CN116425849B

CN116425849B - Recombinant spider silk protein, recombinant spider silk protein mixed fiber, and preparation method and application thereof

Info

Publication number: CN116425849B
Application number: CN202310379098.5A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Beijing Xincheng Zhongke Technology Co ltd
Current assignee: Suzhou Kangchengyuan Technology Partnership Enterprise LP
Priority date: 2023-04-11
Filing date: 2023-04-11
Publication date: 2024-02-06
Anticipated expiration: 2043-04-11
Also published as: CN116425849A

Abstract

The invention relates to a recombinant spider silk protein, a recombinant spider silk protein mixed fiber, a preparation method and application thereof, and relates to the field of proteins, wherein the recombinant spider silk protein mixed fiber comprises at least two recombinant proteins: the invention designs two recombinant spider silk fusion proteins from the bionic point of view by utilizing a genetic engineering technology, and provides a novel method for developing recombinant spider silk protein fibers by utilizing the fusion of a natural spider dragline silk partial sequence and hydrophilic elastin.

Description

Recombinant spider silk protein, recombinant spider silk protein mixed fiber, and preparation method and application thereof

Technical Field

The invention relates to the field of proteins, in particular to recombinant spider silk protein, a recombinant spider silk protein mixed fiber, a preparation method of the recombinant spider silk protein mixed fiber, a biological material and application.

Background

Today, humans are facing the crisis of the exhaustion of non-sustainable resources, and natural polymers are receiving increasing attention due to their abundant sources and sustainability. Spider proteins have received particular attention as a structural protein in many natural polymeric materials in nature, and adult spiders secrete at least 7 protein filaments, of which dragline filaments have been extensively studied for their excellent comprehensive mechanical properties.

Spider dragline silk is produced by cylindrical epithelial cells of the major ampullate gland of the spider, and is a protein fiber with a multi-stage structure formed by at least two protein components (MaSp 1 and MaSp 2) as the core. Most spider dragline silk proteins comprise a central large repetitive core domain flanked by small non-repetitive end domains. The number of repeating core domains and the different types of repeating segments play a decisive role in the material properties of the spider silk. The two end domains play an important role in participating in the storage of spider silk proteins in glands and in the self-assembly process of stretch-forming silk. Depending on the molecular structure of the spider proteins MaSp1 and MaSp2, both spider proteins are believed to contribute in different ways to the mechanical properties of the dragline silk. Among them MaSp1 is considered to be the main source of spider dragline silk stiffness properties, while MaSp2 is considered to contribute significantly to its elastic properties. The spider can obtain fibers with different mechanical properties by mixing the two proteins in different proportions so as to realize different physiological functions.

Because spider silk production is small and difficult to feed on a large scale, artificial spider silk fibers are prepared mainly by expressing recombinant spider silk proteins and performing artificial spinning. However, the mechanical properties of the spider silk fibers synthesized in vitro by the current artificial method are still not comparable with those of natural spider silk. One of the main reasons is that most recombinant spider silk proteins only modify a single MaSp1 or MaSp2 gene sequence, so that little research is done on the formation of heterodimers of two proteins in vivo or blending in vitro, and on the other hand, many recombinant spider silk proteins lack terminal domains during heterologous synthesis, which leads to the difficulty of cross self-assembly of the two proteins and influences fiber properties.

Disclosure of Invention

Technical problem

In view of the above, the technical problem to be solved by the present invention is how to provide a recombinant spider silk protein, a recombinant spider silk protein mixed fiber, a preparation method of the recombinant spider silk protein mixed fiber, a biological material and an application.

The invention develops two proteins by simulating the natural state of the spider, mixes the two proteins in vitro according to the proportion of the natural proteins and fuses and expresses the two proteins in vivo to form fibers, thus having great significance for developing novel assemblies of spider silk proteins and improving the performance of artificial spider silk fibers.

Solution scheme

In order to solve the technical problems, the invention provides the following technical scheme:

in a first aspect, the present invention provides a recombinant spider silk protein comprising at least two recombinant proteins: SP-1-K fusion proteins and SP-2-K fusion proteins;

the SP-1-K fusion protein is any one of the following A1) to A4) proteins:

a1 The amino acid sequence of which comprises: m directly tandem expression forms are shown as [ S1-ELP1] amino acid sequence units, wherein S1 represents an amino acid sequence shown as SEQ ID NO. 1, ELP1 is an elastin-like sequence, and [ S1-ELP1] represents that the S1 sequence and the ELP1 sequence are in tandem; m represents the number of repetitions of the [ S1-ELP1] sequence, m being an integer between 1 and 36;

wherein, the amino acid sequence shown in SEQ ID NO. 1 is as follows:

GPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPGQQGPGQQ

a2 The amino acid sequence of which comprises: a connecting sequence is connected between adjacent amino acid sequence units [ S1-ELP1] in the A1), optionally, the connecting sequence comprises two or four amino acids, and optionally, the connecting sequence is two or four amino acids coded after enzyme cutting at different enzyme cutting sites with the same tail and then connecting; alternatively, the linking sequence is two amino acids; alternatively, the linking sequence may be TS (which is a tail of the same-tail cleavage site SpeI (A/CTAGT) and a head of the same-tail cleavage site NheI (G/CTAGC), and the linking sequence may be generated when the repeating units are linked in the cleavage site manner because of a certain difficulty in direct synthesis of the multiple repeating units, and the cleavage site may be selected according to the requirement, that is, TS may be replaced by other sequences generated by replacing the cleavage site, and generally two or four amino acids may be used;

a3 The amino acid sequence of which comprises: a1 A protein which is obtained by substituting and/or deleting and/or adding one, two or more amino acid residues in the amino acid sequence shown in A2) and has the same or similar properties with the protein shown in A1);

a4 The amino acid sequence of which comprises: introducing a natural spider silk amino domain at the N-terminus of A1) or A2) or A3), and/or introducing a natural spider silk carboxyl domain at the C-terminus of A1) or A2) or A3);

a5 The amino acid sequence of which comprises: introducing a histidine tag at the N-terminal and/or C-terminal of A1) or A2) or A3) or A4);

the SP-2-K fusion protein is any one of the following B1) to B4):

b1 The amino acid sequence of which comprises: n directly tandem expression forms are shown as [ S2-ELP2], wherein S2 represents an amino acid sequence shown as SEQ ID NO. 2; ELP2 is an elastin-like sequence; [ S2-ELP2] represents a tandem of the S2 sequence and the ELP2 sequence; n represents the number of repetitions of the [ S2-ELP2] sequence, n being an integer between 1 and 36;

wherein, the amino acid sequence shown in SEQ ID NO. 2 is as follows:

GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP

b2 The amino acid sequence of which comprises: b1 A connecting sequence is arranged between adjacent amino acid sequence units [ S2-ELP2], and the connecting sequence is two or four amino acids coded after enzyme digestion and then connection of different enzyme digestion sites with the same tail;

b3 The amino acid sequence of which comprises: b1 A protein which is obtained by substituting and/or deleting and/or adding one, two or more amino acid residues in the amino acid sequence shown in the B2) and has the same or similar properties with the protein shown in the A1);

b4 The amino acid sequence of which comprises: b1 The N-terminal of B2) or B3) and/or the C-terminal of B1) or B2) or B3) of a natural spider silk amino domain;

b5 A histidine tag is introduced at the N-and/or C-terminus of B1) or B2) or B3) or B4).

Further, in the SP-1-K fusion protein:

m is an integer between 8 and 24, alternatively an integer between 8 and 16, alternatively an integer between 10 and 14, alternatively 12;

the amino acid sequence of ELP1 comprises several sequences as shown in SEQ ID NO. 3: the expression pattern of the amino acid sequence obtained by concatenating the amino acid sequences shown in VPGXG is (VPGXG) _i Wherein X is lysine or arginine or other amino acid, optionally X is lysine; i is the number of repeats of the sequence SEQ ID NO. 3, i is an integer between 1 and 40, alternatively i is an integer between 3 and 10, alternatively i is an integer between 3 and 8, alternatively i is an integer between 4 and 6, alternatively i is 5.

The amino acid sequence of SEQ ID NO. 3 is: VPGXG, X is lysine or arginine or other amino acid, optionally X is lysine;

alternatively, the amino acid sequence of the SP-1-K fusion protein is expressed in the form of

[GPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPGQQGPGQQ-(VPGKG) _i ] _m Wherein m represents [ S1-ELP1]]Sequence (GPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPGQQGPGQQ- (VPGKG) _i ) M is an integer between 1 and 36, alternatively an integer between 8 and 16, alternatively an integer between 10 and 14, alternatively 12; i is an integer between 3 and 10, alternatively i is an integer between 3 and 8, alternatively i is an integer between 4 and 6, alternatively i is 5.

Alternatively, the amino acid sequence of the amino acid sequence unit [ S1-ELP1] is shown in SEQ ID NO. 4, and the sequence is as follows:

GPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPGQQGPGQQVPGKGVPG KGVPGKGVPGKGVPGKG

further, in the SP-2-K fusion protein:

n is an integer between 8 and 24, alternatively an integer between 8 and 16, alternatively an integer between 10 and 14, alternatively 12;

the amino acid sequence of ELP2 comprises several sequences as shown in SEQ ID NO. 3: the expression pattern of the amino acid sequence obtained by concatenating the amino acid sequences shown in VPGXG is (VPGXG) _j Wherein X is lysine or arginine or other amino acid, optionally X is lysine; j is the number of repetitions of the SEQ ID NO 3 sequence, j is an integer between 1 and 40, alternatively j is an integer between 3 and 10, alternatively j is an integer between 3 and 8, alternatively j is an integer between 4 and 6, alternatively j is 5.

Alternatively, the sequence of the SP-2-K fusion protein may be expressed in the form of

[GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP(VPGKG) _j ] _n Wherein n represents [ S2-ELP2]]Sequence (GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (VPGKG)) _j ) N is an integer between 1 and 36, optionally an integer between 8 and 16, optionally an integer between 10 and 14, optionally 12; j is an integer between 3 and 10, alternatively j is an integer between 3 and 8, alternatively j is an integer between 4 and 6, alternatively j is 5.

Alternatively, the amino acid sequence [ S2-ELP2] of the amino acid sequence unit is shown in SEQ ID NO. 5, and the sequence is as follows:

GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGPVPGKGVPGKGVPGKGVP GKGVPGKG

further, in the A4) protein of the SP-1-K fusion protein and/or the B4) protein of the SP-2-K fusion protein, the natural spider silk amino domain comprises an amino acid sequence as shown in SEQ ID NO. 6.

The amino acid sequence shown in SEQ ID NO. 6 is as follows:

GQANTPWSSKANADAFINSFISAASNTGSFSQDQMEDMSLIGNTLMAAMDNMGGRITPSKLQALDMAFASSVAEIAASEGGDLGVTTNAIADALTSAFYQTTGVVNSRFISEIRSLIGMFAQASANDVYASAGSGSGGGGYGASSASAASASAAAPSGVAYQAPAQAQISFTLRGQQPVS

further, in A4) of the SP-1-K fusion protein and/or B4) of the SP-2-K fusion protein, the carboxy domain of the natural spider silk comprises the amino acid sequence as shown in SEQ ID NO. 7.

The amino acid sequence shown in SEQ ID NO. 7 is as follows:

GAASAAVSVGGYGPQSSSAPVASAAASRLSSPAASSRVSSAVSSLVSSGPTNQAALSNTISSVVSQVSASNPGLSGCDVLVQALLEVVSALVSILGSSSIGQINYGASAQYTQMVGQSVAQALAG

alternatively, the sequence of the SP-1-K fusion protein is shown in SEQ ID NO. 8 or 9.

The sequence of SEQ ID NO 8 (1174 amino acids, which contains an amino domain and a carboxyl domain) is as follows:

the sequence shown in SEQ ID NO. 9 is the underlined tag moiety (982 amino acids) of the sequence shown in SEQ ID NO. 8, which contains the carboxy domain.

Alternatively, the sequence of the SP-2-K fusion protein is shown in SEQ ID NO. 10 or 11.

The sequence of SEQ ID NO 10 (1066 amino acids, containing an amino domain and a carboxyl domain) is as follows:

the sequence shown in SEQ ID NO. 11 is the underlined tag moiety (874 amino acids) of the sequence shown in SEQ ID NO. 10, which contains the carboxy domain.

Further, the weight ratio of the SP-1-K fusion protein to the SP-2-K fusion protein is (0.2-3): 1, optionally (0.6-2): 1, optionally (0.6-1.5): 1, alternatively 1.5:1.

further, the SP-1-K fusion protein and the SP-2-K fusion protein are obtained by in vitro blending or in vivo coexpression of microorganisms.

In a second aspect, there is provided a recombinant spidroin protein mixed fiber comprising the recombinant spidroin protein of the first aspect, which is spun artificially;

optionally, the weight ratio of the SP-1-K fusion protein to the SP-2-K fusion protein is (0.2-3): 1, optionally (0.6-2): 1, optionally (0.6-1.5): 1, alternatively 1.5:1.

in a third aspect, there is provided a method for preparing the recombinant spidroin mixed fiber according to the second aspect, comprising:

and (3) dissolving the SP-1-K fusion protein and the SP-2-K fusion protein in a solvent to prepare a protein solution, extruding the protein solution into a coagulating bath by using a syringe pump, and solidifying the protein solution into nascent fibers.

Further, the solvent is hexafluoroisopropanol or formic acid.

Further, the SP-1-K fusion protein and the SP-2-K fusion protein are mixed according to the weight ratio (0.2-3): 1 in hexafluoroisopropanol to prepare a protein solution, optionally, the mass fraction of the SP-1-K fusion protein and the SP-2-K fusion protein in the protein solution is 150-200 mg/mL, optionally, 150mg/mL, optionally, the SP-1-K fusion protein and the SP-2-K fusion protein are respectively obtained by recombinant escherichia coli expression and then purification.

Further, the SP-1-K fusion protein and the SP-2-K fusion protein are obtained by purification after co-expression in recombinant E.coli, optionally in formic acid solution.

Further, the coagulation bath is 80-100% methanol solution by volume, alternatively 90% methanol solution.

Further, post-stretching of the as-spun fibers is also included: and (3) soaking the nascent fiber in a stretching bath to soften, and stretching the nascent fiber to 2-5 times of the original length to obtain the post-stretched fiber. Alternatively, the stretching bath is a 0-80% methanol solution, alternatively a 50% methanol solution, by volume.

In a fourth aspect, there is provided a biological material associated with the recombinant spidroin according to the first aspect, or the recombinant spidroin mixed fiber according to the second aspect, or the recombinant spidroin mixed fiber prepared by the preparation method according to the third aspect, the biological material being any one of C1) to C4):

c1 Nucleic acid molecules encoding the SP-1-K fusion protein and/or the SP-2-K fusion protein of the recombinant spider silk protein of the first aspect;

c2 An expression cassette comprising C1) said nucleic acid molecule;

c3 A recombinant vector comprising C1) said nucleic acid molecule, or a recombinant vector comprising C2) said expression cassette; alternatively, the recombinant vector adopts pET25b plasmid or pbluescript II ks plasmid;

c4 A recombinant microorganism comprising C1) said nucleic acid molecule, or a recombinant microorganism comprising B2) said expression cassette, or a recombinant microorganism comprising C3) said recombinant vector; alternatively, the recombinant microorganism is a recombinant E.coli, B.subtilis, mammalian cell, yeast cell or insect cell.

In a fifth aspect, there is provided a recombinant spidroin according to the first aspect or the recombinant spidroin mixed fiber according to the second aspect or the recombinant spidroin mixed fiber phase prepared by the preparation method according to the third aspect or the use of the biomaterial according to the fourth aspect, the use comprising any one or more of the following D1) to D5):

d1 A) aerospace field, military field or preparation of biological materials;

d2 A paper product;

d3 A) a film;

d4 A textile;

d5 A) a coating.

Advantageous effects

(1) The invention prepares the fiber by mixing two recombinant spider silk proteins in vitro according to a proportion, researches the advantages of the mixed protein fiber compared with a single protein fiber in terms of breaking strength, young modulus and toughness, and simulates the natural state of a spider by coexpression of the two recombinant spider silk proteins in vivo and self-assembly of the two proteins in escherichia coli by introducing two end structural domains so as to obtain the protein fiber with excellent mechanical properties. Both ways are helpful for realizing mass preparation of protein fibers.

(2) From the bionic point of view, the invention designs two recombinant spider silk fusion proteins by utilizing the genetic engineering technology, utilizes the fusion of a part sequence of a natural spider dragline silk and hydrophilic elastin, and utilizes the escherichia coli expression system to improve the soluble expression (the soluble expression can reach 20 mg/L), thereby providing a novel method for developing recombinant spider silk protein fibers.

(3) The SP-1-K fusion protein and the SP-2-K fusion protein can be mixed in vitro by different proportions, and the comprehensive mechanical properties of the SP-1-K fusion protein and the SP-2-K fusion protein are superior to those of single protein fibers. The recombined fusion spider silk fiber formed by mixing natural spider silk components according to the proportion of 3:2 has toughness comparable with that of natural spider silk.

(4) The invention also carries out fusion expression on the SP-1-K fusion protein and the SP-2-K fusion protein in escherichia coli through disulfide bonds, and simulates the natural state of a spider as far as possible so as to obtain protein fibers with excellent mechanical properties.

(5) The spinning process is simple and convenient, is easy to repeat, can be prepared in a large amount, and is longer-range ordered after post-stretching. Although the organic reagent is used for spinning, the fiber still has biocompatibility and safety, and provides a new material for the application of the fiber in the medical field.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

FIG. 1A schematic diagram of recombinant spidroin and co-expression recombinant spidroin vector construction according to example 1 of the invention; wherein A is a recombinant expression vector of SP-1-K-carboxyl domain protein, B is a recombinant expression vector of SP-2-K-carboxyl domain protein, and C is a recombinant vector fused with amino domain-SP-1-K-carboxyl domain protein and amino domain-SP-2-K-carboxyl domain protein;

FIG. 2 is a schematic diagram of a spinning apparatus used in the present invention;

FIG. 3 is a graph showing the mechanical properties of the SP-1-K-carboxydomain protein of example 1 of the present invention before and after stretching; wherein A is before stretching, and B is after stretching;

FIG. 4 is a graph showing the mechanical properties of the SP-2-K-carboxydomain protein of example 1 of the present invention before and after stretching; wherein A is before stretching, and B is after stretching;

FIG. 5 is a graph showing the mechanical properties of the mixed protein of example 2 of the present invention before and after stretching; wherein A is before stretching, and B is after stretching;

FIG. 6 is a graph showing the mechanical properties of the coexpression recombinant spidroin protein of example 3 of the present invention before and after stretching; wherein A is before stretching, and B is after stretching;

FIG. 7 shows a graph of the in vitro mixed protein fibers and in vivo co-expressed protein fiber cell compatibility of the present invention; wherein A, B, C, D is that A is mixed protein fiber to excite living cells to develop color under 495nm wavelength; b is the color development of dead cells excited by the mixed protein fiber at 539nm wavelength; c is that the coexpression protein fiber excites living cells to develop color under 495nm wavelength; d is the color development of dead cells excited by the coexpression protein fibers at 539 nm.

FIG. 8 shows liver function and kidney function test index chart after the mice of the invention eat in vitro mixed protein fiber.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, protocols, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

Throughout the specification and claims, unless explicitly stated otherwise, the term "comprise" or variations thereof such as "comprises" or "comprising", etc. will be understood to include the stated element or component without excluding other elements or components.

In the following embodiments, the detection method for detecting the mechanical properties includes: and (5) testing the mechanical properties of the fibers by adopting a fiber stretcher. When testing all fibrils, the drawing speed was 6mm/min, the distance between the nips was 3mm, and the breaking strength, toughness and ductility of the fibers were measured, wherein:

the tensile strength at break of the fiber strength is the stress divided by the cross-section, the stress being taken directly from the machine, the cross-section being by the circular formula.

Fiber toughness is the area enclosed by the stress-strain curve. Stress, strain is taken directly from the machine.

Young's modulus is the slope of the stress-strain curve in the linear elastic deformation range. All data treatments were performed using Origin Pro 2016.

In the following examples, the methods for detecting cell compatibility were:

the fiber biocompatibility was verified using mouse embryonic fibroblasts (3T 3). The cultured cells were operated as follows:

1) Cell resuscitation: fetal Bovine Serum (FBS), DMEM was culturedThe substrate is placed in a water bath kettle at 37 ℃ for preheating. And then the 3T3 cells stored in the liquid nitrogen are quickly taken out, and are heated and melted in a water bath kettle at 37 ℃. In an ultra clean bench, 4mL of medium (20% FBS and 80% DMEM) was placed in 25cm ² In a cell culture flask, 1mL of thawed cells were injected thereinto, and cultured in a 5% CO2 incubator at 37 ℃.

2) Cell passage: fetal Bovine Serum (FBS), DMEM medium, PBS solution and pancreatin were first placed in a 37℃water bath for preheating. When the cells were attached to the bottom of the flask and grown to suitable passage, the supernatant was discarded in a super clean bench and washed slowly twice with PBS solution. Then 1mL pancreatin is added, the flask is placed in a constant temperature incubator for 2min, and cells attached to the bottom of the flask are fully digested. Digestion was stopped by adding 2-3mL of medium (10% FBS and 90% DMEM) and cells on the wall were aspirated by blowing. The cell suspension was transferred to a 15mL centrifuge tube, and the supernatant was discarded after centrifugation at 1000rpm at 4℃in the centrifuge. Cells were resuspended in 15mL of medium (10% FBS and 90% DMEM) and then placed in 75cm2 cell culture flasks and incubated at 37℃in a 5% CO2 incubator.

The fibers were fixed in 6-well plates and sterilized in an ultra-clean bench using an ultraviolet lamp for 30min. The cultured cells were digested by the above procedure, centrifuged, and then resuspended in 12mL of medium (10% FBS and 90% DMEM). 2mL of the mixture was poured into a 6-well plate containing fibers, and cultured in a 5% CO2 incubator at 37 ℃. After the cells attach to the surface and periphery of the fiber, the cell activity is verified by using a cell living chromosome staining method.

In the following examples, the cell viability staining method is specifically performed as follows:

the Calcein-AM reagent was diluted with 50. Mu.L of DMSO solution to a final concentration of 1mmol/L. mu.L of the Calcein-AM reagent with the concentration of 1mmol/L is taken in 1mL of PBS solution to obtain PBS-Calcein-AM solution. mu.L of this solution was added to each well, and incubated at 37℃in a 5% CO2 incubator for 20min. And light is prevented during operation. After the completion, 10. Mu.L of Propidium Iodide (PI) was added thereto, and the mixture was cultured in a 5% CO2 incubator at 37℃for 10 minutes. And then observed under a confocal microscope. Calcein-AM was excited at 495nm to emit green light, and PI was excited at 539nm to emit red light.

In the following examples, the method for detecting liver function and kidney function by assay comprises the following steps:

to further verify the safety of the mixed fibers, we wrapped 20cm of fibers in bread and fed to BALB/c mice for 7 consecutive days. We took 1mL whole blood from the mice on day 9. Standing whole blood in a4 degree refrigerator for 12h, centrifuging at 1000rpm for 1min after layering, and collecting upper serum. Serum was commissioned to the biological company for testing.

The construction method of the fusion protein expression vector comprises the following steps:

1) Construction of basic Gene elements: the coding sequence formed by serial connection according to the following sequence: ndeI+NheI+coding sequence of amino acid sequence units+SpeI+ (CAC) ₆ +TAATGA+EcoRI, wherein the NdeI cleavage site is CATATG, the NheI cleavage site is GCTAGC, and the SpeI cleavage site is ACTAGT (CAC) ₆ Is a histidine tag sequence, TAATGA is a stop codon and EcoRI cleavage site is GAATTC. Construction of the basic Gene element [ S1-ELP1] respectively]Basic Gene element [ S2-ELP2]]Specifically:

basic Gene element [ S1-ELP1]]: the coding sequence is formed by serial connection according to the following sequence: ndeI+NheI+SEQ ID NO. 4+SpeI+ (CAC) ₆ +TAATGA+EcoRI。

Basic Gene element [ S2-ELP2]]: the coding sequence is formed by serial connection according to the following sequence: ndeI+NheI+SEQ ID NO. 5 coding sequence of the amino acid sequence +SpeI+ (CAC) ₆ +TAATGA+EcoRI。

The basic gene elements [ S1-ELP1], [ S2-ELP2] are synthesized artificially, for example, by the company Suzhou gold only.

One possible method for the tandem connection of amino acid sequence units is as follows:

1) Obtaining a genetic element 2; the basic gene element (1 or 2) and the pbluescript II ks plasmid (commercially available, abbreviated herein as m 13) were ligated by NheI and SpeI double cleavage to obtain a chimeric protein monomer vector, abbreviated herein as m 13-monomer; the m 13-monomer was digested with NheI and SpeI to obtain gene element 2.

2) Obtaining chimeric protein dimer vector (abbreviated as m 13-dimer vector): the gene element 2 was digested with NheI in the presence of T4 ligase (available from TaKaRa) to obtain the chimeric protein dimer vector (m 13-dimer). Wherein the SpeI cohesive end of the tail of the gene element 2 was ligated to the NheI cohesive end of the linear fragment of m 13-mer, and the junction was changed to ACTAGC (encoded amino acid sequence TS).

3) Obtaining chimeric protein tetramer vector (abbreviated as m 13-tetramer vector): a chimeric protein dimer gene fragment (the chimeric protein dimer gene fragment can be expressed as NheI cohesive end+coding sequence of amino acid sequence unit+ACTAGC+coding sequence of amino acid sequence unit+SpeI cohesive end) is obtained from m 13-dimer by NheI and SpeI double cleavage, and the chimeric protein dimer gene fragment and m 13-dimer subjected to NheI cleavage are subjected to T4 ligase to obtain a chimeric protein tetramer vector (abbreviated as m 13-tetramer vector).

Chimeric protein tetramer vector (abbreviated as m 13-tetramer vector) the chimeric protein tetramer gene fragment obtained by double cleavage with NheI and SpeI can be expressed as: the NheI cohesive end + the coding sequence of the amino acid sequence unit + the coding sequence of the ACTAGC + C-ELP protein unit + the SpeI cohesive end.

4) According to the mode, a chimeric protein octamer vector (m 13-octamer vector for short), a chimeric protein dodecamer vector (m 13-dodecamer vector for short), a chimeric protein twenty-four mer vector (m 13-twenty-four mer vector for short) and other multimeric vectors (collectively called m 13-multimeric vector) can be continuously obtained, and then the multimeric vectors are connected to a pET25b plasmid through double enzyme digestion of NdeI and EcoRI to form an expression vector pET25 b-multimeric, and are ready to be transferred into a recA recombinase defect type escherichia coli BLR (DE 3) expression system for IPTG induction expression.

5) To optimize the protein, the carboxy domain fragment (SpeI+the coding sequence for the amino acid sequence shown in SEQ ID NO: 7+SpeI) was digested with SpeI, and then the m 13-multimeric vector was digested singly with SpeI, under the action of T4 ligase to obtain an m 13-multimeric vector with CTD sequence; then connecting the vector to pET25b plasmid through NdeI and EcoRI double enzyme digestion to form an expression vector pET25 b-multimeric-CTD, and obtaining various expression vectors according to different sequences and numbers of amino acid sequence units, wherein the expression vectors comprise:

to encode the essential gene element [ S1-ELP1]]Constituted dodecamer expression vector pET25b- [ S1-ELP1] ₁₂ CTD (the amino acid sequence shown in SEQ ID NO:4 is adjacent to the amino acid sequence shown in SEQ ID NO:4 is also connected with the amino acid TS between the carboxyl domain and the amino acid sequence shown in SEQ ID NO: 4); the fusion protein expressed by the expression vector is named SP-1-ELP-carboxyl domain (which contains the amino acid sequence shown in SEQ ID NO: 9).

To encode the essential gene element [ S2-ELP2]]Constituted dodecamer expression vector pET25b- [ S2-ELP2] ₁₂ CTD (the amino acid sequence shown in SEQ ID NO:5 is adjacent to the amino acid sequence shown in SEQ ID NO:5 is connected with the amino acid TS, the carboxyl domain and the amino acid sequence shown in SEQ ID NO:5 is also connected with the amino acid TS); the fusion protein expressed by the expression vector is named SP-2-ELP-carboxyl domain (which contains the amino acid sequence shown in SEQ ID NO: 11).

Construction of the Co-expression vector is shown in FIG. 1, in the expression vector pET25b- [ S1-ELP1] ₁₂ -CTD、pET25b-[S2–ELP2] ₁₂ -CTD is augmented with a natural spider silk amino domain encoding the amino acid sequence shown in SEQ ID NO. 6. The specific method comprises the following steps:

in the expression vector pET25b- [ S1-ELP1] ₁₂ -adding an amino domain to CTD: the amino domain fragment encoding the amino acid sequence shown in SEQ ID NO. 6 is subjected to homologous recombination by the PCR technique under the action of a recombinant enzyme ClonII (which is obtained by in vitro recombination reactions catalyzed by a recombinant enzyme only in a position within about 10bp of the vector cut) from Vazyme company, wherein the amino domain fragment encodes the amino acid sequence shown in SEQ ID NO. 6, the nucleotide sequence shown in SEQ ID NO. 12, the nucleotide sequence shown in SEQ ID NO. TTTAACTTTAAGAAGGAGATATACATATGGGTCAAGCGAACACCC), the nucleotide sequence shown in SEQ ID NO. 12, the nucleotide sequence shown in SEQ ID NO. GTACGGACCGCTAGCCATATGACTGCCACCGCCACCGCTACCGCCACCGCCACTTACGGGTTGTTGCCC, and the chimeric protein dodecameric vector (pET 25 b-SP-1-ELP-CTD) are subjected to single cleavage by NdeIThe expression vector pET25b-NTD-SP-1-ELP-CTD with amino domain and carboxyl domain is obtained to obtain fusion protein NTD-SP-1-ELP-CTD, which contains the amino acid sequence shown in SEQ ID NO. 8.

In the expression vector pET25b- [ S2-ELP2] ₁₂ -adding an amino domain to CTD: the method is referred to above, the amino domain fragment of the amino acid sequence shown in SEQ ID NO. 6 is added with homology arms 1 (base sequence shown in SEQ ID NO. 12: TTTAACTTTAAGAAGGAGATATACATATGGGTCAAGCGAACACCC)) and homology arms 3 (base sequence shown in SEQ ID NO. 14) at both ends of the gene sequence of the amino acid sequence shown in SEQ ID NO. 6 by using PCR technology: GCAGCCGCAGAAGAGCCGCTAGCCATATGACTGCCACCGCCACCGCTACCGCCACCGCCACTTACGGGTTGTTGCCCGCG). The expression vector pET25b-NTD-SP-2-ELP-CTD with amino domain and carboxyl domain is obtained, and fusion protein NTD-SP-2-ELP-CTD is obtained, which contains the amino acid sequence shown as SEQ ID NO. 10.

Constructing a co-expression plasmid, carrying out double enzyme digestion on an expression vector pET25b-NTD-SP-1-ELP-CTD by using BgIII enzyme and BamHI enzyme, carrying out single enzyme digestion on pET25b-NTD-SP-2-ELP-CTD by using BgIII enzyme, cloning two proteins onto the same expression vector under the action of T4 ligase purchased from TaKaRa company, and obtaining a recombinant expression vector pET25b-NTD-SP-1-ELP-CTD-NTD-SP-2-ELP-CTD (which contains amino acid sequences shown as SEQ ID NO:8 and 10) shown in figure 1C.

Example 1: preparation of single recombinant spider silk protein fibers

Expression vector pET25b- [ S1-ELP1] ₁₂ -CTD、pET25b-[S2–ELP2] ₁₂ CTD is purified after expression of recA recombinase-deficient engineered E.coli BLR (DE 3) to obtain freeze-dried SP-1-ELP-carboxyl domain fusion spider silk protein and SP-2-K-carboxyl domain fusion spider silk protein, respectively, dissolving the protein proteins in 200 mu L hexafluoroisopropanol according to a protein concentration of 150mg/L, sucking the protein solution by using a 1mL syringe, selecting a needle with an inner diameter of 200 mu m, extruding the protein solution into a 90% methanol aqueous solution (coagulation bath), regulating the advancing speed to 5 mu L/min by using a syringe pump, and collecting fibers by using a rotary drum collector at a linear speed of 0.6m/min (FIG. 2) to obtain primary fibers. Will collectThe obtained fibers were immersed in 50% methanol until the fibers became soft, and then immediately taken out and then stretched to about 200% of the original length to obtain post-stretched fibers, and the mechanical properties of the prepared fibers were examined (FIGS. 3 to 4).

FIG. 3 shows that the SP-1-ELP-carboxyl domain fusion protein has a tensile strength of 102.68.+ -. 5.76MPa and a toughness of 246.9.+ -. 17.9MJ/m before stretching (as-spun fiber) ³ The method comprises the steps of carrying out a first treatment on the surface of the The tensile strength after stretching (post-stretching fiber) is 242.85 + -7.12 MPa, and the toughness is 57.19 + -3.98 MJ/m ³ . Indicating an increase in tensile strength and a decrease in toughness after stretching.

FIG. 4 shows that the SP-2-ELP-carboxyl domain fusion protein has a tensile strength of 142.32.+ -. 7.99MPa and a toughness of 286.82.+ -. 27.28MJ/m before stretching (as-spun fiber) ³ The method comprises the steps of carrying out a first treatment on the surface of the The tensile strength after stretching (post-stretching fiber) was 269.65.+ -. 13.33MPa, and the toughness was 69.34.+ -. 9.61MJ/m ³ . Indicating an increase in tensile strength and a decrease in toughness after stretching.

Example 2: preparation of two recombinant spider silk protein in-vitro mixed fibers

The two proteins SP-1-ELP-carboxyl domain fusion protein and SP-2-K-carboxyl domain fusion protein of example 1 were dissolved in 200. Mu.L hexafluoroisopropanol (protein concentration 150 mg/L) at a weight ratio of 3:2, the mixed protein solution was centrifuged to obtain a supernatant, which was extruded into a 90% methanol solution (coagulation bath) by a syringe, and the advancing speed was adjusted to 5. Mu.L/min by a syringe pump, and the fibers were collected by a drum collector at a linear speed of 0.6m/min to obtain primary fibers. Soaking the collected fiber in 50% methanol until the fiber becomes soft, immediately taking out the fiber, and stretching to about 200% of the original length to obtain post-stretched fiber, and performing mechanical property detection (figure 5)

FIG. 5 shows that the fibers obtained by physically mixing the SP-1-ELP-carboxyl domain fusion protein and the SP-2-K-carboxyl domain fusion protein in a ratio of 3:2 have a tensile strength of 188.18.+ -. 5.03MPa and a detection toughness of 256.8.+ -. 14.72MJ/m before stretching (as-spun fibers) ³ The method comprises the steps of carrying out a first treatment on the surface of the The tensile strength after stretching (post-stretching fiber) was 362.83.+ -. 11.23MPa, and the measured toughness was 174.76.+ -. 9.79MJ/m ³ . Indicating an increase in tensile strength and a decrease in toughness after stretching.

It has also been shown that the tensile strength of the fiber is increased after physical mixing of the two fusion proteins relative to the tensile strength of the single fusion protein.

Example 2A

This example differs from example 2 in that the SP-1-ELP-carboxydomain fusion protein and the SP-2-K-carboxydomain fusion protein are mixed in a 2:3 ratio, and the tensile strength after stretching is 293.45 + -14.33 MPa, toughness 100.36 + -18.02 MPa, and modulus 5.91+ -0.99 GPa.

Example 3: preparation of co-expressed protein fibers in two recombinant spider silk bodies

Expressing the expression vector pET25b-NTD-SP-1-ELP-CTD-NTD-SP-2-ELP-CTD by recA recombinase defect engineering escherichia coli BLR (DE 3), purifying to obtain co-expressed protein, dissolving in 98% formic acid (protein concentration 150 mg/L), centrifuging the mixed protein solution, taking supernatant, extruding the supernatant into methanol solution (coagulation bath) by using a syringe, regulating the advancing speed to 5 mu L/min by using a syringe pump, and collecting fibers at a linear speed of 0.6m/min by using a rotary drum collector to obtain primary fibers. The collected fibers were immersed in 50% methanol until the fibers were softened, and then immediately taken out and then stretched to about 100% of the original length to obtain post-stretched fibers, and the prepared fibers were subjected to mechanical property detection (fig. 6).

FIG. 6 shows that the fibers obtained after coexpression of the fusion proteins of the amino domain-SP-1-ELP-carboxy domain and the amino domain-SP-2-K-carboxy domain have a tensile strength of 232.41.+ -. 5.89MPa and a detection toughness of 355.05.+ -. 30.75MJ/m before stretching (as-spun fibers) ³ The method comprises the steps of carrying out a first treatment on the surface of the The tensile strength after stretching (post-stretching fiber) was 336.21.+ -. 27.37MPa, and the test toughness was 86.93.+ -. 6.11MJ/m ³ . Indicating an increase in tensile strength and a decrease in toughness after stretching.

Example 4: comparison of Performance between protein fibers prepared according to the invention

The invention adopts two 12-polymer recombined spider silk protein fibers with carboxyl, the mechanical properties of the fibers prepared by adjusting the sequence and the mixing proportion are different, and the strength, toughness and modulus properties are compared in table 1;

table 1 properties of the mixed fibers of examples before and after drawing

The comprehensive mechanical property of the mixed fiber is superior to that of single protein fiber. The toughness of the recombined fusion spider silk fiber formed by mixing natural spider silk components according to the proportion of 3:2 can be comparable with that of natural spider silk, and a new thought is provided for exploring and improving the mechanical property of protein fiber.

Example 5: in vitro mixed protein fiber and in vivo coexpression protein fiber cytotoxicity experiment

3T3 cells (mouse embryonic fibroblasts, purchased from Shanghai relay and Bio Inc.) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% diabodies (penicillin and gentamicin). The cytotoxicity of both protein fibers was detected using 3T3 cells. Before the test, the two proteins SP-1-ELP-carboxyl: SP-2-K-carboxyl prepared in example 2 were mixed in a ratio of 3:2 using an adhesive tape autoclaved at a high temperature of 121℃for 30min to prepare protein fibers (FIGS. 7 (A), (B)) and the in vivo co-expressed protein fibers prepared in example 3 (FIGS. 7 (C), (D)) were fixed to the bottom of a 12-well plate, and then sterilized in a UV cabinet for 12 hours. Cells were co-cultured with fibers in 12-well plates at 50000/mL for 24 hours. The culture was removed and washed three times with PBS buffer. 1. Mu.L of Calcein-AM stained active cells and 1. Mu. L Propidium Iodide (PI) stained apoptotic cells were added and incubated for 10-20 min. Two protein fibers and control cells were observed with a confocal laser scanning microscope (LSCM) at an emission wavelength of 495nm AM and 539nm PI. Experiments show that cells can grow on both fibers, which shows that the fibers have good biocompatibility.

Example 6: in vitro mixed protein fiber biosafety experiment

The two proteins of the SP-1-ELP-carboxyl group and the SP-2-K-carboxyl group of the example 2 are mixed according to the proportion of 3:2 to prepare the protein fiber. Spun fibers were fed to BALB/c mice per 20cm of fiber per day, 6 BALB/c mice per day, and body weights were recorded daily. The control group was 6 mice not fed fiber BALB/c, and the body weight was recorded daily. After one week, 1mL of blood was taken from the orbit of the mouse. Serum was obtained by resting in a refrigerator at 4℃for 24 hours. The supernatant was then centrifuged at 3000 rpm in a centrifuge at 4℃for 15 minutes. And detecting liver function and kidney function indexes by using a full-automatic biochemical analyzer. 13 blood biochemical indicators related to liver and kidney function including alanine Aminotransferase (ALT), aspartic acid Aminotransferase (AST), ALT/AST, total Protein (TP), albumin (ALB), globulin (GLOB), albumin/globulin (a/G), alkaline phosphatase (ALP), lactate Dehydrogenase (LDH), γ -glutamyl transferase (GGT), uric Acid (UA), creatinine (CREA) and carbon dioxide (CO 2), and a significant difference between each experimental and control group was calculated, and the experimental and control groups showed no significant difference (p > 0.05) other than alkaline phosphatase (ALP) by assay indicator data. The difference in ALP single index may be caused by individual differences. These results indicate that the mixed protein fibers have good biosafety. This offers the possibility for subsequent use in biomedical and medical devices. (FIG. 8)

The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. Any simple modifications, equivalent variations and modifications of the above-described exemplary embodiments should fall within the scope of the present invention.

Claims

1. A recombinant spider silk protein comprising at least two recombinant proteins: SP-1-K fusion proteins and SP-2-K fusion proteins,

the SP-1-K fusion protein and the SP-2-K fusion protein are prepared by mixing the following components in parts by weight (0.6-1.5): 1 blending or coexpression in a microorganism;

the SP-1-K fusion protein is any one of the following A1), A2), A4) and A5) proteins:

a1 The amino acid sequence of which comprises: m directly tandem expression forms are shown as [ S1-ELP1] amino acid sequence units, wherein S1 represents an amino acid sequence shown as SEQ ID NO. 1, ELP1 is an elastin-like sequence, and [ S1-ELP1] represents that the S1 sequence and the ELP1 sequence are in tandem; m represents the number of repetitions of the [ S1-ELP1] sequence, m being 12; the amino acid sequence [ S1-ELP1] of the amino acid sequence unit is shown as SEQ ID NO. 4;

a2 The amino acid sequence of which comprises: a connecting sequence is connected between adjacent amino acid sequence units [ S1-ELP1] in the A1), and the connecting sequence comprises two or four amino acids;

a4 The amino acid sequence of which comprises: introducing a natural spider silk amino domain at the N-terminus of A1) or A2), and/or introducing a natural spider silk carboxyl domain at the C-terminus of A1) or A2);

a5 The amino acid sequence of which comprises: introducing a histidine tag at the N-terminal and/or C-terminal of A1) or A2) or A4);

the SP-2-K fusion protein is any one of the following B1), B2), B4) and B5):

b1 The amino acid sequence of which comprises: n directly tandem expression forms are shown as [ S2-ELP2], wherein S2 represents an amino acid sequence shown as SEQ ID NO. 2; ELP2 is an elastin-like sequence; [ S2-ELP2] represents a tandem of the S2 sequence and the ELP2 sequence; n represents the number of repetitions of the [ S2-ELP2] sequence, n being 12; the amino acid sequence [ S2-ELP2] of the amino acid sequence unit is shown as SEQ ID NO. 5;

b2 The amino acid sequence of which comprises: a connecting sequence is connected between adjacent amino acid sequence units [ S2-ELP2] in the B1), and the connecting sequence comprises two or four amino acids;

b4 The amino acid sequence of which comprises: introducing a natural spider silk amino domain at the N-terminus of B1) or B2), and/or introducing a natural spider silk carboxyl domain at the C-terminus of B1) or B2);

b5 The amino acid sequence of which comprises: a histidine tag is introduced at the N-terminal and/or C-terminal of B1) or B2) or B4).

2. The recombinant spider silk protein according to claim 1, wherein in the A2) protein of the SP-1-K fusion protein, the connecting sequence is two or four amino acids encoded after enzyme cleavage at different enzyme cleavage sites of the same tail and then connection.

3. The recombinant spider silk protein according to claim 1 or 2, wherein in the B2) protein of the SP-2-K fusion protein, the linking sequence is two or four amino acids encoded after cleavage at different cleavage sites of the same tail and subsequent ligation.

4. The recombinant spider silk protein according to claim 1, wherein the natural spider silk amino domain comprises an amino acid sequence as shown in SEQ ID NO. 6 in the A4) protein of the SP-1-K fusion protein and/or the B4) protein of the SP-2-K fusion protein.

5. The recombinant spider silk protein according to claim 1, wherein in A4) of the SP-1-K fusion protein and/or B4) of the SP-2-K fusion protein, the carboxy domain of the natural spider silk comprises the amino acid sequence shown in SEQ ID NO. 7.

6. The recombinant spider silk protein according to claim 1, wherein the sequence of the SP-1-K fusion protein is shown in SEQ ID NO. 8 or 9.

7. The recombinant spider silk protein according to claim 1, wherein the sequence of the SP-2-K fusion protein is shown in SEQ ID NO. 10 or 11.

8. The recombinant spider silk protein according to claim 1, wherein the weight ratio of SP-1-K fusion protein to SP-2-K fusion protein is 1.5:1 or 2:3.

9. a recombinant spidroin protein mixed fiber comprising the recombinant spidroin protein as defined in any one of claims 1 to 8, which is spun artificially.

10. A method of preparing a recombinant spidroin protein mixed fiber according to claim 9, comprising:

11. The method of claim 10, wherein the solvent is hexafluoroisopropanol or formic acid.

12. The preparation method of claim 10, wherein the SP-1-K fusion protein and the SP-2-K fusion protein are mixed according to the weight ratio of (0.6-1.5): 1 in hexafluoroisopropanol to prepare a protein solution.

13. The preparation method of claim 12, wherein the mass fraction of the SP-1-K fusion protein and the SP-2-K fusion protein in the protein solution is 150-200 mg/mL.

14. The method of claim 12, wherein the mass fraction of the SP-1-K fusion protein and SP-2-K fusion protein in the protein solution is 150 mg/mL.

15. The method according to claim 10, wherein the SP-1-K fusion protein and the SP-2-K fusion protein are obtained by recombinant E.coli expression and purification, respectively.

16. The method according to claim 10, wherein the SP-1-K fusion protein and the SP-2-K fusion protein are obtained by purification after co-expression in recombinant escherichia coli.

17. The method of claim 16, wherein the aqueous solution is dissolved in formic acid.

18. The method of claim 10, wherein the coagulation bath is 80% -100% methanol solution by volume.

19. The method of claim 10, wherein the coagulation bath is a 90% methanol solution.

20. The method of any one of claims 10 to 19, further comprising post-stretching the as-spun fiber: and (3) soaking the nascent fiber in a stretching bath to soften, and stretching the nascent fiber to 2-5 times of the original length to obtain the post-stretched fiber.

21. A biomaterial associated with the recombinant spidroin according to any one of claims 1 to 8, or the recombinant spidroin mixed fiber according to claim 9, or the recombinant spidroin mixed fiber prepared by the preparation method according to any one of claims 10 to 20, characterized in that the biomaterial is any one of C1) to C6):

c1 A nucleic acid molecule encoding the SP-1-K fusion protein and/or SP-2-K fusion protein of a recombinant spider silk protein according to any one of claims 1 to 8;

c2 An expression cassette comprising C1) said nucleic acid molecule;

c3 A recombinant vector comprising C1) said nucleic acid molecule, or a recombinant vector comprising C2) said expression cassette;

c4 A recombinant microorganism comprising C1) said nucleic acid molecule, or a recombinant microorganism comprising C2) said expression cassette, or a recombinant microorganism comprising C3) said recombinant vector;

c5 A recombinant mammalian cell comprising C1) said nucleic acid molecule, or a recombinant mammalian cell comprising C2) said expression cassette, or a mammalian cell comprising C3) said recombinant vector;

c6 A recombinant insect cell comprising the nucleic acid molecule of C1), or a recombinant insect cell comprising the expression cassette of C2), or an insect cell comprising the recombinant vector of C3).

22. The biomaterial of claim 21, wherein in C3) the recombinant vector is a pET25b plasmid or a pbluescript II ks plasmid.

23. The biomaterial of claim 21, wherein in C4) the recombinant microorganism is a recombinant escherichia coli, bacillus subtilis or yeast cell.

24. Use of the recombinant spidroin according to any one of claims 1 to 8 or the recombinant spidroin mixed fiber according to claim 9 or the recombinant spidroin mixed fiber prepared by the preparation method according to any one of claims 10 to 20, comprising any one or several of the following D1) to D5):

d1 A) aerospace field, military field or preparation of biological materials;

d2 A paper product;

d3 A) a film;

d4 A textile;

d5 A) a coating.