Signal peptide for improving expression of recombinant silk fibroin and application

Abstract

The invention discloses a signal peptide for improving expression of recombinant silk fibroin. The amino acid sequence of the signal peptide is shown as SEQ ID NO: 1. The invention also discloses a nucleotide sequence for coding the signal peptide. The invention also discloses a recombinant plasmid, a recombinant transformant, a recombinant expression vector or a recombinant bacterium containing the nucleotide sequence. Compared with the traditional alpha-MF signal peptide, the signal peptide disclosed by the invention has the advantages that the secretory expression quantity of the recombinant silk fibroin is remarkably improved (improved by 4.9 times), and a high-efficiency expression technical scheme is provided for large-scale production of the recombinant silk fibroin.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a signal peptide for improving the expression of recombinant silk fibroin and its application. Background Art

[0002] Silk fibroin (SF), with its excellent cell adhesion properties and ability to promote cell proliferation, differentiation, and migration, has garnered widespread attention in fields such as medicine and skincare. Currently, natural silk fibroin is primarily extracted from silkworm cocoons. However, the extraction process is complex and inefficient, making it difficult to meet the needs of large-scale industrial production.

[0003] With the development of biotechnology, the expression system of exogenous proteins has become increasingly popular among researchers. Pichia pastoris, as an important eukaryotic expression system, is widely used in the field. Among them, the secretion of recombinant proteins in Pichia pastoris is usually guided by signal peptides, among which the α-mating factor signal peptide (α-MF) of Saccharomyces cerevisiae is the most widely used. However, experimental studies have found that the secretion effect of α-MF on recombinant silk fibroin is not very ideal. Therefore, it is of great significance to study how to increase the expression of recombinant silk fibroin by changing the signal peptide. Summary of the Invention

[0004] The present invention addresses the problem of low expression of recombinant silk fibroin in the Pichia pastoris expression system and provides a signal peptide for improving the expression of recombinant silk fibroin. By modifying and optimizing the signal peptide to increase the expression of recombinant silk fibroin, not only the production cost is reduced, but also large-scale production and application are facilitated.

[0005] A first aspect of the present invention provides a signal peptide for improving the expression of recombinant silk fibroin, characterized in that the amino acid sequence of the signal peptide is shown in SEQ ID NO: 1.

[0006] The second aspect of the present invention provides a nucleotide sequence encoding the above-mentioned signal peptide, wherein the nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 3 or its complementary sequence.

[0007] The third aspect of the present invention provides a recombinant plasmid, recombinant transformant, recombinant expression vector or recombinant bacteria containing the above nucleotide sequence.

[0008] The fourth aspect of the present invention provides a use of the above-mentioned signal peptide in increasing the expression level of recombinant silk fibroin.

[0009] A fifth aspect of the present invention provides a method for preparing recombinant silk fibroin, characterized by comprising: using a signal peptide comprising the amino acid sequence shown in SEQ ID NO: 1 to guide the expression of the recombinant silk fibroin in a Pichia pastoris host cell.

[0010] The sixth aspect of the present invention provides a recombinant silk fibroin prepared by the above method.

[0011] A seventh aspect of the present invention provides a use of the above-mentioned recombinant silk fibroin in the preparation of a product for promoting cell proliferation, cell migration and / or repairing skin barrier function.

[0012] An eighth aspect of the present invention provides a use of the recombinant silk fibroin obtained by using the above-mentioned signal peptide in the preparation of a product for promoting cell proliferation, cell migration and / or repairing skin barrier function.

[0013] The ninth aspect of the present invention provides a use of the recombinant silk fibroin obtained by using the above-mentioned recombinant plasmid, recombinant expression vector or recombinant bacteria in the preparation of a product for promoting cell proliferation, cell migration and / or repairing skin barrier function.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] The present invention redesigns the amino acids in the original signal peptide sequence and mutates 6 amino acids therein, mutating Glu (E) to Phe (F), Asp (D) to Ser (S), Glu (E) to Pro (P), Glu (E) to Lys (K), Glu (E) to Pro (P), and Glu (E) to Phe (F). The isoelectric point of the obtained modified signal peptide amino acid sequence is changed from 3.84 to 5.91, and the acidity and alkalinity are changed from strongly acidic to weakly acidic, making it closer to the fermentation environment conditions suitable for the target protein. Therefore, the target protein can pass through the endoplasmic reticulum and Golgi apparatus more smoothly, reducing its retention and degradation in the cell, thereby improving the secretion efficiency of the target protein. Compared with the use of α-MF, the secretory expression amount of the target protein in this solution is increased by 4.9 times.

[0016] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is an electrophoresis diagram of the PCR identification results of GS115 / ZaA-g-Silk recombinant transformants. M represents a marker, and the number followed by # represents the sample number of the GS115 / ZaA-g-Silk recombinant transformant.

[0018] Figure 2This is the electrophoresis diagram of the identification results of the expression product induced by the shake flask of the GS115 / ZaA-g-Silk recombinant transformant. M represents the marker, and the number followed by # represents the sample number of the GS115 / ZaA-g-Silk recombinant transformant.

[0019] Figure 3 The electrophoresis diagram shows the results of shake flask induced expression of GS115 / ZaA-Silk and GS115 / ZaA-g-Silk recombinant transformants.

[0020] Figure 4 Schematic diagram of the proliferation of L929 cells cultured in vitro by the recombinant silk fibroin obtained in Example 1 and the recombinant silk fibroin obtained in Comparative Example 1.

[0021] Figure 5 Schematic diagram of the microscope results of the recombinant silk fibroin obtained in Example 1 and the recombinant silk fibroin obtained in Comparative Example 1 at 0-72h and a data diagram of cell migration rate.

[0022] Figure 6 This is a schematic diagram showing the average transepidermal water loss results of samples prepared from the recombinant silk fibroin obtained in Example 1 and the recombinant silk fibroin obtained in Comparative Example 1 during 0-8 days.

[0023] Figure 7 Schematic diagram of the results of the transepidermal water loss change rate of samples prepared with the recombinant silk fibroin obtained in Example 1 and the recombinant silk fibroin obtained in Comparative Example 1 on the 6th and 8th days. DETAILED DESCRIPTION

[0024] To make the above-mentioned objects, features and advantages of the present invention more clearly understood, the following detailed description of the specific embodiments of the present invention is given in conjunction with the following examples. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and examples, but the protection scope of the present invention is not limited thereto. Any changes in the methods made by those skilled in the art based on these embodiments are all included in the protection scope of the present invention.

[0026] Example 1

[0027] S1: Sequence Design

[0028] 1.1 Signal peptide amino acid sequence

[0029] The amino acid sequence of α-MF in the pPICZaA plasmid was redesigned, and the resulting new signal peptide amino acid sequence is shown in SEQ ID NO: 1.

[0030] 1.2 Silk fibroin peptide (Silk) amino acid sequence

[0031] The amino acid sequence of silk fibroin was obtained from the NCBI website, and stable peptide segments were screened. The obtained silk fibroin amino acid sequence is shown in SEQ ID NO: 2.

[0032] 1.3 Nucleotide sequence

[0033] According to the codon preference of Pichia pastoris, the nucleotide sequences corresponding to the signal peptide amino acid sequence SEQ ID NO:1 and the silk fibroin amino acid sequence SEQ ID NO:2 were encoded, and sequence elements such as enzyme cleavage sites and stop codons were directly added to both ends of the nucleotide sequence to obtain the nucleotide sequence SEQ ID NO:3 corresponding to the signal peptide amino acid sequence and the nucleotide sequence SEQ ID NO:4 corresponding to the silk fibroin amino acid sequence.

[0034] SEQ ID NO: 3:

[0035] 5'-gcggccgcttcgaaacga tgagatttcc ttcaattttt actgcagttt tattcgcagcatcctccgca ttagctgctc cagtcaacac tacaacattc tccgaaacgg cacaaattcc ggctccagctgtcatcggtt actcagattt aaagggggat ttcgatgttg ctgttttgcc attttccaac agcacaaataacgggttatt gtttataaat actactattg ccagcattgc tgctaaacca ttcggggtat ctctcgag-3'

[0036] SEQ ID NO: 4:

[0037]

[0038]

[0039] For the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4, the restriction endonuclease recognition sites of Xho I and Not I are marked in italics at the 5' and 3' ends, respectively, and the bases boxed near the 5' end are the complementary sequences.

[0040] 1.4 Design of primers for bacterial liquid PCR identification

[0041] 5'AOX (i.e., SEQ ID NO: 5):

[0042] 5'-GACTGGTTCCAATTGACAAGC-3'

[0043] 3'AOX (i.e., SEQ ID NO: 6):

[0044] 5'-GCAAATGGCATTCTGACATCC-3'

[0045] S2: Construction of pPICZaA-g-Silk recombinant plasmid

[0046] 2.1 Materials:

[0047] 2× Taq PCR MasterMix (Tiangen Biochemical), T4 DNA Ligase (Thermo), Escherichia coli (E. coli) DH5α competent cells (Tiangen Biochemical), restriction endonuclease Not I Fast Digest (Thermo), restriction endonuclease XhoI Fast Digest (Thermo), agarose gel DNA recovery kit (Tiangen Biochemical), LB medium, signal peptide nucleotide molecule (SEQ ID NO: 3) and silk fibroin nucleotide molecule sequence (SEQ ID NO: 4) were all synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0048] 2.2 Equipment

[0049] PCR instrument (Bio-RAD), horizontal electrophoresis apparatus, centrifuge, and shaker.

[0050] 2.3 Methods

[0051] (1) Prepare the enzyme digestion reaction system in a clean PCR tube as shown in Table 1 and Table 2:

[0052] Table 1. Signal peptide nucleotide restriction enzyme digestion system

[0053] Components Volume (μL) SEQ ID NO:3 6.0 10×Buffer 2.0 NotI 1.0 XOt 1.0 <![CDATA[ddH2O]]> 10.0 A total of 20 μL

[0054] Table 2. Restriction enzyme digestion system of silk fibroin nucleotide molecules

[0055] Components Volume (μL) SEQ ID NO:4 6.0 10×Buffer 2.0 NotI 1.0 XOt 1.0 ddH2O 10.0 A total of 20 μL

[0056] (2) Gently tap the tube wall to mix the solution with a suspension mixer, and centrifuge briefly to concentrate the solution at the bottom of the tube;

[0057] (3) Place the PCR tubes containing the restriction enzyme digestion systems in Tables 1 and 2 in a 37°C water bath for 17 minutes;

[0058] (4) After the enzyme cleavage reaction is completed, the enzyme cleavage product is subjected to 1% agarose gel electrophoresis detection, and the gel is cut and recovered according to the requirements of the agarose gel DNA recovery kit to obtain a recovered product, wherein the recovered product obtained after the signal peptide nucleotide molecule is subjected to the enzyme cleavage reaction is pPICZaA-signal peptide (X+N) 3593bp; the recovered product obtained after the silk fibroin nucleotide molecule is subjected to the enzyme cleavage reaction is Silk (X+N) 1067bp.

[0059] (5) Use T4 DNA ligase to ligate the recovered products. The specific ligation steps are as follows:

[0060] Table 3. T4 ligase ligation system

[0061] Components Volume (μL) pPICZaA-Signal Peptide (X+N) 1.0 Silk(X+N) 9.0 T4 DNA ligase 1.0 10xbuffer 2.0 <![CDATA[ddH2O]]> 7.0 A total of 20 μL

[0062] a. Gently tap the tube wall to mix the solution with a mixer, and centrifuge briefly to concentrate the solution at the bottom of the tube;

[0063] b. Place the PCR tube containing the T4 ligase ligation system in a 22°C thermostat for 3 hours.

[0064] c. After the ligation reaction is completed, the ligation product pPICZaA-g-Silk is obtained, and then the competent bacteria are transformed.

[0065] (6) Use DH5α competent cells to transform pPICZaA-g-Silk. The specific experimental steps are as follows:

[0066] a. Remove one tube of DH5α competent cells (50 μL) stored at -80°C and quickly place it in an ice box. After the cell block thaws, add 5.0 μL of the aforementioned pPICZaA-g-Silk in a clean bench. Gently stir the bottom of the tube with your finger to mix thoroughly. Place the EP tube on an ice box and let it stand for 30 minutes.

[0067] b. Heat shock the EP tube in a 42°C water bath for 90 seconds, then quickly place it in an ice box and let it rest for 3 minutes.

[0068] c. Add 400 μL of sterile LB liquid medium without antibiotics to the EP tube, shake to mix, and incubate in a shaker at 29°C and 220 rpm for 1 hour;

[0069] d. In a clean bench, pipette approximately 200 μL of the incubation solution and spread it evenly onto an LB plate containing 25 μg / mL Zeocin antibiotics.

[0070] e. Place the LB plate upside down in a 37°C incubator and incubate overnight.

[0071] S3. Identification of the recombinant plasmid pPICZaA-g-Silk

[0072] Select the positive transformants that have grown, perform enzyme digestion identification, and sequence the strains that have been correctly identified by enzyme digestion. The specific experimental steps are as follows:

[0073] (1) Positive transformants grown on LB plates containing 25 μg / mL Zeocin were picked, numbered, and inoculated into 5 mL of LB liquid medium containing 25 μg / mL Zeocin. The plates were placed on a shaker and cultured overnight at 200 rpm and 37°C.

[0074] (2) On the second day, 1 mL of the culture medium was taken and the plasmid was extracted according to the instructions of the Tiangen Plasmid Extraction Kit (DP103-02) and eluted with EB.

[0075] (3) The plasmid was extracted and double enzyme digestion reaction was performed using NotI and XhoI. The enzyme digestion reaction system was prepared according to the reaction system in Table 4.

[0076] (4) Gently tap the tube wall to mix the solution with a suspension mixer, centrifuge briefly to concentrate the solution at the bottom of the tube, and place the tube in a 37°C water bath for 17 minutes.

[0077] (5) The digested products were subjected to 1% agarose gel electrophoresis. Based on the electrophoresis results, positive transformants were preliminarily identified. The corresponding bacterial solution was prepared into glycerol tubes at a ratio of 1:1 between bacterial solution and 50% glycerol and stored at -20°C. 15 μL aliquots were prepared for sequencing. The sequencing results were aligned with the designed sequence, and the positive transformants with the correct sequencing results were used for subsequent experiments.

[0078] Table 4. Enzyme digestion identification system

[0079]

[0080]

[0081] S4. Transformation of recombinant plasmid pPICZaA-g-Silk into competent yeast GS115

[0082] 4.1 Materials

[0083] High-purity plasmid mini-preparation kit (Tiangen Biochemical), universal DNA purification and recovery kit (Tiangen Biochemical, DP214-02), restriction endonuclease Sac I Fast Digest (Thermo), Pichia pastoris strain GS115 (Invitrogen).

[0084] 4.2 Equipment

[0085] Water bath, electroporation equipment, biochemical incubator.

[0086] 4.3 Methods

[0087] (1) Extraction of recombinant plasmid pPICZaA-g-Silk

[0088] a. Take 50 μL of the GS115 yeast plasmid solution to be electroporated and inoculate it into 50 mL of LB medium containing 25 μg / mL Zeocin antibiotics;

[0089] b. Take 15 mL of bacterial solution and centrifuge at 12000 rpm for 1 min at room temperature to collect the bacteria;

[0090] c. Extract pPICZaA-g-Silk according to the instructions of the high-purity plasmid mini-preparation kit, elute with 300 μL TB, and store at -20°C.

[0091] (2) Linearization of recombinant plasmid pPICZaA-g-Silk

[0092] pPICZaA-g-Silk was linearized with Sac I. The linearization reaction system is shown in Table 5. The specific experimental steps are as follows:

[0093] Table 5. pPICZaA-g-Silk linearization reaction system

[0094] Components Volume (μL) pPICZaA-Silk 200 10×FastDigestbuffer 80 SacI 12 <![CDATA[ddH2O]]> 508 800 μL in total

[0095] a. Prepare the reaction mixture according to Table 5 in a sterilized 2 mL centrifuge tube. Mix thoroughly, centrifuge briefly, and aliquot the solution into two tubes at 400 μL / tube. Digest the mixture in a 37°C water bath for 20 minutes.

[0096] b. Detect the linearized plasmid by 1% agarose gel electrophoresis; recover the linearized plasmid according to the instructions of a universal DNA purification and recovery kit;

[0097] c. Combine the two tubes of plasmid to a total of 180 μL, dry and concentrate at 37°C to 10-20 μL, and store at -20°C.

[0098] (3) Preparation of GS115 Pichia pastoris competent cells

[0099] a. Pick a single colony of Pichia pastoris strain GS115, inoculate it into 5 mL of sterile YPD liquid medium, and culture it in a shaking incubator at 29°C and 200 rpm for 8 hours;

[0100] b. After 8 hours, aspirate 400 μL of culture medium and inoculate into 95 mL of sterile YPD liquid medium. Incubate overnight at 29°C and 220 rpm in a shaker.

[0101] c. The next day, aspirate 1 mL of culture medium, dilute to 10 mL, adjust to zero with ddH2O, and measure OD600 using a spectrophotometer. If the OD600 value is between 1.1-1.7, the cells can be used for competent cell preparation.

[0102] d. Take 50 mL of culture medium, centrifuge at 3000 rpm at 4°C for 5 minutes, and discard the supernatant;

[0103] e. Resuspend the cells in 30 mL of sterilized ice-cold ddH2O, centrifuge at 3000 rpm for 5 min at 4°C, and discard the supernatant; repeat step 5 twice;

[0104] f. Resuspend the cells in 30 mL of sterilized ice-cold 1 M D-sorbitol, centrifuge at 3000 rpm for 5 min at 4°C, and discard the supernatant; repeat step 7 twice;

[0105] g. Resuspend the cells in 400 μL of sterilized, ice-cold 1 M D-sorbitol to prepare the competent cells. Aliquot the competent cells into 90 μL / EP tube.

[0106] (4) Electrotransformation and screening of linearized plasmids

[0107] Soak a 2mm electric shock cup in 75% ethanol for 30 minutes; rinse three times with anhydrous ethanol, and air dry on a clean bench.

[0108] b. Add 10 μL of linearized plasmid to 90 μL of competent cells, mix well, and then transfer 100 μL of the mixture to a 2 mm electroporation cuvette and pre-cool on ice for 10 minutes.

[0109] c. Set the electric shock parameters: voltage 1500V, capacitance 25uF, resistance 200Ω; quickly wipe the moisture outside the electric shock cup, put it into the electric shock tank, and shock;

[0110] d. Immediately after the electroporation, add 1 mL of 1 M ice-cold D-sorbitol, mix thoroughly, and aspirate the bacterial solution into a 1.5 mL sterile centrifuge tube.

[0111] e. Prepare YPD plates containing 60 μg / mL Zeocin antibiotics for screening: 200 μL of bacterial solution is spread gently on each plate with a sterile applicator and incubated inverted at 29°C for 3-4 days.

[0112] f. Prepare YPD plates and pick all the single colonies grown on the YPD plates containing Zeocin antibiotics in step e with a sterile toothpick to the YPD plates and incubate them inverted at 29°C for 1-2 days.

[0113] S5. Identification of Yeast Recombinant Transformants

[0114] 5.1 Materials

[0115] 2×Taq PCR Mastermix (Tiangen Biochemical). Ruiyuan Bio-Colony PCR Kit

[0116] 5.2 Equipment

[0117] PCR instrument (Bio-RAD), centrifuge, water bath, -80℃ refrigerator.

[0118] 5.3 Methods

[0119] (1) Extraction of transformant genomic DNA

[0120] a. Depending on the number of transformants grown on the YPD plate, pipette 3.0-3.5 μL (e.g., 3.0 μL) of yeast lysate into a PCR tube;

[0121] b. Use a sterile toothpick to pick up an appropriate amount of yeast colonies and suspend them in the yeast lysis buffer. Centrifuge briefly to concentrate the solution at the bottom of the tube. Mix the solution with a microfuge. Heat in a 98°C water bath for 10 minutes.

[0122] c. Centrifuge, aspirate the supernatant (containing genomic DNA) into a sterile centrifuge tube, and store at -20°C.

[0123] (2) PCR identification of recombinant transformant genome

[0124] PCR was performed using the extracted genomic DNA as a template. The strain that amplified the target gene fragment was considered a positive clone, namely, the GS115 / pPICZaA-g-Silk recombinant transformant. The PCR reaction system is shown in Table 6. The specific experimental steps are as follows:

[0125] Table 6. GS115 / pPICZaA-g-Silk recombinant transformant PCR reaction system

[0126]

[0127]

[0128] a. Take a sterilized EP tube, prepare the reaction system according to Table 4-1, mix well, and centrifuge briefly;

[0129] b. Set the following program in the PCR instrument: 94°C, 5 min; 94°C, 30 s (melting), 60°C, 30 s (annealing), 72°C, 1 min (extension), repeat 30 cycles; 72°C, 5 min, 4°C hold;

[0130] c. According to the PCR results, the positive transformants were preliminarily determined ( Figure 1The high-copy transformant with bright and clear target bands (i.e., GS115 / pPICZaA-g-Silk recombinant transformant) was used as a recombinant yeast strain for induced expression; the plate was stored at -4°C.

[0131] Expression of S6.GS115 / pPICZaA-g-Silk recombinant transformants in shake flasks

[0132] 6.1 Materials

[0133] Fermentation medium: BMGY liquid medium, BMMY liquid medium; strain: GS115 / pPICZaA-g-Silk recombinant transformant.

[0134] 6.2 Equipment

[0135] Shaker.

[0136] 6.3 Methods

[0137] (1) Pick a single positive colony and inoculate it into a 250 mL Erlenmeyer flask containing 30 mL of BMGY liquid medium. Incubate at 29°C and 220 rpm in a shaking incubator for 60 h.

[0138] (2) Number the 50 mL centrifuge tubes, transfer 4 mL of bacterial solution to a 50 mL centrifuge tube, add sterile double-distilled water to 30 mL, and centrifuge at room temperature and 3000 rpm for 5 min to collect the bacteria;

[0139] (3) Discard the supernatant and add 30 mL of sterile double-distilled water; centrifuge at 3000 rpm for 5 min at room temperature to collect the cells;

[0140] (4) Discard the supernatant and add 30 mL of BMMY liquid medium to resuspend the cells. Pour the mixed BMMY culture medium into a 250 mL Erlenmeyer flask and culture on a shaker at 29°C and 220 rpm.

[0141] (5) After culturing for 24 hours, add 300 μL of methanol;

[0142] (6) After culturing for 48 hours, add 300 μL of methanol;

[0143] (7) Cultivate for 68 hours, centrifuge, collect the supernatant in the bottle, and store at -20℃.

[0144] (8) SDS-PAGE electrophoresis was used to detect the expression results of the shake flask and confirm the expression of the target protein (recombinant silk fibroin). Figure 2 ).

[0145] S7. Fermentation and purification process

[0146] 7.1 Fermentation process

[0147] The recombinant silk fibroin genetically engineered bacteria obtained by screening were fermented using inorganic salt BSM culture medium as the base material, placed at 29±0.5°C, and shaken at 220rpm. Methanol induction was started when the wet weight of the bacteria in the fermentation tank (10L) reached 180-200mg / mL, and the fermentation was terminated when the wet weight of the target protein bacteria reached 410mg / mL and the wet weight no longer increased.

[0148] 7.2 Purification process

[0149] After the fermentation stopped, the fermentation broth in the fermenter was discharged, 8 L in total, and centrifuged using a cup centrifuge. A total of 7 L of the centrifugal supernatant was collected and purified using a DEAE Sepharose FF (DEAES seplife FF) ion exchange chromatography column. The chromatography column was equilibrated with three volumes of solution A and then loaded with the sample. After loading, the DEAE Sepharose FF filler chromatography column was flushed with solution A until the pH at the outflow end was 6-6.5. Then, 85% solution A + 15% solution B (volume fraction) was used to flush the column to wash impurities, and then eluted with 10 column volumes of 60% solution A + 40% solution B (volume fraction). The eluate was collected, wherein solution A was 10 mmol / L citric acid buffer at pH 6.0 and solution B was solution A containing 1 M NaCl. Finally, the eluate was dialyzed (dialysis bag with a molecular weight cutoff of 10 kDa) for desalting and lyophilization to obtain pure recombinant silk fibroin.

[0150] Comparative Example 1

[0151] The difference between this comparative example and Example 1 is that the comparative example 1 uses the commonly used signal peptide α-MF of pPICZaA for secretory expression of recombinant silk fibroin, and the rest is the same as Example 1.

[0152] 1. Original signal peptide molecule design

[0153] 1.1 Obtaining the amino acid sequence of α-MF

[0154] The amino acid sequence of α-MF is shown below, SEQ ID NO: 7:

[0155] Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser AlaLeu

[0156] Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro AlaGlu

[0157] Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp Val Ala Val LeuPro

[0158] Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Ile Asn Thr Thr Ile AlaSer Ile

[0159] Ala Ala Lys Glu Glu Gly Val Ser Leu Glu Lys Arg

[0160] (MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFD VAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR)

[0161] 1.2 Original signal peptide gene

[0162] According to the codon preference of Pichia pastoris, the nucleotide sequence corresponding to the α-MF amino acid sequence SEQ ID NO: 7 was encoded, and sequence elements such as restriction sites and stop codons were directly added to both ends of the nucleotide sequence to obtain SEQ ID NO: 8 containing the nucleotide sequence corresponding to the α-MF amino acid sequence.

[0163] SEQ ID NO:8:

[0164] 5'-ttcgaaacgatgagatttccttcaatttttactgctgttttattcgcagcatcctccgcattagctgctccagtcaa cactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttatcagatttagaaggggattt cgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctg ctaaagaagaaggggtatctctcgag-3'

[0165] The remaining steps and methods are the same as those in Example 1.

[0166] The recombinant transformant GS115 / pPICZaA-α-Silk obtained in Comparative Example 1 was induced to express, and the expression results of the shake flask were detected by SDS-PAGE electrophoresis to confirm the expression of the target protein ( Figure 3 At the same time, the peak area was compared with the GS115 / pPICZaA-g-Silk electrophoresis results obtained in Example using ImageJ software, and the results are shown in Table 7.

[0167] Table 7: Comparison of expression levels of recombinant silk fibroin strains between Example 1 and Comparative Example 1

[0168]

[0169]

[0170] The results showed that the secretory expression levels of recombinant silk fibroin in the GS115 / pPICZaA-g-Silk recombinant transformant constructed using the modified signal peptide of the present invention and the GS115 / pPICZaA-α-Silk recombinant transformant constructed using α-MF increased by 4.9 times.

[0171] 1. Experimental example of detecting the effect of recombinant silk fibroin on the proliferation of L929 cells

[0172] 1.1 Materials

[0173] The recombinant silk fibroin obtained in Example 1, the recombinant silk fibroin obtained in Comparative Example 1, DMEM medium (containing 10% fetal bovine serum), PBS, 75% alcohol, cell culture flasks, 96-well plates, pipettes, centrifuge tubes, alcohol lamps, 1 mL syringes, 0.22 μL filter membranes, steel rulers, and marker pens; and L929 cells (Shenzhen Haodi Huatuo Biotechnology Co., Ltd.).

[0174] 1.2 Equipment

[0175] Liquid nitrogen tank, clean bench, cell culture incubator, inverted imaging microscope, centrifuge, and microplate reader.

[0176] 1.3 Methods

[0177] 1.3.1 Paving

[0178] L929 cells were plated into a 96-well plate at a cell count of 10^3 / well and cultured for 24 hours. The example group was added with DMEM culture medium containing 10% fetal bovine serum and the recombinant silk fibroin protein (25 μg / mL) obtained in Example 1; the control group was added with DMEM culture medium containing 10% fetal bovine serum and the recombinant silk fibroin protein (25 μg / mL) obtained in Control Example 1, and then placed together in an incubator for incubation. One plate was taken every 24 hours for MTT detection. 1.3.2

[0180] The cell status was recorded using an inverted imaging microscope, and then the culture medium was removed, 50 μL of MTT solution was added to each well, and the cells were incubated at 37°C and 5% CO2 for 2 h. 1.3.3

[0182] Then, the MTT solution was removed, and 100 μL of isopropanol was added to each well, and the mixture was shaken at room temperature for 30 min. The absorbance (OD value) of each well was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA). 1.3.4

[0184] Data recording and calculation

[0185] Analyze the absorbance of recombinant silk fibroin (calculate the average OD value for each concentration), analyze the data using Prism 8, and plot a statistical line graph of the average OD value for each concentration versus time. The larger the OD value, the greater the number of cells, indicating a more pronounced effect on promoting cell proliferation.

[0186] 1.4 Results

[0187] The results are as follows Figure 4 As shown, the recombinant silk fibroin prepared in Example 1 has no toxicity to the growth of L929 cells and can effectively promote the growth and proliferation of L929 cells. It can play a positive role in the cell growth process. The recombinant silk fibroin obtained in Comparative Example 1 also has a certain effect of promoting cell proliferation. Figure 4 It can be seen that within 24 hours, there was no significant change in Comparative Example 1 compared with Example 1 (P=0.4048), within 48 hours, Example 1 significantly promoted cell proliferation compared with Comparative Example 1 (P=0.0292), and within 72 hours, Example 1 extremely significantly promoted cell proliferation compared with Comparative Example 1 (P<0.0001). This shows that compared with Example 1, the comparative example is not as good as Example 1 in promoting cell proliferation. This may be because different signal peptides have a certain effect on protein modification, thereby affecting the performance of the expressed protein.

[0188] 2. Recombinant Silk Fibroin Cell Scratch Assay

[0189] 2.1 Materials

[0190] The recombinant silk fibroin obtained in Example 1, the recombinant silk fibroin obtained in Comparative Example 1, DMEM culture medium, PBS, 75% alcohol, cell culture flasks, 6-well plates, pipettes, centrifuge tubes, alcohol lamps, 1 mL syringes, 0.22 μL filter membranes, steel rulers, and marker pens; and L929 cells (Shenzhen Haodi Huatuo Biotechnology Co., Ltd.).

[0191] 2.2 Equipment

[0192] Liquid nitrogen tank, clean bench, cell culture incubator, inverted microscope, centrifuge, and microplate reader.

[0193] 2.3 Methods

[0194] Plating: L929 cells were seeded into 6-well plates at a density of 2×10^6 cells. After culturing for 24 hours, the cells were streaked with a pipette tip and washed three times with PBS. Subsequently, the recombinant silk fibroin protein obtained in Example 1 (25 μg / mL) and the recombinant silk fibroin protein obtained in Comparative Example 1 (25 μg / mL) were added to serum-free DMEM medium for culture; a blank control group was treated with serum-free DMEM medium alone. Pictures were taken under a microscope at 0, 24, 48, and 72 hours of culture, and the scratch area was measured using Image J software. The cell migration rate of each group was calculated. The greater the cell migration rate, the more significant the effect of promoting cell migration, that is, the stronger the cell repair and healing ability.

[0195] 2.4 Results

[0196] The results are as follows Figure 5 As shown, within 24 hours, there was no significant difference between Comparative Example 1 and Example 1 (P=0.1167), within 48 hours, Example 1 significantly promoted cell migration compared with Comparative Example 1 (P=0.0472), and within 72 hours, Example 1 significantly promoted cell migration compared with Comparative Example 1 (P=0.0155). The results show that the recombinant silk fibroin obtained in Example 1 and the recombinant silk fibroin obtained in Comparative Example 1 both have a relatively obvious effect of promoting cell migration, among which the recombinant silk fibroin obtained in Example 1 has the best effect of promoting cell migration.

[0197] 3. Detection of epidermal water loss by recombinant silk fibroin

[0198] 3.1 Materials

[0199] The recombinant silk fibroin obtained in Example 1, the recombinant silk fibroin obtained in Comparative Example 1, sodium lauryl sulfate, 1% sodium lauryl sulfate solution, a spot tester and corresponding filter paper, a lint-free absorbent dry paper towel, a test shielding box, purified water, and glycerin.

[0200] 3.2 Equipment

[0201] Transepidermal water loss tester (Tewameter™ Hex), analytical balance, single-channel pipette

[0202] 3.3 Methods

[0203] 3.3.1 Preparation of Test Samples: The Example group consisted of the recombinant silk fibroin obtained in Example 1, glycerol, and water, with the mass fraction of the recombinant silk fibroin obtained in Example 1 being 0.05%, the mass fraction of glycerol being 5%, and the purified water making up to 100%. The Comparative Example group consisted of the recombinant silk fibroin obtained in Comparative Example 1, glycerol, and water, with the mass fraction of the recombinant silk fibroin obtained in Comparative Example 1 being 0.05%, the mass fraction of glycerol being 5%, and the purified water making up to 100%. 3.3.2

[0205] This study used a fermented composition containing recombinant silk fibroin as a test sample to investigate its performance in skincare products. The skin barrier function was analyzed using a transepidermal water loss meter to assess whether the test sample enhanced skin repair efficacy. Four groups were selected, each consisting of 14 participants, for the experimental and comparative examples.

[0206] 3.3.3 Steps:

[0207] (1) Establishment of skin barrier damage: 200 μL of sodium dodecyl sulfate solution was injected into the patch test chamber, and the filter paper was soaked. The filter paper was applied to the curved side of the left and right forearms of the subject with low-allergenic tape, 3 on each side. The distance between the edges of adjacent patch test chambers was not less than 3 cm. The patch was evenly applied to the skin with light pressure using the palm of the hand for 24 hours.

[0208] After removing the patch tester, the subjects sat quietly in a standard test environment for 30 minutes (after the indentation disappeared), and the TEWL value at the center of each patch area was measured. Each area was measured once, and the measurement result was expressed as the average of the TEWL values ​​of the three patch areas on a single arm as the initial value.

[0209] During the sitting period, the subjects were not allowed to drink water or beverages, their forearms were exposed and placed in the testing position, they remained relaxed, and avoided touching the patch area.

[0210] Shielding boxes A, B, and C are used in a cycle. After each subject is tested, the used shielding box is immediately turned upside down with the bottom facing upward to restore the environment inside the box to the test environment.

[0211] (2) Determination

[0212] The left and right forearm flexures of the subjects were randomly divided into the sample application side and the control side, and each side contained 3 patch areas to ensure that the sample application side and the control side were statistically balanced.

[0213] Before each measurement, clean the test area with clean water and dry it with a lint-free absorbent paper towel.

[0214] At the set measurement time point, the TEWL value at the center of each patch area was measured, and each area was measured once. The measurement result was expressed as the average TEWL value of the three patch areas in a single arm.

[0215] The test of the same subject must be completed by the same tester using the same instrument. If the subject experiences an adverse skin reaction during sample use, the test should be terminated immediately, the subject should be given appropriate medical treatment, and the adverse reaction should be recorded.

[0216] 3.4 Results

[0217] Transepidermal water loss (TEWL), also known as transepidermal water loss rate, refers to the rate at which water is lost from the dermis through the epidermis per unit time. In human studies, TEWL measurements can reflect changes in skin barrier function. When the skin barrier is intact, the TEWL value is low. When the skin barrier is damaged by physical, chemical, or pathological factors, the TEWL value increases. Therefore, by measuring the change in TEWL values ​​before and after product use, the effectiveness of cosmetics or cosmetic ingredients in improving skin barrier function can be evaluated.

[0218] Depend on Figure 6 , Figure 7 It can be seen that after using the sample for 6 and 8 days, the average value of transepidermal water loss of Example 1 gradually decreased from 17.59 to 13.85, and the value of Comparative Example 1 decreased from 17.37 before use to 16.80 on the 8th day; from the change rate of transepidermal water loss, it can be seen that Example 1 changed from -4.57 on the 6th day to -18.87 on the 8th day, and Comparative Example 1 changed from -1.52 to -3.26. It can be seen from the data changes that the recombinant silk fibroin prepared in Example 1 and Comparative Example 1 has a repairing effect on the skin, but the recombinant silk fibroin expressed in Example 1 has a significantly stronger skin repairing ability than the recombinant silk fibroin using α-MF.

[0219] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and such modifications or substitutions are intended to be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A signal peptide for increasing the expression of recombinant silk fibroin, characterized in that: The amino acid sequence of the signal peptide is shown in SEQ ID NO:

1.

2. A nucleotide sequence encoding the signal peptide according to claim 1, characterized in that: The nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 3 or its complementary sequence.

3. A recombinant plasmid, recombinant transformant, recombinant expression vector or recombinant bacterium containing the nucleotide sequence according to claim 2.

4. Use of the signal peptide according to claim 1 in increasing the expression level of recombinant silk fibroin.

5. A method for preparing recombinant silk fibroin, characterized in that: include: A signal peptide comprising the amino acid sequence shown in SEQ ID NO: 1 is used to direct the expression of recombinant silk fibroin in Pichia pastoris host cells.

6. A recombinant silk fibroin prepared by the method according to claim 5.

7. Use of the recombinant silk fibroin according to claim 6 in the preparation of a product for promoting cell proliferation, cell migration and / or repairing skin barrier function.

8. Use of the recombinant silk fibroin obtained by using the signal peptide according to claim 1 in preparing a product for promoting cell proliferation, cell migration and / or repairing skin barrier function.

9. Use of the recombinant silk fibroin obtained by using the recombinant plasmid, recombinant expression vector or recombinant bacteria as claimed in claim 3 in the preparation of a product for promoting cell proliferation, cell migration and / or repairing skin barrier function.

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