparation method and application thereof.

Background

The spider dragline silk, namely the major ampullate gland silk, shows excellent mechanical properties such as high strength, high toughness and the like, and the mechanical properties of the spider dragline silk are even superior to those of certain current artificial high-performance fibers. Its excellent mechanical properties benefit from the delicate fine structure in the fiber, with the β -sheet crystals responsible for having a very high modulus of elasticity, providing the spider silk with very high tensile strength, while the amorphous α -helix structure is the main source of spider silk's high toughness and ductility. 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, so that spider silk materials become a research hotspot in the academic and industrial fields at present.

The method is limited by natural fighting of spiders, self-protection consciousness, regional aggressiveness and other natural properties, and the mass collection of spider silk by a large-scale culture mode like silk harvest cannot be realized. And the spider silk gland structure and function are very complicated, and the aggregation and self-assembly behaviors of silk protein molecular chains in the spinning process are not completely clear. Therefore, the expression of spider silk proteins (Whittall et al.2020) in bioreactors such as E.coli and yeast by genetic engineering methods and the production of high performance recombinant spider silk (Venkatesan, chen, and Hu 2019) by artificial spinning methods have become a research focus in recent years. Researchers hope to simulate the spinning solution performance and spinning environment in spider silk glands by methods of amino acid sequence optimization, bionic spinning and the like, explore the aggregation and self-assembly behaviors of protein molecular chains in the natural spinning process by researching the physicochemical properties of the spider silk protein molecular chains, and provide references for subsequent optimization and simulation. Oktaviani et al explored the kinetic behavior of Random Coil in spider dope and the formation process of beta-sheet structure (Oktaviani et al 2018); malay and Saric et al investigated the guiding effect of N-/C-domains on the self-assembly behavior of protein molecular chains in the context of intrafilarial spinning (Saric et al 2021; malay et al 2020).

In addition to The fields, in The textile field, the recombinant spider silk still has wide application prospects due to excellent performance, and The recombinant spider silk is used by existing companies and research teams to try to manufacture textiles, such as Biofabric running shoes of Germany original companies AMSilk and Adidas, moon park down jackets developed by The North Face company and Spiber company in a combined manner, however, the products and fabrics still Face The problems of low wet strength, poor durability, irreversible shrinkage when meeting water and The like, so that The product cost is high, the quality does not reach The standard and The market is delayed, and obviously, the problems seriously influence The wide application of The recombinant spider silk in The textile field.

Disclosure of Invention

Aiming at the problems of low wet strength, poor durability, irreversible shrinkage when meeting water and the like of the recombinant spider silk in the prior art, the invention aims to provide a recombinant spider silk protein; the second objective of the invention is to provide a polynucleotide for coding the protein amino acid sequence; the third objective of the invention is to provide a recombinant plasmid containing the polynucleotide DNA sequence; the fourth purpose of the invention is to provide a host bacterium into which the recombinant plasmid is transferred; the fifth object of the present invention is to provide a method for producing a recombinant spider silk protein; the sixth object of the present invention is to provide a method for producing a recombinant spider silk; the seventh object of the present invention is to provide a recombinant spider silk obtained by wet spinning a recombinant spider silk protein or a recombinant spider silk obtained by a method for producing a recombinant spider silk; an eighth object of the present invention is to provide the use of the recombinant spider silk in the field of biomaterials, in the field of high-performance fiber materials or in the field of textile materials. According to the invention, the gene encoding cysteine is used for transforming the gene MaSp2 of the major ampullate gland silk of cercospora Nucifera (Euprosthenops australis), so that firmer disulfide bonds are formed among protein molecular chains to optimize the crystal structure in the fiber, and further the mechanical property and the waterproof property of the fiber are enhanced; the recombinant spider silk protein gene can be efficiently expressed in escherichia coli, and the prepared recombinant spider silk fiber has uniform shape and excellent mechanical strength, thereby developing a prospect for the industrial application of the recombinant spider silk.

The purpose of the invention is realized 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 amino acid sequence of SEQ ID NO: 6. The nucleotide sequence of SEQ ID NO:6 the sequence is as follows:

SHTTPWTNPGLAENFMNSFMQGLSSMPGFTASQLDDMSTIAQSMVQSIQSLAAQGRTSPNKLQALNMAFASSMAEIAASEEGGGSLSTKTSSIASAMSNAFLQTTGVVNQPFINEITQLVSMFAQAGMNDVSAQGGFGQGAGGNAAACAAAAAACAAAQQGGQGGFGGQGQGGFGPGAGSSAACAAAACAAGQGGQGRGGFGQGVTSGGYGYGTSAAAGAGVAAGSYAGAVNRLSSAEAASRVSSNIAAIASGGASALPSVISNIYSGVVASGVSSNEALIQALLELLSALVHVLSSASIGNVSSVGVDSTLNVVQDSVGQYVG

in a second aspect, the present invention also provides a polynucleotide encoding a recombinant spider silk protein amino acid sequence, the polynucleotide having the structure:

the 5 'end of the cysteine-modified major ampullate spidroin MaSp2 gene of the ceroid spider is connected with an N-structural domain, and the 3' end is connected with a C-structural domain.

Among the above polynucleotides, cysteine modification is preferably performed using as a template a gene MaSp2 of a major ampullate gland silk of Nephilus arachnoides (Euprosthenops australis), the gene MaSp2 having the amino acid sequence shown in SEQ ID NO:1, having a total of 213 base pairs, and the sequence of SEQ ID NO:1 the sequence is as follows:

5’-CAAGGAGGATTTGGTCAAGGTGCTGGAGGTAATGCCGCAGCCGCTGCAGCAGCCGCCGCAGCAGC AGCAGCAGCTCAACAAGGTGGTCAAGGTGGTTTTGGAGGACAAGGTCAAGGAGGATTTGGACCTGGAGCAGGAAGTTCTGCAGCTGCAGCCGCTGCAGCAGCAGCAGCTGGTCAAGGTGGACAAGGAAGAGGAGGATTCGGTCAAGGT-3’

in the above-mentioned polynucleotides, preferably, the DNA sequence of the cysteine-modified major ampullate spidroin MaSp2 gene has the sequence of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. It is shown in SEQ ID NO:1 to substitute a certain amount of cysteine gene to obtain an improved DNA sequence with 213 base pairs, wherein the sequence shown in SEQ ID NO:2 the sequence is as follows:

5’-CAAGGAGGATTTGGTCAAGGTGCTGGAGGTAATGCCGCAGCCTGTGCAGCAGCCGCCGCAGCATG TGCAGCAGCTCAACAAGGTGGTCAAGGTGGTTTTGGAGGACAAGGTCAAGGAGGATTTGGACCTGGAGCAGGAAGTTCTGCAGCTTGTGCCGCTGCAGCATGTGCAGCTGGTCAAGGTGGACAAGGAAGAGGAGGATTCGGTCAAGGT-3’

in the above polynucleotide, preferably, the N-domain is selected from the group consisting of a mamm 1 gene of a web spider (Euprosthenops australis), the DNA sequence of the MaSp1 gene having the amino acid sequence of SEQ ID NO:3, having a total of 399 base pairs, the sequence of SEQ ID NO:3 the sequence is as follows:

5’-TCACACACTACACCATGGACAAACCCAGGACTCGCAGAAAACTTCATGAACAGTTTCATGCAAGGCCTGAGCTCGATGCCAGGTTTCACGGCAAGCCAATTGGATGATATGTCAACCATCGCACAATCCATGGTACAGTCAATACAATCCTTGGCGGCACAAGGCAGGACATCACCGAATAAGCTGCAGGCCCTTAACATGGCTTTTGCATCTTCGATGGCAGAAATCGCGGCATCCGAAGAAGGAGGGGGAAGCCTTTCCACCAAAACTAGCTCTATAGCCAGTGCAATGTCCAACGCGTTTCTGCAAACAACTGGAGTGGTAAACCAACCGTTCATAAATGAAATAACTCAGCTCGTTAGCATGTTTGCTCAAGCAGGTATGAATGATGTCAGTGCT-3’

in the above polynucleotide, preferably, the C-domain is selected from the group consisting of araneidae ventricosus (Araneus ventricosus) MiSp gene having a DNA sequence of SEQ ID NO:4, having a total of 360 base pairs, and the sequence of SEQ ID NO:4 the sequence is as follows:

5’-GTTACATCTGGAGGTTACGGATATGGAACCAGTGCAGCTGCAGGAGCTGGAGTTGCAGCAGGTTCATATGCAGGTGCTGTCAATCGCTTGTCTAGTGCTGAAGCTGCCAGTAGAGTATCCTCTAATATTGCAGCTATTGCATCTGGTGGTGCTTCCGCCCTCCCCAGTGTTATTTCAAATATTTACTCAGGTGTCGTTGCTTCTGGTGTTTCTTCTAATGAAGCTCTGATTCAAGCTCTGTTGGAACTCCTTTCCGCACTTGTTCATGTTTTAAGCAGTGCCTCTATCGGTAATGTTAGCTCAGTAGGAGTAGATAGTACATTGAATGTTGTTCAGGATTCAGTAGGCCAATATGTAGGT-3’

in the above-mentioned polynucleotide, preferably, the DNA sequence of the polynucleotide has the sequence of SEQ ID NO:5, i.e. a polynucleotide sequence encoding the amino acid sequence of the recombinant spider silk protein, which polynucleotide (i.e. the gene of interest m.nt2repct) has a total of 972 base pairs, said SEQ ID NO:5 the sequence is as follows:

5’-TCACACACTACACCATGGACAAACCCAGGACTCGCAGAAAACTTCATGAACAGTTTCATGCAAGGCCTGAGCTCGATGCCAGGTTTCACGGCAAGCCAATTGGATGATATGTCAACCATCGCACAATCCATGGTACAGTCAATACAATCCTTGGCGGCACAAGGCAGGACATCACCGAATAAGCTGCAGGCCCTTAACATGGCTTTTGCATCTTCGATGGCAGAAATCGCGGCATCCGAAGAAGGAGGGGGAAGCCTTTCCACCAAAACTAGCTCTATAGCCAGTGCAATGTCCAACGCGTTTCTGCAAACAACTGGAGTGGTAAACCAACCGTTCATAAATGAAATAACTCAGCTCGTTAGCATGTTTGCTCAAGCAGGTATGAATGATGTCAGTGCTCAAGGAGGATTTGGTCAAGGTGCTGGAGGTAATGCCGCAGCCTGTGCAGCAGCCGCCGCAGCATGTGCAGCAGCTCAACAAGGTGGTCAAGGTGGTTTTGGAGGACAAGGTCAAGGAGGATTTGGACCTGGAGCAGGAAGTTCTGCAGCTTGTGCCGCTGCAGCATGTGCAGCTGGTCAAGGTGGACAAGGAAGAGGAGGATTCGGTCAAGGTGTTACATCTGGAGGTTACGGATATGGAACCAGTGCAGCTGCAGGAGCTGGAGTTGCAGCAGGTTCATATGCAGGTGCTGTCAATCGCTTGTCTAGTGCTGAAGCTGCCAGTAGAGTATCCTCTAATATTGCAGCTATTGCATCTGGTGGTGCTTCCGCCCTCCCCAGTGTTATTTCAAATATTTACTCAGGTGTCGTTGCTTCTGGTGTTTCTTCTAATGAAGCTCTGATTCAAGCTCTGTTGGAACTCCTTTCCGCACTTGTTCATGTTTTAAGCAGTGCCTCTATCGGTAATGTTAGCTCAGTAGGAGTAGATAGTACATTGAATGTTGTTCAGGATTCAGTAGGCCAATATGTAGGT-3’

in a third aspect, the present invention also provides a recombinant plasmid, the DNA sequence of which comprises the sequence of the polynucleotide.

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

In a fifth aspect, the present invention also provides a method for the preparation of the recombinant spider silk protein described above, comprising the steps of:

and amplifying the DNA sequence of the polynucleotide by using a primer to obtain a target gene, inserting the target gene into a plasmid vector to obtain a recombinant plasmid, and introducing the recombinant plasmid or the recombinant plasmid into host bacteria for expression to obtain the recombinant spider silk protein.

In the above preparation method, preferably, 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, or a sequence shown in figure 8.

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

5’-CGCGGATCCGCGATGTCACACACTACACCATGGACAAACC-3’

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

5’-CCCAAGCTTGGGACCTACATATTGGCCTACTGAATCCTGA-3’

in the above method for producing a recombinant spider silk protein, the plasmid vector preferably comprises the pET-28a plasmid, but is not limited thereto.

In the above method for producing a recombinant spider silk protein, the host bacteria preferably include, but are not limited to, escherichia coli.

In a sixth aspect, the present invention also provides a method for the preparation of recombinant spider silk comprising the steps of:

purifying the recombinant spider silk protein prepared by the preparation method, centrifuging, filtering and concentrating to obtain a recombinant spider silk spinning protein stock solution;

and extruding the recombinant spider silk spinning protein stock solution by using a wet spinning machine, solidifying the extruded fibers in a coagulating bath, then carrying out hot steam annealing and post-stretching treatment, and drying at room temperature to obtain the recombinant spider silk.

In the above method for producing recombinant spider silk, the recombinant spider silk protein is preferably purified using an eluent containing, as components: 50-80 mM Tris-HCl, pH7.5, 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%.

In the above method for preparing recombinant spider silk, preferably, a 10000MWCO (cut-off molecular weight) centrifugal filter is used to concentrate the recombinant spider silk protein solution, and the mass concentration of the obtained recombinant spider silk spinning solution is 20-25%.

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

In the above method for producing recombinant spider silk, DMSO (dimethyl sulfoxide) is preferably used for the coagulation bath; the time for curing by adopting a coagulating bath is 1-2 h. The DMSO not only can promote protein coagulation to form fibers, but also can provide a weak oxidation environment for proteins so as to promote the formation of disulfide bonds between beta-sheets.

In the above method for producing recombinant spider silk, the hot steam temperature for the hot steam annealing step is preferably 80 to 120 ℃ and the annealing time is preferably 30 to 90 seconds.

In the above-mentioned method for producing a recombinant spider silk, the post-stretching step is preferably carried out at a stretching rate of 1 to 2mm/s and a stretching ratio of 0.5 to 0.8.

In a seventh aspect, the present invention also provides a recombinant spider silk having a β -sheet crystal structure (having compactability); the diameter of the fiber is 50-60 μm, the average breaking stress of the fiber is 30-40 MPa, and the average breaking elongation is 10-20%.

In the above-mentioned recombinant spider silk, the recombinant spider silk is preferably obtained by wet spinning the above-mentioned recombinant spider silk protein or by the above-mentioned method for producing recombinant spider silk.

The beta-sheet crystal of the recombinant spider silk is generally formed by overlapping multiple layers of polyalanine molecules, and the layers are tightly combined through hydrogen bonds. However, in the process of manually simulating spinning, the crystal structure is defective due to the reasons that the concentration of the spinning solution is low, the spinning process cannot be completely simulated, and the like, so that the spinning strength is further reduced. The invention inserts a certain amount of cysteine into a specific position in a polyalanine sequence, aims to form anchoring effect through a disulfide bond formed in a coagulating bath so as to stabilize beta-sheet crystals, and simultaneously can inhibit the self-assembly behavior of polyalanine segments in an aqueous solution so as to improve the concentration of a spinning solution because the cysteine is hydrophilic amino acid.

In an eighth aspect, the invention also provides the use of the recombinant spider silk described above in the field of biomaterials, in the field of fiber materials or in the field of textile materials.

The invention has the beneficial effects that:

the recombinant spider silk protein gene can be efficiently expressed in escherichia coli, the prepared recombinant spider silk fiber has a compact beta-sheet crystal structure and excellent mechanical property and wet strength, the diameter of the fiber is 50-60 mu m, the average breaking stress of the fiber can reach 30-40 MPa, and the average breaking elongation is 10-20%, so that the recombinant spider silk protein gene develops prospects for industrial application of the recombinant spider silk.

Drawings

FIG. 1 is a flow chart of a recombinant plasmid prepared in example 1 of the present invention.

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

FIG. 3 is a graph showing the results of the kinetic simulation of the recombinant spider silk molecules prepared in example 1 of the present invention.

Detailed Description

The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention should not be construed as limiting the implementable scope of the present invention. The raw materials used in the following examples are all commercially available in the conventional art unless otherwise specified; the method is conventional in the art unless otherwise specified.

Example 1:

this example provides a method for producing recombinant spider silk, comprising the steps of:

1. design of recombinant spider silk protein genes (specifically designed sequences entrusted to gene synthesis company):

(1) Cysteine modification was performed using as a template the major ampullate silk MaSp2 gene of the Nephila (Euprosthenops australis) having a total of 213 base pairs and a DNA sequence as shown in SEQ ID NO:1 is shown.

(2) In SEQ ID NO:1 to obtain cysteine-modified dragons major ampullate spidroin MaSp2 gene with 213 base pairs, wherein the DNA sequence of the gene is shown as SEQ ID NO:2, respectively.

(3) In said SEQ ID NO: the 5 'end of the 2 sequence is connected with the N-structural domain, and the 3' end is connected with the C-structural domain to obtain the recombinant spider silk protein gene.

The N-domain is selected from the MaSp1 gene of Nephilus argentea (Euprosthenops australis), with 399 base pairs in total, and the DNA sequence thereof is shown in SEQ ID NO:3, respectively.

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

The finally obtained recombinant spider silk protein gene (M.NT2RePCT) has 972 base pairs in total, and the DNA sequence of the gene is shown as SEQ ID NO:5, respectively.

2. Preparing recombinant plasmid and recombinant spider silk protein:

(1) Converting SEQ ID NO:5 the sequence was amplified by primers to obtain the desired gene, and the plasmid vector pET-28a was digested with HindIII and BamHI restriction enzymes to prepare a digestion vector, which was BamHI buffer at a digestion temperature of 37 ℃. The digestion vector and the gene of interest were purified by agarose gel electrophoresis, and the concentration thereof was determined using a spectrophotometer. A certain amount of the digestion vector and the target gene were added to a microcentrifuge tube so that the ratio of the amount of DNA fragments was 3. And (3) after shaking the centrifugal tube, placing the micro centrifugal tube at 20 ℃ and incubating for 4h 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 DNA sequence of the downstream primer of the primer has the sequence shown in SEQ ID NO: 8; the flow chart for preparing the recombinant plasmid is shown in FIG. 1.

(2) And introducing the recombinant plasmid into escherichia coli for expression to obtain the recombinant spider silk protein. The method specifically comprises the following steps:

100 μ L of Escherichia coli BL21 (DE 3) competent cells were thawed on ice for 10min, 2 μ L of recombinant plasmid was added, ice-bath was carried out for 30min, heat shock was carried out at 42 ℃ for 60s, and ice-bath was carried out for 2min. Adding 900 μ L of LB liquid medium without antibiotics preheated at 37 deg.C into Escherichia coli, shake culturing at 180rpm and 37 deg.C for 45min on a constant temperature shaking table; 100. Mu.L of the culture broth was uniformly spread on LB agar plate with a concentration of 50. Mu.g/mL kanamycin, and cultured overnight at 37 ℃ to obtain an E.coli strain containing the desired gene.

3. Preparation of recombinant spider silk:

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

(2) Pouring the recombinant spider silk spinning protein stock solution into a raw material tank of a wet spinning machine, wherein the specification of an extrusion needle is 30G, the extrusion speed is 10 mu L/min, and DMSO is adopted in a coagulating bath; and (3) extruding the recombinant spider silk fiber, curing for 1 hour in a coagulating bath, taking the fiber out of the coagulating bath after curing, carrying out annealing treatment for 30 seconds at 80 ℃ by hot steam, then carrying out post-stretching, stretching the fiber at a stretching rate of 1mm/s and a stretching ratio of 0.5 time, and drying at room temperature to prepare the recombinant spider silk.

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

The recombinant spider silk was subjected to molecular dynamics simulation, and the simulation result is shown in FIG. 3. The yellow part (i.e. the dark part in the black-white picture) is the optimized beta-sheet crystal part, the gray part (i.e. the light part in the RH 0% of the black-white picture) is an amorphous area structure such as alpha-helix, random coil and the like, and the turquoise part (i.e. the light part in the RH 25% -100% of the black-white picture) is water molecules. As can be seen from fig. 3, in a humidity environment, water molecules invade the amorphous region structure in a limited manner, and with increasing humidity, the water molecules start to invade the β -sheet crystal structure, but due to the protection of the inter-layer disulfide bonds, the β -sheet structure can remain largely intact even in a high humidity environment of 100% RH.

Sequence listing

<110> hong Kong City university

<120> cysteine-reinforced recombinant spider silk and preparation method and application thereof

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<170> PatentIn version 3.5

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<213> Euprosthenops australis

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<223> MaSp2 Gene

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Ser His Thr Thr Pro Trp Thr Asn Pro Gly Leu Ala Glu Asn Phe Met

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

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 has the amino acid sequence of SEQ ID NO: 6.

2. A polynucleotide encoding the recombinant spider silk protein amino acid sequence of claim 1, the polynucleotide having the structure:

3. The polynucleotide of claim 2, wherein the DNA sequence of the cysteine-modified major ampullate spidroin MaSp2 gene has the sequence of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

4. The polynucleotide of claim 2, wherein the N-domain is selected from the group consisting of a mamma nursery spider MaSp1 gene, the DNA sequence of which MaSp1 gene has the sequence of SEQ ID NO:3, and (b) is the sequence shown in the specification.

5. The polynucleotide of claim 2, wherein the C-domain is selected from the group consisting of an isospica grandis gene whose DNA sequence has the sequence of SEQ ID NO:4, or a sequence shown in the figure.

6. The polynucleotide according to any one of claims 2 to 5, wherein the DNA sequence of the polynucleotide has the sequence shown in SEQ ID NO:5, and (c) a sequence shown in (5).

7. A recombinant plasmid having a DNA sequence comprising the sequence of the polynucleotide of any one of claims 2 to 6.

8. A host bacterium into which the recombinant plasmid according to claim 7 has been transferred.

9. A method of producing a recombinant spider silk protein according to claim 1, comprising the steps of:

a target gene is obtained by amplifying the DNA sequence of the polynucleotide according to any one of claims 2 to 6 with a primer, the target gene is inserted into a plasmid vector to obtain a recombinant plasmid, and the recombinant plasmid or the recombinant plasmid according to claim 7 is introduced into a host bacterium and expressed to obtain a recombinant spider silk protein.

10. The method of claim 9, wherein the upstream primer DNA sequence of the primer has the sequence set forth in SEQ ID NO: 7; the DNA sequence of the downstream primer of the primer has the sequence shown in SEQ ID NO:8, or a sequence shown in figure 8.

11. The method of claim 9, wherein the plasmid vector comprises a pET-28a plasmid.

12. The method according to claim 9, wherein the host bacterium comprises Escherichia coli.

13. A method for the preparation of recombinant spider silk comprising the steps of:

purifying the recombinant spider silk protein prepared by the preparation method of any one of claims 9 to 12, centrifuging, filtering and concentrating to obtain a recombinant spider silk spinning protein stock solution;

14. The production method according to claim 13, wherein the recombinant spider silk protein is purified using an eluent comprising the following components: 50-80 mM Tris-HCl, pH7.5, 100-120 mM NaCl, 1-1.5 mM EDTA and 1-2 mM DTT; the mass concentration of the purified recombinant spider silk protein is 2-3%.

15. The preparation method of claim 13, wherein the mass concentration of the recombinant spider silk spinning solution obtained by concentrating the recombinant spider silk protein solution by a 10000MWCO centrifugal filter is 20-25%.

16. The manufacturing method according to claim 13, wherein the extrusion needle of the wet spinning machine has a gauge of 30G; the extrusion speed of the recombinant spider silk spinning protein stock solution is 10-30 mu L/min.

17. The production method according to claim 13, wherein the coagulation bath employs DMSO; the time for curing by adopting a coagulating bath is 1-2 h.

18. The method according to claim 13, wherein the hot steam annealing is performed at a hot steam temperature of 80 to 120 ℃ for an annealing time of 30 to 90 seconds.

19. The production method according to claim 13, wherein the post-stretching treatment step is performed at a stretching rate of 1 to 2mm/s and a stretching ratio of 0.5 to 0.8.

20. A recombinant spider silk having a β -sheet crystal structure; the diameter of the 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 of claim 20, wherein said recombinant spider silk is prepared from the recombinant spider silk protein of claim 1 by wet spinning or by the preparation method of any one of claims 13 to 19.

22. Use of the recombinant spider silk of claim 20 or 21 in the field of biomaterials, in the field of fiber materials or in the field of textile materials.