CN116425849B - A kind of recombinant spider silk protein, recombinant spider silk protein mixed fiber and its preparation method and application - Google Patents

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

A kind of recombinant spider silk protein, recombinant spider silk protein mixed fiber and preparation method thereof application

Technical field

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

Background technique

Today, humanity is facing the crisis of unsustainable resource depletion, and natural polymers are receiving increasing attention due to their rich sources and sustainability. As a structural protein, spider protein has received special attention among many natural polymer materials in nature. Adult spiders secrete at least 7 kinds of protein silks, among which drag silk has been widely studied because of its excellent comprehensive mechanical properties.

Spider drag silk is produced by the cylindrical epithelial cells of the main ampulla gland of spiders. It is a protein fiber with a hierarchical structure formed by at least two protein components (MaSp1 and MaSp2) as the core. Most spider drag silk proteins contain a large central repetitive core domain flanked by small non-repetitive terminal domains. The number of repeating core domains and different types of repeating segments play a decisive role in the material properties of spider silk. The domains at both ends play an important role in the self-assembly process of storage and stretching of spider silk proteins in glands. Based on the molecular structures of the spider silk proteins MaSp1 and MaSp2, both spider proteins are thought to contribute in different ways to the mechanical properties of the drag silk. Among them, MaSp1 is considered to be the main source of the rigid properties of spider dragline silk, while MaSp2 is believed to contribute greatly to its elastic properties. Spiders can obtain fibers with different mechanical properties by mixing two proteins in different proportions to achieve different physiological functions.

Since spiders produce a small amount of silk and are difficult to raise on a large scale, the preparation of artificial spider silk fibers is mainly achieved by expressing recombinant spider silk proteins and performing artificial spinning. However, the mechanical properties of artificial in vitro synthesized spider silk fibers are still not comparable to 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, and there are few studies on the formation of heterodimers or blends of the two proteins in vivo or in vitro. On the other hand, many recombinant spider silk proteins Spider silk proteins lack terminal domains during heterologous synthesis, which makes it difficult for the two proteins to cross-assemble and affect fiber properties.

Contents of the invention

technical problem

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

The present invention develops two proteins by simulating the natural state of spiders, mixes them in vitro according to the natural protein ratio, and fuses and expresses the two proteins in vivo to form fibers. It is very important for developing new spider silk protein assemblies and improving the performance of artificial spider silk fibers. significance.

solution

In order to solve the above technical problems, the present invention provides the following technical solutions:

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

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

A1) Its amino acid sequence includes: m directly connected amino acid sequence units in the form of [S1-ELP1], where S1 represents the amino acid sequence as shown in SEQ ID NO: 1, and ELP1 is an elastin-like sequence. , [S1–ELP1] represents the concatenation of S1 sequence and ELP1 sequence; m represents the repeat number of [S1–ELP1] sequence, m is an integer between 1 and 36;

Among them, the amino acid sequence shown in SEQ ID NO:1 is as follows:

GPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPGQQGPGQQ

A2) Its amino acid sequence includes: a connecting sequence is connected between the adjacent amino acid sequence units [S1-ELP1] in A1), optionally, the connecting sequence includes two or four amino acids, optionally the connecting sequence It is the two or four amino acids encoded after digestion at different enzyme cleavage sites of the same tail and then connected; optionally, the connection sequence is two amino acids; optionally, the connection sequence can be TS (which is the same tail The tail after digestion by SpeI (A/CTAGT) and the head after digestion by NheI (G/CTAGC) are connected again (the encoded amino acid of ACTAGC). Due to the direct synthesis of multiple repeating units, there is a certain Difficulty, this kind of connection sequence will be generated when repeating units are connected by enzyme cleavage sites, and the enzyme cleavage sites can be selected according to needs, that is, TS can also be replaced with other sequences resulting from replacement of enzyme cleavage sites. Generally speaking, two amino acids or four amino acids can be used;

A3) Its amino acid sequence includes: the amino acid sequence represented by A1) or A2) is the same as or similar to the protein represented by A1) obtained by the substitution and/or deletion and/or addition of one, two or more amino acid residues. properties of proteins;

A4) Its amino acid sequence includes: 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) Its amino acid sequence includes: 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) Its amino acid sequence includes: n directly connected amino acid sequence units as shown in [S2-ELP2], where S2 represents the amino acid sequence as shown in SEQ ID NO:2; ELP2 is an elastin-like sequence ; [S2–ELP2] represents the concatenation of S2 sequence and ELP2 sequence; n represents the repeat number of [S2–ELP2] sequence, n is an integer between 1-36;

Among them, the amino acid sequence shown in SEQ ID NO:2 is as follows:

GSSAAAAAAAASGPGGYGPENQGSGPGGYGPGGP

B2) The amino acid sequence includes: There is a connecting sequence between the adjacent amino acid sequence units [S2-ELP2] in B1). The connecting sequence is two codes encoded by digesting different enzyme cleavage sites of the same tail and then connecting them. or four amino acids;

B3) Its amino acid sequence includes: the amino acid sequence represented by B1) or B2) is the same as or similar to the protein represented by A1) obtained by the substitution and/or deletion and/or addition of one, two or more amino acid residues. properties of proteins;

B4) Its amino acid sequence includes: the introduction of a natural spider silk amino domain at the N-terminus of B1) or B2) or B3), and/or the introduction of a natural spider silk carboxyl domain at the C-terminus of B1) or B2) or B3);

B5) Introduce a histidine tag at the N-terminus 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 8-24, optionally an integer between 8-16, optionally an integer between 10-14, optionally 12;

The amino acid sequence of ELP1 includes several amino acid sequences concatenated together as shown in SEQ ID NO: 3: VPGXG, expressed in the form (VPGXG) _i , where X is lysine or arginine or other amino acids, and can be Optionally, An integer between -8, optionally i is an integer between 4-6, optionally i is 5.

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

Alternatively, the amino acid sequence of the SP-1-K fusion protein is expressed as

[GPYGPGASAAAAAAGGYGPSGQQQGPGQQGPGQQGPGQQGPGQQ-(VPGKG) _i ] _m , where m represents the repeat number of the [S1–ELP1] sequence (GPYGPGASAAAAAAGGYGPSGGQQGPGQQGPGQQGPGQQGPGQQ-(VPGKG) _i ), m is an integer between 1-36, optionally between 8-16 is an integer between 10-14, optionally 12; i is an integer between 3-10, optionally i is an integer between 3-8, optionally i is An integer between 4-6, optionally i is 5.

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

GPYGPGASAAAAAAGGYGPGSGQQGPGQQGPGQQGPGQQGPGQQVPGKGVPGKGVPGKGVPGKGVPGKG

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

n is an integer between 8-24, optionally an integer between 8-16, optionally an integer between 10-14, optionally 12;

The amino acid sequence of ELP2 includes several amino acid sequences concatenated together as shown in SEQ ID NO: 3: VPGXG, expressed in the form (VPGXG) _j , where X is lysine or arginine or other amino acids, and can be Optionally, An integer between 8, optionally j is an integer between 4-6, optionally j is 5.

Alternatively, the sequence of the SP-2-K fusion protein is expressed as

[GSSAAAAAAAASGPGGYGPENQGSGPGGYGPGGP(VPGKG) _j ] _n , where n represents the repeat number of the [S2–ELP2] sequence (GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP(VPGKG) _j ), n is an integer between 1-36, optionally between 8-16 Integer, optionally an integer between 10-14, optionally 12; j is an integer between 3-10, optionally j is an integer between 3-8, optionally j is 4- An integer between 6, optionally 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:

GSSAAAAAAAASGPGGYGPENQGSGPGGYGPGGPVPGKGVPGKGVPGKGVP 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 contains the amino acid sequence 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 carboxyl domain of the natural spider silk contains the amino acid sequence 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 amino domain and carboxyl domain) is as follows:

The sequence shown in SEQ ID NO:9 is the underlined portion (982 amino acids) of the sequence of SEQ ID NO:8, which contains the carboxyl 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, which contains amino domain and carboxyl domain) is as follows:

The sequence shown in SEQ ID NO:11 is the underlined portion (874 amino acids) of the sequence of SEQ ID NO:10, which contains the carboxyl domain.

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

Further, the SP-1-K fusion protein and SP-2-K fusion protein are fusion proteins obtained by blending in vitro or co-expressing in microorganisms.

In a second aspect, a recombinant spider silk protein mixed fiber is provided, which includes the recombinant spider silk protein described in the first aspect, which is artificially spun;

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

In a third aspect, a method for preparing the recombinant spider silk protein mixed fiber described in the second aspect is provided, which includes:

Dissolve the SP-1-K fusion protein and SP-2-K fusion protein in a solvent to prepare a protein solution, and use a syringe pump to extrude the protein solution into a coagulation bath to solidify into nascent fibers.

Further, the solvent is hexafluoroisopropanol or formic acid.

Further, the SP-1-K fusion protein and SP-2-K fusion protein are dissolved in hexafluoroisopropanol at a weight ratio (0.2-3): 1 to prepare a protein solution. Alternatively, the SP- The mass fraction of 1-K fusion protein and SP-2-K fusion protein in the protein solution is 150-200 mg/mL, optionally 150 mg/mL. Alternatively, the SP-1-K fusion protein and SP -2-K fusion proteins were expressed and purified through recombinant Escherichia coli.

Further, the SP-1-K fusion protein and SP-2-K fusion protein are obtained by co-expression in recombinant Escherichia coli and then purified, optionally, dissolved in formic acid solution.

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

Further, it also includes post-drawing of the virgin fiber: after soaking the virgin fiber in a stretching bath to soften it, it is stretched to 2 to 5 times its original length to obtain a post-drawn fiber. Optionally, the stretching bath is a 0-80% methanol solution by volume, optionally a 50% methanol solution.

The fourth aspect provides biological materials related to the recombinant spider silk protein described in the first aspect, or the recombinant spider silk protein mixed fiber described in the second aspect, or the recombinant spider silk protein mixed fiber prepared by the preparation method described in the third aspect. , the biological material is any one of C1) to C4):

C1) Nucleic acid molecules encoding the SP-1-K fusion protein and/or SP-2-K fusion protein of the recombinant spider silk protein described in the first aspect;

C2) an expression cassette containing the nucleic acid molecule described in C1);

C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); optionally, the recombinant vector uses pET25b plasmid or pbluescript II ks plasmid;

C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in C3); optionally, the recombinant microorganism is recombinant Escherichia coli, subtilis, mammalian cells, yeast cells or insect cells.

The fifth aspect provides a recombinant spider silk protein described in the first aspect or a recombinant spider silk protein mixed fiber described in the second aspect or a recombinant spider silk protein mixed fiber phase prepared by the preparation method described in the third aspect or a third aspect. The uses of biological materials described in the four aspects include any one or more of the following D1) to D5):

D1) Aerospace field, military field or preparation of biological materials;

D2) Paper products;

D3) thin film;

D4) Textiles;

D5) Coating.

beneficial effects

(1) The present invention makes fibers by mixing two recombinant spider silk proteins in vitro in proportion, and studies the advantages of the mixed protein fibers over single protein fibers in terms of breaking strength, Young's modulus, and toughness. Secondly, By co-expressing two recombinant spider silk proteins in vivo, introducing two terminal domains, and self-assembling the two proteins in E. coli, the natural state of spiders is simulated to obtain protein fibers with excellent mechanical properties. Both methods help achieve large-scale preparation of protein fibers.

(2) From the perspective of bionics, the present invention uses genetic engineering technology to design two recombinant spider silk fusion proteins, using partial sequences of natural spider traction silk to fuse with hydrophilic elastin-like proteins to improve soluble expression when using the E. coli expression system. (Soluble expression can reach 20mg/L), providing a new method for the development of recombinant spider silk protein fibers.

(3) The SP-1-K fusion protein and SP-2-K fusion protein of the present invention can be mixed in different proportions in vitro, and their comprehensive mechanical properties are better than those of single protein fibers. The recombinant fused spider silk fiber formed by mixing natural spider silk components in a ratio of 3:2 has a toughness comparable to that of natural spider silk.

(4) The present invention also fuses and expresses SP-1-K fusion protein and SP-2-K fusion protein in E. coli through disulfide bonds, simulating the natural state of spiders as much as possible to obtain protein fibers with excellent mechanical properties.

(5) The spinning process of the present invention is simple, easy to repeat, can be prepared in large quantities, and is more orderly in the long run after post-stretching. Although organic reagents are used for spinning, it is still biocompatible and safe, providing new materials for fiber applications in the medical field.

Description of drawings

One or more embodiments are exemplified by the pictures in the corresponding drawings, and these exemplary illustrations do not constitute limitations to the embodiments. The word "exemplary" as used herein means "serving as an example, example, or illustrative." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or superior to other embodiments.

Figure 1. Schematic diagram of the construction of recombinant spider silk protein and co-expression vector for recombinant spider silk protein in Example 1 of the present invention; wherein, A is the recombinant expression vector of SP-1-K-carboxyl domain protein, and B is SP-2 -The recombinant expression vector of K-carboxy domain protein, C is the recombinant vector of the fusion of amino domain-SP-1-K-carboxy domain protein and amino domain-SP-2-K-carboxy domain protein;

Figure 2. Schematic diagram of the spinning device used in the present invention;

Figure 3. Mechanical property test curve chart of the SP-1-K-carboxy domain protein of Example 1 of the present invention before and after stretching; where A is before stretching and B is after stretching;

Figure 4. Mechanical property test curve chart of the SP-2-K-carboxyl domain protein of Example 1 of the present invention before and after stretching; wherein, A is before stretching, and B is after stretching;

Figure 5. Mechanical property test curve chart of the mixed protein before and after stretching according to Example 2 of the present invention; where A is before stretching and B is after stretching;

Figure 6. Mechanical property test curve chart of the co-expressed recombinant spider silk protein before and after stretching according to Example 3 of the present invention; where A is before stretching and B is after stretching;

Figure 7. Cytocompatibility diagram of the present invention's in vitro mixed protein fiber and in vivo co-expressed protein fiber; wherein, A, B, C, and D are respectively A is the mixed protein fiber exciting living cells to develop color at a wavelength of 495 nm; B is The mixed protein fiber excites dead cells to develop color at a wavelength of 539nm; C is a co-expressed protein fiber that excites living cells to develop color at a wavelength of 495 nm; D is a co-expressed protein fiber to excite dead cells at a wavelength of 539 nm to develop color.

Figure 8. Laboratory index chart of liver function and kidney function after mice of the present invention consumed mixed protein fiber in vitro.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. embodiment. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

In addition, in order to better explain the present invention, numerous specific details are given in the following detailed description. It will be understood by those skilled in the art that the present invention may be practiced without certain specific details. In some embodiments, raw materials, solutions, methods, means, etc. that are well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

Unless expressly stated otherwise, throughout the specification and claims, the term "comprises" or its variations such as "comprises" or "comprising" will be understood to include the stated elements or components, and to Other elements or other components are not excluded.

In the following examples, the detection method for mechanical property testing is: using a fiber tensile instrument to test fiber mechanical properties. When testing all fibrils, the tensile speed is 6mm/min, and the distance between clamps is 3mm. The breaking strength, toughness and ductility of the fibers are tested, among which:

Fiber strength breaking strength The tensile force at break is the stress divided by the cross-section. The stress is obtained directly from the machine and the cross-sectional area uses the circular formula.

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

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

In the following examples, the detection method for cytocompatibility detection is:

Mouse embryonic fibroblasts (3T3) were used to verify fiber biocompatibility. To culture cells, proceed as follows:

1) Cell recovery: First, place fetal bovine serum (FBS) and DMEM culture medium in a 37°C water bath to preheat. Then quickly take out the 3T3 cells stored in liquid nitrogen and heat and melt them in a 37°C water bath. In a clean bench, place 4 mL of culture medium (20% FBS and 80% DMEM) into a 25 cm ² cell culture flask, then inject 1 mL of thawed cells into it, and culture it in a 37°C, 5% CO2 incubator.

2) Cell passaging: First, place fetal bovine serum (FBS), DMEM medium, PBS solution, and trypsin in a 37°C water bath to preheat. When the above-mentioned cells are attached to the bottom of the culture flask and grown to be suitable for passage, discard the supernatant and slowly wash twice with PBS solution in a clean bench. Add 1 mL of trypsin and place the culture bottle in a constant-temperature incubator for 2 minutes to fully digest the cells attached to the bottom of the culture bottle. Add another 2-3 mL of culture medium (10% FBS and 90% DMEM) to stop digestion, and pipet off the cells on the wall. Then transfer the cell suspension to a 15 mL centrifuge tube, centrifuge in a centrifuge at 4°C, 1000 rpm for 5 min and discard the supernatant. Resuspend the cells in 15 mL of culture medium (10% FBS and 90% DMEM) and place them in a 75 cm2 cell culture flask, and culture them in a 37°C, 5% CO2 incubator.

The fibers were fixed in a 6-well plate and sterilized using ultraviolet light in a clean bench for 30 minutes. The cultured cells were digested according to the above steps, centrifuged, and resuspended in 12 mL of culture medium (10% FBS and 90% DMEM). Then, 2 mL per well was injected into a 6-well plate containing fiber, and cultured in a 37°C, 5% CO2 incubator. After the cells attach to and around the fiber surface, cell viability is verified using cell live-dead staining.

In the following examples, the specific operation of the live and dead cell staining method is as follows:

Dilute the Calcein-AM reagent with 50 μL of DMSO solution to a final concentration of 1 mmol/L. Add 5 μL of Calcein-AM reagent with a concentration of 1 mmol/L into 1 mL of PBS solution to obtain a PBS-Calcein-AM solution. Add 20 μL of this solution to each well and incubate for 20 min in a 37°C, 5% CO2 incubator. Protect from light during operation. After completion, add 10 μL of propidium iodide (PI) and incubate for 10 min in a 37°C, 5% CO2 incubator. Then observe under a confocal microscope. Calcein-AM is excited at a wavelength of 495nm to make it emit green light, and PI is excited at a wavelength of 539nm to make it emit red light.

In the following examples, the detection methods for liver function and kidney function tests are:

To further verify the safety of mixed fiber, we wrapped 20cm of fiber in bread and fed it to BALB/c mice for 7 consecutive days. We took 1mL of whole blood from mice on the 9th day. Let the whole blood rest for 12 hours in a refrigerator at 4 degrees Celsius. After stratification, centrifuge at 1000 rpm for 1 minute and take the upper serum. The serum was entrusted to a biological company for testing.

Methods for constructing expression vectors for fusion proteins:

1) Construct basic gene elements: Coding sequences concatenated in the following order: NdeI+NheI+coding sequence of amino acid sequence units+SpeI+(CAC) ₆ +TAATGA+EcoRI, where the NdeI enzyme cleavage sites are CATATG and NheI enzyme cleavage sites The site is GCTAGC, the SpeI restriction site is ACTAGT, (CAC) ₆ is the histidine tag sequence, TAATGA is the stop codon and the EcoRI restriction site is GAATTC. Construct basic genetic elements [S1–ELP1] and basic genetic elements [S2–ELP2] respectively, specifically:

Basic gene element [S1–ELP1]: is a coding sequence concatenated in the following order: NdeI+NheI+coding sequence of the amino acid sequence shown in SEQ IDNO:4+SpeI+(CAC) ₆ +TAATGA+EcoRI.

Basic gene element [S2–ELP2]: is a coding sequence concatenated in the following order: NdeI+NheI+coding sequence of the amino acid sequence shown in SEQ IDNO:5+SpeI+(CAC) ₆ +TAATGA+EcoRI.

The basic genetic elements [S1–ELP1] and [S2–ELP2] are artificially synthesized, for example, by Suzhou Jinweizhi Company.

A possible concatenation method of amino acid sequence units is as follows:

1) Obtain genetic element 2; connect the basic genetic element (1 or 2) and pbluescript II ks plasmid (commercially available, referred to as m13 here) through NheI and SpeI double enzyme digestion to obtain a chimeric protein monomer vector, referred to as m13 -monomer; m13-monomer was digested with NheI and SpeI to obtain gene element 2.

2) Obtain the chimeric protein dimer vector (referred to as the m13-dimer vector): combine the genetic element 2 and the m13-monomer digested by NheI under the action of T4 ligase (purchased from TaKaRa Company), and obtain Chimeric protein dimer vector (m13-dimer). Among them, the SpeI sticky end at the tail of gene element 2 is connected to the NheI sticky end of the m13-monomer linear fragment, and the junction becomes ACTAGC (the encoded amino acid sequence is TS).

3) Obtaining the chimeric protein tetramer vector (referred to as the m13-tetramer vector): Obtain the chimeric protein dimer gene fragment (chimeric protein dimer) from the m13-dimer through double enzyme digestion with NheI and SpeI. The body gene fragment can be expressed as: NheI sticky end + coding sequence of amino acid sequence unit + ACTAGC + coding sequence of amino acid sequence unit + SpeI sticky end), chimeric protein dimer gene fragment and m13-dimer digested by NheI , under the action of T4 ligase, a chimeric protein tetramer vector (referred to as m13-tetramer vector) was obtained.

The chimeric protein tetramer gene fragment obtained by double digestion of NheI and SpeI with the chimeric protein tetramer vector (referred to as m13-tetramer vector) can be expressed as: NheI sticky end + coding sequence of the amino acid sequence unit + ACTAGC + amino acid The coding sequence of the sequence unit + ACTAGC + the coding sequence of the amino acid sequence unit + ACTAGC + the coding sequence of the C-ELP protein unit + SpeI sticky end.

4) According to the above method, chimeric protein octamer vectors (referred to as m13-octamer vectors), chimeric protein dodecamer vectors (referred to as m13-dodecamer vectors), and chimeric protein twenty-four Polymer vectors (collectively referred to as m13-polymer vectors) and other multimeric vectors (referred to as m13-tetramer vectors) are then connected to the pET25b plasmid through NdeI and EcoRI double enzyme digestion to form the expression vector pET25b-polymer vector. The polymer is ready to be transferred into the recA recombinase-deficient E. coli BLR (DE3) expression system for IPTG-induced expression.

5) In order to optimize the protein, the carboxyl domain fragment (SpeI + the coding sequence of the amino acid sequence shown in SEQ ID NO: 7 + SpeI) was digested with SpeI, and then the m13-polymer vector was digested with SpeI enzyme. Under the action of T4 ligase, the m13-polymer vector with CTD sequence is obtained; then it is connected to the pET25b plasmid through double enzyme digestion with NdeI and EcoRI to form the expression vector pET25b-polymer-CTD. According to the sequence of the amino acid sequence unit Depending on the quantity, a variety of expression vectors are available, including:

The dodecamer expression vector pET25b-[S1–ELP1] ₁₂ -CTD composed of encoding the basic gene element [S1–ELP1] (the connecting amino acid between the adjacent amino acid sequences shown in SEQ ID NO:4 is TS , there is also a connecting amino acid TS between the carboxyl domain and the amino acid sequence shown in SEQ ID NO:4); the fusion protein expressed by this expression vector is named SP-1-ELP-carboxyl domain (which contains the amino acid sequence shown in SEQ ID NO:9 the amino acid sequence shown).

The dodecamer expression vector pET25b-[S2-ELP2] ₁₂ -CTD composed of encoding the basic gene element [S2-ELP2] (the connecting amino acid between the adjacent amino acid sequences shown in SEQ ID NO:5 is TS , there is also a connecting amino acid TS between the carboxyl domain and the amino acid sequence shown in SEQ ID NO:5); the fusion protein expressed by this expression vector is named SP-2-ELP-carboxyl domain (which contains the amino acid sequence shown in SEQ ID NO:11 the amino acid sequence shown).

The construction of the co-expression vector is shown in Figure 1. The amino acid sequence encoding the amino acid sequence shown in SEQ ID NO: 6 is added to the expression vector pET25b-[S1–ELP1] ₁₂ -CTD and pET25b-[S2–ELP2] ₁₂ -CTD. The amino domain of natural spider silk. The specific method is as follows:

Add an amino domain to the expression vector pET25b-[S1–ELP1] ₁₂ -CTD: use PCR technology to encode the amino domain fragment encoding the amino acid sequence shown in SEQ ID NO:6 The amino acid sequence of the gene sequence is coupled with homology arm 1 (the base sequence is shown in SEQ ID NO: 12: TTTAACTTTAAGAAGGAGATATACATATGGGTCAAGCGAACACCC) and homology arm 2 (the base sequence is shown in SEQ ID NO: 13: GTACGGACCGCTAGCCATATGACTGCCACCGCCACCGCTACCGCCACCGCCACTTACGGGTTGTTGCCC), Then, the chimeric protein dodecamer vector (pET25b-SP-1-ELP-CTD) was digested with NdeI enzyme, and the homologous recombination reaction catalyzed by recombinase in vitro was carried out using the recombinase ClonExpress II purchased from Vazyme Company. (can be carried out at a position within ~10 bp near the nick of the vector), perform homologous recombination, and obtain the expression vector pET25b-NTD-SP-1-ELP-CTD with amino domain and carboxyl domain, and obtain the fusion protein NTD- SP-1-ELP-CTD, which contains the amino acid sequence shown in SEQ ID NO:8.

Add the amino domain to the expression vector pET25b-[S2–ELP2] ₁₂ -CTD: the method is as above, and use PCR technology to add the amino domain fragment encoding the amino acid sequence shown in SEQ ID NO:6 to the amino domain fragment encoding the amino acid sequence shown in SEQ ID NO The gene sequence of the amino acid sequence shown in :6 is added to both ends of the homology arm 1 (the base sequence is shown in SEQ ID NO: 12: TTTAACTTTAAGAAGGAGATATACATATGGGTCAAGCGAACACCC)), and the homology arm 3 (the base sequence is shown in SEQ ID NO: 14 Display: GCAGCCGCAGAAGAGCCGCTAGCCATATGACTGCCACCGCCACCGCTACCGCCACCGCCACTTACGGGTTGTTGCCCGCG). The expression vector pET25b-NTD-SP-2-ELP-CTD with amino domain and carboxyl domain was obtained, and the fusion protein NTD-SP-2-ELP-CTD was obtained, which contains the amino acid shown in SEQ ID NO:10 sequence.

To construct a co-expression plasmid, the expression vector pET25b-NTD-SP-1-ELP-CTD was double-digested with BgIII enzyme and BamHI enzyme, and then pET25b-NTD-SP-2-ELP-CTD) was digested with BgIII enzyme. Under the action of T4 ligase purchased from TaKaRa Company, the two proteins were cloned into the same expression vector to obtain the recombinant expression vector pET25b-NTD-SP-1-ELP-CTD-NTD-SP-2 as shown in Figure 1C. -ELP-CTD (which contains the amino acid sequence shown in SEQ ID NO: 8, 10).

Example 1: Preparation of single recombinant spider silk protein fiber

The expression vectors pET25b-[S1–ELP1] ₁₂ -CTD and pET25b-[S2–ELP2] ₁₂ -CTD were respectively expressed in recA recombinase-deficient engineered E. coli BLR (DE3) and then purified to obtain lyophilized SP-1. -ELP-carboxy domain fused spider silk protein and SP-2-K-carboxyl domain fused spider silk protein are respectively dissolved in 200 μL hexafluoroisopropanol at a protein concentration of 150 mg/L. Use a 1mL syringe to draw the protein solution, and select Use a needle with an inner diameter of 200 μm, and then squeeze the protein solution into a 90% methanol aqueous solution (coagulation bath), and use a syringe pump to adjust the propulsion speed to 5 μl/min, and use a rotating drum collector to collect the fibers at a linear speed of 0.6 m/min. (Figure 2), primary fibers are obtained. The collected fibers were soaked in 50% methanol until the fibers became soft, and then the fibers were taken out and immediately stretched to about 200% of the original length to obtain the post-stretched fibers, and the mechanical properties of the prepared fibers were tested (Figure 3- 4).

Figure 3 results show that the tensile strength of SP-1-ELP-carboxyl domain fusion protein before stretching (primary fiber) is 102.68±5.76MPa and the toughness is 246.9±17.9MJ/m ³ ; after stretching (post-stretching) The tensile strength of the fiber is 242.85±7.12MPa, and the toughness is 57.19±3.98MJ/m ³ . It shows that after stretching, the tensile strength increases and the toughness decreases.

Figure 4 results show that the tensile strength of SP-2-ELP-carboxyl domain fusion protein before stretching (primary fiber) is 142.32±7.99MPa and the toughness is 286.82±27.28MJ/m ³ ; after stretching (post-stretching) The tensile strength of the fiber is 269.65±13.33MPa, and the toughness is 69.34±9.61MJ/m ³ . It shows that after stretching, the tensile strength increases and the toughness decreases.

Example 2: Preparation of two kinds of recombinant spider silk protein mixed fibers in vitro

The two proteins in Example 1, SP-1-ELP-carboxy domain fusion protein and SP-2-K-carboxy domain fusion protein, were dissolved in 200 μL hexafluoroisopropanol at a weight ratio of 3:2 ( Protein concentration 150mg/L), centrifuge the mixed protein solution, take the supernatant and squeeze it into 90% methanol solution (coagulation bath) with a syringe, and use a syringe pump to adjust the propulsion speed to 5μl/min, and use a rotating drum collector to The fibers are collected at a linear speed of 0.6m/min to obtain primary fibers. The collected fibers were soaked in 50% methanol until the fibers became soft, and then the fibers were taken out and immediately stretched to about 200% of their original length to obtain post-stretched fibers. The prepared fibers were tested for mechanical properties (Figure 5)

Figure 5 results show that the tensile strength of fibers obtained by physically mixing SP-1-ELP-carboxy domain fusion protein and SP-2-K-carboxy domain fusion protein in a ratio of 3:2 before stretching (primary fiber) The tensile strength after stretching (post-stretched fiber) is 362.83±11.23MPa, and the tested toughness is 174.76±9.79MJ/m ^{3 .} It shows that after stretching, the tensile strength increases and the toughness decreases.

It was also shown that physical mixing of the two fusion proteins resulted in an increase in the tensile strength of the fibers relative to a single fusion protein.

Example 2A

The difference between this example and Example 2 is that the SP-1-ELP-carboxyl domain fusion protein and SP-2-K-carboxyl domain fusion protein are mixed at a ratio of 2:3, and the tensile strength after stretching is 293.45 ±14.33MPa, toughness 100.36±18.02MPa, modulus 5.91±0.99GPa.

Example 3: Preparation of two recombinant spider silk co-expressed protein fibers in vivo

The expression vector pET25b-NTD-SP-1-ELP-CTD-NTD-SP-2-ELP-CTD was expressed in recA recombinase-deficient engineered E. coli BLR (DE3) and purified to obtain the co-expressed protein, which was dissolved in 98 % formic acid (protein concentration 150mg/L), centrifuge the mixed protein solution, take the supernatant and squeeze it into the methanol solution (coagulation bath) with a syringe, and use a syringe pump to adjust the propulsion speed to 5μl/min, and collect it with a rotating drum The machine collects fibers at a linear speed of 0.6m/min to obtain primary fibers. The collected fibers were soaked in 50% methanol until the fibers became soft, and then the fibers were taken out and immediately post-stretched to about 100% of their original length to obtain post-stretched fibers. The prepared fibers were tested for mechanical properties (Figure 6).

The results of Figure 6 show that the fiber obtained after co-expression of the fusion protein of the amino domain-SP-1-ELP-carboxyl domain and the amino domain-SP-2-K-carboxyl domain, before stretching (primary fiber) The tensile strength is 232.41±5.89MPa, and the tested toughness is 355.05±30.75MJ/m ³ ; the tensile strength after stretching (post-stretched fiber) is 336.21±27.37MPa, and the tested toughness is 86.93±6.11MJ/m ³ . It shows that after stretching, the tensile strength increases and the toughness decreases.

Example 4: Performance comparison between protein fibers prepared by the present invention

The present invention uses two kinds of 12-mer recombinant spider silk protein fibers with carboxyl groups. The mechanical properties of the fibers prepared by adjusting the sequence and mixing ratio are different. A comparison of their strength, toughness, and modulus properties is shown in Table 1;

Table 1 Performance of hybrid fibers before and after stretching in each embodiment

The comprehensive mechanical properties of the mixed fibers are better than those of single protein fibers. The toughness of the recombinant fused spider silk fiber formed by mixing natural spider silk components in a ratio of 3:2 is comparable to that of natural spider silk, which provides new ideas for exploring and improving the mechanical properties of protein fibers.

Example 5: Cytotoxicity experiment of mixed protein fibers in vitro and co-expressed protein fibers in vivo

3T3 cells (mouse embryonic fibroblasts, purchased from Shanghai Jihe Biological Company) were cultured in DMEM supplemented with 10% fetal calf serum and 1% double antibodies (penicillin and gentamicin). 3T3 cells were used to detect the cytotoxicity of two protein fibers. Before the test, the two proteins of SP-1-ELP-carboxy and SP-2-K-carboxy prepared in Example 2 were mixed in a ratio of 3:2 using tape that had been sterilized at high temperature at 121°C for 30 minutes to make protein fiber ( Figure 7 (A), (B)) and the in vivo co-expression protein fibers prepared in Example 3 (Figure 7 (C), (D)) were fixed on the bottom of a 12-well plate, and then sterilized in a UV cabinet for 12 hours. The cells were injected into a 12-well plate at 50,000 cells/mL and co-cultured with fibers for 24 hours. Remove the culture medium and wash three times with PBS buffer. Add 1 μL Calcein-AM to stain active living cells and 1 μL propidium iodide (PI) to stain apoptotic cells and incubate for 10-20 minutes. The two protein fibers and control cells were observed using laser confocal scanning microscopy (LSCM) under AM emission wavelengths of 495 nm and PI of 539 nm. Experiments show that cells can grow on both fibers, indicating that the fibers have good biocompatibility.

Example 6: In vitro biological safety experiment of mixed protein fibers

The two proteins of Example 2, SP-1-ELP-carboxy: SP-2-K-carboxy, were mixed in a ratio of 3:2 to prepare protein fibers. The spun fibers were fed to BALB/c mice with 20 cm fiber per day, and 6 BALB/c mice were fed every day, and their body weights were recorded every day. The control group consisted of 6 BALB/c mice that were not fed fiber, and their body weight was recorded every day. One week later, ≈1 mL of blood was taken from the mouse orbit. Serum was obtained by resting in a refrigerator at 4°C for 24 hours. Then centrifuge at 3000 rpm in a 4°C centrifuge for 15 minutes and take the supernatant. Use a fully automatic biochemical analyzer to detect liver and kidney function indicators. 13 blood biochemical indicators related to liver and kidney function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), ALT/AST, total protein (TP), albumin (ALB), Globulin (GLOB), albumin/globulin (A/G), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transferase (GGT), uric acid (UA), creatinine (CREA) and carbon dioxide (CO2), and calculate the significant difference between each experimental group and the control group. From the laboratory index data, it can be seen that except for alkaline phosphatase (ALP), there is no significant difference in other indicators between the experimental group and the control group. Difference (p>0.05). The difference in the single index of ALP may be caused by individual differences. These results indicate that the hybrid protein fibers have good biosafety. This opens up possibilities for subsequent applications in biomedicine and medical devices. (Figure 8)

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and illustration. These descriptions are not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teachings. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical applications, thereby enabling others skilled in the art to make and utilize various exemplary embodiments of the invention and various different applications. Choice and change. Any simple modifications, equivalent changes and modifications made to the above exemplary embodiments should fall within the protection scope of the present invention.

Claims (24)

1. Recombinant spider silk protein, which contains at least two recombinant proteins: SP-1-K fusion protein and SP-2-K fusion protein, The SP-1-K fusion protein and SP-2-K fusion protein are fusion proteins obtained by blending in vitro at a weight ratio (0.6~1.5):1 or co-expressing in microorganisms; The SP-1-K fusion protein is any one of the following A1), A2), A4), and A5) proteins: A1) Its amino acid sequence includes: m directly connected amino acid sequence units in the form of [S1-ELP1], where S1 represents the amino acid sequence as shown in SEQ ID NO: 1, and ELP1 is an elastin-like sequence. , [S1–ELP1] represents the concatenation of S1 sequence and ELP1 sequence; m represents the repeat number of [S1–ELP1] sequence, m is 12; the amino acid sequence of the amino acid sequence unit [S1–ELP1] is as shown in SEQ ID NO: 4; A2) Its amino acid sequence includes: there is a connecting sequence between adjacent amino acid sequence units [S1–ELP1] in A1), and the connecting sequence includes two or four amino acids; A4) Its amino acid sequence includes: 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) Its amino acid sequence includes: introducing a histidine tag at the N-terminus and/or C-terminus of A1) or A2) or A4); The SP-2-K fusion protein is any one of the following B1), B2), B4), and B5): B1) Its amino acid sequence includes: n directly connected amino acid sequence units as shown in [S2-ELP2], where S2 represents the amino acid sequence as shown in SEQ ID NO:2; ELP2 is an elastin-like sequence ; [S2–ELP2] represents the concatenation of S2 sequence and ELP2 sequence; n represents the repeat number of [S2–ELP2] sequence, n is 12; the amino acid sequence of the amino acid sequence unit [S2–ELP2] is as shown in SEQ ID NO: 5; B2) Its amino acid sequence includes: a connecting sequence is connected between the adjacent amino acid sequence units [S2–ELP2] in B1), and the connecting sequence includes two or four amino acids; B4) Its amino acid sequence includes: 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 includes: introducing a histidine tag at the N-terminus and/or C-terminus of B1) or B2) or B4). 2. The recombinant spider silk protein according to claim 1, characterized in that in the A2) protein of the SP-1-K fusion protein, the connecting sequence is digested with different enzyme cleavage sites of the same tail and then connected. Coded for two or four amino acids. 3. The recombinant spider silk protein according to claim 1 or 2, characterized in that in the B2) protein of the SP-2-K fusion protein, the connecting sequence is digested with different enzyme cleavage sites of the same tail and then Two or four amino acids encoded when linked. 4. The recombinant spider silk protein according to claim 1, characterized in that in the A4) protein of SP-1-K fusion protein and/or the B4) protein of SP-2-K fusion protein, the amino structure of natural spider silk The domain contains the amino acid sequence shown in SEQ ID NO:6. 5. The recombinant spider silk protein according to claim 1, characterized in that in A4) of the SP-1-K fusion protein and/or B4) of the SP-2-K fusion protein, the carboxyl domain of the natural spider silk Contains 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 as 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 as shown in SEQ ID NO: 10 or 11. 8. The recombinant spider silk protein according to claim 1, wherein the weight ratio of the SP-1-K fusion protein and SP-2-K fusion protein is 1.5:1 or 2:3. 9. A recombinant spider silk protein mixed fiber, which includes the recombinant spider silk protein according to any one of claims 1 to 8, and is artificially spun. 10. A method for preparing recombinant spider silk protein mixed fiber according to claim 9, characterized in that it includes: Dissolve the SP-1-K fusion protein and SP-2-K fusion protein in a solvent to prepare a protein solution, and use a syringe pump to extrude the protein solution into a coagulation bath to solidify into nascent fibers. 11. The preparation method according to claim 10, characterized in that the solvent is hexafluoroisopropanol or formic acid. 12. The preparation method according to claim 10, characterized in that the SP-1-K fusion protein and SP-2-K fusion protein are dissolved in hexafluoroisopropanol according to a weight ratio (0.6~1.5): 1 Prepare protein solution. 13. The preparation method according to claim 12, characterized in that the mass fraction of the SP-1-K fusion protein and SP-2-K fusion protein in the protein solution is 150~200 mg/mL. 14. The preparation method according to claim 12, characterized in that 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 preparation method according to claim 10, characterized in that the SP-1-K fusion protein and SP-2-K fusion protein are respectively expressed and purified by recombinant Escherichia coli. 16. The preparation method according to claim 10, characterized in that the SP-1-K fusion protein and SP-2-K fusion protein are obtained by co-expression in recombinant Escherichia coli and then purified. 17. The preparation method according to claim 16, characterized in that it is dissolved in formic acid solution. 18. The preparation method according to claim 10, characterized in that the coagulation bath is a methanol solution of 80%-100% by volume. 19. The preparation method according to claim 10, characterized in that the coagulation bath is a 90% methanol solution. 20. The preparation method according to any one of claims 10 to 19, characterized in that it also includes post-stretching of the nascent fiber: after soaking the nascent fiber in a stretching bath to soften it, it is stretched to its original length. 2~5 times, and then stretch the fiber. 21. Recombinant spider silk protein mixed with the recombinant spider silk protein according to any one of claims 1 to 8, or the recombinant spider silk protein mixed fiber according to claim 9, or the recombinant spider silk protein prepared by the preparation method according to any one of claims 10 to 20 Mixed fiber-related biomaterials, characterized in that the biomaterials are 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 the recombinant spider silk protein according to any one of claims 1 to 8; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2); C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3); C5) A recombinant mammalian cell containing the nucleic acid molecule described in C1), or a recombinant mammalian cell containing the expression cassette described in C2), or a mammalian cell containing the recombinant vector described in C3); C6) A recombinant insect cell containing the nucleic acid molecule described in C1), or a recombinant insect cell containing the expression cassette described in C2), or an insect cell containing the recombinant vector described in C3). 22. The biological material according to claim 21, characterized in that in C3), the recombinant vector adopts pET25b plasmid or pbluescript II ks plasmid. 23. The biological material according to claim 21, characterized in that in C4), the recombinant microorganism is recombinant Escherichia coli, Bacillus subtilis or yeast cells. 24. A recombinant spider silk protein according to any one of claims 1 to 8 or a recombinant spider silk protein mixed fiber according to claim 9 or a recombinant spider silk protein prepared by the preparation method according to any one of claims 10 to 20 The use of mixed fibers includes any one or more of the following D1) to D5): D1) Aerospace field, military field or preparation of biological materials; D2) Paper products; D3) thin film; D4) Textiles; D5) Coating.