CN117164721A - Fusion spider silk protein, spider silk protein fiber and application thereof - Google Patents
Fusion spider silk protein, spider silk protein fiber and application thereof Download PDFInfo
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Abstract
The invention discloses a fusion spider silk protein, spider silk protein fiber and application thereof, wherein the fusion spider silk protein is formed by fusion expression of a segment of sequence S in recombinant spider silk protein; the amino acid sequence of the recombinant spider silk protein comprises an N-terminal sequence, a middle sequence M and a C-terminal sequence, wherein the amino acid sequence of the N-terminal sequence is shown as SEQ ID NO. 1; the amino acid sequence of the intermediate sequence M is shown as SEQ ID NO. 2; the amino acid sequence of the C-terminal sequence is shown as SEQ ID NO. 3; the amino acid sequence of the sequence S is shown as SEQ ID NO.4, and the sequence S is expressed in a fusion way between the intermediate sequence M and the C-terminal sequence. Compared with the spider silk fiber obtained by spinning the fusion spider silk protein containing the S sequence and the spider silk fiber obtained by recombining the spider silk protein without the S sequence, the mechanical strength of the fiber is obviously improved to 3-4 times; can be widely applied to the fields of medicine, chemical industry or military industry.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a fusion spider silk protein, spider silk protein fiber and application thereof.
Background
The spider silk has excellent mechanical property and biocompatibility, is an excellent biological material, and has great potential application value in the fields of medical engineering, military aviation and the like; however, since the spiders are solitary organisms and can be eaten in the same kind, the spiders are difficult to feed on a large scale, the spinning quantity of the spiders is far less than that of silkworms, and the spider silk fibers cannot be obtained on a large scale; thus severely hampering the production of natural spider silk fibers and their large-scale use in life.
Along with the development of biotechnology, people begin to explore the preparation of bionic spider silk by means of genetic engineering to solve the problem that spider silk fibers are difficult to obtain on a large scale. However, the spider species and the produced silk fibers are various, and the molecular weight of the complete spider silk protein is large, so that the whole spider silk protein coding gene is large and the GC content in the sequence is high. Therefore, it is difficult for general expression vectors to accomplish their full-length expression; in addition, the ratio of gene repeats of spidroin is high and the properties of different types of spidroin fibers are believed to be determined by the repeats. The spider silk protein only containing one middle repeat region has relatively high expression level, but the fiber obtained by spinning has relatively poor mechanical properties, so that the application of the spider silk protein in the fields of bioengineering, aerospace and the like is greatly limited.
The Chinese patent publication No. CN105755025A discloses a preparation method of recombinant spider silk protein, which takes the full-length coding gene of major ampullate hypo gland silk (Misp) and the complete repeat region of piriform gland silk (PySp) as templates, adopts fusion PCR technology to amplify the NT end gene fragment MiSp-NT of the hypo ampullate gland silk MiSp by PCR for splicing, and then obtains the gene fragment PySp-Rp of the complete repeat region by PCR amplification; and is connected with a pET28a vector to construct and recombine and transfer into BL21 for expression. The method directly adopts the N-terminal non-repetitive region and one repetitive region of PySp in the full-length coding gene of MiSp to prepare the recombinant spider silk protein. However, the mechanical properties of the spider silk protein synthesized by a repeated region are not high, and are much weaker than those of natural spider silk fibers, so that it is necessary to develop a spider silk protein which can form spider silk fibers with stronger mechanical properties, and thus, the artificial spider silk protein fibers with excellent mechanical properties are prepared.
Disclosure of Invention
The invention aims to develop a fusion spider silk protein through genetic engineering, and the spider silk protein fiber with higher mechanical strength is prepared by using the protein. It is another object of the present invention to provide a method for preparing the fusion spider silk protein and its use.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a fusion spider silk protein formed by fusion expression of a segment of sequence S in a recombinant spider silk protein; the amino acid sequence of the recombinant spider silk protein comprises an N-terminal sequence, a middle sequence M and a C-terminal sequence, wherein the amino acid sequence of the N-terminal sequence is shown as SEQ ID NO. 1; the amino acid sequence of the intermediate sequence M is shown as SEQ ID NO. 2; the amino acid sequence of the C-terminal sequence is shown as SEQ ID NO. 3; the amino acid sequence of the sequence S is shown as SEQ ID NO.4, and the sequence S is expressed in a fusion way between the intermediate sequence M and the C-terminal sequence.
The fusion spider silk protein is formed by inserting a section of sequence S into the recombinant spider silk protein, and the S sequence is inserted between the intermediate sequence M and the C end sequence of the recombinant spider silk protein, so that the mechanical strength of the prepared spider silk protein fiber is obviously improved.
Further, the amino acid sequence of the fusion spider silk protein is shown as SEQ ID NO. 5.
Further, the fusion spider silk protein is prepared by the following method:
s1, performing codon optimization according to the codon preference of escherichia coli, and obtaining a gene segment NMSC for expressing the fusion spider silk protein through gene synthesis, wherein the nucleotide sequence of the gene segment NMSC is shown as SEQ ID NO. 6;
s2, inserting the gene segment NMSC into a vector through enzyme digestion, and constructing to obtain a recombinant vector;
s3, transforming the recombinant vector into receptor cells, inducing the receptor cells to fuse the expression of gene segment NMSC of the spider silk protein, and collecting inclusion bodies of the fused spider silk protein to obtain the fused spider silk protein.
Preferably, the vector is inserted by cleavage as described in step S2, and the vector is plasmid PET-32a.
Preferably, the restriction enzymes used for the cleavage in step S2 are NdeI and XhoI enzymes.
Preferably, the recipient cell in step S3 is e.coli BL21 (DE 3).
Preferably, after the recombinant vector is transformed into the recipient cell in step S3, the recipient cell is inoculated into an LB medium containing ampicillin resistance to verify whether the NMSC gene fragment is expressed.
Preferably, the induction in step S3 is performed by adding IPTG as an inducer to the medium in which the recipient cells are located.
Preferably, after the induction in step S3 is completed, the bacterial cells need to be disrupted.
The invention also claims a nucleotide sequence for expressing the fusion spider silk protein.
The invention also claims an expression vector comprising the nucleotide sequence.
The invention also claims a receptor cell comprising the above expression vector.
The invention also provides application of the sequence S in improving the mechanical strength of recombinant spider silk protein fibers, and spider silk protein fibers prepared from the fusion spider silk proteins are formed by fusion expression of the sequence S into the recombinant spider silk proteins; the amino acid sequence of the fusion spider silk protein is shown as SEQ ID NO. 5.
Since spidroin fibers with improved mechanical strength can be prepared from the fusion spidroin described above, the invention is also claimed:
a spider silk protein fiber comprising the fusion spider silk protein described above.
The preparation method of the spider silk protein fiber comprises the steps of preparing the fusion spider silk protein into completely dehydrated protein powder, dissolving the protein powder by using a solvent capable of dissolving the protein powder to prepare a spinnable fiber solution, and carrying out wet spinning on the spinnable fiber solution to obtain the spider silk protein fiber, wherein the solvent capable of dissolving the protein powder is 100% hexafluoroisopropanol HFIP.
Further, the preparation method of the fully dehydrated protein powder comprises the following steps: dialyzing the fusion spider silk protein into pure water, and freeze-drying a pure water solution containing the fusion spider silk protein to remove all water to obtain completely dehydrated protein powder.
The invention also claims the application of the spider silk protein fiber in preparing or being used as medical suture, carbon nano tube material and textile.
Compared with the prior art, the invention has the beneficial effects that:
(1) Compared with the recombinant spider silk protein prepared by only using a gene segment of one middle region of the minor ampullate gland silk of the natural spider silk protein, the spider silk protein fiber prepared by the fusion spider silk protein has obviously improved mechanical strength which is 3-4 times that of the spider silk protein fiber prepared by the recombinant spider silk protein.
(2) The sequence S is inserted into the recombinant spider silk protein to form fusion spider silk protein, so that the spider silk protein fiber is prepared by the fusion spider silk protein, the mechanical strength of the spider silk protein fiber can be obviously improved, and the spider silk fiber can be applied to the fields of medicine, chemical industry or military industry, in particular to the application in preparing or serving as medical suture lines, carbon nano tube materials and textiles.
Drawings
FIG. 1 is a schematic diagram showing the construction process of the recombinant spidroin expression vector PE-NMC and the fusion spidroin expression vector PE-NMSC according to the invention.
FIG. 1A shows recombinant spider silk protein expression vector PE-NMC; b represents a fusion spider silk protein expression vector PE-NMSC.
FIG. 2 is a graph showing the results of purification of recombinant spidroin NMC and fusion spidroin NMSC expression. NMC means recombinant spider silk proteins expressed by NMC gene fragments; NMSC represents fusion spider silk proteins expressed by NMSC gene fragments.
Fig. 3 is a schematic representation of the wet spinning process of recombinant and fusion spider silk proteins NMCs.
In fig. 3, reference 1 indicates a syringe fixing and self-advancing device; 2 represents a syringe; 3 represents a spinning dope; 4 represents a coagulation bath; 5 represents a silk fiber; 6 denotes a winding device.
Fig. 4 is a sample of silk fibers prepared from recombinant spider silk protein NMC and fusion spider silk protein NMSC.
NMC in FIG. 4 represents a silk fiber sample prepared from recombinant spider silk protein NMC; NMSC represents a silk fiber sample prepared by fusion of spider silk protein NMSC.
Fig. 5 is a graph of mechanical strength measurements of silk fibers of recombinant spidroin NMC and fusion spidroin NMSC.
NMC in FIG. 5 represents silk fibers prepared from recombinant spider silk proteins expressed by NMC gene fragments; NMSC represents silk fibers made from fusion spider silk proteins expressed by NMSC gene fragments.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only 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.
The following examples relate to the nucleotide or amino acid sequences as follows:
example 1 fusion spider silk protein NMSC and preparation thereof
A fusion spider silk protein, which is formed by fusion expression of a sequence S in recombinant spider silk protein; the amino acid sequence of the fusion spider silk protein comprises an N-terminal sequence, a middle sequence M and a C-terminal sequence, wherein the sequence of the N-terminal sequence is shown as SEQ ID NO. 1; the sequence of the intermediate sequence M is shown as SEQ ID NO. 2; the sequence of the C-terminal sequence is shown as SEQ ID NO. 3; the amino acid sequence of the sequence S is shown as SEQ ID NO.4, the sequence S is inserted between the intermediate sequence M and the C-terminal sequence of the recombinant spider silk protein, and the amino acid sequence of the fusion spider silk protein is shown as SEQ ID NO. 5.
The fusion spider silk protein is prepared by the following method:
s1, taking a full-length gene sequence of major-abdomen garden spider minor ampullate gland silk as a template, carrying out codon optimization according to the codon preference of escherichia coli, and obtaining a gene segment NMSC for expressing the fusion spider silk protein through gene synthesis, wherein the nucleotide sequence of the gene segment NMSC is shown as SEQ ID NO.6, and the nucleotide sequence corresponding to the S sequence is shown as SEQ ID NO. 8;
s2, inserting a gene segment NMSC into a vector by using restriction enzymes NdeI and XhoI, wherein the vector is plasmid PET-32a, and constructing to obtain a recombinant vector;
s3, transforming the recombinant vector into receptor cells E.coli BL21 (DE 3), inoculating the receptor cells into LB culture medium containing ampicillin resistance, adding inducer IPTG into the culture medium where the receptor cells are located, inducing the expression of gene segment NMSC of fusion spider silk protein of the receptor cells, collecting escherichia coli thalli, removing supernatant after crushing, collecting inclusion bodies containing the fusion spider silk protein, and dissolving through 8M urea after washing to obtain the fusion spider silk protein.
The method comprises the following specific steps:
(1) Optimally synthesizing fusion gene NMSC according to E.coli codons, inserting the fusion gene NMSC into a vector PET-32a through NdeI and XhoI cleavage sites, and constructing a fusion spider silk protein expression vector PE-NMSC as shown in FIG. 1B; wherein the sequence of the gene fragment NMSC is shown in SEQ ID NO. 6.
(2) The plasmid is transferred into a competent cell of E.coli BL21 (DE 3), and is shaken and cultured in LB medium containing ampicillin, IPTG with the final concentration of 1mM is added, and the E.coli BL21 (DE 3) is induced to express fusion spider silk protein at room temperature of 25 ℃ overnight.
(3) After the induction is finished, E.coli BL21 (DE 3) bacterial liquid is collected, a high-pressure homogenizer is used for crushing the E.coli, the supernatant is discarded, and the obtained inclusion body protein is repeatedly washed through precipitation of washing liquid to obtain pure fusion inclusion body protein, and the precipitation is collected to obtain the fusion spider silk protein existing in the form of inclusion body.
(4) Adding the fusion spider silk protein into Tris solution containing 8M urea, dissolving inclusion body protein through urea denaturation, dialyzing to double distilled water to remove urea, various salt particles and other impurities, wherein a 10K MWCO dialysis bag with the brand number of 68035 of the Simer yarn is selected as a dialysis bag, and dialyzing to obtain the fusion spider silk protein solution. Comparative example 1 recombinant spider silk protein and preparation thereof
A recombinant spider silk protein; the amino acid sequence of the recombinant spider silk protein comprises an N-terminal sequence, a middle sequence M and a C-terminal sequence, wherein the sequence of the N-terminal sequence is shown as SEQ ID NO. 1; the sequence M is shown as SEQ ID NO. 2; the sequence of the C-terminal sequence is shown as SEQ ID NO.3, and the amino acid sequence of the recombinant spider silk protein is shown as SEQ ID NO. 9.
The recombinant spider silk protein is prepared by the following method:
s1, carrying out code optimization by taking the full-length gene sequence of major-abdominal-garden spider minor ampullate gland silk as a template to obtain a gene segment NMC for expressing recombinant spider silk protein, wherein the nucleotide sequence of the gene segment NMC is shown as SEQ ID NO. 7;
s2, inserting a gene segment NMC into a vector by using restriction enzymes NdeI and XhoI, wherein the vector is plasmid PET-32a, and constructing to obtain a recombinant vector;
s3, transforming the recombinant vector into receptor cells E.coli BL21 (DE 3), inoculating the receptor cells into LB culture medium containing ampicillin resistance, adding IPTG into the culture medium where the receptor cells are positioned, inducing the expression of gene segment NMC of the recombinant spider silk protein of the receptor cells, and collecting inclusion bodies of the recombinant spider silk protein to obtain the recombinant spider silk protein.
The method comprises the following specific steps:
(1) Optimally synthesizing fusion gene NMC according to the escherichia coli codon, inserting the fusion gene NMC into a vector PET-32a through NdeI and XhoI enzyme cutting sites, and constructing a recombinant spider silk protein expression vector PE-NMC as shown in figure 1A; wherein the NMC sequence of the gene fragment is shown in SEQ ID NO. 7.
(2) Transferring the plasmid into a culture solution containing E.coli BL21 (DE 3), adding IPTG with a final concentration of 1mM, and inducing E.coli BL21 (DE 3) to produce recombinant spider silk protein at room temperature of 25 ℃.
(3) After the induction is finished, E.coli BL21 (DE 3) bacterial liquid is collected, a high-pressure homogenizer is used for crushing the E.coli, the supernatant is discarded, and the obtained inclusion body protein is repeatedly washed through precipitation of washing liquid to obtain pure fusion inclusion body protein, and the precipitation is collected to obtain the recombinant spider silk protein existing in the form of inclusion body.
(4) Adding the recombinant spider silk protein into Tris solution containing 8M urea, and dissolving inclusion body protein through urea denaturation to obtain recombinant spider silk protein solution.
EXAMPLE 2 preparation of spider silk protein fibers from fusion of spider silk proteins
Freezing and drying the fusion spider silk protein solution obtained in the example 1 at the temperature of minus 80 ℃ to obtain protein powder, wherein the freeze drying time is not less than 48 hours for thorough freeze drying; after lyophilization, the completely dried protein powder was dissolved in 100% hexafluoroisopropanol to prepare a spinning dope with a fusion spider silk protein concentration of 8% w/v, and the artificial spider silk protein fibers were obtained by a wet spinning apparatus.
The wet spinning process is shown in figure 3, a spinning solution 3 with the concentration of the fusion spider silk protein of 8% w/v is placed in a syringe 2, the flow rate is controlled to be 10ul/min through a syringe fixing and automatic propelling device 1, the syringe 2 adopts a 1mL specification, and the needle head model is 29G; the spinning dope 3 is pressurized and flows into a coagulation bath 4, and is gradually coagulated into a silk fiber 5, and the silk fiber 5 is separated from the coagulation bath 4 by the rotation of a winding device 6, so that the spider silk protein fiber prepared by the wet spinning of the fusion spider silk protein is obtained.
Comparative example 3 (spider silk protein fibers prepared from comparative example 1 protein)
The preparation method is the same as in example 2, except that the recombinant spidroin protein solution prepared in comparative example 1 is used as a raw material for lyophilization and wet spinning is performed to obtain the spidroin protein fiber.
Example 3 detection of protein by Polyacrylamide gel electrophoresis and its purity
1. Experimental method
The proteins obtained in example 1 and comparative example 1 were examined for purity by polyacrylamide gel (SDS-PAGE) electrophoresis. The specific operation steps are as follows:
(1) And (3) performing expansion culture on 500mL of escherichia coli respectively containing recombinant plasmids PE-NMC and PE-NMSC, adding 1mM IPTG to induce expression for 16h at 25 ℃, centrifuging at 3500rpm for 30min at 4 ℃, collecting thalli, adding 15mL of 10mM Tris buffer solution, placing at-80 ℃ for freezing for 1h, thawing at room temperature, and crushing by a high-pressure homogenizing machine to obtain crushed thalli.
(2) The crushed thalli of the previous step is centrifuged for 20min at 4 ℃ and 12000rpm, the supernatant is discarded, the sediment is collected and washed for 3 times to obtain pure NMC and inclusion body protein sediment of NMSC.
(3) Dissolving inclusion body protein with Tris buffer containing 8M urea, dialyzing to double distilled water, completely dialyzing, standing at-80deg.C for overnight, and standing in a freeze dryer for 48 hr or more to obtain NMC and NMSC lyophilized powder.
(4) The lyophilized powder was dissolved in Tris solution containing 8M urea, dissolved at room temperature for 10min, and centrifuged at 12000rpm for 5min to collect the supernatant.
(5) Taking 40ul of supernatant after centrifugation, adding 10ul of 5xSDS-loading, boiling for 10min at 100 ℃, taking 10ul of sample, and carrying out SDS-PAGE electrophoresis, coomassie brilliant blue staining and decolorization.
2. Experimental results
The electrophoresis result is shown in figure 2, and the figure 2 shows that NMC and NMSC proteins are successfully obtained, and the electrophoresis result shows that the molecular weight of the proteins is consistent with the theoretical value, and the subsequent spinning and mechanical strength performance test can be performed. Example 4 test of mechanical Strength of spider silk protein fibers
1. Experimental method
The artificial spider silk protein fibers prepared in example 2 and comparative example 3 were subjected to a mechanical strength test, and the mechanical properties tested were as follows: the stress and strain were measured as follows:
(1) The fiber structure was checked with an optical microscope to ensure uniformity and fiber diameter was measured with Image J software.
(2) Preparing a sample for performance test: two ends of the fiber were adhered to a paper frame having a length of 20mm, and 15 or more samples were prepared for each protein.
(3) Performance testing mechanical properties of synthetic fibers were measured on an INSTRON-3365 (dual arm materials tester) from INSTRON corporation, usa, at 20±2 ℃ and humidity of about 65±5%. These above samples were drawn at a constant strain rate of 10mm/min, a gauge length of 20mm, an initial load strength of 10uN, and at least 10 samples were tested for each type of silk fiber.
2. Experimental results
Samples for mechanical strength performance test as shown in fig. 4, the silk fibers obtained by spinning of protein NMC and NMSC were adhered to paper frames at both ends, respectively, and 15 silk fibers were obtained for each protein sample. As shown in fig. 5, as can be seen from fig. 5, the fusion spidroin NMSC obtained in the present invention has significantly improved mechanical properties compared with the recombinant spidroin NMC not containing S fragment, and the NMSC fiber (fusion spidroin fiber) exhibits significantly higher strength and toughness than the NMC fiber (recombinant spidroin fiber). The average strength of NMSC fibers can reach 624MPa, which is about 3.2 times that of NMC fibers (198 MPa), over most artificial recombinant spider silk fibers. The sequence S is inserted into the recombinant spider silk protein to form a fusion spider silk protein, so that the spider silk protein fiber is prepared by the fusion spider silk protein, and the mechanical strength of the spider silk protein fiber can be remarkably improved. Therefore, the spider silk protein fiber can be applied to tensile materials, can be widely applied to the fields of medicine, chemical industry or military industry, and the like, and particularly can be prepared or used as medical suture lines, carbon nano tube materials, textiles, and the like.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A fusion spider silk protein, characterized in that the fusion spider silk protein is formed by fusion expression of a segment of sequence S in recombinant spider silk protein; the amino acid sequence of the recombinant spider silk protein comprises an N-terminal sequence, a middle sequence M and a C-terminal sequence, wherein the amino acid sequence of the N-terminal sequence is shown as SEQ ID NO. 1; the amino acid sequence of the intermediate sequence M is shown as SEQ ID NO. 2; the amino acid sequence of the C-terminal sequence is shown as SEQ ID NO. 3; the amino acid sequence of the sequence S is shown as SEQ ID NO.4, and the sequence S is expressed in a fusion way between the intermediate sequence M and the C-terminal sequence.
2. The fusion spider silk protein of claim 1, wherein the amino acid sequence of the fusion spider silk protein is shown in SEQ ID NO. 5.
3. The fusion spider silk protein according to claim 1 or 2, which is prepared by the following method:
s1, performing codon optimization according to the codon preference of escherichia coli, and obtaining a gene segment NMSC for expressing the fusion spider silk protein through gene synthesis, wherein the nucleotide sequence of the gene segment NMSC is shown as SEQ ID NO. 6;
s2, inserting the gene segment NMSC into a vector through enzyme digestion, and constructing to obtain a recombinant vector;
s3, transforming the recombinant vector into receptor cells, inducing the receptor cells to fuse the expression of gene segment NMSC of the spider silk protein, and collecting inclusion bodies of the fused spider silk protein to obtain the fused spider silk protein.
4. A nucleotide sequence for expressing the fusion spider silk protein of any one of claims 1-3.
5. An expression vector comprising the nucleotide sequence of claim 4.
6. A recipient cell comprising the expression vector of claim 5.
7. Use of the sequence S according to any one of claims 1 or 2 for increasing the mechanical strength of a spidroin fibre, characterized in that the sequence S is expressed fusion into the recombinant spidroin according to claim 1 to form a fusion spidroin, a spidroin fibre produced from the fusion spidroin; the amino acid sequence of the fusion spider silk protein is shown as SEQ ID NO. 5.
8. A spidroin fiber comprising the fusion spidroin protein according to any one of claims 1 to 3.
9. A spider silk protein fiber, characterized in that the preparation method comprises the steps of preparing the fusion spider silk protein according to any one of claims 1-3 into completely dehydrated protein powder, preparing a spinnable fiber solution by dissolving the protein powder by using a solvent capable of dissolving the protein powder, and carrying out wet spinning on the spinnable fiber solution to obtain the spider silk protein fiber, wherein the solvent capable of dissolving the protein powder is 100% hexafluoroisopropanol.
10. Use of the spidroin fibres according to any one of claims 8 or 9 in the preparation or as medical sutures, carbon nanotube materials, textiles.
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| CN202310962802.XA CN117164721A (en) | 2023-08-02 | 2023-08-02 | Fusion spider silk protein, spider silk protein fiber and application thereof |
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| CN202310962802.XA CN117164721A (en) | 2023-08-02 | 2023-08-02 | Fusion spider silk protein, spider silk protein fiber and application thereof |
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