CN114000352B - Recombinant protein composite fiber, preparation method and application thereof - Google Patents

Recombinant protein composite fiber, preparation method and application thereof Download PDF

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CN114000352B
CN114000352B CN202111274876.1A CN202111274876A CN114000352B CN 114000352 B CN114000352 B CN 114000352B CN 202111274876 A CN202111274876 A CN 202111274876A CN 114000352 B CN114000352 B CN 114000352B
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recombinant protein
val pro
gly val
pro gly
gly lys
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CN114000352A (en
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刘凯
赵来
张洪杰
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Tsinghua University
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    • D06M13/402—Amides imides, sulfamic acids
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Abstract

The invention provides a recombinant protein composite fiber, which is obtained by covalently crosslinking recombinant protein and nano-cellulose; the recombinant protein has a sequence of MGAGPGVG [ (VPGKG)9VPGVG]n‑1(VPGKG)9VPGWPHHHHHH, n is 2, 4, 8 or 16; the recombinant protein is used for improving the mechanical property of the nano-cellulose; the mass ratio of the nano-cellulose to the recombinant protein is (5-50): 1; the covalent cross-linking is first treated with EDC/NHS and then reacted with recombinant protein or polylysine. The invention also provides a preparation method and application of the recombinant protein composite fiber.

Description

Recombinant protein composite fiber, preparation method and application thereof
Technical Field
The invention belongs to the technical field of composite fiber preparation, and particularly relates to a preparation method and application of a recombinant protein composite fiber.
Background
Cellulose is a natural polymer material with rich sources, and the main sources of the cellulose comprise wood, plant fiber, bacteria and tunica animals and plants. Cellulose is a polymer chain formed by glucose condensation, the polymerization degree of the cellulose reaches 1000-30000, and the length of the cellulose is more than 500-15000 nm. The structure with rigid crystal region and non-rigid amorphous region is formed between molecular chains through hydrogen bonds, and the rigid structure can provide strong support for plant cells so as to maintain the basic morphology of the cells. According to the literature, the theoretical strength of the crystal region can reach 75GPa, and the elastic modulus can reach 150 GPa. However, the high-strength cellulose cannot simultaneously have the characteristic of high toughness, and most of the fiber prepared based on the cellulose reported in the prior literature has the toughness of 20MJ/m3The following.
The nano-cellulose is nano-sized cellulose prepared by a physical method, a chemical method, a biological method and the like, and can be well dispersed in water to form stable colloid. The nano-cellulose not only has the characteristics of high strength and good biocompatibility of cellulose, but also further shows the characteristics of nano-particles such as high specific surface area and better transparency. However, the problem of poor toughness is not solved by the nanocrystallization of cellulose.
Disclosure of Invention
The invention aims to provide a recombinant protein composite fiber, a preparation method and application thereof.
The invention provides a recombinant protein composite fiber, which is obtained by covalently crosslinking recombinant protein and nano-cellulose;
the recombinant protein has a sequence of MGAGPGVG [ (VPGKG)9VPGVG]n-1(VPGKG)9VPGWPHHHHHH, n is 2, 4, 8 or 16; the recombinant protein is used for improving the mechanical property of the nano-cellulose;
the mass ratio of the nano-cellulose to the recombinant protein is (5-50): 1;
the covalent cross-linking is first treated with EDC/NHS and then reacted with recombinant protein or polylysine.
Preferably, the nano-cellulose is prepared by a mechanical method after cellulose wood pulp is treated by a TEMPO oxidation method; the concentration of the nano-cellulose is 5-20 mg/mL.
The present invention provides a method for preparing a recombinant protein composite fiber as described above, comprising the steps of:
A) mixing the recombinant protein solution and the nano-cellulose, and then extruding and molding in an acid solution to obtain a molded fiber;
B) reacting the formed fiber in a buffer solution containing an EDC/NHS cross-linking agent, and then reacting the reacted fiber in a buffer solution containing recombinant protein or polylysine to obtain recombinant protein composite fiber;
alternatively, the first and second electrodes may be,
C) collecting a plurality of the formed fibers to form single-stranded fibers, reacting the single-stranded fibers in a buffer solution containing an EDC/NHS cross-linking agent according to the method in the step B), and then reacting the reacted fibers in a buffer solution containing recombinant protein or polylysine to obtain the recombinant protein composite fiber.
Preferably, the recombinant protein is obtained according to the following steps:
culturing K in LB culture solution and TB culture solution18、K36、K72、K144Engineering bacteria, crushing bacteria, collecting supernatant, purifying with nickel column, ion exchange column and desalting column to obtain the target recombinant protein.
Preferably, the extrusion molding is carried out in a hydrochloric acid solution with the pH value of 0.5-2 in the step A); the flow rate of the extrusion is 10-150 mu L/min.
Preferably, the buffer solution containing the EDC/NHS cross-linking agent is MES buffer solution containing the EDC/NHS cross-linking agent; the reaction time is 24-48 hours.
Preferably, the buffer solution containing the recombinant protein or the polylysine is a PBS buffer solution containing the recombinant protein or the polylysine, the concentration of the recombinant protein or the polylysine is 10-20 mg/mL, and the reaction time is 2-4 hours.
The present invention provides the use of recombinant protein complex fibers as described above in tissue suturing and cell culture.
Preferably, the recombinant protein complex fiber is sutured to the damaged tissue, which includes one or more of the small intestine, liver, bladder, skin, spleen and muscle of the animal.
Preferably, the recombinant protein composite fiber is used as a scaffold for attaching adherent cells to culture cells, and the cells comprise human-derived bone marrow mesenchymal stem cells or mouse-derived bone marrow mesenchymal stem cells.
The invention provides a recombinant protein composite fiber, which is obtained by covalently crosslinking recombinant protein and nano-cellulose; the recombinant protein has a sequence of MGAGPGVG [ (VPGKG)9VPGVG]n-1(VPGKG)9VPGWPHHHHHH, n is 2, 4, 8 or 16; the recombinant protein is used for improving the mechanical property of the nano-cellulose; the mass ratio of the nano-cellulose to the recombinant protein is (5-50): 1; the covalent cross-linking is firstly processed by EDC/NHS and then reacts with recombinant protein or polylysine.
The invention has the following beneficial effects:
(1) the mechanical properties, especially toughness and ductility, of the nano-cellulose fiber are improved by using a covalent crosslinking method of ELP recombinant protein rich in lysine and nano-cellulose.
(2) The composite fiber prepared by the recombinant protein and the nano-cellulose has excellent mechanical properties and good biocompatibility.
(3) The single-stranded composite fiber obtained by post-treatment and twisting can be applied to tissue suturing and cell culture, and the application range of the composite fiber is expanded.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a graph of tensile testing of single-purity nanocellulose in example 1 of the present invention;
FIG. 2 is a drawing test graph of the nano-cellulose/recombinant protein composite fiber after post-treatment in example 1 of the present invention
FIG. 3 is a drawing test graph of a post-treated nano-cellulose/natural bovine serum albumin composite fiber of comparative example 1 of the present invention;
FIG. 4 shows the mechanical properties of the recombinant protein composite nanofibers according to examples 1 to 3 of the present invention;
FIG. 5 shows the mechanical properties of the recombinant protein composite nanofibers according to examples 5 to 7 of the present invention;
FIG. 6 shows the mechanical properties of the recombinant protein composite nanofibers according to examples 8 to 9 of the present invention;
FIG. 7 is a graph showing the effect of skin suture of single fibers in example 10 of the present invention;
FIG. 8 shows the cell culture conditions of single fibers in example 10 of the present invention.
Detailed Description
The invention provides a recombinant protein composite fiber, which is obtained by covalently crosslinking recombinant protein and nano-cellulose;
the recombinant protein has a sequence of MGAGPGVG [ (VPGKG)9VPGVG]n-1(VPGKG)9VPGWPHHHHHH, n is 2, 4, 8 or 16; the recombinant protein is used for improving the mechanical property of the nano-cellulose;
when n is 2, the sequence of the ELP recombinant protein is MGAGPGVG (VPGKG)9VPGVG(VPGKG)9VPGWPHHHHHH, as shown in SEQ ID No. 1;
when n is 4, the sequence of ELP recombinant protein is MGAGPGVG [ (VPGKG)9VPGVG]3(VPGKG)9VPGWPHHHHHH, as shown in SEQ ID No. 2;
when n is 8, the sequence of ELP recombinant protein is MGAGPGVG [ (VPGKG)9VPGVG]7(VPGKG)9VPGWPHHHHHH, as shown in SEQ ID No. 3;
when n is 16, the sequence of the ELP recombinant protein is MGAGPGVG [ (VPGKG)9VPGVG]15(VPGKG)9VPGWPHHHHHH, as shown in SEQ ID No. 4;
the mass ratio of the nano-cellulose to the recombinant protein is (5-50): 1;
the covalent cross-linking is first treated with EDC/NHS and then reacted with recombinant protein or polylysine.
The sequence used in the present invention is MGAGPGVG [ (VPGKG)9VPGVG]n-1(VPGKG)9VPGWPHHHHHH ELP recombinant protein (K18, K36, K72, K144) engineering bacteria are constructed by laboratory and cultured K18、K36、K72、K144Engineering bacteria and inducing them to express target protein. And removing foreign proteins by using a nickel column, an ion exchange column and a desalting column, and freeze-drying to obtain the recombinant protein with higher purity. Specifically, "Engineered Near-innovative Fluorescent proteins Assemblies for Robust Bioimaging and Therapeutic Applications [ J]Advanced Materials,2020,32(17), "or" Ultra-strongbio-glue from genetic engineered polypeptides [ J]K in Nature communications,2021,12(1)18、K36、K72、K144And (4) engineering bacteria.
In the invention, the nano-cellulose is preferably nano-cellulose prepared by a mechanical method after cellulose wood pulp is treated by a TEMPO oxidation method; the concentration of the nano-cellulose is 5-20mg/mL, preferably 10-15 mg/mL.
In the invention, the mass ratio (dry weight) of the nano-cellulose to the recombinant protein is preferably (5-50): 1, more preferably (10 to 40): 1, such as 5: 1. 10: 1. 15: 1. 20: 1. 25: 1. 30: 1. 35: 1. 40: 1. 45, and (2) 45: 1. 50: 1 is preferably a range value having any of the above numerical values as an upper limit or a lower limit.
The invention also provides a preparation method of the recombinant protein composite fiber, namely a method for improving the mechanical property of the nano cellulose fiber by using the recombinant protein, which comprises the following steps:
firstly, the expression and purification of recombinant protein and the preparation of nano-cellulose are carried out
1) Expression and purification of recombinant proteins
Culturing K with LB culture solution18、K36、K72Or K144And 7-8 hours of engineering bacteria, transferring the engineering bacteria into a TB culture solution after the OD value of the bacteria solution reaches 3-4, inducing the bacteria to express target protein by IPTG when the OD value reaches 0.6-0.8, and culturing for 10-12 hours after induction. Centrifuging at 5000-6000 rpm for 15 minutes, discarding the supernatant, and keeping the precipitate. Resuspending the thallus with a nickel buffer solution without imidazole and adding DNA enzyme with a final concentration of 5-6 mug/ml, 1-2 mg/ml lysozyme and 20-30 mmol/LMgCl2And crushing the bacterial liquid after the heavy suspension by using a high-pressure crusher, and then centrifuging for 30-40 minutes at 10000-11000 rpm. Collecting supernatant, and filtering to remove solid impurities.
And then purifying by using a nickel affinity chromatography column, a cation exchange column and a desalting column in sequence, and freeze-drying the purified target protein for later use.
2) Preparation of nanocellulose
The invention utilizes a TEMPO oxidation method to treat cellulose wood pulp and then utilizes a mechanical method to prepare nano-cellulose. The method comprises the following specific steps:
adding ultrapure water into wood pulp, stirring overnight, then adding an aqueous solution of TEMPO (2,2,6, 6-tetramethylpiperidine oxide) and NaBr, uniformly mixing with the wood pulp, adjusting the pH of the uniformly mixed solution to be about 10 by using NaOH, dropwise adding NaClO, reacting, adding ethanol for precipitation after the reaction is finished, centrifuging, washing the precipitate with water, finally, uniformly mixing with the ultrapure water, and treating by using a mechanical method to obtain the nano-cellulose.
In the invention, the wood pulp is preferably birch, cedar or pine wood pulp, and the mass ratio of TEMPO to NaBr is (1-10): 25, more preferably (3-8): 25, most preferably 4: 25; the concentration of NaOH is preferably 0.1-1.0 mol/L, more preferably 0.3-0.8 mol/L, and most preferably 0.5-0.6 mol/L.
According to the method, NaClO is preferably added dropwise within half an hour, and the dosage relationship of the NaClO is preferably 5-15 mmol/g wood pulp, and more preferably 10mmol/g wood pulp. After NaClO is added, the reaction is carried out for 2-5 hours, preferably 3-4 hours, and the pH is preferably kept at 10.5 in the reaction process.
In the invention, the mechanical treatment to obtain the nano-cellulose is specifically mechanical treatment for 10min by a high-pressure homogenizer at 1000 bar. .
In the invention, the nano-cellulose obtained by the method is in a colloidal state, and the concentration of the nano-cellulose is preferably 5-20mg/mL, and preferably 10-15 mg/mL.
After the recombinant protein and the nano-cellulose are obtained, the recombinant protein and the nano-cellulose are mixed, then the mixed solution is extruded and molded in an acid solution, and a collector is used for collecting and drying the mixed solution to obtain the molded fiber.
In the invention, the mass ratio of the nano-cellulose to the recombinant protein is preferably (5-50): 1, more preferably (10 to 40): 1, such as 5: 1. 10: 1. 15: 1. 20: 1. 25: 1. 30: 1. 35: 1. 40: 1. 45, and (2) 45: 1. 50: 1 is preferably a range value having any of the above numerical values as an upper limit or a lower limit.
In the present invention, the acid solution is preferably a hydrochloric acid solution, the pH value of the acid solution is preferably 0.5-2, more preferably 0.5-1, the present invention preferably enables the above-mentioned extrusion using a syringe with a syringe pump, and the flow rate of the extrusion is preferably 10-150. mu.L/min, more preferably 30-120. mu.L/min, most preferably 50-100. mu.L/min, and specifically may be 10. mu.L/min, 20. mu.L/min, 30. mu.L/min, 40. mu.L/min, 50. mu.L/min, 60. mu.L/min, 70. mu.L/min, 80. mu.L/min, 90. mu.L/min, 100. mu.L/min, 110. mu.L/min, 120. mu.L/min, 130. mu.L/min, 140. mu.L/min, 150. mu.L/min, 160. mu.L/min, 170. mu.L/min, preferably, the above arbitrary value is a range value having an upper limit or a lower limit.
Drying the collected formed fibers, wherein the drying temperature is preferably 20-25 ℃; the drying time is preferably 0.5 to 2 hours, and more preferably 1 to 1.5 hours.
The present invention treats the dried shaped fiber with EDC/NHS first to activate the carboxyl group of nanocellulose, and then further reacts with the amino group of polylysine or the amino group of recombinant protein to form an amide bond, with recombinant protein being more preferred. The method comprises the following specific steps:
soaking the formed fiber in MES buffer solution dissolved with EDC/NHS for activation, wherein the molar ratio of EDC to NHS is preferably (2-5): 1, more preferably (3-4): 1; the mass ratio of EDC to NHS to nanocellulose is preferably (320-80): (48-12): 1, more preferably (100 to 80): (15-12): 1; the pH value of the MES buffer solution is 6, the reaction time is 24-48 hours, and the reaction temperature is preferably 20-25 ℃.
Then, the activated fiber is transferred to a PBS buffer solution containing recombinant protein or polylysine for amidation reaction.
In the invention, the concentration of the recombinant protein or polylysine in the PBS buffer solution of the recombinant protein or polylysine is preferably 10-20 mg/mL, more preferably 13-15 mg/mL, and the pH value of the PBS buffer solution is preferably 7.2-7.4; the reaction temperature is preferably 20-25 ℃; the reaction time is preferably 2 to 4 hours.
And finally, standing the composite fiber in ultrapure water for 2-5 hours, and drying to obtain the recombinant protein composite fiber.
The single recombinant protein composite fiber prepared by the scheme can be used as a scaffold for adherent cell attachment for cell culture, or a plurality of (such as 10-35) recombinant protein composite fibers can be collected after being formed in a hydrochloric acid solution, and a single-stranded recombinant protein composite fiber is formed after amidation reaction and can be used for suturing damaged tissues after drying.
Based on the method, the invention also provides the application of the recombinant protein composite fiber in tissue suturing and cell culture.
In the present invention, the tissue includes one or more of small intestine, liver, bladder, skin, spleen and muscle of the animal.
The cells comprise human-derived bone marrow mesenchymal stem cells or mouse-derived bone marrow mesenchymal stem cells.
The invention provides a recombinant protein composite fiber, which is obtained by covalently crosslinking recombinant protein and nano-cellulose; the recombinant protein has a sequence of MGAGPGVG [ (VPGKG)9VPGVG]n-1(VPGKG)9VPGWPHHHHHH, n is 2, 4, 8 or 16; the recombinant protein is used for improving the mechanical property of the nano-cellulose; the mass ratio of the nano-cellulose to the recombinant protein is (5-50): 1; the covalent cross-linking is first treated with EDC/NHS and then reacted with recombinant protein or polylysine.
The invention has the following beneficial effects:
(1) the mechanical properties, especially toughness and ductility, of the nano-cellulose fiber are improved by using a covalent crosslinking method of ELP recombinant protein rich in lysine and nano-cellulose.
(2) The composite fiber prepared by the recombinant protein and the nano-cellulose has excellent mechanical properties and good biocompatibility.
(3) The single-stranded composite fiber after post-treatment and twisting can be applied to tissue suturing and cell culture, and the application range of the composite fiber is expanded.
In order to further illustrate the present invention, the following examples are provided to describe the recombinant protein composite fiber, its preparation method and application in detail, but should not be construed as limiting the scope of the present invention.
Example 1
Preparation of nanocellulose
1g of birch, cedar or pine pulp is weighed out and stirred overnight with 60ml of ultrapure water. Weighing 16mgTEMPO and 100mgNaBr, dissolving with 40ml of ultrapure water, and mixing the dissolved solution with wood pulp. The pH of the wood pulp was adjusted to 10 with 0.5mol/L NaOH. NaClO7.5ml was added dropwise over a half hour period with a syringe, reacted for 3h and the pH was maintained at 10.5. Then 300ml ethanol is added for precipitation, and the mixture is centrifuged at 6000rpm for 15 minutes, and the precipitate is left. Washed with deionized water and centrifuged 3 times. Finally, the mixture is evenly mixed with ultrapure water and treated by a mechanical method to obtain the nano-cellulose. 1ml of nano-cellulose is taken for freeze-drying, and the mass after freeze-drying is weighed to determine the concentration. The concentration range is 5-20 mg/ml.
1ml of nanocellulose with a concentration of 10mg/ml was taken with a pipette. The nanocellulose solution was injected with a 1ml syringe into a hydrochloric acid solution at pH 0.5 for moulding. The flow rate of the injection pump was 80. mu.l/min.
The formed fibers were picked up and collected using a collector, and the nanocellulose fibers were dried at room temperature for 2 hours.
And randomly selecting 3 nano cellulose fibers for mechanical tensile test. The mechanical tensile test results are shown in fig. 1. The strength of the pure nano cellulose fiber is 300-400 MPa, the ductility is 3-6%, and the toughness is 9-14 MJ/m3。
Expression and purification of recombinant proteins
Laboratory-constructed K was cultured with 100ml LB broth144And (3) 7-8 hours of engineering bacteria, transferring the engineering bacteria into 1LTB culture solution after the OD value of the bacteria solution reaches 3-4, inducing the bacteria to express target protein by IPTG when the OD value reaches 0.6-0.8, and culturing for 10-12 hours after induction. Centrifuge at 6000rpm for 15 minutes, discard the supernatant, retain the precipitate. Resuspending the cells in imidazole-free nickel buffer and adding DNase at a final concentration of 5. mu.g/ml, 1mg/ml lysozyme, 30mmol/LMgCl2The resuspended suspension was crushed by a high-pressure crusher and then centrifuged at 11000rpm for 30 minutes. Collecting supernatant, and filtering to remove solid impurities. Preparing a nickel lysine buffer solution and a nickel electrophoresis buffer solution. And (3) purifying the protein by using a purifier, firstly installing a nickel affinity chromatography column and cleaning the system, balancing the nickel column by using lysis buffer solution, and loading the sample after UV280 is stable at the flow rate of 10-20 ml/min. After loadingEquilibration with lysis buffer was continued until the UV280 value stabilized. Linearly eluting the sample, collecting the sample when UV280 is peaked until the collection of the absorption peak is completed, wherein the flow rate is 10-20ml/min, and the collection time is 30-50 min. Purifying by the same method using cation exchange column and desalting column in turn, wherein the desalting column can be purified with ultrapure water without buffer solution. And (5) obtaining the target protein after purification and freeze-drying for later use.
Preparation of recombinant protein composite nanofiber
(1) Taking 1ml of 10mg/ml nanocellulose by using a pipette gun, adding the nanocellulose into a centrifuge tube, weighing 10mg of recombinant protein, dissolving the recombinant protein with 2ml of ultrapure water, wherein the concentration of the recombinant protein is 5mg/ml, and uniformly mixing 200 mu l of recombinant protein solution with the nanocellulose. Wherein the ratio of cellulose to recombinant protein is 10:1
(2) The mixed solution was injected into a hydrochloric acid solution having a pH of 0.5 by a 1ml syringe with the aid of a syringe pump, molded and collected by a collector at a flow rate of 80. mu.l/min. The fibers were dried at room temperature for 2 hours.
(3) MES4.265g and NaCl5.844g were weighed and dissolved in 200ml of ultrapure water, and the pH of the buffer was adjusted to 6. EDC800mg and NHS120mg (molar ratio EDC: NHS 4: 1) were weighed and dissolved in 40ml prepared MES buffer. And putting the dried composite fiber into MES buffer solution containing EDC/NHS for reaction for 24 hours.
(4) Weighing KH2PO40.24g、NaHPO41.44g, NaCl8g, and KCl0.2g were dissolved in 1L of ultrapure water, and the pH was adjusted to 7.4. 40mg of the recombinant protein was weighed and dissolved in 30ml of PBS buffer solution. The complex fiber activated with EDC/NHS was transferred to PBS buffer containing recombinant protein, and amidation reaction was carried out for 2 hours.
(5) The composite fiber was then transferred to 40ml of ultrapure water and allowed to stand for 3 hours.
(6) The composite fiber was taken out from the ultrapure water and dried at room temperature for 2 hours.
And randomly selecting 3 dried composite fibers for mechanical tensile test. The mechanical tensile test chart is shown in figure 2. The results show that the mechanical properties of the composite fiber are comprehensively and obviously improved. The strength reaches 400-600 MPa, and the toughnessUp to 30-40 MJ/m3And ductility is 8% -13%.
Comparative example 1
A recombinant protein composite nanofiber was prepared according to the method of example 1, except that the recombinant protein of example 1 was replaced with natural bovine serum albumin.
The mechanical tensile test chart of the nanocellulose/natural bovine serum albumin composite fiber is shown in fig. 3, and the comparison between fig. 2 and fig. 3 shows that the mechanical property of the nanocellulose/recombinant protein is significantly higher than that of the pure nanocellulose and the nanocellulose/natural bovine serum albumin composite fiber.
Examples 2 to 3
A recombinant protein composite nanofiber was prepared according to the method of example 1, except that EDC: NHS ═ 8: 1 (molar ratio), EDC in example 3: NHS ═ 16: 1 (molar ratio).
The mechanical properties of the fibers of examples 1-3 are shown in fig. 4, and it can be seen from fig. 4 that the mechanical properties of the fibers are not significantly improved by performing post-treatment on the composite fibers with a plurality of EDC/NHS with different ratios after drying, and the ratio of EDC to NHS is higher than 4: 1.
Examples 4 to 7
Preparing a recombinant protein composite nanofiber according to the method in the example 1, except that the dried composite fiber is put into MES buffer solution containing EDC/NHS to react for 2 hours in the example 4;
in example 5, the dried composite fiber is put into MES buffer solution containing EDC/NHS to react for 12 hours;
in example 6, the dried composite fiber is put into MES buffer solution containing EDC/NHS to react for 24 hours;
in example 7, the dried composite fiber is put into MES buffer solution containing EDC/NHS to react for 48 hours;
the mechanical property comparison is shown in fig. 5. The result shows that the mechanical property of the composite fiber is improved to a certain extent along with the prolonging of the post-treatment time within 24 hours, but when the post-treatment time exceeds 24 hours, the post-treatment time is further prolonged, and the mechanical property of the composite fiber is reduced.
Examples 8 to 9
A recombinant protein composite nanofiber was prepared according to the method of example 1, except that the amount of polylysine used was 80mg in example 8 and 160mg in example 9.
The comparison of the mechanical properties is shown in FIG. 6. It can be seen that the increase in the content of polylysine has no significant effect on the improvement in mechanical properties.
Example 10
Individual composite fibers were prepared according to the method of example 1, and 20 composite fibers were repeatedly collected together to form a single strand fiber before the composite fibers were dried in step (2). The single strand fibers were dried for 2 hours.
The dried single-strand fibers were subjected to a crosslinking post-treatment in accordance with steps (3) to (6) in example 1.
And (4) trimming two ends of the prepared single-strand fiber to be flush to prepare the suture, wherein the surface of the suture is smooth.
A skin damage model is prepared by utilizing an experimental pig, and the prepared suture is utilized to suture the skin damage. The physical diagram is shown in figure 7.
Culturing human mesenchymal stem cells by using DMEM culture solution, digesting the cells for 2 minutes by using trypsin when the cells are about 80 percent of a culture bottle full of the cells, transferring the cells to a 1ml culture dish containing single-strand composite fibers, and adding the DMEM culture solution to co-culture for 24 hours.
And observing the growth condition of the human mesenchymal stem cells on the composite fiber by a confocal microscope. As shown in particular in figure 8.
According to the embodiment, the mechanical property of the composite fiber after covalent crosslinking with the ELP recombinant protein is comprehensively and obviously improved compared with that of a pure nano cellulose fiber. In addition, the composite fiber has better biocompatibility and can be applied to tissue injury suturing and cell culture, so that the application range of the composite fiber is expanded.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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Claims (10)

1. A recombinant protein composite fiber is obtained by covalently crosslinking recombinant protein and nano-cellulose;
the recombinant protein has a sequence of MGAGPGVG [ (VPGKG)9VPGVG]n-1（VPGKG）9VPGWPHHHHHH, n is 2, 4, 8 or 16; the recombinant protein is used for improving the mechanical property of the nano-cellulose;
the mass ratio of the nano-cellulose to the recombinant protein is (5-50): 1;
the covalent cross-linking is first treated with EDC/NHS and then reacted with recombinant protein or polylysine.
2. The recombinant protein composite fiber according to claim 1, wherein the nanocellulose is prepared by a mechanical method after cellulose wood pulp is treated by a TEMPO oxidation method; the concentration of the nano-cellulose is 5-20 mg/mL.
3. The method for preparing a recombinant protein composite fiber according to claim 1, comprising the steps of:
A) mixing the recombinant protein and the nano-cellulose, and then extruding and molding in an acid solution to obtain a molded fiber;
B) reacting the formed fiber in a buffer solution containing an EDC/NHS cross-linking agent for 24 hours, and then reacting the reacted fiber in a buffer solution containing recombinant protein or polylysine to obtain recombinant protein composite fiber;
alternatively, the first and second electrodes may be,
C) collecting a plurality of the formed fibers to form single-stranded fibers, reacting the single-stranded fibers in a buffer solution containing an EDC/NHS cross-linking agent for 24 hours according to the method in the step B), and then reacting the reacted fibers in a buffer solution containing recombinant protein or polylysine to obtain the recombinant protein composite fiber.
4. The method according to claim 3, wherein the recombinant protein is obtained by the steps of:
culturing K in LB culture solution and TB culture solution18、K36、K72、K144Engineering bacteria, crushing bacteria, collecting supernatant, purifying with nickel column, ion exchange column and desalting column to obtain the target recombinant protein.
5. The preparation method according to claim 3, wherein the step A) is extrusion molding in a hydrochloric acid solution with a pH value of 0.5-2; the flow rate of the extrusion is 10-150 mu L/min.
6. The method according to claim 3, wherein the buffer solution containing EDC/NHS crosslinking agent is MES buffer solution containing EDC/NHS crosslinking agent.
7. The method according to claim 6, wherein the buffer solution containing the recombinant protein or the polylysine is a PBS buffer solution containing the recombinant protein or the polylysine, the concentration of the recombinant protein or the polylysine is 10-20 mg/mL, and the reaction time is 2-4 hours.
8. Use of the recombinant protein complex fiber of claim 1 in cell culture.
9. The use of claim 8, wherein the recombinant protein composite fiber is used as a scaffold for anchorage dependent cell culture of cells, including human-derived mesenchymal stem cells or murine-derived mesenchymal stem cells.
10. A tissue suture prepared from the recombinant protein conjugate fiber of claim 1.
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