Collagen-like material and application thereof

Abstract

The invention discloses a collagen-like material and application thereof, and belongs to the fields of biology and genetic engineering. The invention designs a self-assembled collagen mimic peptide (collagen-like) with functions of promoting blood coagulation, cell regeneration and osteoblast differentiation, and realizes recombinant expression and fermentation production of the collagen-like in escherichia coli. The prepared collagen-like material can form hydrogel at the concentration of more than or equal to 0.08 g / L. The invention also verifies the effects of the collagen-like compound on the aspects of promoting blood coagulation, cell regeneration and osteoblast differentiation. The collagen-like compound provided by the invention shows the blood coagulation capability superior to that of a commercial blood coagulation agent rat tail collagen I, and has huge value and potential in the fields of cosmetics, medicine and tissue engineering.

Description

Technical Field

[0001] The invention relates to a collagen-like substance and application thereof, belonging to the biological field and the genetic engineering field. Background Art

[0002] Collagen accounts for one-third of human protein. They are widely distributed in bones, muscles and blood. There are 28 subtypes of human collagen, most of which are type I, type II and type III. Collagen is an important biomaterial for cosmetics, drug delivery, nutrition, and tissue engineering. The integrity of collagen structure is very important to support its mechanical function and stability. Collagen is composed of Gly-XY repeating sequences, which are the basic units of triple helix, where X and Y are usually proline and hydroxyproline. Collagen becomes tropocollagen by self-assembly to form a triple helical structure, which fundamentally supports the formation of collagen fibers. Collagen fibers in turn support mechanical function under physiological conditions and give collagen properties that make it an ideal biocompatible material for biomedical applications.

[0003] Natural collagen has inherent limitations such as poor stability and immunogenicity, low mechanical strength and weak biological activity. Therefore, the characteristics of natural collagen can be captured by simulating the structural hierarchy of natural collagen to construct collagen-like structures to overcome the limitations of natural collagen. The formation of collagen triple helix promotes the assembly of higher-order oligomers, which is triggered by non-covalent interactions between molecules to form a stable supramolecular structure.

[0004] However, it is still challenging to design a controllable collagenoid that can mimic natural collagen and self-assemble into a triple helix. The self-assembly of collagen is crucial to its biological function. The GXY repeat sequence is the basic unit that supports the formation of a triple helix, and the self-assembly of collagen is determined by the content of charged residues and the degree of proline hydroxylation. In addition, incubation conditions such as temperature, solvent and concentration can also affect collagen self-assembly. Therefore, it is important to design a collagenoid that can self-assemble into a triple helix.

[0005] Existing recombinant proteins are mainly produced by four major expression systems: prokaryotic (E. coli) expression system, Pichia pastoris expression system, mammalian cell expression system, and insect cell (baculovirus) expression system. There are few studies on mammalian cell expression, and mammalian cell expression has high cost and low yield, and is mostly used for the expression and production of high-value pharmaceutical proteins. In addition to high cost and low yield, the insect cell (baculovirus) expression system is very different from human cells after translation. Therefore, the mass production of collagen is mainly completed by the prokaryotic (E. coli) expression system and the Pichia pastoris expression system. Summary of the invention

[0006] In order to solve the above problems, the sequence generation model (ColDiff) of the diffusion model is used in the present invention to learn the amino acid sequence characteristics from natural collagen to generate functional collagen-like substances, and the corresponding collagen-like substances are expressed through Escherichia coli and Pichia pastoris recombinant protein expression systems to prepare collagen products with the ability to induce blood coagulation, osteoblast differentiation and cell adhesion.

[0007] The present invention provides a collagen-like substance comprising the amino acid sequence shown in (a) or (b):

[0008] (a) GVKGYRGAPGENGEDGLQGFPGLKGEPGIQ (SEQ ID NO: 1);

[0009] (b) GVKGYRGAO GENGEDGLQGFOGLKGEOGIQ (SEQ ID NO. 2).

[0010] The present invention provides a gene encoding the collagen-like sequence.

[0011] In one embodiment, the nucleotide sequence of the gene is shown as SEQ ID NO.3.

[0012] The present invention also provides a recombinant expression vector, which contains a gene encoding the collagen-like sequence.

[0013] In one embodiment, the recombinant expression vector includes but is not limited to pET series, Duet series, pGEX series, pHY300, pHY300PLK, pPIC3K, pPIC9K or pTrc series vectors.

[0014] In one embodiment, the pET series vectors include pET-24a(+), pET-28a(+), pET-29a(+), and pET-30a(+); the Duet series vectors include pRSFDuet-1 and pCDFDuet-1; and the pTrc series vectors include pTrc99a.

[0015] The present invention also provides a recombinant microbial cell expressing the collagen-like protein or containing the gene.

[0016] In one embodiment, the host of the microbial cell includes, but is not limited to, Escherichia coli, Bacillus subtilis or Pichia pastoris.

[0017] In one embodiment, the Escherichia coli includes Escherichia coli JM109 (DE3), Escherichia coli HMS174 (DE3), Escherichia coli BL21 (DE3), Escherichia coli Rostta2 (DE3), Escherichia coli Rosttagami (DE3), Escherichia coli Rostta2 (DE3), Escherichia coli DH5α, Escherichia coli W3110 and / or Escherichia coli K12.

[0018] In one embodiment, the recombinant microorganism uses Escherichia coli as a host to express a collagen-like protein having an amino acid sequence shown in SEQ ID NO.2.

[0019] In one embodiment, the recombinant microorganism uses Escherichia coli BL21 (DE3) as a host and pET-22b (+) as a vector to express the gene sequence shown in SEQ ID NO.3.

[0020] The present invention also provides a method for preparing the collagen-like substance, wherein the method utilizes the recombinant microbial cell to ferment and produce the collagen-like substance.

[0021] In one embodiment, the recombinant microbial cells are cultured at 37°C for 10-12 hours to obtain a seed solution, which is then inoculated into 50 mL of TB medium at an inoculum volume of 0.1%-2% (v / v) and cultured until the fermentation solution OD reaches 0. 600 When the value reached 1, IPTG with a final concentration of 0.1 mM was added for induction. After 20 h of induction at 20°C, the fermentation was terminated and the collagen in the cells was collected.

[0022] In one embodiment, the cells are further subjected to cell wall disruption treatment.

[0023] In one embodiment, the supernatant is collected after cell disruption, and the supernatant is purified to obtain collagen.

[0024] The invention also provides a medicine containing the collagen-like substance.

[0025] The invention also provides application of the collagen-like substance in preparing products for promoting blood coagulation.

[0026] In one embodiment, the product includes, but is not limited to, a drug.

[0027] In one embodiment, the product includes but is not limited to hemostatic materials, medical dressings, wound care materials, surgical materials, drug delivery carriers, artificial synthetic skin materials, etc.

[0028] The present invention also provides the use of the collagen-like substance in preparing a medicine for promoting cell regeneration and / or promoting osteoblast differentiation.

[0029] The present invention also provides the use of the collagen-like substance in preparing injection filling materials, regenerative tissue engineering scaffolds and bone repair materials.

[0030] The invention also provides application of the collagen-like substance in preparing food, health products or cosmetics.

[0031] Beneficial effects:

[0032] (1) The present invention establishes a series of models with the assistance of computers to design functional self-assembling collagen mimetic peptides (collagen-like peptides), and obtains polypeptide sequences with self-assembly ability based on the designed models.

[0033] (2) The present invention achieved recombinant expression of the designed polypeptide in Escherichia coli and verified that the recombinantly expressed protein can self-assemble into a triple helical structure and form a hydrogel at a minimum concentration of 0.8 mg / mL.

[0034] (3) The present invention also provides the use of the prepared collagen-like substance in promoting coagulation, cell regeneration and osteoblast differentiation, showing coagulation ability superior to that of the commercial coagulant rat tail collagen I, and has great value and potential in the fields of cosmetics, medicine and tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the process of establishing the diffusion model of self-assembled collagen-like; (A) The diffusion model is developed based on amino acid sequence learning, and the diffusion model is used to learn the characteristics of collagen and generate collagen-like; (B) The accuracy of the diffusion model is evaluated by calculating the proportion of continuous GXY repeating sequences in the generated sequence; (C) The accuracy of the model is obtained based on ColNet cross-validation. The accuracy is evaluated by calculating the Pearson correlation between the predicted Tm value and the actual Tm value; (D) The Gaussian distribution of the predicted Tm value and the actual Tm value.

[0036] Figure 2 This is the verification result of collagen-like self-assembly to form triple helix; among them, (A) the correlation between predicted and actual Tm values; (B) the CD spectra of independently designed collagen-like, which are sample 1, sample 9, sample 18, sample 23, sample 24, sample 31, sample 32, sample 34, sample 36, sample 39, sample 40, sample 43, sample 6, and sample 35.

[0037] Figure 3The results of recombinant expression of the independently designed collagen-like. Among them; (A) SDS-PAGE of the collagen-like recombinantly expressed by E. coli; the collagen-like sample E3 is numbered 3; "↑" represents the fermentation supernatant, and "↓" represents the fermentation precipitate; (B) SDS-PAGE of the collagen-like purified from E. coli expression; "Before" and "After" refer to the sample E3 before and after heat treatment.

[0038] Figure 4 The CD spectra of recombinantly expressed collagen-like proteins and the transmission electron microscopy (TEM) and scanning electron microscopy (SEM) results of collagen-like samples; wherein, (A) the sample E3 expressed by Escherichia coli was prepared into a sample protein with a concentration of 0.2 mg / mL and incubated at 4°C, and then observed by CD spectroscopy; (B) the collagen-like protein with a concentration of 1 mg / mL was incubated at 4°C for 12 hours and then observed by transmission electron microscopy; (C) the collagen sponge made by freeze-drying the collagen-like sample was observed under a scanning electron microscope.

[0039] Figure 5 The validation results of the hydrogel prepared by using the collagen-like substance; wherein, (A) the oscillatory shear rheological analysis results of collagen-like substances of different concentrations; (B) the changes of the storage modulus G' and loss modulus G" of the collagen-like substance with a sample concentration of 5 mg / mL.

[0040] Figure 6 It is the coagulation ability of collagen-like samples; among them, (A) the cell proliferation rate of L929 cells after adding 2 mg / mL collagen-like samples and commercial rat tail collagen I; (B) the swelling rate of hydrogel samples cultured with mouse blood and the samples of hydrogel before and after blood culture.

[0041] Figure 7 In vitro blood coagulation test results.

[0042] Figure 8 Results of in vivo blood coagulation experiment.

[0043] Fig. 9 The results are verified by cell proliferation and adhesion ability. DETAILED DESCRIPTION

[0044] Example 1 Design of collagen-like sequences

[0045] (1) Generate collagen-like proteins by combining generative and supervised models

[0046] ColDiff is a variant of the diffusion model developed by the inventor team for the de novo generation of functional promoter sequences. A training set was constructed by collecting 28 natural collagens, which retrieved amino acid sequences with a length of 30 amino acids and continuous "GXY" repeating units from natural collagens. A total of 7270 sequences were obtained. After evaluation, the above sequences were all able to form triple helices naturally. ColDiff is a diffusion model that integrates the features of UNet for feature extraction on the encoder side and sample size recovery on the decoder side. The collagen sequence features are extracted using the one-hot encoding method, and Gaussian noise is gradually added. The diffusion model is used to learn the noise addition and denoising process. Recover the original sequence based on the input sequence ( Figure 1 A), network architecture analysis using deep learning modules such as CNN, ResNet and SA. The quality of the generated sequence is evaluated by querying the continuous GXY repetition part within the generated sequence. The ResNet network is used to solve the gradient vanishing problem by skipping connections and summing subsequent layers, and self-attention is used to link partial information to the overall information. These two networks are used on the encoder side, and the sampled information is used on the decoder side.

[0047] The best model was able to generate continuous GXY repeats at >92% after 50 training epochs ( Figure 1 B). After adjusting the training parameters, a total of 25,600 sequences were generated. The sequence logo analysis showed that the logos of the generated sequences were very similar to the input sequences, with continuous GPO repeats as obvious logos and charged residues as minor logos. At the same time, reported functional motifs such as GAOGEN, GLKGEN, and GLOGEN were found in the generated sequences. Overall, the generated collagenoids were very similar to the collagens in the training set.

[0048] In order to study the transition between triple helix and disordered states and investigate the amino acid sequence features that affect triple helix formation, a supervised model ColNet was used for collagen Tm prediction. The ColNet network is integrated by ResNet and self-attention through network optimization. ColNet learns collagen features and predicts Tm values ​​by treating the process as a regression prediction problem. The learning rate and batch size are optimized based on the mean squared error between the predicted and true Tm values. The accuracy of the model is defined as the Pearson correlation between the predicted and true Tm values. The final supervised model achieved 95% (r 2 =90.2%) Pearson correlation ( Figure 1 C). The generated collagenoids and collagen from the training set have similar melting temperature Gaussian distributions, with the highest points of the two correlation curves being 23.5°C and 23.8°C, respectively ( Figure 1D), demonstrating a high correspondence between the generated collagenoid and native collagen.

[0049] (2) Characterization of the self-assembly ability of the designed collagen-like

[0050] The self-assembly of collagen into triple helices can fundamentally support its mechanical properties. The formation of triple helical structure is the most important characteristic of collagen fibril formation, network formation and other collagen formation processes. Here, triple helix formation was analyzed by evaluating the CD curve. The positive peak at 220-222nm indicates the formation of collagen triple helical structure, and the collagen-like Tm value can also reflect the formation and unwinding of triple helices. Based on the Tm and functional domains of ColDiff-generated sequences, we selected 50 sequences with Tm varying between 3-37℃ as training sets, of which 30 sequences reported functional domains (Table 1)37. 33 synthetic samples out of 50 sequences presented a positive peak at 220-222nm, indicating that the success rate of ColDiff was 66%. The predicted Tm value and the true Tm value have a Pearson correlation of 0.8 ( Figure 2 A), indicating that the combined generation network and Tm prediction module is highly effective for generating self-assembling collagen-like structures.

[0051] At the same time, the effect of temperature on the formation of triple helix collagen was also studied. Since 25℃ and 37℃ are most relevant to bioengineering applications, we tested the triple helix formation effect of 15 samples with Tm values ​​>25℃ and 3 samples with Tm values ​​>37℃. The results showed that samples with Tm values ​​higher than the incubation temperature by >5℃ can stably maintain triple helix formation ( Figure 2 B), confirming the temperature-dependent transition from triple helix to disordered state. The above results indicate that not all consecutive GXY repeats can assemble into triple helices and that the self-assembly process depends on the incubation temperature.

[0052] Table 1 Collagen-like sequences for sequence and property verification

[0053]

[0054]

[0055] Example 2 Recombinant expression of collagen in Escherichia coli

[0056] The recombinant plasmid pET-22b(+) was constructed in E.coli BL 21(DE 3) using plasmid pET-22b(+). Since E. coli lacks post-translational modification, it is difficult to achieve effective expression of collagen sequences containing proline (P). Therefore, when the sequences shown in Table 1 are expressed in E. coli, proline (P) needs to be replaced with pyrrolysine (O). Preliminary experiments have shown that replicating the amino acid fragment of the collagen-like polypeptide up to 16 times is conducive to the recombinant expression of collagen-like polypeptides in E. coli. Genes encoding collagen-like polypeptides (e.g., the polypeptide sequence shown in SEQ ID NO.2 is encoded by the gene shown in SEQ ID NO.3) were optimized and synthesized by Genscript (Nanjing, China) based on the collagen-like polypeptide sequences shown in Table 1. The genes were cloned into pET-22b(+) through the NdeI-BlpI sites and transformed in E. coli. A single colony was inoculated into Luria-Bertani medium as a seed culture, incubated at 37°C, 220 rpm in a shaker for 10 h, and then inoculated into TB medium supplemented with ampicillin at a final concentration of 50 μg / mL. 600 When it reached 1.0, IPTG was added at a final concentration of 0.1 mM to induce protein expression, and the culture was continued for 20 h in a shaker at 20°C and 220 rpm, and the bacterial cell culture fluid was collected.

[0057] Example 3 Extraction and purification of collagen from Escherichia coli

[0058] Collect the bacteria obtained by culture in Example 2, add Tris buffer at a volume ratio of 1:1 (for example: add 50 mL of crushing buffer to the bacteria obtained from 50 mL of fermentation broth), crush the bacteria by a high-pressure homogenizer, and collect the supernatant by centrifugation. Protein purification was performed using affinity chromatography using a His-Trap column (GE Healthcare, New York, USA). Collagen was eluted by linear gradient elution using 50 mM Tris-HCl and 100 mM NaCl (pH 7.5) supplemented with 500 mM imidazole, and then gel filtered using a Superdex 75 column (GE Healthcare) and eluted with PBS (pH 7.5). The collagen solution can restore its natural structure after high-temperature induced unwinding, so the purified sample was protease cleaved to remove the His tag, then heated to 85°C, cooled, centrifuged and dialyzed to remove contaminating proteins and cleaved tags to obtain high-purity collagen ( Figure 3 B).

[0059] Example 4 Characterization of structural properties of collagen

[0060] The sample obtained by the E. coli fermentation in Example 3 was subjected to SDS-PAGE analysis. The recombinant collagen expressed by E. coli can be expressed in a soluble form ( Figure 3 A), the sample E3 region showed an obvious band, and the protein was confirmed to be the target protein by Maldi-Tof.

[0061] The samples prepared in Example 3 were tested as follows:

[0062] (1) Circular dichroism (CD) detection

[0063] The collagenoid prepared in Example 3 was prepared into a collagenoid solution with a concentration of 0.2 mg / mL in PBS (pH 7.5) and incubated at 4°C for 12 h before CD measurement. CD spectra were recorded at 4°C using a Photophysics Chirascan instrument equipped with a Peltier temperature controller (Model 110-OS; Hellma, Shanghai, China) and a quartz cuvette with an optical path length of 1 mm (Model 110-OS; Hellma). The scanning wavelength range was 190-250 nm. The changes in ellipticity at 220 nm during heating at 0-70°C and 1°C / min were recorded, and the Tm value was calculated based on the melting curve.

[0064] CD was performed on the expressed protein to confirm triple helix formation. Sample E3 purified from E. coli showed a positive peak at 220-222 nm ( Figure 4 A), demonstrating that it can self-assemble into a triple helical structure.

[0065] (2) Transmission electron microscopy (TEM) detection

[0066] The collagen-like PBS (pH 7.5) prepared in Example 3 was formulated into a collagen-like solution with a concentration of 1 mg / mL and incubated at 4°C. The obtained solution was loaded onto a copper grid, allowed to absorb for 1 minute, and the excess solvent was removed using filter paper. The samples were negatively stained with 0.75% phosphotungstic acid before imaging using a Hitachi H-7650 electron microscope (Hitachi, Tokyo, Japan). The sample width was evaluated using ImageJ (https: / / imagej.net / downloads). Sample E3 was able to detect the formation of nanofibers ( Figure 4 B).

[0067] (3) Scanning electron microscope (SEM) inspection

[0068] The morphology and microstructure of collagen sponges were characterized using SEM SU8220 (Hitachi). The collagen-like gel was prepared into a sponge by freeze drying. The dried samples were loaded onto a SEM disk and imaged.

[0069] Sample E3 was found to be porous ( Figure 4 C).

[0070] Example 5 Preparation of hydrogel using collagen-like

[0071] The formation of collagen hydrogel is very important for the development of biocompatible materials for cell adhesion and proliferation. The formation of a triple helix structure is the basis, followed by the formation of nanofibers, and finally the formation of a gel. Here, the gelation test was performed on the samples that can form a triple helix. The samples prepared in Example 3 were dissolved in PBS solution at different concentrations (e.g., 0.8 mg / mL, 1 mg / mL, 2 mg / mL) and incubated at 4°C. After incubation at 4°C for 8 hours, sample E3 with a minimum concentration of 0.8 mg / mL could form a hydrogel ( Figure 5 A). The storage modulus G' and loss modulus G" at low deformation amplitude were measured to determine the elasticity and viscosity of the hydrogel. For the above samples, the storage modulus was more than twice that before treatment, indicating that all tested samples formed hydrogels. Increasing the concentration to 5 mg / mL and incubating for up to 7 days resulted in the formation of hydrogels in the samples ( Figure 5 B) Hydrogels are brittle and can be easily converted to the liquid phase by shaking or increasing the temperature.

[0072] According to the above method, the sample concentration was further increased from 1 mg / mL to 50 mg / mL. The results showed that increasing the protein concentration could increase the rigidity of the hydrogel, so that the obtained hydrogel no longer transformed from a gel state to a liquid flow state when shaken.

[0073] Example 6 Cytotoxicity Detection of Collagen

[0074] The collagenoids prepared in Examples 2 to 3 and commercial rat collagen I (Merck, Shanghai, China) were treated by freeze drying at -60°C and 0.01-0.1 MPa for 24 h to prepare collagen sponges for L929 fibroblasts (ATCC, China) to evaluate their toxicity. The culture medium contained 10% (v / v) fetal bovine serum (Ausbian, Australia) and 1% (v / v) penicillin-streptomycin. L929 cells were cultured for 48 hours and 10 cells were added to each well. 4The cells were transferred to a new culture plate at a cell density of 10 cells. The cells were allowed to adhere for 12 hours, and then 1 mL of culture medium supplemented with 2 mg / mL collagen sponge was added. The cells were cultured for 48 hours, centrifuged, the solution was discarded, and the cell number was measured using a CCK-8 kit (Sangon). The proliferation rate of L929 cells was evaluated by using the following cell counting formula: Proliferation rate (%) = (total number of cells after culture) / (total number of cells before culture). When the selected samples were added to the culture medium at a concentration of 2 mg / mL, the cell proliferation rate ranged from 139.2% to 220.8% ( Figure 6 A), indicating that all tested samples were non-toxic.

[0075] Example 7 Application of collagen in promoting blood coagulation

[0076] Collagen is believed to perform dual functions by supporting blood coagulation and wound healing. They can promote platelet adhesion by binding to platelet receptors and subsequently induce endogenous hemostasis mechanisms to help wound healing. Collagen also supports the migration of fibroblasts and macrophages to promote tissue regeneration. Sample E3, which can be quickly assembled into a hydrogel prepared in Examples 2 to 3, was used for in vitro and in vivo coagulation experiments, respectively.

[0077] (1) In vitro coagulation ability test

[0078] Collagenoids were prepared according to the method of Examples 2 to 3. Sample E3 and commercial rat tail collagen I (Merck, Shanghai, China) were dissolved in PBS solution at a concentration of 1 mg / mL to prepare collagenoids and collagen I samples, and platelet-poor plasma was supplemented to a concentration of 2 mg / mL during the culture period. The platelet-poor plasma was centrifuged at 2000×g for 10 min to extract the supernatant. After culturing at 25°C for 30 min and centrifuging at 2000×g for 20 min, the supernatant was collected and the cells were counted using a CCK-8 kit. The coagulation rate was calculated by the following formula: Blood coagulation rate (%) = 1-(cells from the supernatant after culture) / (total cells before culture) Collagenoids were used to prepare hydrogels to determine the swelling rate. The hydrogels were cultured with mouse blood (Gibco, Shanghai, China) at 25°C and rinsed with PBS. Excess liquid was removed using filter paper, and the initial weight of the hydrogel and the weight after blood swelling were recorded to calculate the swelling rate. In addition, the collagen sponge was mixed with anticoagulated mouse blood, wherein the concentration of collagen in the mixed solution was 2 mg / ml, and 10 mM CaCl2 was added to the mixture. The coagulation test was performed at 37°C for 10 min, and the absorbance at 540 nm before and after coagulation was recorded to evaluate the coagulation rate. Swelling rate (%) = 1-(weight of hydrogel before culture) / (weight of hydrogel after culture)

[0079] The results are as follows Figure 6 , Figure 7 The results showed that after treatment at 25°C for 30 minutes, sample E3 induced 96% blood cell coagulation. In contrast, COL 1 only induced 81% cell coagulation ( Figure 6 A, Figure 7 ).

[0080] (2) In vivo coagulation ability test

[0081] All experiments for in vivo coagulation assays were performed in accordance with ethical rules for animal treatment. 1 cm tail tip of 7-8 week old mice was cut off. Collagenoid and collagen I samples were prepared at 2 mg / mL and applied to the wound, and PBS was applied to the wound as a negative control. The process was recorded by a digital camera.

[0082] The results showed that after application to the wound for 10 min, sample E3 showed the strongest coagulation ability and showed better coagulation ability than commercial rat tail collagen I (Merck, Shanghai, China) ( Figure 8 ). Therefore, this type of collagen appears to be a robust in vivo coagulation component. The swelling properties of collagen-like substances also affect the hemostatic ability. Collagen-like sample E3 (which can quickly form hydrogels at low concentrations) and commercial collagen I were prepared into hydrogels with a concentration of 20 mg / mL. The blood retention capacity of the samples was then evaluated by weighing the co-cultured mouse blood and the selected samples. After incubation for 6 minutes, the swelling ratio of the sample hydrogels varied between 25% and 59% ( Figure 6 B). In addition, platelet adhesion was observed on the surface of all sample hydrogels, further confirming that collagen has hemostatic ability ( Figure 6 B).

[0083] Example 8 Collagen-like Promotes Cell Regeneration

[0084] Collagen assembles into ribbon-like fibers that support bone formation and matrix mineralization, and collagen fibers can induce stem cell proliferation and differentiation, thereby promoting tissue regeneration.

[0085] (1) Cell proliferation and adhesion

[0086] The cell adhesion and cell regeneration abilities of sample E3 were tested as follows: 1×10 4 Mouse embryonic osteoblasts (MC3T3 cells) were cultured using Gibco MEM (Invitrogen, Shanghai, China), 10% (v / v) fetal bovine serum, and 1% (v / v) penicillin-streptomycin (supplemented with 2 mg / mL lyophilized collagen or collagen I). The cell proliferation rate was recorded using a CCK-8 kit during 5 days of culture. For the cell adhesion test, 2 mL of collagen and collagen I samples were supplemented to culture the cells. During the cell adhesion test, 1×104 Pre-cultured MC-3T3 cells were added to the well plate, cultured for 12 hours to allow cell adhesion, washed with PBS, added with culture medium, and counted using CCK-8 kit. Cell adhesion (%) = 1-(weight of CLP sponge before culture) / (weight of CLP sponge after culture)

[0087] The collagen sponge for MC-3T3 cell culture was prepared by freeze drying according to the method of Example 6. BSA and commercial collagen I were used as negative and positive controls, respectively. During the first 3 days of culture, the designed collagen and collagen I supplemented with 2 mg / mL promoted cell proliferation more effectively than BSA, and the proliferation was similar on the 5th day ( Fig. 9 A).

[0088] As described above, cell adhesion analysis was performed by adding 2 mg / mL of collagenoid and collagen I during the culture of MC-3T3 cells. After incubation, the adhered cells were resuspended before cell counting. The results showed that after 6 hours of incubation, collagen I and sample E3 collagenoid promoted a 3-fold higher cell adhesion rate than BSA ( Fig. 9 B). The cell area of ​​sample E3 collagen is >88% larger than that of the collagen sample when BSA is used alone ( Fig. 9 C, 9D). These results indicate that the designed collagenoids can promote cell adhesion.

[0089] (2) Detection of collagen-like cells promoting osteoblast differentiation

[0090] The genes VCL and ACTN are reported to be associated with the formation of focal adhesion sites and occupancy of cytoplasmic actin-binding proteins, and the mRNA levels of VCL and ACTN can be used to evaluate osteoblast differentiation. The ability of the collagen-like sample E3 in promoting osteoblast differentiation was tested as follows, with commercial rat tail collagen I (Merck, Shanghai, China) as a positive control. The gene expression levels of MC-3T3 cells under the aforementioned different culture conditions were measured by real-time quantitative PCR (RT-qPCR). Total RNA was extracted from MC-3T3 cells using TRIzol reagent (Invitrogen). RNA was reverse transcribed into cDNA using the One Step RT-qPCR kit (Sangon) and the QuantStudio 3RT-qPCR instrument (Thermo Fisher, Shanghai, China). The primers are shown in Table 2, and the gene expression levels were referenced to GAPDH. The results showed that samples E3 and collagen I showed the ability to upregulate VCL and ACTN, and the mRNA levels of the given two genes were 2 times higher than those using BSA ( Fig. 9B) These results indicate that the designed collagenoids can promote cell differentiation.

[0091] Table 2 RT-qPCR primers

[0092]

[0093] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A collagen-like substance, characterized in that: Containing the amino acid sequence shown in (a) or (b): (a)GVKGYRGAPGENGEDGLQGFPGLKGEPGIQ; (b)GVKGYRGAOGENGEDGLQGFOGLKGEOGIQ.

2. A gene encoding the collagen-like protein according to claim 1.

3. A recombinant expression vector containing the gene according to claim 2.

4. A recombinant microbial cell expressing the collagen-like substance of claim 1 or containing the gene of claim 2.

5. A recombinant Escherichia coli, characterized in that The collagen-like protein of claim 1 is expressed by using Escherichia coli BL21 (DE3) as a host and pET-22b (+) as a vector.

6. A method for preparing the collagen-like substance according to claim 1, characterized in that: The collagen-like substance is produced by fermentation using the recombinant microbial cell described in claim 4 or the recombinant Escherichia coli described in claim 5.

7. A medicament containing the collagen-like substance according to claim 1.

8. Use of the collagen-like substance according to claim 1 in the preparation of a product for promoting blood coagulation.

9. Use of the collagen-like substance according to claim 1 in the preparation of a drug for promoting cell regeneration and / or osteoblast differentiation, or in the preparation of a biomaterial, characterized in that: The biomaterials include but are not limited to injectable filling materials, regenerative tissue engineering scaffolds, and bone repair materials.

10. Use of the collagen-like substance according to claim 1 in the preparation of food, health products or cosmetics.

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