Recombinant human collagen type i silk material with cell proliferation function and application thereof

Abstract

The application discloses a silk material with a cell proliferation function and application thereof, the functional material is expressed by a human type I collagen truncated fragment in a middle silk gland of a domestic silkworm, a sericin promoter is used as a tissue-specific promoter to drive specific expression of an exogenous gene in the middle silk gland, and finally the exogenous gene is combined with sericin protein and is secreted to outside of the body, the exogenous protein can be separated after a certain treatment of a cocoon, and the expression product can keep biological activity. The silk material is used to prepare a hydrogel, has a function of promoting wound healing, and can be applied to aspects of promoting skin regeneration and repair, bone graft substitutes, cartilage reconstruction, artificial skin and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of materials, in particular to a silk material with cell proliferation function, and also relates to the application of the silk material. BACKGROUND

[0002] Collagen is the most abundant protein in mammalian tissues, accounting for about 30% of the total protein content of the body. It not only forms a bridge between cells, but also provides many important biological functions, such as protection, support, repair and nutrient transport. It is also an important component of the extracellular matrix (ECM) and plays a key role in helping skin, cartilage, tendon, ligament and internal organs maintain their structure and shape, and is a coordinator of many biological processes. In addition to its structural role, collagen can interact with various macromolecules such as integrins, decorin, fibronectin, heparin and matrix metalloproteinases (MMPs), which are very important in regulating tissue regeneration. Fibroblasts, chondroblasts, osteoblasts, odontoblasts, myoblasts, adipocytes, endothelial cells and some epithelial cells can all secrete collagen. With its special structure, diverse functions, biodegradability, low immunogenicity and cell proliferation and differentiation promoting properties, collagen is an ideal biological medical and beauty and skin care material. As a biopolymer, collagen can also be mixed with other hydrophilic polymers to form high-quality hydrogels and biodegradable scaffolds, and the preparation of collagen-chitosan blends has become a focus of scientific attention.

[0003] Collagen contains a typical triple helix structure, which is a complex structure formed by 3 alpha chains twisted into a triple helix, providing cell adhesion and receptor recognition sites for collagen, and is a key factor for collagen to exert biological activity. The triple helix structure of collagen is derived from the Gly-Xaa-Yaa tripeptide repeat sequence in its primary structure, where Xaa is often proline and Yaa is often hydroxyproline. In the triple helix domain, there are three polypeptide (alpha) chains, each with a left-handed conformation, and they are twisted to form a right-handed superhelix. The length of this triple helix domain varies depending on the type of collagen and can be continuous or interrupted by non-helical domains. The assembly process of the triple helix of collagen is strictly regulated, and the procollagen chains are selectively combined in a type-specific manner. This is particularly important when different collagens are co-expressed in the same tissue. There are at least 28 different types of collagen in the human body, forming a highly specialized family of glycoproteins.

[0004] Collagen type I is an α1)2α2 heterotrimer transmembrane protein, and the α1 chain has a total length of 1464 AA and a molecular weight of about 138 KD. Collagen type I is the most abundant collagen protein, which is widely distributed in connective tissues such as skin, skeleton, ligament and tendon, and accounts for about 85% of the total collagen content in the skin. Compared with other types of collagen proteins, the fiber of collagen type I is relatively thick and hard, which is equivalent to a collagen skeleton and plays a main supporting role, and endows the skin with toughness and elasticity. Collagen type I is involved in cell proliferation, differentiation, migration and signal transmission, and plays an important role in tissue support and repair. It has the functions of promoting skin regeneration and repair, maintaining the integrity and mechanical characteristics of bones, and is applied to bone graft substitutes, guided tissue regeneration in dentistry, cartilage reconstruction, artificial skin and the like in tissue engineering, and is applied to filling injections and skin care products and the like in the cosmetic field.

[0005] The silkworm transgenic silk gland bioreactor is an expression system for expressing exogenous genes in the silkworm silk gland. The middle silk gland (MSG) of the silkworm is mainly responsible for the synthesis and secretion of sericin, and the sericin promoter can drive the specific expression of the exogenous gene in the middle silk gland, and finally combine with the sericin protein to be secreted to the outside of the body. After a certain treatment is performed on the cocoon, the exogenous protein can be separated and purified, and the expression product can maintain biological activity. In this study, through transgenic technology, human collagen type I (Col I) is successfully expressed in the middle silk gland of the silkworm and secreted into the sericin layer of the cocoon. SUMMARY

[0006] Therefore, one of the purposes of the present application is to provide a silk material with cell proliferation function; the second purpose of the present application is to provide a preparation method of the Col I / sericin freeze-dried powder with cell proliferation function; the third purpose of the present application is to provide the Col I / sericin freeze-dried powder prepared by the preparation method; the fourth purpose of the present application is to provide a preparation method of a material for promoting wound healing, and the fifth purpose of the present application is to provide the material for promoting wound healing prepared by the preparation method; and the sixth purpose of the present application is to provide the application of the material.

[0007] To achieve the above purposes, the present application provides the following technical solutions.

[0008] 1. A silk material with cell proliferation function, wherein the silk material is a truncated fragment of human collagen type I expressed in the middle silk gland of the silkworm and secreted into the sericin layer of the cocoon, and the obtained silk has cell proliferation function; the nucleotide of the truncated fragment of human collagen type I is shown in positions 7-1578 of SEQ ID NO. 1.

[0009] Preferably, the silk material is obtained by connecting the truncated fragment of human type I collagen shown in SEQ ID NO. 1 into the BamHI and NotI enzyme cutting sites of psl1180[hr3PSer1spRedSer1PA], then cutting with AscI, and connecting into the pBac[3xp3DsRedaf] vector cut with the same enzyme, and the obtained vector is used for microinjection to screen transgenic silkworms, and the silk secreted by the transgenic silkworms is the silk with the function of promoting cell proliferation.

[0010] 2. A preparation method of Col I / silk sericin freeze-dried powder with the function of promoting cell proliferation, comprising the following steps: crushing silk, adding 1 million to 3 million U / L of bacillus subtilis protease, and hydrolyzing at 40-45°C for at least 3 hours.

[0011] 3. Col I / silk sericin freeze-dried powder prepared by the preparation method.

[0012] 4. A preparation method of a material for promoting wound healing, comprising the following steps: crushing silk expressing a truncated fragment of human type I collagen, extracting silk sericin containing the truncated fragment of human type I collagen by urea method, removing urea by dialysis, and inducing gel formation at low temperature to obtain the material for promoting wound healing.

[0013] Preferably, the urea method extraction is carried out in 8M urea 50mM Tris-HCl solution, 80°C water bath extraction for 60 min, then 13500 rpm centrifugation for 10 min, and taking the supernatant.

[0014] Preferably, the silk bath ratio in the urea method extraction is 60-70 mg / mL.

[0015] Preferably, the dialysis is carried out at 4-25°C with deionized water for at least 3 days.

[0016] 5. The material for promoting wound healing prepared by the preparation method.

[0017] 6. Application of the Col I / silk sericin freeze-dried powder or the material in preparing a material for promoting skin regeneration and repair, bone graft substitutes, cartilage reconstruction or artificial skin.

[0018] The beneficial effects of the present application are that the present application provides a silk material with cell proliferation function, the functional material is expressed by a truncated fragment of human type I collagen in the middle silk gland of the domestic silkworm, the sericin promoter is used as a tissue-specific promoter to drive the specific expression of the exogenous gene in the middle silk gland, and finally combined with the sericin protein and secreted to the outside of the body, the exogenous protein can be separated and purified after a certain treatment of the cocoon, and the expression product can maintain biological activity. The silk material is used to make a hydrogel, which has the effect of promoting wound healing, so it can be used as a function of promoting skin regeneration and repair, maintaining the integrity and mechanical characteristics of bone, and applied to bone graft substitutes in tissue engineering, guided tissue regeneration in dentistry, cartilage reconstruction, artificial skin, and the like, and applied to filling injections and skin care products in the cosmetic field. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to make the purpose, technical scheme and beneficial effects of the present application clearer, the present application provides the following drawings for illustration:

[0020] Figure 1 is a structural diagram of a recombinant Col I expression vector;

[0021] Figure 2 is a screening of a recombinant Col I positive domestic silkworm;

[0022] Figure 3 is a screening of a recombinant Col I positive domestic silkworm;

[0023] Figure 4 is a Col I Western Blot detection;

[0024] Figure 5 is a Col I expression amount detection;

[0025] Figure 6 is a Col I / sericin hydrogel;

[0026] Figure 7 is a mouse wound healing situation;

[0027] Figure 8 is a 14th day mouse wound skin HE staining and Masson staining;

[0028] Figure 9 is a subtilisin hydrolysis of cocoon sericin protein;

[0029] Figure 10 is a Col I / sericin freeze-dried powder;

[0030] Figure 11 is the influence of Col I / sericin on cell survival rate. DETAILED DESCRIPTION

[0031] The present application will be further described below in connection with the drawings and specific embodiments so that those skilled in the art can better understand the present application and implement it. The embodiments are not intended to limit the present application.

[0032] Example 1, Selection of Col I fragment

[0033] The full length of Col I alpha 1 chain contains 1464 amino acids. The 179-1192 AA is the helical region, which is the main functional region of Col I. The 228-751 AA region in the helical region contains multiple motifs with high affinity to integrin, such as DGEA, RGD, GER, KGD, which are predicted to play an important role in the biological function of Col I.

[0034] Example 2, Obtaining of Col I transgenic silkworm strain

[0035] The amino acid sequence of human Col I protein NP_000079.2 was obtained from Uniprot. According to the codon bias in the translation process of Bombyx mori, the base sequence was optimized, and a Bam HI restriction site was added at the N terminus of the sequence, and a 6×His tag and a Not I restriction site were added at the C terminus for the construction of an expression vector and subsequent detection. The recombinant Col I protein gene with a 6×His tag was artificially synthesized, and the sequence is shown in SEQ ID NO. 1.

[0036]

[0037] The synthetic sequence was connected to the BamHI and NotI enzyme cutting sites of sl1180[hr3PSer1spRedSer1PA], and then connected to the pBac[3xp3DsRedaf] vector cut by AscI, which was abbreviated as pBac[3xp3DsRed-Col I-S]. The result of nucleic acid electrophoresis after AscI cutting showed that the expression vector was successfully constructed, and the structure thereof is shown in Figure 1 The wild type (WT) eggs of the non-diapause Bombyx mori strain were microinjected, and positive individuals were obtained through fluorescence screening. The recombinant collagen type I was extracted from the positive eggs of the domestic silkworm and observed for specific bands through Western blot detection, and further mass spectrometry was used to detect the successful expression of the recombinant collagen type I.

[0038] Example 3, screening of recombinant Col I positive domestic silkworms

[0039] Before microinjection, an auxiliary plasmid pHA3PIG was added to the pBac[3xp3DsRed-Col I-S] plasmid, and the two plasmids were mixed at a mass ratio of 1:1, and the eggs laid by female moths were fixed on a glass slide. The eggs were injected using a microinjection instrument, and fresh eggs laid within two hours were selected to increase the success rate of transgenic. Each injected egg was sealed with non-toxic glue. The injected eggs were placed in a 26°C environment for development, and G0 generation larvae were hatched after about 10 days. The G0 generation moths were mated with each other or with wild type moths to obtain F1 generation eggs. The F1 generation eggs were hatched to the seventh day, and the in-egg larvae were observed under a fluorescence microscope. The transgenic silkworms successfully introduced into the silk glue expression system emitted specific red fluorescence under excitation light. The transgenic silkworm larvae and moths after hatching also had this property. Figure 2

[0040] Example 4, recombinant expression and identification of Col I in Bombyx mori

[0041] Extraction of Col I cocoon silk glue: Clean cocoon was quickly frozen in liquid nitrogen, and then completely pulverized into powder (A). An appropriate amount of cocoon powder was weighed, and then immersed in 8M urea and 50mM Tris-HCL solution at a bath ratio of 20mg / mL. The solution was extracted at 80°C for 45min, and then centrifuged at 13500rpm for 10min. The supernatant was carefully aspirated to obtain a silk glue sample containing recombinant protein (B). Figure 3 Figure 3

[0042] ​​​Western Blot experiment was performed with the extracted Col I sericin solution as described above, using WT silkworm cocoon sericin as control, and incubated with HIS antibody, and the results are shown in Figure 4 The results show that Col I silkworm sericin has a clear specific band at about 70KD, indicating that recombinant Col I silkworm successfully expresses type I collagen and is secreted into silkworm cocoon silk glue. After the above extracted Col I sericin extract solution was pretreated by mass spectrometry, the types of proteins and amino acid sequences contained in the extract solution were detected and identified, and the results are shown in Table 1. The mass spectrometry results show that the sericin extract solution contains type I collagen, and 10 characteristic peptide segments of type I collagen are identified, further verifying that the transgenic silkworm successfully expresses type I collagen and can be extracted from silkworm cocoons. The Col I sericin solution was subjected to SDS-PAGE experiment with standard concentration BSA, and standard curve was prepared using standard concentration BSA, and gray scale analysis detected that each g of silkworm cocoon contained about 16.13 mg of type 1 collagen, and the results are shown in Figure 5 .

[0043] Table 1, mass spectrometry identified Col I characteristic peptides in the sample

[0044] Sequence Proteins GFPGERGVQGPPGPAGPR sp|P02452|CO1A1_HUMAN Collagen alpha-1(Ⅰ) GLPGERGRPGAPGPAGAR sp|P02452|CO1A1_HUMAN Collagen alpha-1(Ⅰ) GVQGPPGPAGPR sp|P02452|CO1A1_HUMAN Collagen alpha-1(Ⅰ) GERGFPGERGVQGPPGPAGPR sp|P02452|CO1A1_HUMAN Collagen alpha-1(Ⅰ) GARGEPGPTGLPGPPGER sp|P02452|CO1A1_HUMAN Collagen alpha-1(Ⅰ) GEPGPTGLPGPPGERGGPGSR sp|P02452|CO1A1_HUMAN Collagen alpha-1(Ⅰ) GARGEPGPTGLPGPPGERGGPGSR sp|P02452|CO1A1_HUMAN Collagen alpha-1(Ⅰ) GEPGPTGLPGPPGER sp|P02452|CO1A1_HUMAN Collagen alpha-1(Ⅰ) GERGFPGER sp|P02452|CO1A1_HUMAN Collagen alpha-1(Ⅰ) GRPGAPGPAGAR sp|P02452|CO1A1_HUMAN Collagen alpha-1(Ⅰ)

[0045] Example 5, preparation of Col I / silk glue hydrogel

[0046] In order to verify the activity and function of recombinant Col I, Col I / silk glue hydrogel was prepared and the effect on wound healing was explored, and the specific method is as follows: clean silkworm cocoons were crushed and immersed in 8M urea 50mM Tris-HCl solution, extracted at 80℃ water bath for 60min, centrifuged at 13500rpm for 10min, and the supernatant was taken. After the obtained silk glue solution was dialyzed against deionized water at 4~25℃ for 3 days to remove urea, it was placed at 4℃ to induce gel formation.

[0047] In order to explore the effect of different silkworm cocoon addition amount on the formation of silk glue hydrogel, 20mg / ml, 30mg / ml, 40mg / ml, 50mg / ml, 60mg / ml and 70mg / ml were selected to prepare Col 1 / silk glue hydrogel, and the results are shown in Figure 6 . The results show that the bath ratio of 60mg / ml and 70mg / ml can obtain solid silk glue hydrogel. The Col 1 / silk glue hydrogel prepared with 70mg / ml bath ratio was selected for subsequent experiments.

[0048] Example 6, Col I / silk glue hydrogel promotes wound healing in mice

[0049] Full-thickness skin wounds were made on the back of mice on day 0, and divided into 3 groups, 3 mice in each group. The model group was not treated; the sericin group and the Col I / sericin group were treated with WT sericin hydrogel and Col I / sericin hydrogel prepared from WT cocoon, respectively, from day 1. The wound healing was observed by taking photos on day 0, 1, 5, 7, and 14, and the skin at the wound site was taken on day 14 for histological analysis. Figure 7 ).

[0050] From the fifth day, the wound area of the Col I / sericin group was significantly smaller than that of the model group and the sericin group, and the healing speed was faster. The HE staining results of the wound skin on day 14 showed that granulation tissue promoting the generation of new skin appeared at the wound site in the three groups. The granulation tissue in the sericin group and the Col I / sericin group was more than that in the model group, and the cells in the Col I / sericin group were more closely arranged and more new blood vessels were generated under the skin. Figure 8 The Masson staining results showed that the content of new collagen at the wound site in the Col I / sericin group was more than that in the sericin group and the model group, and was more closely arranged.

[0051] Example 7, Production of Recombinant Col I

[0052] Col I was obtained from cocoon by enzymatic hydrolysis and freeze-drying. According to the BIOPEP-UWM enzymatic hydrolysis simulation analysis, all 16 functional sites of Col I, including DGEA, RGD, GER, and KGD, could be retained after hydrolysis by subtilisin, which maximized the retention of Col I efficacy. Therefore, 1 million to 3 million U / L of subtilisin was used to hydrolyze at 40-45°C for at least 3 hours. The effects of hydrolysis at different times are shown in Figure 9 The silk fibroin containing Col I extracted from cocoon by enzymatic hydrolysis was freeze-dried to obtain Col I / sericin freeze-dried powder, and the results are shown in Figure 10 .

[0053] Example 8, CCK8 Assay of Col I / sericin

[0054] The Col I / sericin freeze-dried powder was dissolved in water to prepare Col I / sericin solutions of different concentrations, with WT sericin solution as a control. After co-culturing with Hacat cells for 24 hours, CCK8 experiment was performed to detect the effect of Col I / sericin on cell survival rate, and the results are shown in Figure 11The results showed that Col I / silk sericin had the effect of promoting cell proliferation at the concentration of 0.125%-0.0625%. The cell survival rate was 187% at the concentration of 0.0625%, which was significantly higher than that of the WT group. It was shown that Col I / silk sericin powder had no cytotoxicity, good biocompatibility and cell proliferation activity, which was consistent with the above-mentioned results of Col I promoting wound healing in mice.

[0055] The above-described embodiments are only preferred embodiments of the present application, and the protection scope of the present application is not limited thereto. Any equivalent replacement or transformation made by those skilled in the art based on the present application is within the protection scope of the present application. The protection scope of the present application is subject to the claims.

Claims

1. A silk material with cell proliferation-promoting function, characterized in that: The silk material is made by expressing a truncated fragment of human type I collagen in the silk glands of the silkworm in the middle part of the silkworm and secreting it into the sericin layer of the cocoon. The resulting silk has the function of promoting cell proliferation. The nucleotides of the truncated fragment of human type I collagen are shown in positions 7 to 1578 of SEQ ID NO.

1.

2. The silk material with cell proliferation-promoting function according to claim 1, characterized in that: The silk material is obtained by ligating a truncated fragment of human type I collagen as shown in SEQ ID NO.1 into the BamHI and NotI restriction sites of psl1180[hr3PSer1spRedSer1PA], then digesting it with AscI, and finally ligating it into the pBac[3xp3DsRedaf] vector that has been digested with the same restriction enzymes. The resulting vector is used for microinjection to screen transgenic silkworms. The silk secreted by the transgenic silkworms is the silk that promotes cell proliferation.

3. The method for preparing the silk material with cell proliferation-promoting function as described in claim 1, characterized in that, The process includes the following steps: pulverizing silk and adding 1 million to 3 million U / L of Bacillus subtilis protease, followed by hydrolysis at 40 to 45°C for at least 3 hours.

4. The Col I / sericite lyophilized powder prepared by the preparation method described in claim 3.

5. A method for preparing a material that promotes wound healing, characterized in that, The process includes the following steps: pulverizing the silk material with cell proliferation-promoting function as described in claim 1 or 2, extracting sericin containing human type I collagen fragments using the urea method, removing urea by dialysis, and then inducing gel formation at low temperature to obtain a material that promotes wound healing.

6. The method for preparing the material for promoting wound healing according to claim 5, characterized in that: The urea extraction method involves extracting the solution in 8M urea in a 50 mM Tris-HCl solution at 80°C for 60 min, followed by centrifugation at 13500 rpm for 10 min, and collecting the supernatant.

7. The method for preparing the wound-healing material according to claim 5 or 6, characterized in that: The silk bath ratio in the urea extraction method is 60~70 mg / mL.

8. The method for preparing the wound-healing material according to claim 5, characterized in that: The dialysis is performed at 4-25°C using deionized water for at least 3 days.

9. A material for promoting wound healing prepared by the preparation method according to any one of claims 5 to 7.

10. The use of the Col I / silicone lyophilized powder of claim 4 or the material of claim 9 in the preparation of materials that promote skin regeneration and repair, bone graft substitutes, cartilage reconstruction or artificial skin.

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