Application of silkworm silk glue protein sericin 4 repetitive motif in improvement of silk and sericin hydrogel performance and method of silkworm silk glue protein sericin 4 repetitive motif in improvement of silk and sericin hydrogel performance

Abstract

The invention discloses an application of a silkworm silk glue protein sericin 4 repetitive motif in improvement of silk and sericin hydrogel performance and a method thereof, a second repetitive region of a sericin 4 gene is completely expressed in a silkworm cocoon sericin layer through cloning and micro-injection, a bred transgenic strain improves the silk mechanical property, the sericin hydrogel performance is remarkably improved, and the sericin protein sericin 4 repetitive motif can be used as a sericin protein sericin 4 repetitive motif. The material is expected to be applied to biological materials and tissue medical engineering.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of a silkworm sericin protein sericin4 repeating motif in improving the properties of silk and sericin hydrogels, and also to a method for improving the properties of silk. Background Art

[0002] Silk is the earliest and most widely used natural animal protein fiber by humans. Silkworms (Bombyx mori) produce different types of silk fibers at different stages of their development. Silkworm larvae undergo four molts before forming cocoons, resulting in the fifth instar larvae. The first and last instar silks secreted by silkworms at the beginning and end of each instar are collectively referred to as small silk. Small silk and cocoon silk have distinct sericin compositions, suggesting that different sericin components may play distinct biological roles in small and cocoon silk. Previous research by the inventors has shown that small silk sericin has a more β-sheet structure and greater viscosity than cocoon sericin. The unique composition of silk proteins is a fundamental factor in determining the superior properties of silk. This suggests that the unique sericin components of small silk may significantly contribute to its exceptional mechanical and adhesive properties. Further in vitro and in vivo experiments have demonstrated that the sericin 4 (Ser4) protein is key to the improved mechanical properties of small silk. Researchers generally believe that the highly repetitive motifs of silk proteins determine their biological properties. Sericin proteins are primarily composed of highly ordered repetitive motifs. Analysis has revealed that the Ser4 protein contains two large repetitive regions, but whether Ser4 influences the mechanical properties of silk through these repetitive regions remains unclear. Furthermore, Ser4 has a large molecular weight exceeding 250 kDa, making it difficult to express, purify, and utilize. Finding a sericin repetitive region protein with a small molecular weight and excellent mechanical properties would be of great value to the application and expansion of sericin.

[0003] With the publication of the silkworm genome map and the establishment of a molecular breeding system, the molecular and technological foundations for genetic improvement and material innovation in silk have been laid. In recent years, silk properties and genetic manipulation of silkworms have become a hot topic in silk protein research. Since Japanese researchers established a silkworm transgenic technology system based on the piggyBac transposon in 2003, transgenic overexpression systems for incremental gene expression in various silkworm tissues have also been widely used. Transgenic overexpression technology is a highly effective method for modifying the silk gland and silk properties of silkworms. Unlike gene editing, transgenic overexpression technology recombinantly expresses functional exogenous target proteins in specific silkworm tissues, making it relatively gentle and unlikely to significantly affect the silkworm's physical constitution or silk properties. Using these genetic manipulation systems, researchers have successfully modified silk properties, endowed silk with new biological functions, and expanded the application of silk. Therefore, using the silk gland transgenic incremental expression system to study the influence of sericin repeat motifs on silk properties is of great significance for obtaining high-performance silk and sericin materials. Summary of the Invention

[0004] In light of this, one objective of the present invention is to provide an application of a repeating motif of the silkworm sericin protein sericin4 to improve the properties of silk and sericin hydrogels. This invention utilizes the overexpression of the second repeating region of the sericin protein sericin4, unique to small silkworms, in sericin to obtain silk with superior mechanical properties. A second objective of the present invention is to provide a method for improving the properties of silkworm silk. A third objective of the present invention is to provide silk with enhanced properties produced by the method. A fourth objective of the present invention is to provide a sericin hydrogel with enhanced properties produced by the method.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: 1. Application of a Bombyx mori sericin protein sericin4 repeating motif in improving the properties of silk and sericin hydrogels, wherein the amino acid sequence of the Bombyx mori sericin protein sericin4 repeating motif is shown in SEQ ID NO. 4.

[0006] In some embodiments of the present invention, the nucleotide sequence encoding the Bombyx mori sericin sericin4 repeating motif is shown as SEQ ID NO.3.

[0007] In some embodiments of the present invention, the property is strength or stiffness among mechanical properties.

[0008] 2. A method for improving the properties of silkworm silk, comprising overexpressing a repeating motif of the Bombyx mori sericin protein, Sericin 4, in Bombyx mori. The resulting transgenic individuals produce silk with improved properties. The amino acid sequence of the repeating motif of the Bombyx mori sericin protein, Sericin 4, is shown in SEQ ID NO. 4.

[0009] In some embodiments of the present invention, the overexpression method is to construct an overexpression transgenic vector containing the silkworm sericin 4 repeating motif, and then inject the transgenic vector into silkworm eggs. After raising the G0 generation, mating and laying eggs, the G1 generation silkworm eggs are fluorescently screened to successfully obtain transgenic positive individuals.

[0010] In some embodiments of the present invention, the transgenic vector further contains the promoter of the Sericin1 gene specifically expressed in the middle silk gland of the silkworm, the Hr3 enhancer, and the poly A terminator sequence of the Sericin1 gene.

[0011] 3. Silk with improved properties produced by the method.

[0012] 4. A sericin hydrogel with improved properties prepared by the method.

[0013] The beneficial effects of the present invention are as follows: through cloning and vector construction, the present invention realizes the expression of the Ser4 second repeat region protein in the sericin layer of silkworm cocoons, and the cultivated strain has the following characteristics: 1) It was first determined that the second repeat region of sericin Ser4 in the young silkworm stage affects silk properties; 2) Improvements in silk mechanical properties were achieved without the introduction of exogenous proteins; 3) The mechanical properties of sericin hydrogels prepared from silkworms overexpressing Ser4-rp2 can be significantly improved, and are expected to be applied in the fields of tissue engineering and biomaterials. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration: Figure 1 It is the amino acid sequence of different repeat regions of Ser4 protein.

[0015] Figure 2 Alphafold2 three-dimensional structure prediction for different repeat regions of Ser4 protein.

[0016] Figure 3Schematic diagram of the transgenic vector and the screening and molecular detection of positive individuals (A. Schematic diagram of the transgenic vector containing the Ser1 repeat region and the Ser4 repeat region; B. Screening of positive individuals; 3xp3 is the eye-specific promoter; DsRed is the red fluorescent protein; SV40 is the stop codon sequence; hr3CQ is the enhancer; Ser1 is the promoter region of the sericin1 gene; Ser1PA is the poly A termination sequence of the sericin1 gene; C. SDS-PAGE and Western Blot detection of transgenic silk Ser1-rp2; D. SDS-PAGE and Western Blot detection of transgenic silk Ser4-rp2).

[0017] Figure 4 Purification of Ser1-rp2 and Ser4-rp2 proteins. (A. Purification process for target proteins from silkworm cocoons; B. SDS-PAGE analysis of purified proteins).

[0018] Figure 5 Infrared spectral analysis of silk (AC. Deconvolution analysis of the amide I band in the FTIR spectra of silk from WT, Over-Ser1-rp2, and Over-Ser4-rp2 strains; (○) original data, (─) overall fitting curve, (┄) peak fitted by Gaussian function; D. β-sheet structure content of silk from each strain).

[0019] Figure 6 Mechanical properties testing of WT, Over-Ser1-rp2 and Over-Ser4-rp2 silk lines (A. Stress-strain curve of WT silk; B. Stress-strain curve of Over-Ser1-rp2 silk line; C. Stress-strain curve of Over-Ser4-rp2 silk line; D. Average mechanical curve of WT, Over-Ser1-rp2 and Over-Ser4-rp2 silk lines).

[0020] Figure 7 Analysis of silk mechanical parameters (A. stress; B. strain; C. Young's modulus; D. toughness; significant difference analysis: ns p ≥ 0.05, *** p < 0.001).

[0021] Figure 8 Preparation and mechanical property testing of sericin hydrogel (A. sericin hydrogel; B. hydrogel compression performance curve; C. hydrogel compression strength. Significant difference analysis: ns p ≥ 0.05, *** p < 0.001). DETAILED DESCRIPTION

[0022] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0023] Example 1 Sericin Ser4 in young silkworms can improve the mechanical properties of silk. So what causes the differences in performance between different sericins? Existing studies have shown that the primary structure of silk protein largely determines its performance. Among them, the functional modularity of silk protein sequences has been relatively clear. The highly repetitive regions in silk protein determine its biological properties. We further analyzed the amino acid sequence of the sericin Ser4 repeat unit. The Ser4 protein also has two repeat regions. The first repeat region consists of 54 amino acids repeated 12 times, and the second repeat region consists of 40 amino acids repeated 8 times ( Figure 1 ).

[0024] Further three-dimensional structure prediction of the two repeat regions of Ser4 was performed using Alphafold2 (https: / / robetta.bakerlab.org / ). The structure of the first repeat region of Ser4 (Ser4-rp1) is primarily composed of helices and random coils, while the second repeat region of Ser4 (Ser4-rp2) is composed of a regular stack of β-sheets (Figure 2). β-sheet structure plays a decisive role in the mechanical properties of silk, so we speculate that Ser4 influences silk mechanical properties through its second repeat region. The three-dimensional structure prediction results indicate that Ser4-rp2 is primarily composed of β-sheets.

[0025] To prove that the prediction result is correct, we used the second repeat module of Ser1 (Ser1 repeat2, SEQ ID NO.1-2) as a control and constructed a transgenic overexpression vector of the second repeat module of Ser4 (Ser4 repeat2, SEQ ID NO.3-4) in the middle silk gland ( Figure 3, A). 3xp3 is the eye-specific promoter, DsRed is the red fluorescent protein, SV40 is the stop codon, Ser1 is the promoter of the sericin1 gene, and Ser1PA is the poly A terminator sequence of the sericin1 gene. To facilitate subsequent protein isolation and purification, a 6×His tag was added to the front of the expression sequence. The constructed transgenic vector was extracted and ultrapure plasmid was injected into silkworms. After rearing the G0 generation, mating, and egg laying, G1 generation eggs were further screened for fluorescence to successfully obtain transgenic individuals positive for the Ser1-rp2 and Ser4-rp2 genes (Figure 3, B). These lines are designated as Over-Ser1-rp2 and Over-Ser4-rp2.

[0026] To confirm that Ser1-repeat2 and Ser4-repeat2 proteins were successfully expressed in silk, we collected cocoons from the wild-type control D9L and the two overexpression lines, dissolved the silk with lithium thiocyanate for protein quantification, and then performed SDS-PAGE and Western blot analysis. The results showed that Ser1-rp2 and Ser4-rp2 proteins were successfully expressed in the cocoons ( Figure 3 , C, D). We Figure 4 Recombinant sericin proteins Ser1-rp2 and Ser4-rp2 were isolated and purified from silkworm cocoons using method A. After urea extraction at 80°C for 1 hour, the sericin extract was collected. The supernatant was centrifuged, filtered, and passed through a nickel affinity chromatography column. Elution was then performed with 20, 50, 100, 250, 500 mM, and 1 M imidazole. The eluate and sericin stock solution were then collected and analyzed by SDS-PAGE. Figure 4 , B shows that in the 250 mM imidazole eluate, we obtained very pure Ser1-rp2 and Ser4-rp2 proteins.

[0027] Example 2 We previously predicted that the Ser4-rp2 protein is mainly composed of a β-sheet structure. To verify this result, we used Fourier transform infrared spectroscopy (FTIR) to analyze the secondary structure of silkworms from WT, Over-Ser1-rp2, and Over-Ser4-rp2 strains. Figure 5, AC is the result of deconvolution analysis of amide I band. The secondary structure content in each silk was calculated based on the peak area. The results showed that the average β-sheet content of the Over-Ser4-rp2 strain was 24.6%, significantly higher than the 21.81% of the wild-type control; while the average β-sheet content of the Over-Ser1-rp2 strain was 22.7%, which was not significantly different from the wild-type. There was no significant difference in the helical / random coil content of the three strains of silk. The β-turn structure content of the Over-Ser4-rp2 strain was 17.45%, significantly lower than the 20% of the wild-type; while the average β-turn content of the Over-Ser1-rp2 strain was 19.15%, which was not significantly different from the wild-type ( Figure 5 , D).

[0028] Infrared spectroscopy results are consistent with 3D structural predictions, demonstrating that overexpression of the Ser4-rp2 protein increases the β-sheet content of silk. β-sheet structure is considered a primary factor influencing the mechanical properties of silk. Therefore, our discovery that the second repeat module of the Ser4 protein differs from the repeat region of the Ser1 protein may explain the improved properties of the Ser4 protein.

[0029] To demonstrate the effect of the sericin repeat module on silk properties, we tested the mechanical properties of silk from two transgenic strains and a wild-type control strain. The results showed that the strength of the silk from the Over-Ser4-rp2 strain was significantly higher than that of the WT and Over-Ser1-rp2 strains, while its ductility was slightly lower than that of the WT and OverSer4-rp1 strains ( Figure 6 ). Statistical analysis of the mechanical properties of silk revealed that the strength of Over-Ser4-rp2 silk increased by 37% and the Young's modulus increased by 48% compared to WT silk, which is consistent with the infrared spectroscopy results. The higher β-sheet content of Over-Ser4-rp2 silk gives the silk excellent strength and stiffness; while the reduced β-turn structure content causes the ductility of Over-Ser4-rp2 silk to decrease by 22% compared to WT. There is no significant difference in the mechanical parameters between Over-Ser1-rp2 and WT silk, which further illustrates that Ser1-rp2 has no effect on silk performance, while Ser4-rp2 gives the silk better strength and stiffness by forming a β-sheet structure ( Figure 7 ).

[0030] Example 3 To prepare sericin hydrogels, silkworm cocoons were frozen in liquid nitrogen and ground into cocoon powder using a high-speed rotary mill. Equal amounts of cocoon powder from different strains were weighed and extracted with 8 M urea at a concentration of 120 mg / mL at 80°C for 50 min. Undissolved silk fibroin was removed by filtration using gauze and multiple layers of filter paper to obtain a sericin solution. The sericin solution was centrifuged three times at 13,000 g for 6-10 min each at 25°C, and the supernatant was retained in a clean beaker for later use. Wild-type and transgenic sericin solutions were added to a 10 kDa dialysis bag and dialyzed overnight at 4°C with stirring in PBS. The dialyzate was changed every 6 hours for a total of 4 days, ultimately forming a solid hydrogel. Pieces of wild-type D9L silk sericin hydrogel, Over-Ser1-repeat2, and Over-Ser4-repeat2 silk sericin hydrogels with a diameter of 8 mm and a height of 0.8 cm were cut and placed on a stage ( Figure 8 , A). A hydrogel dynamometer was used to test the mechanical properties of the hydrogels at a compression rate of 40 mm / min. The results showed that the compressive strength of the Over-Ser4-rp2 silk sericin hydrogel increased by 48.99%, while there was no significant difference between the Over-Ser1-rp2 and WT hydrogels (Figure 8, B, C).

[0031] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. Application of the silkworm sericin protein sericin4 repeat motif in improving the properties of silk and sericin hydrogels, characterized by: The amino acid sequence of the silkworm sericin sericin4 repeating motif is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that: The nucleotide sequence encoding the Bombyx mori sericin sericin4 repeating motif is shown in SEQ ID NO.

3.

3. The use according to claim 1, characterized in that: The performance is strength or stiffness among mechanical properties.

4. A method for improving the properties of silkworm silk, characterized by: The silkworm sericin protein sericin4 repeating motif is overexpressed in silkworms, and the silk produced by the transgenic positive individuals obtained is silk with improved performance. The amino acid sequence of the silkworm sericin protein sericin4 repeating motif is shown in SEQ ID NO.

4.

5. The method according to claim 4, characterized in that: The overexpression method comprises constructing an overexpression transgenic vector containing a repeating motif of silkworm sericin sericin4, injecting the transgenic vector into silkworm eggs, raising the G0 generation, allowing the moths to mate and lay eggs, and fluorescently screening the G1 generation silkworm eggs to successfully obtain transgenic positive individuals.

6. The method according to claim 5, characterized in that: The transgenic vector also contains the promoter of the Sericin1 gene specifically expressed in the middle silk gland of the silkworm, the Hr3 enhancer and the poly A termination sequence of the Sericin1 gene.

7. Silk with improved properties obtained by the method according to any one of claims 4 to 6.

8. A sericin hydrogel with improved properties prepared by the method according to any one of claims 4 to 6.

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